Synthesis and Evaluation of PEG-PR for Water Flux Correction in an In Situ Rat Perfusion Model.

Phenol red (PR) is a widely used marker for water flux correction in studies of in situ perfusion, in which intestinal absorption usually leads to the underestimation of results. In this paper, we propose a novel marker polyethylene glycol (PEG)-PR (i.e., PR modified by PEGylation) with less permeability and evaluate its application in an in situ perfusion model in rats. PEG-PR was synthesized by the chemical conjunction of polyethylene glycol-4k/5k (PEG-4k/5k) and PR. The synthesized PEG-PR was then characterized using 1H-NMR, 13C-NMR, ultraviolet (UV), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) analyses. The low permeability of PEG-PR was assessed using everted gut sac (EGS) methods. The apparent permeability coefficients (Papp, 3-8 × 10-7 cm/s) of PEG4k/5k-PR exhibited a nearly 15-fold reduction compared to that of PR. The different concentrations of PEG4k/5k-PR did not contribute to the Papp value or cumulative permeable percentage (about 0.02-0.06%). Furthermore, the larger molecular weight due to PEGylation (PEG5k-PR) enhanced the nonabsorbable effect. To evaluate the potential application of the novel marker, atenolol, ketoprofen, and metoprolol, which represent various biopharmaceutics classification system (BCS) classes, were selected as model drugs for the recirculation perfusion method. The water flux corrected by PEG4k/5k-PR reflected the accuracy due to the nonabsorbable effect, while the effective intestinal membrane permeability (Peff) of atenolol corrected by PEG4k/5k-PR showed a statistically significant increase (p < 0.05) in different intestinal segments. In conclusion, PEG-PR is a promising marker for the permeability estimation when using the in situ perfusion model in rats.

D-Malate decreases renal content of α-ketoglutarate, a driving force of organic anion transporters OAT1 and OAT3, resulting in inhibited tubular secretion of phenolsulfonphthalein, in rats.

d-Malate inhibits a Krebs cycle enzyme and the tubular transport of α-ketoglutarate, an intermediate of the Krebs cycle and the driving force for rat organic anion transporter 1 (rOAT1) and rOAT3 in the kidney. This study examined the effects of d-malate on the rat organic anion transport system. The uptake of 6-carboxyfluorescein by HEK293 cells expressing rOAT1 or rOAT3 was not affected by d-malate and l-malate. Up to 60 min after the intravenous injection of phenolsulfonphthalein (PSP), a typical substrate of the renal organic anion transporters, as a bolus to rats, 47.1% of the dose was recovered in the urine, and its renal clearance was estimated to be 8.60 ml/min/kg. d-Malate but not l-malate interfered with its renal excretion, resulting in the delayed elimination of PSP from plasma. No effect of d-malate was recognized on creatinine clearance or the expression level of rOAT3 in the kidney cortex. d-Malate increased the plasma concentration of α-ketoglutarate. In addition, the compound greatly stimulated the renal excretion of α-ketoglutarate, implying that d-malate inhibited its reabsorption. The content of α-ketoglutarate was significantly decreased in the kidney cortex of rats administered d-malate. Collectively, this study shows that d-malate abrogates the tubular secretion of PSP, and the reduction of the renal content of α-ketoglutarate was proposed to be one of the mechanisms. A relationship between the reabsorption of α-ketoglutarate and the basolateral uptake of organic anion in the kidney is suggested.

[Rat prostate glandular epithelial cells cultured in vitro and their barrier function].

