Near-infrared imaging of in vivo performance of orally administered solid forms to rats: Feasibility study with indocyanine green.

This study demonstrates the applicability of near-infrared (NIR) imaging to evaluating in vivo oral formulation performance. As a NIR probe and model drug, indocyanine green (ICG) and acetaminophen (ACE) were selected, respectively. The fluorescence intensity of ICG greatly increased upon dissolution, with the dissolved ICG passing through the gastrointestinal tract over time. Both compounds (0.05 mg of ICG and 0.5 mg of ACE) were encapsulated in gelatin and hydroxypropyl methylcellulose (HPMC) capsules in the solid form. In vitro, the HPMC capsules showed a disintegration lag time, a feature that was not observed for the gelatin capsules. After oral administration of each capsule to rats, blood samples were collected, followed by fluorescent imaging of the abdominal region. At 0.25 h after HPMC capsule administration, the fluorescence area and intensity were significantly small and relatively weak compared to that of the gelatin capsule. These tendencies resulted from the difference in capsule disintegration times, leading to a change in gastric emptying, which corresponded well with the initial time profile of the plasma concentration of ACE. These results indicate that possibility of NIR imaging with ICG to evaluate in vivo performance of orally administered formulations.

The effects of degree and duration of supersaturation on in vivo absorption profiles for highly permeable drugs, dipyridamole and ketoconazole.

The prediction of oral absorption from a supersaturating drug delivery system (SDDS) remains a significant challenge. Here we evaluated the effects of the degree and duration of supersaturation on in vivoabsorption for dipyridamole and ketoconazole. Various dose concentrations of supersaturated suspensions were prepared by a pH shift method, and in vitro dissolution and in vivo absorption profiles were determined. For dipyridamole, the duration of supersaturation decreased with the increase of the dose concentration owing to rapid precipitation. For ketoconazole, the initially constant dissolved concentrations due probably to the liquid-liquid phase separation (LLPS) as a reservoir were observed at high dose concentrations. However, the LLPS did not delay the peak plasma concentration of ketoconazole in rats, indicating that drug molecules were immediately released from the oil phase to the bulk aqueous phase. For both model drugs, the degree of supersaturation, but not the duration of supersaturation, correlated with systemic exposure, indicating quick drug absorption before precipitation. Therefore, the degree of supersaturation is an important parameter compared with the duration of supersaturation for enhancing the in vivo absorption of highly permeable drugs. These findings would help develop a promising SDDS.

Contribution analysis of metabolic tissues on systemic exposure of an active metabolite after oral administration of verapamil using a stable isotope-labeled compound.

The present study illustrates the advantage of an isotope-IV study for the contribution analysis of metabolic tissues on systemic exposure of metabolites. A model parent drug, verapamil (VER), and its metabolite, norverapamil (Nor-VER), were used. This isotope-IV study used rats with and without the pre-treatment of the CYP inhibitor 1-aminobenzotriazole (ABT), was performed by the oral administration of VER (1 mg/kg) combined with the intravenous administration of stable isotope-labeled VER (VER-d6, 0.005 mg/kg). Plasma concentration profiles of both compounds and respective metabolites (Nor-VER, Nor-VER-d6) were then evaluated by LC-MSMS. VER oral availability was increased, and the systemic clearance decreased, in addition, the relative systemic exposure of Nor-VER and Nor-VER-d6 was increased by ABT pre-treatment. PK analyses revealed that, in ABT untreated rats, most Nor-VER in systemic circulation originated from the intestinal absorption process. ABT pre-treatment increased the contribution ratio to the systemic exposure of Nor-VER from the hepatic metabolism of systemically circulated VER, and decreased the contribution ratio of intestinal metabolism. These findings indicated that the isotope-IV study may be useful for considering the PK profile of metabolites.

The impact of surface area per volume (SA/V) ratio on drug transport from supersaturated solutions of ketoconazole.

