Biopharmaceutics classification and intestinal absorption of chikusetsusaponin IVa

The objective of this study was to investigate the absorption behavior of chikusetsusaponin IVa (CHS‐IVa) in the rat intestine using single‐pass intestinal perfusion (SPIP) and to classify CHS‐IVa into the biopharmaceutics classification system (BCS). The equilibrium solubility of CHS‐IVa was determined by the shaker method. The absorption mechanism of CHS‐IVa in the intestine was studied by comparing the P_eff of different concentrations of CHS‐IVa. The intestinal site dependence of CHS‐IVa absorption was studied by comparing the P_eff of the same concentration of CHS‐IVa in different intestinal segments. The relationship between CHS‐IVa and intestinal efflux protein was studied by perfusion with an efflux protein inhibitor. The permeability of CHS‐IVa was investigated by comparing the P_eff of CHS‐IVa and the reported value. The solubility of CHS‐IVa over the pH range 1.0–7.5 was 14.4 ± 0.29 to 16.9 ± 0.34 mg/ml. The P_eff of CHS‐IVa in the duodenum was 1.76 × 10^?3 to 2.00 × 10^?3 cm/min. The P_eff of CHS‐IVa in the jejunum was 1.26 × 10^?3 to 1.39 × 10^?3 cm/min. The P_eff of CHS‐IVa in the ileum was 1.25 × 10^?3 to 1.31 × 10^?3 cm/min. The P_eff of CHS‐IVa in the colon was 1.02 × 10^?3 to 1.08 × 10^?3 cm/min. There was no statistical difference of the P_eff in the four segments at different CHS‐IVa concentrations. The P_eff of CHS‐IVa (0.07, 0.7 and 7.0 mg/ml) were all notably smaller than the reported P_eff (3.00 × 10^?3 cm/min) in the jejunum. The P_eff of CHS‐IVa was not influenced by verapamil (P‐gp inhibitor), indomethacin (MRP inhibitor) and pantoprazole (BCRP inhibitor). CHS‐IVa was classified as high solubility, low permeability and BCS III. The main absorptive tracts were the upper intestinal tracts and the rank order of intestinal permeability was duodenum > jejunum ≈ ileum > colon. The transport mechanism of CHS‐IVa in all intestinal segments might be primarily passive transport. CHS‐IVa was not a substrate of P‐gp, MRP and BCRP.     

Relevance of PepT1 in the Intestinal Permeability and Oral Absorption of Cefadroxil

Purpose

To determine the contribution of intestinal PepT1 on the permeability and oral absorption of the β-lactam antibiotic drug cefadroxil.

Methods

The effective permeability (P _eff ) of cefadroxil was evaluated in wild-type and PepT1 knockout mice following in situ single-pass intestinal perfusions. The plasma concentration-time profiles of cefadroxil were also examined after oral gavage.

Results

The P _eff (cm/s) of cefadroxil in wild-type mice was 0.49?×?10^?4 in duodenum, 0.80?×?10^?4 in jejunum, 0.88?×?10^?4 in ileum and 0.064?×?10^?4 in colon. The P _eff (cm/s) in PepT1 knockout mice was significantly reduced in small intestine, but not in colon, as shown by values of 0.003?×?10^?4, 0.090?×?10^?4, 0.042?×?10^?4 and 0.032?×?10^?4, respectively. Jejunal uptake of cefadroxil was saturable (Km?=?2–4 mM) and significantly attenuated by the sodium-proton exchange inhibitor 5-(N,N-dimethyl)amiloride. Jejunal permeability of cefadroxil was not affected by L-histidine, glycine, cephalothin, p-aminohippurate or N-methylnicotinamide. In contrast, cefadroxil permeability was significantly reduced by glycylproline, glycylsarcosine, or cephalexin. Finally, PepT1 ablation resulted in 23-fold reductions in peak plasma concentrations and 14-fold reductions in systemic exposure of cefadroxil after oral dosing.

Conclusions

The findings are definitive in demonstrating that PepT1 is the major transporter responsible for the small intestinal permeability of cefadroxil as well as its enhanced oral drug performance.     