OBJECTIVE: To culture rat prostate glandular epithelial cells and study their barrier functions in vitro. METHODS: Rat prostate glandular epithelial cells were cultured in vitro. The expression of the tight junction protein claudin-1 was determined by immunohistochemistry, the structure and composition of the epithelial cells observed under the inverted microscope and transmission electron microscope. The transepithelial electrical resistances (TEERs) were monitored with the Millicell system. The permeability of the prostate glandular epithelial cells was assessed by the phenol red leakage test. RESULTS: Compact monolayer cell structures were formed in the prostate glandular epithelial cells cultured in vitro. Immunohistochemistry showed the expression of the tight junction protein claudin-1 and transmission electron microscopy confirmed the formation of tight junctions between the adjacent glandular epithelial cells. The TEERs in the cultured prostate glandular epithelial cells reached the peak of about (201.3 ± 3.5) Ω/cm2 on the 8th day. The phenol red leakage test manifested a decreased permeability of the cell layers with the increase of TEERs. CONCLUSION: The structure and function of rat prostate glandular epithelial cells are similar to those of brain capillary endothelial cells, retinal capillary endothelial cells, and intestinal epithelial cells. In vitro cultured prostate glandular epithelial cells have the barrier function and can be used as a model for the study of blood prostate barrier in vitro.

Lithium Interferes with the Urinary Excretion of Phenolsulfonphthalein in Rats: Involvement of a Reduced Content of α-Ketoglutarate, the Driving Force for Organic Anion Transporters OAT1 and OAT3, in the Kidney Cortex.

AIMS: Lithium is effective in the treatment for bipolar disorder and is known to influence renal functions. The purpose of this study was to examine the effects of lithium on the renal organic anion transport system. METHODS: Pharmacokinetic experiments using rats and Western blotting were conducted. RESULTS: Until 60 min after the intravenous injection of phenolsulfonphthalein, a typical substrate of the renal organic anion transporters, as a bolus, 41.2% of the dose was recovered in the urine, and the infusion of lithium chloride dose-dependently interfered with its renal excretion. No significant changes were observed in the expression levels of rat organic anion transporters rOAT1 and rOAT3, and the content of α-ketoglutarate, the driving force for both transporters, was significantly decreased in the kidney cortex of rats administered lithium. CONCLUSION: These findings represented the dysfunction of the renal organic anion transport system in rats by lithium. As the mechanism, the reduced driving force for rOAT1 and rOAT3 was suggested to be involved at least in part.

Low-dose probenecid selectively inhibits urinary excretion of phenolsulfonphthalein in rats without affecting biliary excretion.

Renal organic anion transport systems play an important role in the excretion of anionic drugs and toxic compounds. Probenecid has been used as a potent inhibitor of urinary and biliary excretion of anionic compounds mediated by transporters such as organic anion transporters and multidrug resistance-associated protein 2 (Mrp2). The purpose of this study was to optimize the dose of probenecid required for selective inhibition of urinary excretion of anionic compounds in rats, without inhibition of biliary excretion. Phenolsulfonphthalein (PSP), a model anionic compound that is excreted in urine and bile, was intravenously administered to rats after intraperitoneal injection of different doses of probenecid (0, 0.2, 2, 10, 100, 200 and 400 mg kg(-1) ). Treatment with 100, 200 or 400 mg kg(-1) probenecid decreased both renal clearance (CLr ) and biliary clearance (CLb ) of PSP, whereas 0.2 mg kg(-1) probenecid did not have any effect. Probenecid administered at doses of 2 and 10 mg kg(-1) decreased only CLr . The median effective doses of probenecid for inhibiting CLr and CLb were 0.925 and 23.9 mg kg(-1) , respectively. These data suggest that a low dose of probenecid selectively inhibits urinary excretion of PSP that may be mediated by organic anion transporters, without affecting biliary excretion that may be mediated by Mrp2.

Developmental expression of renal organic anion transporters in rat kidney and its effect on renal secretion of phenolsulfonphthalein.

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.

[A method for simultaneous assay of propulsion and absorption in small intestine].