The purpose of this study aimed to evaluate the impact of the surface area per volume (SA/V) ratio on drug transport from two supersaturated solutions (SSs) of ketoconazole with and without hydroxypropyl methylcellulose (HPMC), used as a precipitation inhibitor. In vitro dissolution, membrane permeation with two SA/V ratios, and in vivo absorption profiles for both SSs were determined. For the SS without HPMC, a two-step precipitation process due to the liquid-liquid phase separation was observed; the constant concentration with approximately 80 % of the dissolved amount was maintained for the first 5 min and subsequently decreased between 5 and 30 min. For the SS with HPMC, a parachute effect was observed; the constant concentration with approximately 80 % dissolved amount was maintained for more than 30 min and decreased very slowly thereafter. Assessment of the SA/V ratio using in vitro and in vivo models demonstrated that when the SA/V ratio was small, the SS with HPMC resulted in a significantly higher permeated amount than the SS without HPMC. In contrast, when the SA/V ratio was large, the HPMC-mediated parachute effect on drug transport from SSs was attenuated, both in vitro and in vivo. The parachute effect by HPMC decreased as the SA/V ratio increased, and the performance of supersaturating formulations would be overestimated by in vitro studies with small SA/V ratios.

Effects of Ingested Water Volume on Oral Absorption of Fenofibrate, a BCS Class II Drug, from Micronized and Amorphous Solid Dispersion Formulations in Rats.

In this study, we investigated the effects of ingested water volume on the oral absorption of fenofibrate (FEN) with several formulations to confirm the applicability of rats for oral formulation screening. Oral absorption of suspended crystalline FEN was significantly improved by increasing ingested water volume (from 0.5 to 2 mL). FEN absorption improvement by particle size reduction and the linearity in oral absorption by dose escalation suggested that the rate-limiting step of FEN absorption in rats was the dissolution rate, consistent with that in humans. When FEN, as an amorphous solid dispersion (ASD) formulation, was suspended in water followed by immediate administration, oral FEN absorption was significantly higher than when administered in crystalline form and was not influenced by the differences in ingested water volume. Oral absorption of FEN from encapsulated ASD formulation in 1 or 2 mL of water was comparable with that of the suspension form. However, 0.5 mL of water significantly reduced the oral absorption of the solid ASD FEN formulation. These results indicate that to improve the oral absorption of poorly water-soluble drugs when performing a preclinical study with rats, 1 mL of water is the minimum preferable ingested volume to evaluate in vivo formulation performance.

Maximizing the Oral Bioavailability of Poorly Water-Soluble Drugs Using Novel Oil-Like Materials in Lipid-Based Formulations.

Lipid-based formulations, such as self-microemulsifying drug-delivery systems (SMEDDSs), are promising tools for the oral delivery of poorly water-soluble drugs. However, failure to maintain adequate aqueous solubility after coming into contact with gastrointestinal fluids is a major drawback. In this study, we examined the use of a novel cinnamic acid-derived oil-like material (CAOM) that binds drugs with a high affinity through π-π stacking and hydrophobic interactions, as an oil core in a SMEDDS for the oral delivery of fenofibrate in rats. The use of the CAOM in the SMEDDS resulted in an unprecedented enhancement in fenofibrate bioavailability, which exceeded the bioavailability values obtained using SMEDDSs based on corn oil, a conventional triglyceride oil, or Labrasol, an enhancer of intestinal permeation. Further characterization revealed that the CAOM SMEDDS does not alter the intestinal permeability and has no inhibitory activity on P-glycoprotein-mediated drug efflux. The results reported herein demonstrate the strong potential of CAOM formulations as new solubilizers for the efficient and safe oral delivery of drugs that have limited water solubility.

In vitro-in vivo correlation in the effect of cyclodextrin on oral absorption of poorly soluble drugs.

In this study the concentration effect of 2-Hydroxypropyl-beta-cyclodextrin (HP-ßCyD) on oral drug absorption of the BCS class II drugs Danazol (DNZ) and Albendazole (ABZ) was evaluated. In vitro permeation of solutions and suspension systems was compared with their in vivo intestinal absorption in rats and their in vitro-in vivo correlation assessed. In solutions excess amounts of HP-ßCyD decreased both in vitro permeation and in vivo absorption due to the decrease in free drug concentration, as expected. However, in suspension systems the contribution of HP-ßCyD by drug complexation was found to be altered by further rate limiting steps for membrane permeation and intestinal absorption of each drug. In vitro permeation of DNZ was rate-limited by the diffusion into the unstirred water layer (UWL), while that of ABZ was rate-limited by the permeation across the lipid membrane. For the in vivo intestinal absorption, both drugs were rate-limited by the dissolution rate from undissolved drug. These differences in the rate-limiting process were considered to cause discrepancies in the result of in vitro and in vivo assays. In conclusion, it is quite important to understand the rate limiting process of oral absorption of the target drug for designing oral liquid formulations containing cyclodextrins.