Concentration- and Region-dependent Intestinal Permeability of Fluvastatin in the Rat

The purpose of this study was to investigate the mechanisms of transport of fluvastatin across the intestinal mucosa in various regions of the intestine in the rat. In-situ single-pass perfusions of the jejunum, ileum and colon were performed and the effective permeability (P_eff) of fluvastatin, antipyrine and D-glucose were assessed in each region, at three different perfusate fluvastatin concentrations (1.6, 16 and 160 μM). The effect of lovastatin acid on the bi-directional transport of fluvastatin across the ileal mucosa was also studied. The P_eff of fluvastatin was found to be dependent both on the intestinal region and on the concentration in the intestinal lumen (P < 0.001). Fluvastatin had the lowest P_eff (0.55±0.10 times 10^?4 cm s^?1) in the jejunum at 1.6 μM, and the highest P_eff (1.0±0.16 times 10^?4 cm s^?1) in the colon at 160 μM. The highest concentration of fluvastatin increased the average absorption of water from the intestine by 209% (P < 0.05), and the average P_eff of D-glucose by 29% (P < 0.05). The presence of excess lovastatin acid (100 μM, compared with fluvastatin 1.6 μM) at the luminal side increased the average absorption of water by 218% (P < 0.001), and the P_eff of fluvastatin in the ileum and the colon by 44 and 50%, respectively (P < 0.05). The presence of lovastatin acid on the luminal side in the ileum also increased the blood-to-lumen transport (exsorption) of fluvastatin by 43% (P < 0.001). The increased intestinal absorption of fluvastatin at higher concentrations does not suggest that substantial absorption occurs by any carrier-mediated process in the absorptive direction. The increased bi-directional transport when lovastatin acid was added to the lumen suggests that fluvastatin is not a P-glycoprotein substrate. Instead, the concentration-dependent increase in the absorption of fluvastatin, water and D-glucose suggests a direct effect of fluvastatin on the transcellular passive transport.     

The Absence of Stereoselective P-Glycoprotein-mediated Transport of R/S-Verapamil Across the Rat Jejunum

We have studied the potential stereoselective transport and metabolism of R/S-verapamil in rat jejunum, in-situ. A regional single-pass perfusion of the rat jejunum was performed on 24 rats in six separate groups. The effective permeability (P_eff) was assessed for three different concentrations of verapamil, 4, 40 and 400 mg L^?1. The P_eff of each enantiomer was also determined at 400 mg L^?1 when chlorpromazine (10 mM) was added to the perfusion solution. Two other groups of rats received R/S-verapamil as an intravenous infusion and the intestinal secretion and metabolism were studied by simultaneously perfusing the jejunum with a control or with chlorpromazine (10 mM) added. The concentrations in the outlet perfusate of each enantiomer of verapamil and norverapamil were assayed with HPLC. R/S-Verapamil is a high permeability drug in the proximal rat small intestine throughout the luminal concentration range studied and complete intestinal absorption was expected. There was an increase of P_eff from 0.42 times 10^?4 cm s^?1 to 0.80 times 10^?4 cm s^?1 (P < 0.05) at concentrations from 4 to 400 mg L^?1, respectively. The observed concentration-dependent jejunal P_eff and fraction absorbed (P < 0.05) of R/S-verapamil is consistent with the saturation of an efflux mechanism. When chlorpromazine (a P-glycoprotein inhibitor/substrate) was added the jejunal P_eff increased to 1.47 times 10^?4 cm s^?1. There was no difference between the P_eff of the two enantiomers in any of these experiments. The efflux of R/S-norverapamil into the rat jejunum was high after intravenous administration of R/S-verapamil, suggesting extensive metabolism in the enterocyte. In conclusion, both R/S-verapamil enantiomers are P-glycoprotein substrates, but there is no stereoselective transport of R/S-verapamil in the rat jejunum. The results also suggests that R/S-norverapamil is formed inside the enterocytes.     