OBJECTIVE: To develop a method for simultaneous assay of propulsion and absorption in small intestine. METHODS: The mice were administrated through gastric tube with mixed reagents containing 0.12% phenol red, D-xylose (1.25%, 2.5% and 5%) and 15% gelatin. The influence of phenol red on D-xylose absorption and the influence of D-xylose on small intestine propulsion rate were investigated by measuring serum concentration of D-xylose with phloroglucinol method. RESULTS: At 10 min, no significant difference was found between 5% D-xylose mixed reagent group and 5% D-xylose control. At 15 min, small intestine propulsion rate in 5% D-xylose mixed reagent group, but not in 2.5% and 1.25% D-xylose mixed reagent groups, was significantly higher than in phenol red control (P<0.05). CONCLUSION: Gastric administration of mixed reagent containing 0.12% phenol red, 5% D-xylose and 15% gelatin can simultaneously assay propulsion and absorption of small intestine in mice.

Effects of metabolic acidosis on expression levels of renal drug transporters.

PURPOSE: In the renal proximal tubular cells, various transporters play important roles in the secretion and reabsorption of drugs. When metabolic acidosis is induced, a number of adaptive changes occur in the kidney. The purpose of this study was to clarify the changes of drug transporters under the acidosis and the effects of these changes on urinary drug excretion. METHODS: Wistar/ST rats were given 1.5% NH4Cl in tap water for 48 h to induce the acidosis. Pharmacokinetics of PSP or metformin was evaluated. In addition, expression levels of drug transporters were examined by Western Blotting. RESULTS: The renal clearance of PSP was markedly decreased, whereas the creatinine clearance and renal clearance of metformin were unchanged. Furthermore, Western blots indicated that the protein expression level of organic anion transporter (OAT) 3 was decreased. In contrast to OAT3 levels, OAT1 and organic cation transporter (OCT) 2 levels were unaffected. An immunohistochemical analysis showed that the OAT3 protein in the proximal tubules was localized in the basolateral membrane both of the normal and the acidosis rats. CONCLUSION: The decrease of renal excretion of anionic drugs during metabolic acidosis might be partly due to a reduction in the level of OAT3 protein.

Investigating Intestinal Permeability of Bortezomib Using a Validated HPLC-UV Method.

Bortezomib (BTZ), as a proteasome inhibitor, has been used for treatment of patients with relapsed/refractory multiple myeloma and mantle cell lymphoma. BTZ is available for intravenous injection or subcutaneous administration. In this study, for evaluating the potential of BTZ oral delivery, intestinal permeability of BTZ was determined using in situ single-pass intestinal perfusion (SPIP) technique and the perfused solutions were analyzed using a validated HPLC-UV method. The chromatographic separation was performed using a C18 column via isocratic mode at a flow rate of 0.5 mL/min at 270 nm. The mobile phase was a mixture of methanol/deionized water (50:50% v/v) with 0.1% glacial acetic acid. The results indicated that calibration curves were linear (r2 ˃0.99) in a concentration range of 1.65-5 µg/mL for BTZ and 8.33-25 µg/mL for phenol red. A limit of quantitation of 1.03 and 6.67 µg/mL was obtained for BTZ and phenol red, respectively. The recovery values were in the range of 96.5-105.4% for BTZ, and 88-99.2% for phenol red. The relative standard deviations (RSD) were ≤4.9% for BTZ and ≤7% for phenol red. Stability studies indicated that the working standard solution is stable over a period of 48 h at room temperature. Finally, an effective permeability (Peff) value of (3.36±0.5)×10-5 cm/sec (mean±SEM) was achieved for BTZ. Moreover, it was predicted that BTZ belongs to the biopharmaceutical class ІІІ.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Human oral drugs absorption is correlated to their in vitro uptake by brush border membrane vesicles.

Brush border membrane vesicles (BBMV) were prepared from the rabbit small intestine for testing drug absorption potency through the enterocyte's apical membrane, which is an important compartment for drug oral absorption. Some modifications have been made to the traditional vesicle assay for adapting it to the 96-well plate format. The accumulation of 23 reference drugs was measured, and the data showed a good correlation with human oral absorption with a correlation coefficient R=0.853 (P<0.001), with the exception of a few false positive results. As the measured drug absorption may contain a membrane/protein binding component as well as drug uptake into vesicles, these two fractions can be discriminated by changing extravesicular osmolarity using different mannitol concentrations. This model can be applied for evaluating drug absorption rate/mechanisms, and helping drug selection in early drug research and development.