An enteric polymer mitigates the effects of gastric pH on oral absorption of poorly soluble weak acid drugs from supersaturable formulations: A case study with dantrolene.

This study demonstrated that an enteric polymer can mitigate the effects of gastric pH on the oral absorption of a poorly water-soluble weak acid drug, dantrolene (DNT). An amorphous solid dispersion (ASD) of DNT with hydroxypropyl methylcellulose (HPMC) acetate succinate (ASD-HPMCAS) was prepared as the enteric released ASD (ER-SF). ASD with HPMC (ASD-HPMC) and DNT sodium salt were also used as immediate-release supersaturable formulations (IR-SFs) with and without water-soluble polymer, respectively. In vivo study with rats and in vitro study with a dissolution/permeation (D/P) system were performed to evaluate oral DNT absorption from each formulation under normal and high gastric pH conditions in rats and humans, respectively. The oral absorption of DNT from both IR-SFs in rats with a high gastric pH was significantly higher than that in rats with a normal gastric pH. In contrast, ASD-HPMCAS attenuated the difference in oral absorption between normal and high gastric pH conditions with significant improvement of DNT absorption. In vivo results implied that an enteric polymer delayed the onset of dissolution until after gastric emptying. ASD-HPMCAS generated supersaturation in the small intestine irrespective of gastric conditions, which was supported bythe in vitrostudy using the D/P system. This study suggested that an enteric polymer is useful to mitigate the inter- and intra-individual differences in oral absorption of poorly water-soluble weak acid drugs.

Species differences in the drug-drug interaction between atorvastatin and cyclosporine: In vivo study using a stable isotope-IV method in rats and dogs.

In this study, drug-drug interaction (DDI) between atorvastatin (ATV) and cyclosporine (CsA) was kinetically analyzed using a stable isotope-IV method in rats and dogs. Obtained results were compared with the clinical data quoted from literatures to clarify the species difference in DDI both qualitatively and quantitatively. ATV only or ATV with CsA was orally administered to rats or dogs, and at 90 minutes after administration, a small amount of deuterium labeled ATV (ATV-d5) was intravenously injected. Assuming that ATV-d5 exhibits the same pharmacokinetic (PK) profile with ATV, PK parameters for absorption and elimination of ATV were calculated. Plasma levels of orally administered ATV were significantly enhanced by co-administration of CsA both in rats, dogs and humans, resulted in 9.8, 31, and 8.7-fold increase in systemic exposure calculated as AUCpo. High intensity of the DDI in dogs was mainly attributed to the marked decrease of the intrinsic hepatic clearance (to 1/10 of the control), which was induced by the inhibition of hepatic uptake of ATV via organic anion transporting polypeptide 1B1 (OATP1B1). CsA also affected the absorption of ATV form GI tract. Absorbed fraction of ATV into portal vein (calculated as Fa*Fg) was increased almost same extent in rats and dogs (around 3.0-fold) by co-administration of CsA. Inhibition of efflux transport via breast cancer resistance protein as well as the intestinal metabolism mediated by CYP enzymes contributed to the DDI occurred in the intestinal tract. In conclusion, PK analysis on the DDI between ATV and CsA in rats and dogs clearly demonstarted the factors to cause species differences in the extent of DDI. This type of quantitative analysis of DDIs in both small and large animals can be a great help to predict the extent of DDI in humans in the clinical study.

In Vivo Fluid Volume in Rat Gastrointestinal Tract: Kinetic Analysis on the Luminal Concentration of Nonabsorbable FITC-Dextran After Oral Administration.

Previously, 1 mL of purified water (hyposmotic) or saline (isosmotic) which dissolved 200 µM of FITC-dextran (FD-4), a nonabsorbable marker, was orally administered to rats, and luminal concentration-time profile of FD-4 was directly measured. In this study, at first, luminal FD-4 concentration was measured after oral administration of 0.5 mL of FD-4 purified water solution (200 µM). Then, kinetic analysis was conducted to calculate the fluid volume that passed through each segment of the gastrointestinal tract (Vfluid), based on the luminal FD-4 concentration-time profiles obtained from 3 different administration groups. In the group of 1 mL purified water administration, most of administered water was absorbed quickly from the duodenum and upper jejunum, whereas group of saline administration (1 mL) showed only a little amount of absorbed in the upper small intestine. In 0.5 mL purified water group, Vfluid in the stomach was approximately half compared to that in 1 mL purified water group. However, for small intestine, almost the same values of Vfluid were obtained regardless of the dose-volume. Our findings are valuable to improve the quality of in vitro predictive dissolution tools or in silico simulation for predicting oral drug absorption.

Impact of Dietary Intake of Medium-Chain Triacylglycerides on the Intestinal Absorption of Poorly Permeable Compounds.

The present study sought to demonstrate the effect of dietary intake of medium-chain triacylglycerides (MCTs) on the intestinal absorption of a poorly permeable compound of intermediate molecular weight (FITC-dextran 4000 [FD-4]). As a model of MCTs, C8-C12 fatty acid triacylglyceride (COCONAD ML) was mainly used, and the dose strength of each triglyceride was set with consideration of the dietary ingestion dose (12.5 mg/rat). When FD-4 with MCTs dispersed in fasted state simulated intestinal fluid containing surfactants was administered into the rat jejunum, the intestinal absorption of FD-4 was significantly higher than when administered with a similar solution with or without corn oil (long-chain triglycerides). The effects of pretreatment by MCT lipolysis, inhibition of endogenous lipases, and different dose timings of MCTs and FD-4 on the intestinal absorption of FD-4 indicated that medium-chain fatty acids, such as caprylic acid and capric acid, released from MCTs by lipolysis in the small intestine significantly enhanced the intestinal absorption of FD-4, but the effect was transient. In addition, a similar effect was observed when MCTs were dispersed in soymilk, although large interindividual variation was detected. These findings suggested that dietary intake of MCTs might affect the intestinal absorption of poorly permeable compounds.

Analysis of the Complicated Nonlinear Pharmacokinetics of Orally Administered Telmisartan in Rats Using a Stable Isotope-IV Method.

This study aimed to kinetically analyze the nonlinear absorption and systemic exposure of telmisartan (TEL) after oral administration to rats by using a stable isotope-IV method. Rats were orally administered different dose of TEL, followed by the intravenous injection of 0.005 mg/kg of deuterium-labeled TEL (TEL-d3). Assuming that TEL-d3 shows same pharmacokinetic properties with TEL, systemic clearance (CLtot), oral bioavailability (Foral), and intestinal and hepatic availability (Fa*Fg, Fh) of TEL were calculated in each individual rat. AUCpo of TEL increased disproportionately with dose and showed a sigmoid-type relation, indicating the involvement of multi-nonlinear processes in oral absorption of TEL. Fa*Fg of TEL increased with dose at the low-dose range while decreased at the high-dose range. In contrast, Fh increased and CLtot decreased significantly in the middle range (2 to 6 mg/kg). As main factors of nonlinearity, saturations of solubility, efflux transport in the intestine, and the hepatic uptake of TEL were indicated. In conclusion, this study demonstrated a high possibility of a stable isotope-IV method to characterize complicated pharmacokinetic properties of oral drugs in animals, which can help to consider the future risks in their clinical use.

In Vitro Assessment of Supersaturation/Precipitation and Biological Membrane Permeation of Poorly Water-Soluble Drugs: A Case Study With Albendazole and Ketoconazole.

This study aimed to elucidate the relationship between supersaturation and precipitation and the effect of a supersaturated state on drug membrane permeation. Stock solutions of albendazole (ALB) and ketoconazole (KTZ) dissolved in dimethyl sulfoxide (0.1-50 mg/mL) were diluted 100-fold with buffer solution (pH 6.8, 37°C). In the case of ALB, a supersaturated state and immediate precipitation were observed at 10 µg/mL or less and 20 µg/mL or higher, respectively. When KTZ was used, at an initial concentration of 200 µg/mL or higher, precipitation was observed, although the dissolved concentration remained at approximately 120 µg/mL for at least 30 min. These dissolved concentrations of ALB and KTZ related to approximately 10-fold and 14-fold over the saturated solubility from respective bulk powder. An in vitro permeation study implied that the rate of drug permeation across a biological membrane increased with increasing supersaturation. These results suggested favorable strategies for development of a supersaturable formulation could depend on the precipitation properties of the drug. Immediate- and controlled-release forms might be suitable for supersaturable formulations for KTZ and ALB, respectively.

Quantitative Analysis of the Transporter-Mediated Drug-Drug Interaction Between Atorvastatin and Rifampicin Using a Stable Isotope-IV Method.

This study aims to investigate the drug-drug interactions (DDIs) between orally administered atorvastatin (ATV) and rifampicin (RIF) in rats. The isotope-IV method was used for the analysis of the increased systemic exposure (AUCpo) of ATV, in which a small amount of deuterium-labeled ATV (ATV-d5) was intravenously injected after oral administration of ATV. By assuming ATV-d5 showed same pharmacokinetic properties with ATV, this method enabled to calculate the systemic clearance (CLtot) and the oral bioavailability (Foral) of ATV for each individual rat in a single experiment. RIF was orally pretreated to rats to inhibit the organic anion transporting polypeptide 1B1 (OATP1B1). From the analysis using pharmacokinetic parameters in each rat, it was revealed that the AUCpo of ATV increased depending on the plasma level of RIF, showing that the interindividual difference in the absorption of RIF caused the large variability in the extent of DDI. Furthermore, it was indicated that not only the decrease in CLtot but also the increase in Foral caused the significant increase in the AUCpo of ATV. In conclusion, the isotope-IV method possesses various advantages over the conventional method for the analysis of DDIs which affects both absorption and elimination processes of oral drugs.

Increase in the systemic exposure of primary metabolites of Midazolam in rat arising from CYP inhibition or hepatic dysfunction.

The main purpose of this study is to demonstrate the possibility of increase in the systemic exposure of drug metabolites by CYP-inhibition or acute hepatitis. Midazolam (MDZ) was used as a model substrate of CYP3A and 1-aminobenzotriazole (ABT) was used as a CYP-inhibitor. After oral pretreatment with ABT, MDZ was intravenously injected to rats and the plasma profiles of MDZ and its primary metabolites, 1'-hydroxy MDZ and 4-hydroxy MDZ, were observed. In the ABT-pretreatment rats, plasma AUCs of both metabolites were much larger than those in control rats, demonstrating a higher systemic exposure of metabolites under CYP-inhibited condition. Furthermore, kinetic analysis revealed that the amount of both metabolites entered into the systemic circulation increased significantly (about 5-times). Increases in the systemic exposure of the primary metabolites of MDZ were also observed in the acute hepatitis rats induced by CCl4-pretreatment. As underlying mechanisms, it was speculated that ABT inhibited the subsequent metabolism of primary metabolites of MDZ in the hepatocytes and enhanced their release to the systemic circulation. In vitro study with rat liver microsomes supported this speculation. In conclusion, this study showed the complexity of PK profiles of drug metabolites, which might lead to new aspects on their safety issue.

Quantitative analysis of pharmacokinetic profiles of verapamil and drug-drug interactions induced by a CYP inhibitor using a stable isotope-labeled compound.

The purpose of the present study is to demonstrate a useful approach (isotope-IV method) for analyzing drug-drug interactions (DDIs) following the oral administration of drugs using stable isotope-labeled compounds. Verapamil hydrochloride (VER) was used as a drug model. Deuterium-labeled VER (VER-d6, 0.005 mg/kg) was intravenously administered to rats with or without a pre-treatment with 1-aminobenzotriazole (ABT, 100 mg/kg), a potent CYP inhibitor, 1.5 h after the oral administration of VER (1 mg/kg). PK parameters such as AUCpo, AUCiv, and CLtot were evaluated after the oral and intravenous administration of VER from the plasma concentration-time profiles of VER and VER-d6 in each rat. The oral bioavailability (F) of VER in rats was calculated as 0.02 ± 0.01 and was significantly increased to 0.45 ± 0.24 by the pre-treatment with ABT. Further PK analyses revealed that CYP-mediated metabolism was more strongly inhibited by ABT in the intestine (Fg) than in the liver (Fh). These results were consistent with those obtained using the conventional method in which oral and intravenous administration studies were performed using different rat groups. Therefore, the isotope-IV method is effective for performing PK analyses including DDIs after the oral administration of drugs.

Effects of gastric pH on oral drug absorption: In vitro assessment using a dissolution/permeation system reflecting the gastric dissolution process.

The aim of the present study was to evaluate the effects of gastric pH on the oral absorption of poorly water-soluble drugs using an in vitro system. A dissolution/permeation system (D/P system) equipped with a Caco-2 cell monolayer was used as the in vitro system to evaluate oral drug absorption, while a small vessel filled with simulated gastric fluid (SGF) was used to reflect the gastric dissolution phase. After applying drugs in their solid forms to SGF, SGF solution containing a 1/100 clinical dose of each drug was mixed with the apical solution of the D/P system, which was changed to fasted state-simulated intestinal fluid. Dissolved and permeated amounts on applied amount of drugs were then monitored for 2h. Similar experiments were performed using the same drugs, but without the gastric phase. Oral absorption with or without the gastric phase was predicted in humans based on the amount of the drug that permeated in the D/P system, assuming that the system without the gastric phase reflected human absorption with an elevated gastric pH. The dissolved amounts of basic drugs with poor water solubility, namely albendazole, dipyridamole, and ketoconazole, in the apical solution and their permeation across a Caco-2 cell monolayer were significantly enhanced when the gastric dissolution process was reflected due to the physicochemical properties of basic drugs. These amounts resulted in the prediction of higher oral absorption with normal gastric pH than with high gastric pH. On the other hand, when diclofenac sodium, the salt form of an acidic drug, was applied to the D/P system with the gastric phase, its dissolved and permeated amounts were significantly lower than those without the gastric phase. However, the oral absorption of diclofenac was predicted to be complete (96-98%) irrespective of gastric pH because the permeated amounts of diclofenac under both conditions were sufficiently high to achieve complete absorption. These estimations of the effects of gastric pH on the oral absorption of poorly water-soluble drugs were consistent with observations in humans. In conclusion, the D/P system with the gastric phase may be a useful tool for better predicting the oral absorption of poorly water-soluble basic drugs. In addition, the effects of gastric pH on the oral absorption of poorly water-soluble drugs may be evaluated by the D/P system with and without the gastric phase.

Interaction with Mixed Micelles in the Intestine Attenuates the Permeation Enhancing Potential of Alkyl-Maltosides.

The purpose of the present study was to investigate the interaction of intestinal permeation enhancers with lipid and surfactant components present in the milieu of the small intestine. Maltosides of different chain lengths (decyl-, dodecyl-, and tetradecyl-maltoside; DM, DDM, TDM, respectively) were used as examples of nonionic, surfactant-like permeation enhancers, and their effect on the permeation of FD4 across Caco-2 monolayers was monitored. To mimic the environment of the small intestine, modified versions of fasted and fed state simulated intestinal fluid (FaSSIFmod, FeSSIFmod6.5, respectively) were used in addition to standard transport media (TM). Compared to the buffer control, 0.5 mM DDM led to a 200-fold permeation enhancement of FD4 in TM. However, this was dramatically decreased in FaSSIFmod, where a concentration of 5 mM DDM was necessary in order to elicit a moderate, 4-fold, permeation enhancement. Its capacity to promote permeation was diminished further when FeSSIFmod6.5 was employed. Even when cells were exposed to a concentration of 5 mM, no significant permeation enhancement of FD4 was observed. Analogous effects were observed in the case of DM and TDM, with slight deviations on account of differences in their critical micelle concentration (CMC). This observation was corroborated by calculating the amount of maltoside monomer versus micellar bound maltoside in FaSSIFmod and FeSSIFmod6.5, which demonstrated a reduced amount of free monomer in these fluids. To evaluate the in vivo significance of our findings, DDM solutions in TM, FaSSIFmod, and FeSSIFmod6.5 were used for closed intestinal loop studies in rats. Consistent with the results found in in vitro permeation studies, these investigations illustrated the overwhelming impact of sodium taurocholate/lecithin micelles on the permeation enhancing effect of DDM. While DDM led to a 20-fold increase in FD4 bioavailability when it was applied in TM, no significant permeation enhancement was seen in FaSSIFmod/FeSSIFmod6.5. Collectively, these investigations highlight the importance of using biorelevant media when evaluating the potency of permeation enhancers. In doing so, this ensures improved correlations between in vitro and in vivo studies and thus enables an early and more accurate assessment of promising permeation enhancers.

Specificity of lectin-immobilized fluorescent nanospheres for colorectal tumors in a mouse model which better resembles the clinical disease.

We have been investigating an imaging agent that enables real-time and accurate diagnosis of early colorectal cancer at the intestinal mucosa by colonoscopy. The imaging agent is peanut agglutinin-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) chains encapsulating coumarin 6. Intracolonically-administered lectin-immobilized fluorescent nanospheres detect tumor-derived changes through molecular recognition of lectin for the terminal sugar of cancer-specific antigens on the mucosal surface. The focus of the present study was to evaluate imaging abilities of the nanospheres in animal models that reflect clinical environments. We previously developed an orthotopic mouse model with human colorectal tumors growing on the mucosa of the descending colon to better resemble the clinical disease. The entire colon of the mice in the exposed abdomen was monitored in real time with an in vivo imaging apparatus. Fluorescence from the nanospheres was observed along the entire descending colon after intracolonical administration from the anus. When the luminal side of the colon was washed with phosphate-buffered saline, most of the nanospheres were flushed. However, fluorescence persisted in areas where cancer cells were implanted. Histological evaluation demonstrated that tumors were present in the mucosal epithelia where the nanospheres fluoresced. In contrast, no fluorescence was observed when control mice, without tumors were tested. The lectin-immobilized fluorescent nanospheres were tumor-specific and remained bound to tumors even after vigorous washing. The nanospheres nonspecifically bound to normal mucosa were easily removed through mild washing. These results indicate that the nanospheres combined with colonoscopy, will be a clinically-valuable diagnostic tool for early-stage primary colon carcinoma.

In vitro-in vivo correlation of the effect of supersaturation on the intestinal absorption of BCS Class 2 drugs.

The aim of this study was to establish an in vitro method for evaluating the effect of supersaturation on oral absorption of poorly water-soluble drugs in vivo. Albendazole, dipyridamole, gefitinib, and ketoconazole were used as model drugs. Supersaturation of each drug was induced by diluting its stock solution by fasted state simulated intestinal fluid (FaSSIF) (solvent-shift method), then dissolution and precipitation profile of the drug was observed in vitro. The crystalline form of the precipitate was checked by differential scanning calorimetry (DSC). For comparison, control suspension was prepared by suspending a drug powder directly into FaSSIF (powder-suspending method). In vivo intestinal absorption of the drug was observed in rats by determined the plasma concentration after intraduodenal administration of drug suspensions. For all drugs, suspensions prepared by solvent-shift method showed significantly higher dissolved concentration in vitro than that prepared by powder-suspending method, clearly indicated the induction of supersaturation. DSC analysis revealed that crystalline form of the precipitate profoundly affects the extent and the duration of supersaturation. A rat in vivo study confirmed that the supersaturation of these drugs increased the fraction absorbed from the intestine, which corresponded well to the in vitro dissolution and precipitation profile of drugs except for ketoconazole. For ketoconazole, an in vivo absorption study was performed in rats pretreated with 1-aminobenzotriazole, a potent inhibitor of CYP mediated metabolism. CYP inhibition study suggested that the high luminal concentration of ketoconazole caused by supersaturation saturated the metabolic enzymes and further increased the systemic exposure of the absorbed drug. The additional effects of supersaturation on the absorption of ketoconazole are consistent with previous studies in humans under differing gastric pH conditions. In conclusion, effects of supersaturation on the intestinal absorption of poorly water-soluble drugs could be predicted from in vitro dissolution and a precipitation study. However if supersaturation affects the pharmacokinetic profiles of drugs, such as a first-pass metabolism, a combination with in vivo study should be required to evaluate its impact on oral bioavailability.