Exogenous ketone ester delays CNS oxygen toxicity without impairing cognitive and motor performance in male Sprague-Dawley rats.

Hyperbaric oxygen (HBO2) is breathing >1 atmosphere absolute (ATA; 101.3 kPa) O2 and is used in HBO2 therapy and undersea medicine. What limits the use of HBO2 is the risk of developing central nervous system (CNS) oxygen toxicity (CNS-OT). A promising therapy for delaying CNS-OT is ketone metabolic therapy either through diet or exogenous ketone ester (KE) supplement. Previous studies indicate that KE induces ketosis and delays the onset of CNS-OT; however, the effects of exogeneous KE on cognition and performance are understudied. Accordingly, we tested the hypothesis that oral gavage with 7.5 g/kg induces ketosis and increases the latency time to seizure (LSz) without impairing cognition and performance. A single oral dose of 7.5 g/kg KE increases systemic ß-hydroxybutyrate (BHB) levels within 0.5 h and remains elevated for 4 h. Male rats were separated into three groups: control (no gavage), water-gavage, or KE-gavage, and were subjected to behavioral testing while breathing 1 ATA (101.3 kPa) of air. Testing included the following: DigiGait (DG), light/dark (LD), open field (OF), and novel object recognition (NOR). There were no adverse effects of KE on gait or motor performance (DG), cognition (NOR), and anxiety (LD, OF). In fact, KE had an anxiolytic effect (OF, LD). The LSz during exposure to 5 ATA (506.6 kPa) O2 (≤90 min) increased 307% in KE-treated rats compared with control rats. In addition, KE prevented seizures in some animals. We conclude that 7.5 g/kg is an optimal dose of KE in the male Sprague-Dawley rat model of CNS-OT.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.

Safety Assessment of Saccharide Esters as Used in Cosmetics.

This is a safety assessment of 40 saccharide ester ingredients as used in cosmetics. The saccharide esters are reported to function in cosmetics as emollients, skin-conditioning agents, fragrance ingredients, and emulsion stabilizers. The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the relevant data for these ingredients. The Panel concluded that the saccharide esters are safe in cosmetics in the present practices of use and concentrations described in this safety assessment.

Extracellular-Matrix-Anchored Click Motifs for Specific Tissue Targeting.

Local presentation of cancer drugs by injectable drug-eluting depots reduces systemic side effects and improves efficacy. However, local depots deplete their drug stores and are difficult to introduce into stiff tissues, or organs, such as the brain, that cannot accommodate increased pressure. We present a method for introducing targetable depots through injection of activated ester molecules into target tissues that react with and anchor themselves to the local extracellular matrix (ECM) and subsequently capture systemically administered small molecules through bioorthogonal click chemistry. A computational model of tissue-anchoring depot formation and distribution was verified by histological analysis and confocal imaging of cleared tissues. ECM-anchored click groups do not elicit any noticeable local or systemic toxicity or immune response and specifically capture systemically circulating molecules at intradermal, intratumoral, and intracranial sites for multiple months. Taken together, ECM anchoring of click chemistry motifs is a promising approach to specific targeting of both small and large therapeutics, enabling repeated local presentation for cancer therapy and other diseases.

In Vivo Ester Hydrolysis as a New Approach in Development of Positron Emission Tomography Tracers for Imaging Hypoxia.

Hypoxia is an important biochemical and physiological condition associated with uncontrolled growth of tumor. Measurement of hypoxia in tumor tissue may be useful in characterization of tumor progression and monitoring drug treatment. [18F]FMISO is the most widely employed radiotracer for imaging of hypoxic tissue with positron emission tomography (PET). However, it showed relatively low uptake in hypoxic tissues, which led to low target-to-background contrast in PET images. To overcome these shortcomings, two novel 2-fluoroproprioic acid esters, nitroimidazole derivatives 2-fluoropropionic acid 2-(2-nitro-imidazol-1-yl)-ethyl ester (FNPFT, [19F]5) and 2-fluoropropionic acid 2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester (FMNPFT, [19F]8), were prepared and tested. Radiolabeling of [18F]5 and [18F]8 was accomplished in 45 min (radiochemical purity >95%, the decay-corrected radiochemical yield of [18F]5 was 11 ± 2%, and that of [18F]8 was 13 ± 2%, n = 5). In vitro cell uptake studies using EMT-6 tumor cells showed that both radiotracers [18F]5 and [18F]8 displayed significantly higher uptake in hypoxic cells than those under normoxic condition, while 2-[18F]fluoropropionic acid (2-[18F]FPA) displayed no difference. Biodistribution studies in mice bearing EMT-6 tumor showed that [18F]5, [18F]8, and 2-[18F]FPA displayed similar tumor and major organ uptakes. Tumor uptake values for all three agents were higher than those of [18F]FMISO, respectively ( P < 0.05). This is likely due to a rapid in vivo hydrolysis of [18F]5 and [18F]8 to their metabolite, 2-[18F]FPA. Micro PET imaging studies in the same EMT-6 implanted mice tumor model also demonstrated that both [18F]5 and [18F]8 displayed similar tumor uptake comparable to that of 2-[18F]FPA. In conclusion, two new fluorine-18 labeled nitroimidazole derivatives, [18F]5 and [18F]8, showed good tumor uptakes in mice bearing EMT-6 tumor. However, in vivo biodistribution results suggested that they were more likely reflect the predominance of in vivo produced metabolite, 2-[18F]FPA, which may not be related to tumor hypoxic condition.

Coadministration of the prostaglandin F2α receptor antagonist preterm labour drug candidate OBE022 with magnesium sulfate, atosiban, nifedipine and betamethasone.

AIMS: To investigate presence or absence of clinically relevant drug interactions (pharmacokinetic and safety/tolerability) of OBE022 with standard-of-care medicines for preterm labour, enabling coadministration and further clinical development. METHODS: Part A: open-label, randomized, 3-period crossover assessing coadministration of single doses of OBE022 (1100 mg) and MgSO4 . Part B: open-label, single-sequence crossover assessing the interactions following administration of OBE022 (1000 mg/day) at steady state coadministered with single doses of atosiban, nifedipine and betamethasone. Twenty-five healthy nonpregnant women of reproductive age were enrolled (Part A: n = 12; Part B: n = 13). RESULTS: OBE022, alone or in combination with standard-of-care medications, was well tolerated. Headache and dizziness were the most frequently reported adverse events; dizziness occurred more often with the nifedipine/OBE022 combination. There were no clinically significant pharmacokinetic interactions when coadministered with MgSO4 . Co-administration had no notable effect on atosiban exposure. Atosiban reduced exposure to OBE002 (peak concentration [Cmax ] 22%, area under the concentration-time curve [AUC] 19%). Coadministration with betamethasone slightly increased betamethasone exposure (Cmax  + 18%, AUC +27%) and OBE002 exposure (Cmax  + 35%, AUC +15%). These changes were not considered clinically significant. Coadministration with nifedipine slightly increased OBE002 exposure (Cmax  + 29%, AUC +24%) and markedly increased nifedipine exposure (Cmax by 2-fold and AUC by 2-fold), which may be clinically significant. CONCLUSIONS: The use of OBE022, a PGF2α antagonist prodrug, in combination with standard-of-care medicines may provide new treatment alternatives for preterm labour. All tested combinations were well tolerated. Nifedipine doses could potentially be reduced or staggered when coadministered with OBE022.

Safety Assessment of Sorbitan Esters as Used in Cosmetics.

The Cosmetic Ingredient Review Expert Panel (Panel) assessed the safety of 20 sorbitan esters; this report included sorbitan esters that were reviewed in 1985 and 2002, as well as 3 previously unreviewed sorbitan esters (sorbitan undecylenate, sorbitan sesquicaprylate, and sorbitan palmate). Most of the sorbitan esters are reported to function in cosmetics as surfactant-emulsifying agents. The Panel reviewed the data from previous sorbitan ester reports, as well as additional data included in this report, to determine the safety of these ingredients. The Panel concluded that the sorbitan esters included in this safety assessment are safe in cosmetics in the present practices of use and concentration.

Pharmacokinetics, safety and tolerability of OBE022, a selective prostaglandin F2α receptor antagonist tocolytic: A first-in-human trial in healthy postmenopausal women.

AIMS: Preterm birth remains a significant risk for later disability. The selective inhibition of the prostaglandin F2α receptor has significant advantages for a tocolytic. The prodrug OBE022 and its metabolite OBE002 are novel prostaglandin F2α receptor antagonists under development for treating preterm labour. METHODS: We performed a prospective, first in human, Phase I, dose escalation, placebo-controlled, randomized trial at a clinical trial site in the UK. Placebo, single ascending doses of 10, 30, 100, 300, 1000 or 1300 mg, and multiple ascending doses over 7 days of 100, 300 or 1000 mg day-1 ; were administered to postmenopausal female volunteers. Food interaction was additionally evaluated. RESULTS: Subjects tolerated OBE022 well at all single and multiple doses. No clinically relevant changes in safety parameters were shown and there were no serious adverse events. Observations showed that prodrug OBE022 was readily absorbed and rapidly converted into its equally active stable metabolite OBE002. The plasma level of OBE002 rose with increasing doses, reaching exposure levels that were anticipated to be clinically relevant within 1 h following administration. There was no clinically significant food interaction, with peak exposures reduced to 80% and area under the curve staying bioequivalent. The mean half-life of OBE002 ranged between 8 and 11 h following administration of a single dose and 22-29 h after multiple doses. CONCLUSIONS: Administration of OBE022 was safe and had favourable pharmacokinetic characteristics and no clinically relevant interaction with food. Our results allow further investigation of OBE022 in preterm labour patients.

Analysis of lanthionine ketimine ethyl ester in mouse serum, whole blood and tissues using ultrahigh-pressure liquid chromatography/tandem mass spectrometry.

RATIONALE: Preclinical studies in the search for treatments for several neurodegenerative diseases have identified lanthionine ketimine (LK) and its monoethyl ester derivative (LKE) as potential candidates. An ultrahigh-pressure liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) assay was developed to evaluate bioavailability by measuring these compounds in mouse serum, whole blood and brain tissue. METHODS: Following administration of LKE to mice for 3 days in chow at 300 ppm, the animals were sacrificed, and LKE was extracted from serum, whole blood and brain tissues through protein precipitation using cold methanol. To enhance chromatographic separation and electrospray ionization, LK was methylated using diazomethane. Separations were carried out using C18 reversed-phase UHPLC, and quantitative measurements were obtained using on-line triple-quadruple mass spectrometry with positive ion electrospray ionization, collision-induced dissociation and selected reaction monitoring. Tolbutamide was used as internal standard. RESULTS: LKE showed good recovery ranging from 77-90% in serum and 82-88% in brain tissue. An eight-point standard curve ranging from 0.005 to 4.6 µM was linear (R2 0.998). The average LKE detected in mouse serum was 277.42 nM, while the concentration in whole blood was 38 nM. Neither LK nor LKE was detected in brain tissues. CONCLUSIONS: A rapid quantitative method to measure LKE in mouse serum, whole blood and brain tissues using UHPLC/MS/MS was developed and validated following FDA guidelines. This method is suitable for bioavailability and pharmacokinetic studies.

Pharmacokinetic studies of naproxen amides of some amino acid esters with promising colorectal cancer chemopreventive activity.

Naproxen (nap) is belonging to Non-steriodal anti-inflammatory drugs (NSAIDs) group of drugs that characterized by their free carboxylic group. The therapeutic activity of nap is usually accompanied by GI untoward side effects. Recently synthesized naproxen amides of some amino acid esters prodrugs to mask the free carboxylic group were reported. Those prodrugs showed a promising colorectal cancer chemopreventive activity. The current study aims to investigate the fate and hydrolysis of the prodrugs kinetically in different pH conditions, simulated gastric and intestinal fluids with pHs of 1.2, 5.5 and 7.4 in vitro at 37 °C. The effect of enzymes on the hydrolysis of prodrugs was also studied through incubation of these prodrugs at 37 °C in human plasma and rat liver homogenates. The pharmacokinetic parameters of selected prodrugs and the liberated nap were studied after oral and intraperitoneal administration in male wistar rats. The results showed the hydrolysis of naproxen amides of amino acid esters to nap through two steps first by degradation of the ester moiety to form the amide of nap with amino acid and the second was through the degradation of the amide link to liberate nap. The two reactions were followed and studied kinetically where K1 and K2 (rate constants of degradation) is reported. The hydrolysis of prodrugs was faster in liver homogenates than in plasma. The relative bioavailability of the liberated nap in vivo was higher in case of prodrug containing ethyl glycinate moiety than that occupied l-valine ethyl ester moiety. Each of nap. prodrugs containing ethyl glycinate and l-valine ethyl ester moieties appears promising in liberating nap, decreasing direct GI side effect and consequently their colorectal cancer chemopreventive activity.

Involvement of CYP4F2 in the Metabolism of a Novel Monophosphate Ester Prodrug of Gemcitabine and Its Interaction Potential In Vitro.

Compound-3 is an oral monophosphate prodrug of gemcitabine. Previous data showed that Compound-3 was more potent than gemcitabine and it was orally active in a tumor xenograft model. In the present study, the metabolism of Compound-3 was investigated in several well-known in vitro matrices. While relatively stable in human and rat plasma, Compound-3 demonstrated noticeable metabolism in liver and intestinal microsomes in the presence of NADPH and human hepatocytes. Compound-3 could also be hydrolyzed by alkaline phosphatase, leading to gemcitabine formation. Metabolite identification using accurate mass- and information-based scan techniques revealed that Compound-3 was subjected to sequential metabolism, forming alcohol, aldehyde and carboxylic acid metabolites, respectively. Results from reaction phenotyping studies indicated that cytochrome P450 4F2 (CYP4F2) was a key CYP isozyme involved in Compound-3 metabolism. Interaction assays suggested that CYP4F2 activity could be inhibited by Compound-3 or an antiparasitic prodrug pafuramidine. Because CYP4F2 is a key CYP isozyme involved in the metabolism of eicosanoids and therapeutic drugs, clinical relevance of drug-drug interactions mediated via CYP4F2 inhibition warrants further investigation.

Synthesis, stability and bioavailability of astaxanthin succinate diester.

BACKGROUND: We synthesized astaxanthin succinate diester (ASD), a novel astaxanthin (AST) derivate, with succinic anhydride and free AST. ASD was purified and characterized using silica gel column chromatography and spectrometry, respectively. RESULTS: The ASD final synthesis rate was 82.63%. A stability test revealed a high AST and ASD retention rate at pH 5.0-7.0. ASD showed better stability than did AST under acidic conditions. Both sample ions showed lower retention rates under Fe2+ and Fe3+ states. The ASD metabolic curve showed serum and liver area under the curve from 0 h to time t (AUC0-t ) values of 45.05 ± 4.58 and 120.38 ± 23.66 µg h-1  mL-1 , respectively. The long-term accumulation was significantly higher in the ASD group than in the AST group, which showed higher accumulation in the heart, muscle and spleen than in other tissues in vivo. CONCLUSION: The thermal stability and bioavailability of ASD were higher than that of the non-esterified free AST and common free AST, respectively. Additionally, AST accumulation in different tissues of the ASD group was multifold higher than that of free AST. These results prove that ASD may serve as a better source of AST for human nutrition than does free AST. © 2017 Society of Chemical Industry.

Oral pharmacokinetic interaction of ester rich fruit juices and pharmaceutical excipients with tenofovir disoproxil fumarate in male Wistar rats.

1. The aim of this study was to evaluate the role of intestinal esterases on the absorption process of tenofovir disoproxil fumarate (TDF). 2. The esterase inhibition capacity of fruit juices (FJs) rich in ester linkages and pharmaceutical excipients (having ester bonds) was performed in vitro by incubating TDF with each FJ and excipient in the intestinal washings. The ex vivo everted gut sac model was also used to evaluate the absorption enhancement capacity of these FJs and excipients. Single-dose oral pharmacokinetic studies were performed by concomitant administration of TDF with each of the selected FJs and excipients. 3. The in vitro and ex vivo studies showed that cremophor-EL and all FJs prevented the metabolism of TDF with grapefruit juice (GFJ) having the highest level of inhibition. Further, the permeability flux of the monoester form of tenofovir was increased by 113% and 212% by cranberry juice (CBJ) and GFJ, respectively. The in vivo studies also showed that both CBJ and GFJ enhanced the oral bioavailability of TDF as the AUC was increased by 24% and 97%, respectively. 4. These results indicate that the prevention of the metabolic conversion of TDF to its monoester form is crucial in increasing the oral absorption of TDF.

Optimization of potency and pharmacokinetics of tricyclic indole derived inhibitors of HCV NS5B polymerase. Identification of ester prodrugs with improved oral pharmacokinetics.

HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current efforts are directed towards identification of novel direct acting antiviral targeting different mechanism of actions which could become part of all oral therapies. We recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase that bound to the enzyme close to the active site. In this manuscript we describe further optimization of potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a dimethyl ethyl amino ester prodrug.

Identification of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives and their orally active prodrug esters as dual-acting alpha4-beta1 and alpha4-beta7 receptor antagonists.

N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several derivatives were identified as very potent dual-acting α4-integrin, α4ß1 and α4ß7 antagonists. Investigation of a limited number of prodrug esters led to the discovery of the ethyl ester prodrug 42, which demonstrated good intestinal fluid stability and good permeability. Despite low solubility, 42 gave acceptable blood levels of 30 when dosed orally in non-human primates. Additionally, 42 had an overall excellent profile and was selected for clinical trials. Investigation of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives led to the discovery of several very potent dual-acting α4-integrin antagonists. Ethyl ester prodrug 42 advanced to human clinical trials based on the excellent intestinal fluid stability, good permeability and superior efficacy in non-human primates.

Preclinical pharmacokinetic analysis of armillarisin succinate ester in mouse plasma and tissues by LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated to determine the concentration of armillarisin succinate ester in mouse plasma and tissues, used for preclinical evaluation. Bavachin was employed as the internal standard. Separation was performed on a 3.5 µm Zorbax SB-C(18) column (30 × 2.1 mm), with a mobile phase consisting of methanol and aqueous 20 mm ammonium acetate. Both analyte and internal standard were determined using electrospray ionization and the MS data acquisition was via selected ion monitoring in negative scanning mode. Quantification was performed using the transitions m/z 333 → 233 and 323 → 221 for armillarisin succinate ester and internal standard, respectively. The method was validated with respect to linearity, accuracy, precision, recovery and stability. This assay has been successfully applied to a pharmacokinetic and tissue distribution study after intravenous injection of ASE in mouse in a dose of 10 mg/kg.

Enzymatic Strategies for the Preparation of Pharmaceutically Important Amino Acids through Hydrolysis of Amino Carboxylic Esters and Lactams.

The most relevant lipase-catalyzed strategies for the synthesis of pharmaceutically important cyclic and acyclic α-, ß- and γ-amino carboxylic acid enantiomers through hydrolysis of the corresponding amino carboxylic esters and lactams, over the last decade are overviewed. A brief Introduction part deals with the importance and synthesis of enantiomeric amino acids, and formulates the objectives of the actual work. The strategies are presented in the Main Text, in chronological order, classified as kinetic, dynamic kinetic and sequential kinetic resolution. Mechanistic information of the enzymatic transformations is also available at the end of this overview. The pharmacological importance of the enantiomeric amino acids is given next to their synthesis, in the Main Text, and it is also illustrated in the Conclusions and Outlook sections.

Phytosterol ester processing in the small intestine: impact on cholesterol availability for absorption and chylomicron cholesterol incorporation in healthy humans.

Phytosterols (plant sterols and stanols) can lower intestinal cholesterol absorption, but the complex dynamics of the lipid digestion process in the presence of phytosterol esters (PEs) are not fully understood. We performed a clinical experiment in intubated healthy subjects to study the time course of changes in the distribution of all lipid moieties present in duodenal phases during 4 h of digestion of meals with 3.2 g PE (PE meal) or without (control meal) PE. In vitro experiments under simulated gastrointestinal conditions were also performed. The addition of PE did not alter triglyceride (TG) hydrolysis in the duodenum or subsequent chylomicron TG occurrence in the circulation. In contrast, cholesterol accumulation in the duodenum aqueous phase was markedly reduced in the presence of PE (-32%, P < 0.10). In vitro experiments confirmed that PE reduces cholesterol transfer into the aqueous phase. The addition of PE resulted in a markedly reduced presence of meal-derived hepta-deuterated cholesterol in the circulation, i.e., in chylomicrons (-43%, PE meal vs. control; P < 0.0001) and plasma (-54%, PE meal vs. control; P < 0.0001). The present data show that addition of PE to a meal does not alter TG hydrolysis but displaces cholesterol from the intestinal aqueous phase and lowers chylomicron cholesterol occurrence in humans.

Utilisation of iodine from different sources by sows and their progeny.

OBJECTIVES: The aim of the study was to compare iodine utilization from different sources by sows and their progeny and the levels of T3 and T4 in their serum. DESIGN: Pregnant Czech Large White × Landrace sows were fed with an experimental KPK diet (a diet for lactating sows) 14 days before parturition until weaning (at a piglet age of 28 days). In group A (n=50, 10 sows, 40 piglets) the feed was supplemented with KI (0.6 mg of iodine per kg of feed). Iodine enriched alga Chlorella spp. (0.6 mg of iodine per kg of feed) was used as a supplement in group B (n=50, 10 sows, 40 piglets). In group C (n=50, 10 sows, 40 piglets) the sows were injected i.m. with IFAE at a dose of 100 mg of iodine per sow. Iodine, T3 and T4 were measured in each group for comparison of iodine utilization. RESULTS: The use of IFAE resulted in higher serum concentrations in sows compared to KI and alga. In contrast, iodine concentrations in milk and piglets were lower when IFAE were used. We found a wide variation in the concentrations of T3 and T4 in the serum of piglets in all groups. CONCLUSION: Our results indicate a good utilization of iodized oil by sows. However, its transfer into milk is lower compared to the other iodine sources.

Identification of carboxylesterases expressed in rat intestine and effects of their hydrolyzing activity in predicting first-pass metabolism of ester prodrugs.

Carboxylesterases (CESs) located in the intestine play an unique role in the absorption of many drugs especially ester prodrugs. In order to determine the expression and hydrolyzing activity of CESs isozymes (CES1 and CES2) located in rat intestine, the activities of CES1 and CES2 were evaluated by the intestinal S9 incubation with imidapril and irinotecan (CPT-11), the substrates of CES1 and CES2, respectively. The distribution characteristics of CES1, CES2, Pregnane X Receptor (PXR) and Constitutive Androstane Receptor were analyzed by real-time polymerase chain reaction (RT-PCR) or Western blot. