Depressive Symptoms and Overperception of Airflow Obstruction in Older Adults With Asthma.

OBJECTIVE: Older adults are at increased risk for depression and poor asthma outcomes. We examined whether depressive symptoms are associated with overperception of airflow obstruction and a pattern of worse asthma control, but not pulmonary function. METHODS: We recruited a cohort of adults with asthma 60 years and older in East Harlem and the Bronx, New York. Baseline measures included the Geriatric Depression Scale, Asthma Control Questionnaire, and Mini Asthma Quality of Life Questionnaire. Spirometry was conducted at baseline to assess pulmonary function. Perception of airflow obstruction was assessed for 6 weeks following baseline by participants entering estimates of peak expiratory flow (PEF) into a programmable peak flow meter followed by PEF blows. Participants were blinded to actual PEF values. The percentage of time that participants were in the overperception zone was calculated as an average. RESULTS: Among the 334 participants (51% Hispanic, 25% Black), depressive symptoms were associated with overperception of airflow obstruction (ß = 0.14, p = .029), worse self-reported asthma control (ß = 0.17, p = .003), and lower asthma-related quality of life (ß = -0.33, p < .001), but not with lung function (ß = -0.01, p = .82). Overperception was also associated with worse self-reported asthma control (ß = 0.14, p = .021), but not lung function (ß = -0.05, p = .41). CONCLUSIONS: Depressive symptoms were associated with greater perceived impairment from asthma, but not pulmonary function. Overperception of asthma symptoms may play a key role in the relationship between depression and asthma outcomes in older adults.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate-methotrexate (MTX)-was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Comparative in vitro release and clinical pharmacokinetics of leuprolide from Luphere 3M Depot, a 3-month release formulation of leuprolide acetate.

A 3-month depot formulation of leuprolide acetate (Luphere 3M Depot) with a mean microsphere diameter of 22.3 µm was prepared aseptically by spray-drying glacial acetic acid solution of the drug and polylactic acid, and lyophilization in a d-mannitol solution. The encapsulation efficiency and loading content of the drug in the Luphere 3M Depot were 94.7% and 9.92% (w/w), respectively. The in vitro release of leuprolide from the depot was substantially delayed and the release profile was similar to that of Lucrin Depot (Abbott Korea, Korea). The safety and pharmacokinetics of leuprolide were investigated over a period of 42 days in 20 prostate cancer patients following a subcutaneous injection of Luphere 3M or Lucrin Depot suspensions (leuprolide acetate dose of 11.25 mg) in a multi-center, randomized, single dose, parallel study. Both formulations were well tolerated by the patients and no serious adverse effects were observed during and after the study. No significant differences were observed in the maximum serum concentration (Cmax) and area under the curve (AUClast) of leuprolide between the two formulations. The results suggest comparable safety and efficacy profiles of Luphere 3M Depot and Lucrin Depot in clinical situations.

Addition of amino acid moieties to lapatinib increases the anti-cancer effect via amino acid transporters.

Anti-cancer agents delivered to cancer cells often show multi-drug resistance (MDR) due to expulsion of the agents. One way to address this problem is to increase the accumulation of anti-cancer agents in cells via amino acid transporters. Thus, val-lapatinib and tyr-lapatinib were newly synthesized by adding valine and tyrosine moieties, respectively, to the parent anti-cancer agent lapatinib without stability issues in rat plasma. Val-lapatinib and tyr-lapatinib showed enhanced anti-cancer effects versus the parent lapatinib in various cancer cell lines, including human breast cancer cells (MDA-MB-231, MCF7) and lung cancer cells (A549), but not in non-cancerous MDCK-II cells. A glutamine uptake study revealed that both val-lapatinib and tyr-lapatinib, but not the parent lapatinib, inhibited glutamine transport in MDA-MB-231 and MCF7 cells, suggesting the involvement of amino acid transporters. In conclusion, val-lapatinib and tyr-lapatinib have enhanced anti-cancer effects, likely due to an increased uptake of the agents into cancer cells via amino acid transporters. The present data suggest that amino acid transporters may be an effective drug delivery target to increase the uptake of anti-cancer agents, leading to one method of overcoming MDR in cancer cells.