Periplocymarin is a potential natural compound for drug development: highly permeable with absence of P‐glycoprotein efflux and cytochrome P450 inhibitions

Periplocymarin, a cardiac glycoside isolated from Periploca sepium (P. sepium) and Periploca graeca (P. graeca), is a potential anti‐cancer compound. The aim of the study was to investigate the potential for periplocymarin to interact with P‐glycoprotein (P‐gp) and to inhibit cytochrome P450s known to be expressed in the human small intestine. The in vitro and in situ permeability of periplocymarin were studied using Madin‐Darby canine kidney (MDCK‐II‐WT) cells transfected with or without the human multidrug resistance (MDR1) gene and the single‐pass perfused rat intestinal model. The cell system exhibited high functional activity and a net efflux ratio (NER) of 4.32 after transport of Rhodamine 123 (R123) (the P‐gp substrate). Periplocymarin is highly permeable (P_app > 10 × 10^‐6 cm/s; P_eff(rat) > 5.09 × 10^‐5 cm/s) and independent of P‐gp influences. The NER at 100 μm periplocymarin (0.8) was unchanged in the presence of cyclosporine A (a non‐specific P‐gp inhibitor) (0.82). In the single‐pass intestinal model, the P_{eff (rat)} of 5 µg/ml periplocymarin (5.490 × 10^‐5 cm/s) did not change in the presence of cyclosporine A (5.394 × 10^‐5 cm/s). In the R123‐inhibition assay, periplocymarin did not competitively inhibit P‐gp. The inhibitory potential of periplocymarin on cytochrome P450 (CYP450s) was also studied. Periplocymarin (5, 50 μm ) did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4. Thus, periplocymarin is unlikely to encounter drug–drug interactions with P‐gp and CYP450s. Periplocymarin could be taken forward for further studies in drug development to test bioavailability, Phase II enzyme interactions and additional transporter interactions. Copyright © 2014 John Wiley & Sons, Ltd.     

In situ intestinal permeability and in vivo oral bioavailability of celecoxib in supersaturating self-emulsifying drug delivery system

In order to characterize the in situ intestinal permeability and in vivo oral bioavailability of celecoxib (CXB), a poorly water-soluble cyclooxygenase (COX)-2 inhibitor, various formulations including the self-emulsifying drug delivery system (SEDDS) and supersaturating SEDDS (S-SEDDS) were compared. The S-SEDDS formulation was obtained by adding Soluplus as a precipitation inhibitor to SEDDS, composed of Capryol 90 as oil, Tween 20 as surfactant, and Tetraglycol as cosurfactant (1:4.5:4.5 in volume ratio). An in situ single pass intestinal perfusion study in rats was performed with CXB-dissolved solutions at a concentration of 40 μg/mL. The effective permeability (P_eff) of CXB in the control solution (2.5 v/v% Tween 20-containing PBS) was 6.39 × 10^?5 cm/s. The P_eff value was significantly increased (P < 0.05) by the lipid-based formulation, yielding 1.5- and 2.9-fold increases for the SEDDS and S-SEDDS solutions, respectively, compared to the control solution. After oral administration of various formulations to rats at the equivalent dose of 100 mg/kg of CXB, the plasma drug level was measured by LC–MS/MS. The relative bioavailabilities of SEDDS and S-SEDDS were 263 and 355 %, respectively, compared to the CXB suspension as a reference. In particular, S-SEDDS revealed the highest C_max and the smallest T_max, indicating rapid and enhanced absorption with this formulation. This study illustrates the potential use of the S-SEDDS formulation in the oral delivery of poorly water-soluble compounds.     

Mechanistic study of absorption and first‐pass metabolism of GL‐V9, a derivative of wogonin

GL‐V9, a derivative of wogonin, has potent anti‐cancer activity. The absorption and metabolism of this compound have not been investigated systematically. This study aims to illustrate the pharmacokinetic characters of GL‐V9 by exploring its metabolic status under different administration routes. To further clarify the absorption mechanism of GL‐V9, an in situ single‐pass perfusion model and a Caco‐2 cell monolayer model were used. Meanwhile, a microsomal incubation system was used to evaluate the enzyme kinetic parameters. In vivo, the obtained gastrointestinal availability (F_a × F_g) was 21.28 ± 5.38%. The unmetabolized fraction in the gut wall (F_{gut wall}) was 98.59 ± 9.74%, while the hepatic bioavailability (F_h) was 29.11 ± 5.22%. These results indicated that poor absorption and extensive metabolism may contribute greatly to the low bioavailability of GL‐V9. The effective permeability (P_eff) in the duodenum and jejunum was 1.34 ± 0.50 × 10^?4 and 0.90 ± 0.27 × 10^?4 cm/s, respectively. The high permeability of GL‐V9 indicated that other unknown factors (such as metabolism) may account for its systemic exposure problem. Studies in rat liver microsomal (RLMs) confirmed this hypothesis, and the Cl_{int, CYP450s and UGT} of GL‐V9 was 0.20 ml/min/mg protein. In conclusion, these results suggest that GL‐V9 possesses higher permeability than wogonin and the metabolism of GL‐V9 is related to its disposition in rat intestine and liver.     

Chrysosplenetin inhibits artemisinin efflux in P-gp-over-expressing Caco-2 cells and reverses P-gp/MDR1 mRNA up-regulated expression induced by artemisinin in mouse small intestine

Context: CYP3A4 and P-gp together form a highly efficient barrier for orally absorbed drugs and always share the same substrates. Our previous work revealed that chrysosplenetin (CHR) significantly augmented the rat plasma level and anti-malarial efficacy of artemisinin (ART), partially due to the uncompetitive inhibition effect of CHR on rat CYP3A. But the impact of CHR on P-gp is still unknown.

Objective: The present study investigates whether CHR interferes with P-gp-mediated efflux of ART and elucidates the underlying mechanism.

Materials and methods: P-gp-over-expressing Caco-2 cells were treated with ART (10?μM) or ART-CHR (1:2, 10:20?μM) in ART bidirectional transport experiment. ART concentration was determined by UHPLC-MS/MS method. Healthy male ICR mice were randomly divided into nine groups (n?=?6) including negative control (0.5% CMC-Na solution, 13?mL/kg), ART alone (40?mg/kg), verapamil (positive control, 40?mg/kg), ART-verapamil (1:1, 40:40?mg/kg), CHR alone (80?mg/kg), ART-CHR (1:0.1, 40:4?mg/kg), ART-CHR (1:1, 40:40?mg/kg), ART-CHR (1:2, 40:80?mg/kg) and ART-CHR (1:4, 40:160?mg/kg). The drugs were administrated intragastrically for seven consecutive days. MDR1 and P-gp expression levels in mice small intestine were examined by performing RT-PCR and western blot analysis. ABC coupling ATPase activity was also determined.

Results: After combined with CHR (1:2), P_app (AP-BL) and P_app (BL-AP) of ART changed to 4.29?×?10?^??⁸ (increased 1.79-fold) and 2.85?×?10?^??^8?cm/s (decreased 1.24-fold) from 2.40?×?10?^??⁸ and 3.54?×?10?^??^8?cm/s, respectively. Efflux ratio (P_BA/P_AB) declined 2.21-fold (p?p?p?Discussion and conclusion: These results confirm that CHR inhibited P-gp activity and reverse the up-regulated P-gp and MDR1 levels induced by ART. It suggested that CHR potentially can be used as a P-gp reversal agent to obstruct ART multidrug resistance.     

Physicochemical characteristics and gastrointestinal absorption behaviors of S-propargyl-cysteine,a potential new drug candidate for cardiovascular protection and antitumor treatment

Abstract

1. As a potential new drug candidate for cardiovascular protection and antitumor treatment, the physicochemical properties, gastrointestinal (GI) absorption behaviors and mechanisms of S-propargyl-cysteine (SPRC) were investigated in this study.

2. SPRC exhibited favorable solubility in aqueous media. The log P and log D values were low (≤1.93?±?0.08). The pK_a in the acidic and basic regions was 2.08?±?0.02 and 8.72?±?0.03, respectively. The isoelectric point was 5.40?±?0.02. SPRC was stable in the rat GI fluids, and showed no obvious adsorption and metabolism in the rat GI tract.

3. SPRC displayed poor gastric absorption and favorable intestinal absorption in the rat in situ GI perfusion model. Absorption rate constants (k_a), hourly absorption percentage (P) and apparent permeability coefficient (P_app) of SPRC in the small intestine were ≥0.77?±?0.06?h^?1, 59.25?±?4.02% and (7.99?±?0.88)?×?10^?5?cm/s, respectively. Absorption of SPRC exhibited a certain dependence on physiological pH and absorption region. Absorption of SPRC was not inhibited by l-methionine and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid.

4. SPRC showed favorable oral absorption. It can be categorized as a BCS class I drug. The membrane pore transport appeared to be one of the predominant absorption modes for SPRC.     

Utility of in vitro and in vivo systems for studying the permeability of capsaicin and nonivamide through different intestinal regions

1.?The present study determined and compared the permeability of capsaicin and nonivamide along the length of the intestine in rats. Accordingly, the purpose was to evaluate this synthetic analog as a clinical substitute for capsaicin..

2.?Permeabilities of capsaicin and nonivamide were measured in experiments utilizing Ussing chambers and in vivo methods. Capsaicin concentrations were examined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

3.?Both capsaicin (0.80?×?10^?6?cm/s) and nonivamide (0.22?×?10^?6?cm/s, p?>?0.05) had poor permeabilities across the jejunal membrane. The permeability of nonivamide (10.12?×?10^?6?cm/s) was significantly greater than that of capsaicin (5.34?×?10^?6?cm/s, p??6?cm/s) across the colonic membrane was markedly lower than that of capsaicin (14.48?×?10^?6?cm/s, p?in vitro study, the drug concentration-time curve of nonivamide was significantly higher in the ileum (F?=?14.18, p?F?=?11.86, p?4.?The results demonstrate that capsaicin and nonivamide exhibit varying permeabilities across several different intestinal tissues. The relevance of such extended investigations to healthcare is underscored by the lower cost of nonivamide versus capsaicin, along with potential application in prevention and management of the disease.     

Differential induction of midazolam metabolism in the small intestine and liver by oral and intravenous dexamethasone pretreatment in rat

1. Midazolam is metabolized in the rat by CYP3A enzymes to 4-OH-midazolam (4-OH-MDZ) and 1'-OH-midazolam (1'-OH-MDZ). The induction of midazolam metabolism was studied in male Wistar rats treated with dexamethasone (50?mg kg^?1 day^?1) during 4 days via the oral or intravenous routes. Microsomes were prepared from the liver and the proximal small intestine and in vitro metabolism of midazolam was determined. In addition, CYP3A1- and CYP3A2-like protein levels were measured by gel electrophoresis and immunoblotting. 2. The V_max's (mean SEM) for 4-OH-MDZ and 1'-OH-MDZ formation were much lower in intestinal (0.078 ± 0.002 and 0.074 ± 0.002 µM min^?1 mg^?1 protein, respectively) compared with hepatic microsomes prepared from the uninduced rat (0.870 ± 0.007 and 0.310 ± 0.020 µM min^?1 mg^?1 protein, respectively). Induction by oral or intravenous dexamethasone pretreatment led to significant increases in V_max for 4-OH-MDZ and 1'-OH-MDZ by both intestinal and hepatic microsomes. Oral dexamethasone pretreatment via the oral route resulted in a more pronounced increase in V_max compared with intravenous administration of the inducer. 3. CYP3A1 and CYP3A2 protein levels in liver microsomes were significantly increased following oral (3.7- and 3.2-fold, respectively) or intravenous (2.6- and 2.1-fold, respectively) pretreatment with dexamethasone. On the contrary, only oral dexamethasone pretreatment resulted in a significant change in intestinal CYP3A2-like protein (7.3-fold). A slight difference in the migration distance of the immunoreactive band for CYP3A2 was also observed for intestinal microsomes. 4. These results suggest that intestinal CYP3A enzymes in the rat differ from hepatic CYP3A1 and CYP3A2. They also demonstrate that systemic dexamethasone administration can induce intestinal microsome activity.     

Positioning of Rifampicin in the Biopharmaceutics Classification System (BCS)

The position of rifampicin with respect to the biopharmaceutics classification system (BCS) was explored on the basis of its aqueous solubility and intestinal permeability. The aqueous solubility was determined between pH 1 and 7 by the conventional shake-flask method. Permeability coefficients of rifampicin and the US FDA listed low (furosemide, ranitidine) and high permeable (caffeine, naproxen) model drugs were determined in the three segments of the rat intestine by employing the everted gut sac model. The samples were analyzed by HPLC. The maximum human single dose of rifampicin (600 mg) was insoluble in 250 ml of aqueous media between pH 3 and 7. The determined apparent permeability coefficient (Papp) values for rifampicin were 4.856×10^?6, 2.117×10^?6, and 2.149×10^?6 cm/sec in the rat duodenum, jejunum and ileum, respectively. These values were similar to those of the low permeable drugs, ranitidine and furosemide, for which Papp values were determined to be 1.767×10^?6?2.426×10^?6 and 2.469×10^?6?3.008×10^?6 cm/sec, respectively. The determined values for high permeable model drugs, viz., caffeine and naproxen, were ～10–20 folds higher than rifampicin or even ranitidine and furosemide. The study suggests that rifampicin is a drug with low solubility and even low intestinal permeability and hence qualifies to be classified in BCS Class IV, instead of Class II where it is being categorized presently. The contention is supported by the reported data on the permeability of the drug through cell monolayers, the mass balance and the absolute bioavailability values in the literature, and the data for rifampicin according to “Lipinski's rule of five”.     

Bioavailability of caffeic acid in rats and its absorption properties in the Caco-2 cell model

Context: Caffeic acid (CA) is widely distributed in edible plants, and it is beneficial to human health by exerting various biological effects. The potential pharmacological activities of CA are dependent on its absorption in the gastrointestinal tract.

Objective: To investigate the bioavailability of CA in rats and its absorption properties in the Caco-2 cell model.

Materials and methods: A sensitive LC-MS/MS method was successfully applied to determine CA in rat plasma, perfusate, and Hank's balanced salt solution (HBSS). The absolute bioavailability (F_abs) of CA was obtained after i.v. (2?mg/kg) or i.g. administration (10?mg/kg) to rats. Blood samples (approximately 250?µL) were collected from the jugular vein catheter. Pharmacokinetic parameters were calculated using the 3P97 software (version 2.0 PK software; Chinese Society of Mathematical Pharmacology, Anhui, China). The intestinal absorption of CA was explored by the in situ vascularly perfused rat intestinal preparation. CA (5?mg/kg) was administered into the duodenum. Samples (250?µL) were collected from reservoir at specific times, and the same volume fresh perfusate was replaced. The Caco-2 cell model was applied to measure the permeability of CA from the apical to basolateral side (A?→?B) and from the basolateral to apical side (B?→?A).

Results: The absolute bioavailability (F_abs) of CA was 14.7%, and its intestinal absorption was 12.4%. The P_{app A→B} values of CA were ranging from (4.87?±?1.72)?×?10^?7?cm/s to (5.05?±?0.66)?×?10^?7?cm/s as the concentration varied from 5 to 15?µg/mL.

Conclusion: CA was shown to have low oral bioavailability in rats, low intestinal absorption, and poor permeability across Caco-2 cells.     

Intestinal permeability and its relevance for absorption and elimination

Human jejunal permeability (P_eff) is determined in the intestinal region with the highest expression of carrier proteins and largest surface area. Intestinal P_eff are often based on multiple parallel transport processes. Site-specific jejunal P_eff cannot reflect the permeability along the intestinal tract, but they are useful for approximating the fraction oral dose absorbed. It seems like drugs with a jejunal P_eff > 1.5 x 10^–4 cm s^–1 will be completely absorbed no matter which transport mechanism(s) are utilized. Many drugs that are significantly effluxed in vitro have a rapid and complete intestinal absorption (i.e. >85%) mediated by passive transcellular diffusion. The determined jejunal P_eff for drugs transported mainly by absorptive carriers (such as peptide and amino acid transporters) will accurately predict the fraction of the dose absorbed as a consequence of the regional expression. The data also show that: (1) the human intestinal epithelium has a large resistance towards large and hydrophilic compounds; and (2) the paracellular route has a low contribution for compounds larger than approximately molecular weight 200. There is a need for more exploratory in vivo studies to clarify drug absorption and first-pass extraction along the intestine. One is encouraged to develop in vivo perfusion techniques for more distal parts of the gastrointestinal tract in humans. This would stimulate the development of more relevant and complex in vitro absorption models and form the basis for an accurate physiologically based pharmacokinetic modelling of oral drug absorption.     

The Effect of a Drug-delivery System Consisting of Soybean Phosphatidyl Choline and Medium-chain Monoacylglycerol on the Intestinal Permeability of Hexarelin in the Rat

The aim of this study was to investigate if the effective in-situ permeability (P_eff) of a new growth hormone-releasing peptide, hexarelin, along rat intestine was enhanced by a lipid matrix drug-delivery system comprising a mixture of soybean phosphatidyl choline and medium-chain monoacylglycerol (PC-MG). The study was performed with and without a protease inhibitor, Pefabloc SC. To enable better understanding of the mechanism of action of this delivery system we also studied the uptake of a small hydrophilic molecule, atenolol. PC-MG at a concentration of 15 mmol L^?1 increased the jejunal P_eff of hexarelin approximately 20-fold, both in the presence and absence of Pefabloc SC, whereas P_eff was not increased in the ileum and colon. PC-MG had no effect on the jejunal, ileal and colonic P_eff of atenolol. Complete recovery of the non-absorbable molecule PEG 4000 showed that functional intestinal viability was maintained in all experiments. Although the results obtained in this study are promising, pharmacokinetic and toxicological studies are required to investigate if this delivery system is a suitable and safe candidate for improving the oral bioavailability of hexarelin.     

Intestinal transport of bis(12)-hupyridone in Caco-2 cells and its improved permeability by the surfactant Brij-35

The objective of the present study was to elucidate the mechanisms of intestinal transport of bis(12)‐hupyridone (B12H) to predict its oral bioavailability. The effect of the B12H concentration and the contribution of the drug efflux transporters, P‐glycoprotein (P‐gp or ABCB1) and multidrug resistance‐associated proteins (MRPs or ABCC) on B12H absorption were measured and evaluated using the human intestinal epithelial Caco‐2 cell monolayer in the presence of transporter inhibitors. The results indicated that B12H was absorbed in a dose‐dependent manner at concentrations ranging from 132 to 264 µM . However, only apical efflux was observed in the directional transport studies for B12H below 88 µM (P_app(AP‐to‐BL): virtually zero; P_app(BL‐to‐AP): 1.591 ± 0.071 × 10^?5 cm s^?1). P‐gp and mixed P‐gp/MRP inhibitors significantly increased the absorptive transport (P_app(AP‐to‐BL)) to 0.619 ± 0.018 × 10^?5 and 0.608 ± 0.025 × 10^?5 cm s^?1, respectively, while decreasing secretory transport (P_app(BL‐to‐AP)) by >75%. A multiple‐MRP inhibitor, probenecid, increased the P_app(AP‐to‐BL) to 0.329 ± 0.015 × 10^?5 cm s^?1 while decreasing the P_app(BL‐to‐AP) by 50%. Another multiple‐MRP inhibitor, indomethacin, only modestly decreased the P_app(BL‐to‐AP) by ～30% and had no effect on the absorptive transport (P_app(AP‐to‐BL): virtually zero). In addition, the effect of various pharmaceutical excipients (e.g. Pluronic F‐68, Tween‐80 and Brij‐35) on B12H transport was determined and compared. Among them, Brij‐35 effectively enhanced B12H absorption at a concentration lower than its critical micelle concentration (CMC, 60 µM ). Therefore, Brij‐35 can be used as a potential enhancer to improve intestinal absorption of B12H for oral administration. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison Between Permeability Coefficients in Rat and Human Jejunum

Purpose. Our main aim is to determine the effective intestinal permeability (P_eff) in the rat jejunum in situ for 10 compounds with different absorption mechanisms and a broad range of physico chemical properties, and then compare them with corresponding historical human in vivo P_eff values. Methods. The rat P_eff coefficients are determined using an in situ perfusion model in anaesthetized animals. The perfusion flow rate used is 0.2 ml/min, which is 10 times lower than that used in humans. The viability of the method is assessed by testing the physiological function of the rat intestine during perfusions. Results. The P_eff for passively absorbed compounds is on average 3.6 times higher in humans compared to rats (P_eff,man = 3.6 · P_eff,rat + 0.03·10^–4; R^2 = 1.00). Solutes with carrier-mediated absorption deviate from this relationship, which indicates that an absolute scaling of active processes from animal to man is difficult, and therefore needs further investigation. The fraction absorbed of drugs after oral administration in humans (fa) can be estimated from 1 – e^–(2·P _eff,man ^·t _res ^/r·2.8). Conclusions. Rat and human jejunum P_eff estimates of passively absorbed solutes correlate highly, and both can be used with precision to predict in vivo oral absorption in man. The carrier-mediated transport requires scaling between the models, since the transport maximum and/or substrate specificity might differ. Finally, we emphasize the absolute necessity of including marker compounds for continuous monitoring of intestinal viability.     

Studies on a new potential dopaminergic agent: in vitro BBB permeability,in vivo behavioural effects and molecular docking evaluation

2-Amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (DA-PHEN) has been previously synthesized to obtain a potential prodrug capable of release dopamine (DA) into CNS. However, DA-PHEN could act per se as a dopaminergic drug. In this study, the permeability transport (P_e), obtained by parallel artificial permeability assay (PAMPA), indicated a low passive transcellular transport (P_e?=?0.32?±?0.01?×?10^?6?cm/s). Using the Caco-2 cell system, the P_{app AP-BL} in absorptive direction (3.36?±?0.02?×?10^?5?cm/s) was significantly higher than the P_{app BL-AP} in secretive direction (1.75?±?0.07?×?10^?5?cm/s), suggesting a polarized transport. The efflux ratio (P_{app AP-BL}/P_{app BL-AP}?=?0.52?±?0.02) indicated a low affinity of DA-PHEN to efflux carriers. The forced swim test highlighted a reduction of immobility time in both pre-test and test sessions (p?<?0.0001), with an exacerbation in the number of headshakes and divings in the pretest (p?<?0.0001). Morris water maze strengthened the hypothesis that DA-PHEN induces adaptive responses to environmental challenges which are involved on cognitive functions (DA-PHEN versus CTR: escape latency; p?<?0.001; distance swum p?<?0.001, time spent on target quadrant p?<?0.001), without any change in locomotor activity for the administered dose. The molecular docking revealed the interaction of DA-PHEN with the identified D1 site mapping human brain receptor.     

Pharmacokinetic characterization of CK2 inhibitor CX-4945

Over-expression of protein kinase CK2 is highly linked to the survival of cancer cells and the poor prognosis of patients with cancers. CX-4945, a potent and selective orally bioavailable ATP-competitive inhibitor of CK2, inhibits the oncogenic cellular events such as proliferation and angiogenesis, and the increase of tumor growth in mouse xenograft model. In this study, the pharmacokinetic information about CX-4945 was provided; at 10 μM, CX-4945 with high stability in human and rat liver microsome exhibited low percentage of inhibition (<10 %) in CYP450 isoforms (1A2, 2C19, 3A4), but considerable inhibition (~70 %) in CYP450 2C9 and 2D6. In hERG potassium channel inhibition assay, CX-4945 exhibited relatively low inhibition rate. Additionally, CX-4945 showed high MDCK cell permeability (>10 × 10^?6 cm/s) and above 98 % of plasma protein binding in the rat. After intravenous administration, Vss (1.39 l/kg) and extremely low CL (0.08 l/kg/h) were observed. Moreover, orally administrated CX-4945 showed high bioavailability (>70 %) and these data might be related to the MDCK cell permeability results.