Circadian Clock Is Involved in Regulation of Hepatobiliary Transport Mediated by Multidrug Resistance-Associated Protein 2.

There has been a growing interest in circadian regulation of the expression and function of drug transporters. In this study, we investigated circadian rhythm in the expression and function of multidrug resistance-associated protein 2 (Mrp2) in mouse liver and involvement of circadian clock in their regulations by using the circadian clock genes (period 1 and period 2) knockout mice. The mRNA and protein expression of Mrp2, P-glycoprotein, and breast cancer resistance protein was measured in the mouse liver at different times of the day. Circadian variation of hepatobiliary excretion of phenolsulfonphthalein, a model substrate of Mrp2, was also investigated in mice. Circadian oscillation of Mrp2 protein expression was clearly observed in the mouse liver with levels down at the light phase and up at the dark phase. The cumulative biliary excretion and biliary clearance of phenolsulfonphthalein from the liver to the bile was 2.37- and 1.74-fold greater in mice administered during the dark phase than in those administered during the light phase, respectively. The circadian oscillation in mRNA expression of Mrp2 disappeared in period 1 and period 2 double knockout mice. These results suggest that the expression and function of Mrp2 show the circadian rhythm, controlled by circadian clock genes.

Development of the isolated dual perfused rat liver model as an improved reperfusion model for transplantation research.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

The molecular assembly of the ionic liquid/aliphatic carboxylic acid/aliphatic amine as effective and safety transdermal permeation enhancers.

In spite of numerous advantages, transdermal drug delivery systems are unfeasible for most drugs because of the barrier effect of the stratum corneum. Ionic liquids were recently used to enhance transdermal drug delivery by improving drug solubility. In the present study, safe and effective ionic liquids for transdermal absorption were obtained as salts generated by a neutralization reaction between highly biocompatible aliphatic carboxylic acids (octanoic acid or isostearic acid) and aliphatic amines (diisopropanolamine or triisopropanolamine) (Medrx Co., Ltd., 2009). The mechanism of skin permeability enhancement by ionic liquids was investigated by hydrophilic phenol red and hydrophobic tulobuterol. Further, the skin permeation enhancing effect was remarkably superior in the acid excess state rather than the neutralization state. Infrared absorption spectrum analysis confirmed that ionic liquids/aliphatic carboxylic acid/aliphatic amine are coexisting at all mixing states. In the acid excess state, ionic liquids interact with aliphatic carboxylic acids via hydrogen bonds. Thus, the skin permeation enhancing effect is not caused by the ionic liquid alone. The "liquid salt mixture," referred to as a complex of ingredients coexisting with ionic liquids, forms a molecular assembly incorporating hydrophilic drug. This molecular assembly was considered an effective and safety enhancer of transdermal drug permeation.

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red.

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.

Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.

Phenolsulfonphthalein transport by potential-sensitive urate transport system.

The purpose of this study was to elucidate the transporter-mediated secretion systems for phenolsulfonphthalein in brush-border membranes. In human and rat renal brush-border membranes, a potential-sensitive transport system has been shown to be involved in the efflux of organic anions. The uptake of phenolsulfonphthalein into rat renal brush-border membrane vesicles was stimulated by an inside-positive membrane potential. This potential-sensitive uptake of phenolsulfonphthalein was inhibited by probenecid, pyrazinoate and urate. p-Aminohippurate had no effect on the potential-sensitive uptake of phenolsulfonphthalein. Moreover, urate competitively inhibited the uptake of phenolsulfonphthalein. On the other hand, the uptake of phenolsulfonphthalein was slightly increased in the presence of an outward Cl- gradient. These results suggest that phenolsulfonphthalein has high affinity for the potential-sensitive urate transport system but has low affinity for an anion exchanger.

Characterization of secretory intestinal transport of phenolsulfonphthalein.

It is known that secretory transport limits the oral bioavailability of certain drugs. However, there is little information on the secretion of anionic compounds in the intestine. Phenolsulfonphthalein (PSP) and p-aminohippuric acid (PAH) have been used widely as substrates for organic anion transport systems. PAH is transported in the secretory direction in the intestine. It is possible that PSP and PAH share the same transport system at the mucosal membrane. The purpose of this study was to characterize the transport system for PSP in the intestine. In the jejunum, the serosal-to-mucosal permeation rate of PSP was significantly reduced in an ATP-depleted condition, whereas a significant difference was not observed in the ileum. Some multidrug resistance-associated protein 2 (Mrp2) inhibitors inhibited PSP permeation in the jejunum. However, pravastatin, a substrate of Mrp2, did not inhibit the PSP permeation. The jejunal secretory transport of pravastatin was significantly reduced in an ATP-depleted condition and by addition of probenecid, but PSP did not affect the jejunal permeation of pravastatin. These results suggest that PSP is secreted into the intestinal lumen by Mrp2-like transporter and that two Mrp2 substrates, PSP and pravastatin, are likely to be transported by different transport systems at the mucosal membrane.

Delivery advantage to the unilateral kidney by direct drug application to the kidney surface in rats and pharmacokinetic verification based on a physiological model.

The objective of this study was to evaluate the drug delivery advantage to the unilateral kidney by direct drug application to the rat kidney surface based on a physiological pharmacokinetic model. Under anesthesia, a cylindrical diffusion cell (i.d. 6 mm, area 0.28 cm(2)) was attached to the right kidney surface in rats. Phenolsulfonphthalein (PSP), an organic anion chosen as a model compound, was added into the diffusion cell. The free PSP concentration in the right (applied) kidney after application to the right kidney surface at a dose of 1 mg was significantly higher than that of the left (non-applied) kidney until 60 min after application. Similarly, the urinary excretion rate of free PSP from the applied kidney was much faster than that from the non-applied kidney, with a 2.6 times larger excreted amount in 240 min. These results imply the possibility that a considerable drug delivery advantage to the unilateral kidney could be obtained after direct absorption from the kidney surface. This tendency was also observed at the other application doses of 0.3 and 1.5 mg. On the other hand, fluorescein isothiocyanate dextran (Mw 4400, FD-4) was equally excreted into the urine from each kidney and the renal concentrations in the applied and non-applied kidneys were almost the same, possibly due to the involvement of passive transport for the absorbed FD-4, i.e. glomerular filtration. The computer simulations of free PSP concentrations in the plasma and each kidney based on a physiological model after kidney surface application were consistent with the respective experimental data. Moreover, the delivery advantage of kidney surface application of PSP was verified by its comparison with other routes such as i.v. and i.a. administrations.

Absorption characteristics of model compounds from the small intestinal serosal surface and a comparison with other organ surfaces.

We examined the absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FD-4, MW 4400; FD-10, MW 9500; FD-40, MW 40 500) as model compounds through the small intestinal serosal surface. After application to the rat small intestinal serosal surface using a cylindrical diffusion cell, each compound was absorbed at different rates. The absorption ratios in 6 h after PSP, FD-4, FD-10 and FD-40 application were calculated to be 89.2, 34.6, 14.9 and 2.1% of dose, respectively. Elimination profiles of PSP, FD-4 and FD-10 from the small intestinal serosal surface obeyed first-order kinetics. Moreover, we calculated the apparent permeability coefficient P(app) for comparison to other organ surfaces. The kidney had the highest absorption efficiency, as shown by having more than 1.5 times significantly higher P(app) values of PSP, FD-4 and FD-10. Similar to the other organ surfaces, a correlation was observed between the P(app) of the small intestine and the molecular weight of these hydrophilic compounds. In addition, the small intestine is likely to contribute largely to hydrophilic compound absorption from the peritoneal cavity, judging from absorption clearance, CL(a), calculated using the peritoneal organ surface area.