The Immune-Stimulating and Anti-Diabetic Effects of Allium hookeri Leaves Grown in a Plant Factory with Artificial Lights in Immunosuppressed Obese C57BL/6 Mice.

We investigated the immune-stimulating and anti-diabetic effects of Allium hookeri leaves grown in a plant factory with artificial lights. The immunomodulatory effects of A. hookeri leaves' ethanol extracts were evaluated with immune-related hematological factors in blood, the proliferation of splenocytes, NK cell activity, IgG and cytokine levels, and their mechanisms in immunosuppressed obese mice. Anti-diabetic effects were determined by the inhibitory activity against α-amylase and α-glucosidase in vitro and fasting blood glucose levels and biochemical factors in the serum of immunosuppressed obese mice. A. hookeri leaf extracts increased WBC and LYM counts, the proliferation of splenocytes, and serum IgG and IL-1ß concentrations compared to those of the NC group, which was used as a negative control. A. hookeri leaf extracts also improved serum HDL levels while they decreased the activities of digestive enzymes, fasting blood glucose, and biochemical factors (ALT, AST, T-Chol, TG, LDL, and GLU). The expressions of IL-1ß, JNK, c-Jun, p65, and iNOS in the thymus of immunosuppressed mice were activated by the treatment of A. hookeri leaf extracts. The results suggest that A. hookeri leaves grown in a plant factory with artificial lights also have immune-stimulatory and anti-diabetic effects and can be used as novel functional supplements to control related diseases and to improve public health.

Transport of gemifloxacin, a 4th generation quinolone antibiotic, in the Caco-2 and engineered MDCKII cells, and potential involvement of efflux transporters in the intestinal absorption of the drug.

The oral (po) bioavailability of gemifloxacin mesylate in rats and its possible association with efflux transporters was investigated. The apparent permeabilities (Papp) of gemifloxacin across the Caco-2 cell monolayer were 1.20 ± 0.09 × 10(-5) cm/s for apical to basal (absorptive) transport, and 2.13 ± 0.6 × 10(-5) cm/s for basal to apical (secretory) transport for a 5-500 µM concentration range, suggesting the involvement of a carrier-mediated efflux in the secretory transport. The secretory transport in Caco-2 cells was significantly decreased by MRP2 (MK571) and BCRP (Ko143) inhibitors. The secretory transport was distinct in MDCKII/P-gp, MDCKII/MRP2 and MDCKII/BCRP cells, and the affinity was highest for MRP2, followed by BCRP and P-gp. The efflux was significantly decreased by verapamil and Ko143, but not significantly by MK571. The comparative po bioavailability in rats was increased by the preadministration of Ko143 (four-fold), MK571 (two-fold) and verapamil (two-fold). Efflux transporters appeared to significantly limit the bioavailability of gemifloxacin in rats, suggesting their possible contribution to the low bioavailability of the drug in the human (70%).

The limited intestinal absorption via paracellular pathway is responsible for the low oral bioavailability of doxorubicin.

1. Doxorubicin exhibited dose-independent pharmacokinetics after intravenous (5-20 mg/kg) and oral (20-100 mg/kg) administration to rats. Nearly all (82.1-99.7%) of the orally administered doxorubicin remained unabsorbed, and the hepatic first-pass extraction ratio and oral bioavailability of doxorubicin were approximately 0.5% and 1%, respectively. Based on these results, it is likely that the primary factor responsible for the low oral bioavailability of doxorubicin is the limited intestinal absorption, rather than the CYP3A4-mediated first-pass metabolism. 2. Moreover, the in vitro transport and cellular uptake studies using Caco-2 cell monolayers have revealed that doxorubicin crosses the intestinal epithelium primarily via the paracellular pathway (accounting for 85.6% of the overall absorptive transport) probably due to its physicochemical properties (hydrophilic cation; pKa = 9.67, log P = -0.5). These results suggest that P-glycoprotein (P-gp)-mediated efflux activity does not play a significant role in limiting the intestinal absorption of doxorubicin, attenuating the absorptive transport by only 5.56-13.2%. 3. Taken together, the present study demonstrated that the limited and paracellular intestinal absorption of doxorubicin was a major factor responsible for its low oral bioavailability, restricting the role of CYP3A4-mediated first-pass metabolism and P-gp-mediated efflux.

Diffuse alveolar hemorrhage due to ketorolac tromethamine.

Drug-induced lung disease (DILD) is a common but frequently missed diagnosis. Therefore, a high index of clinical suspicion and familiarity with the clinical syndromes associated with DILD are important in making the diagnosis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the mostly commonly used classes of medications. NSAIDs are safe when used at prescribed doses. Side effects from use of NSAIDs are not uncommon and can affect almost every organ system in the body. NSAIDs are notorious for causing pulmonary toxicity, the common ones being bronchospasm and hypersensitivity reactions. Diffuse alveolar hemorrhage (DAH) secondary to NSAIDs is uncommon. Here, we report a case of DAH secondary to the use of ketorolac tromethamine.

An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.

The Biopharmaceutics Classification System (BCS) was developed to facilitate estimation of the in vivo pharmacokinetic performance of drugs from human intestinal permeability and solubility. However, the measurement of human in vivo intestinal permeability, unlike that of solubility, is problematic and inefficient. Thus, rat in vitro intestinal permeability results obtained via the Ussing chamber technique are often used instead. However, these data could be unreliable due to difficulty in maintaining the viability of the dissected intestinal membrane in the Ussing chamber. Therefore, a more efficient method to obtain a reliable in vitro permeability is mandatory. Here, we propose a new approach by introducing a novel factor called the permeability ratio (PR). Basically, PR is a rat in vitro intestinal permeability obtained from the Ussing chamber, which is then corrected by the permeability of lucifer yellow, a paracellular permeability marker. To prove the validity of the method, 12 model drugs representing different BCS classes were tested, and the correlation with human in vivo intestinal permeability was high. More importantly, the new method perfectly classified all 12 model drugs. The results indicate that PR is a reliable factor with high correlation to human in vivo intestinal permeability, which can further be used to accurately predict the BCS classification.

Acquired perforating dermatosis: a clinical and dermatoscopic correlation.

Acquired Perforating Dermatosis (APD) is a perforating disease characterized by transepidermal elimination of dermal material [1,2]. This disease usually develops in adulthood. APD has been reported to occur in association with various diseases, but is most commonly associated with dialysis-dependent chronic renal failure (CRF) or diabetes mellitus (DM) [1,2,3,4]. Morton et al found that APD occurs in up to 10% of patients undergoing hemodialysis [5]. Additionally, Saray et al found that sixteen of twenty-two cases with APD were associated with CRF [3].

Poly-L-arginine and dextran sulfate-based nanocomplex for epidermal growth factor receptor (EGFR) siRNA delivery: its application for head and neck cancer treatment.

PURPOSE: A poly-L-arginine (PLR) and dextran sulfate (DEX)-based nano-sized polyelectrolyte complex (nanocomplex) was developed for epidermal growth factor receptor (EGFR) siRNA delivery for the treatment of head and neck cancer. METHODS: PLR and DEX-based nanocomplex including EGFR siRNA was prepared and characterized. In vitro cellular uptake efficiency and EGFR gene silencing effect of nanocomplex including EGFR siRNA were evaluated in Hep-2 and FaDu cells. Its in vivo anti-tumor efficacy was also assessed in FaDu tumor xenografted mouse model. RESULTS: The weight ratio of polymer:RNA was 15:1 and a nanocomplex system consisting of <200 nm in mean diameter and a positive surface charge was prepared. According to the results of confocal laser scanning microscopy (CLSM) and flow cytometry analyses, the PLR-DEX complex exhibited the best cellular uptake efficiency of EGFR siRNA in Hep-2 and FaDu cells, which led to the highest EGFR gene silencing efficiency in both cell lines. PLR-DEX/EGFR siRNA complex exhibited efficient tumor growth inhibition and EGFR silencing effect in a tumor xenografted mouse model. CONCLUSION: PLR and DEX-based nanocomplex containing EGFR siRNA was successfully developed. The new formulation was effective in EGFR gene silencing and tumor growth inhibition in head and neck cancer cells.

Liver cancer targeting of Doxorubicin with reduced distribution to the heart using hematoporphyrin-modified albumin nanoparticles in rats.

PURPOSE: To evaluate the usefulness of hematoporphyrin (HP)-modification of the surface of doxorubicin (DOX)-loaded bovine serum albumin (BSA) nanoparticles (NPs) in the liver cancer-selective delivery of DOX. METHODS: HP-modified NPs (HP-NPs) were prepared by conjugation of amino groups on the surface of NPs with HP, a ligand for low density lipoprotein (LDL) receptors on the hepatoma cells. In vitro cellular accumulation of DOX, in vivo biodistribution of DOX, safety, and anti-tumor efficacy were evaluated for HP-NPs. RESULTS: Cytotoxicity and accumulation of DOX were in the order of HP-NPs>NPs>solution form (SOL). Cellular uptake from HP-NPs was proportional to the expression level of LDL receptors on the cells, indicating possible involvement of LDL receptor-mediated endocytosis (RME) in uptake. The "merit index," an AUC ratio of DOX in liver (target organ) to DOX in heart (major side effect organ) following iv administration of HP-NPs to hepatoma rats, was 132.5 and 4 times greater compared to SOL and NPs, respectively. The greatest suppression of body weight decrease and tumor size increase was observed for iv-administered HP-NPs in tumor-bearing mice. CONCLUSIONS: HP modification appears to be useful in selective delivery of NP-loaded DOX to tumors.

Hyaluronic acid derivative-based self-assembled nanoparticles for the treatment of melanoma.

PURPOSE: Hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs) were developed for the targeted delivery of doxorubicin (DOX), and their antitumor efficacy for melanoma was evaluated. METHODS: DOX-loaded HACE-based self-assembled NPs were prepared and their physicochemical properties were characterized. The in vitro cytotoxicity of HACE was measured using an MTS-based assay. The cellular uptake efficiency of DOX into mouse melanoma B16F10 cells was assessed by confocal laser scanning microscopy and flow cytometry. Tumor growth and body weight were monitored after the intratumoral and intravenous injection of DOX-loaded NPs into a B16F10 tumor-bearing mouse model. RESULTS: DOX-loaded NPs, with a mean diameter of ~110 nm, a narrow size distribution, and high drug entrapment efficiency, were prepared. A sustained DOX release pattern was shown, and drug release was enhanced at pH 5.5 compared with pH 7.4. The cytotoxicity of HACE to B16F10 cells was negligible. It was assumed that DOX was taken up into the B16F10 cells through receptor-mediated endocytosis. A significant inhibitory effect was observed on tumor growth, without any serious changes in body weight, after the injection of DOX-loaded NPs into the B16F10 tumor-bearing mouse model. CONCLUSIONS: DOX-loaded HACE-based NPs were successfully developed and their antitumor efficacy against B16F10 tumors was demonstrated.

Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay.

The proper orientation and lateral spacing of antibody molecules are a crucial element for an on-chip immunoassay in which the antibody or its antigen-binding fragments are immobilized on a solid surface. We covalently immobilized a modified protein G (Cys-protein G: protein G with only an N-terminal cysteine) on a dendron-coated surface to control its orientation and lateral spacing simultaneously. The cysteine-specific immobilization of Cys-protein G through the N-terminal cysteine resulted in 2.2-fold higher binding efficiency of Cys-protein G to IgG(2a) capture antibody than its random immobilization via lysine residues. The lateral spacing of 3.2 nm due to the surface modification with the 9-acid dendron molecule contributed to a 1.5-fold increase in the antibody-binding ability of Cys-protein G. Topographic images of atomic force microscopy exhibited a uniform coverage of Cys-protein G molecules immobilized on the thiol-reactive 9-acid dendron surface and homogeneous distribution of antibody bound to Cys-protein G. In the sandwich immunoassay, the control of the orientation of Cys-protein G led to 10-fold higher detection capability for rIL-2 compared with the randomly oriented protein G. The synergistic advantage of the unidirectional orientation and homogeneous lateral spacing of Cys-protein Gs on the dendron-coated surface can be applied to the development of more sensitive and reproducible antibody microarrays.

Antifibrotic effect of MMP13-encoding plasmid DNA delivered using polyethylenimine shielded with hyaluronic acid.

The imbalanced expression of matrix metalloproteinases (MMPs) is associated with liver fibrosis, one of the most common chronic liver diseases. Enhanced expression of MMPs by gene therapy is emerging as a promising antifibrotic strategy, but the effectiveness of this approach depends on reliable systems for delivering MMP genes. Here, we evaluated a newly designed hyaluronic acid (HA)-shielded delivery system for systemic administration of plasmid DNA encoding MMP13 (pMMP13), and tested whether the enhanced expression of MMP13 ameliorates liver fibrosis in mice. In the CCl(4)-induced liver fibrosis model, systemic administration of pMMP13 using HA and polyethylenimine (PEI) significantly increased the expression of MMP13 and reduced collagen deposition. Moreover, following delivery of pMMP13 in a HA-shielded PEI complex, the serum levels of aspartate transaminase were reduced to levels approaching those in untreated normal mice. These results indicate that the delivery of pMMP13 using HA-shielded PEI enhances the efficiency of MMP13 expression in the liver, and highlight the potential of pMMP13 gene therapy as an antifibrotic strategy.

Enhanced intracellular accumulation of a non-nucleoside anti-cancer agent via increased uptake of its valine ester prodrug through amino acid transporters.

The phenomenon known as multiple-drug resistance, whereby anti-cancer agents are expelled from cancer cells, makes it necessary to develop methods that will reliably increase the accumulation of anti-cancer agents within cancer cells. To accomplish this goal, a new model compound, Val-SN-38, was synthesized by introducing valine to SN-38, an active ingredient of irinotecan. Val-SN-38 improved intracellular accumulation approximately 5-fold in MCF7 cells, compared with SN-38, and rather than changes in membrane permeability, the amino acid transporter ATB(0,+) played a role, whereas the dipeptide transporter PEPT1 did not. Other sodium-dependent amino acid transporters, namely ATA1, ATA2, and ASCT2, were unexpectedly involved in the uptake of Val-SN-38 as well. The efflux of Val-SN-38 by major efflux transporters was variably changed, but not significantly. In summary, the enhanced accumulation of Val-SN-38 in cancer cells was due to augmented uptake via various amino acid transporters. The results of the present study make a compelling argument in favour of a prodrug concept that can improve intracellular accumulation and take advantage of amino acid transporters without significantly inducing multiple-drug resistance.

Saturable sinusoidal uptake is rate-determining process in hepatic elimination of docetaxel in rats.

Identifying kinetic determinants of hepatic elimination of drugs would be crucial for better understanding its pharmacokinetics and predicting drug interactions. Present study investigated the kinetics of sinusoidal uptake of docetaxel and its impact on the overall hepatic elimination of docetaxel in rats. The non-renal clearance (CL(NR); hepatic elimination) of docetaxel were significantly reduced by co-administration of intravenous rifampicin, a potent inhibitor of organic anion transporting peptides (OATPs; Oatps), at a dose of 20 mg/kg. Docetaxel uptake into isolated rat hepatocytes was found to be temperature/concentration/energy-dependent, saturable, and reduced by Oatps inhibitors (rifampicin and bromosulfophthalein). Moreover, docetaxel uptake into perfused rat liver was significantly reduced in the presence of 10-µM rifampicin. However, docetaxel metabolism in rat hepatic microsome was not affected by rifampicin at less than 50 µM. Based on the comparison of intrinsic clearances related to hepatic clearance, it can be suggested that sinusoidal uptake could be the rate-determining process in the overall hepatic elimination of docetaxel in rats.

Fluorescence detection of Zabofloxacin, a novel fluoroquinolone antibiotic, in plasma, bile, and urine by HPLC: the first oral and intravenous applications in a pharmacokinetic study in rats.

PURPOSE: To develop an HPLC method using fluorescence detection for the pharmacokinetic evaluation of levels of zabofloxacin, a novel broad spectrum fluoroquinolone antibiotic, in the plasma, bile and urine of rats. METHODS: A simple reversed-phase HPLC method using a C18 column with fluorescence detection was developed and validated for the simultaneous determination of zabofloxain and enrofloxacin as an internal standard. The plasma sample was treated with methanol for protein precipitation, and treatment of the bile and urine samples included deproteinization and extraction using chloroform. The applicability of the developed assay method to pharmacokinetic studies of zabofloxacin in rats was examined. Zabofloxacin was intravenously and orally administered to rats at a dose of 20 mg/kg. RESULTS: The limits of quantification (LOQ) was determined to be 50 ng/mL for the plasma with acceptable linearity ranging from 50 to 25,000 ng/mL (R>0.999), and 0.5 µg/mL for the bile and urine samples with acceptable linearity ranging from 0.5 to 100 µg/mL (R>0.999). The validation parameters for zabofloxacin were found to be acceptable according to FDA assay validation (2001). While zabofloxacin in plasma and urine has been stable in all tested handling conditions, it has been unstable in bile during freeze-thaw cycles for 24 h at room temperature. Following intravenous and oral administration of zabofloxacin to rats at a dose of 20 mg/kg, concentration was quantifiable in plasma for up to 8 h. The bioavailability of zabofloxacin was 27.7%, and it was excreted into bile and urine at about 8% each per oral administration. CONCLUSIONS: These observations suggest that a validated assay can be used in pharmacokinetic studies of zabofloxacin in small animals. Due to the limited stability of zabofloxcin in rat bile, freeze-thaw cycles or prolonged handling at room temperature is not recommended. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Pharmacokinetics and first-pass elimination of metoprolol in rats: contribution of intestinal first-pass extraction to low bioavailability of metoprolol.

1. The pharmacokinetics of metoprolol after intravenous (IV) (0.5, 1, and 2 mg/kg) and oral (1, 2, and 5 mg/kg) administration, and the intestinal and hepatic first-pass extraction of metoprolol after IV, intraportal, and intraduodenal (1 and 2 mg/kg) administration were comprehensively assessed in rats. 2. Metoprolol exhibited dose-independent pharmacokinetics after IV administration, and dose-dependent pharmacokinetics after oral administration probably due to the saturable first-pass extraction of metoprolol. At doses where metoprolol exhibited dose-independent pharmacokinetics (1 and 2 mg/kg), complete absorption (>99.2%) and low F (<0.245) after oral administration were observed. The intestinal and hepatic first-pass extraction ratio (E(G) and E(H), respectively) of metoprolol were approximately 0.45 and 0.60, respectively (equivalent to approximately 45% and 30% of orally administered dose, respectively), suggesting considerable contribution of intestinal first-pass extraction to the low F of metoprolol in rats. 3. The E(G) in rats was predicted from in vitro clearance and/or permeability data utilizing the Q(Gut) model and well-stirred model (0.347 and 0.626, respectively). The predicted E(G) values were in good agreement with the observed in vivo E(G) (0.492-0.443), suggesting the utility of the prediction of in vivo intestinal first-pass extraction from the in vitro clearance using intestinal microsomes.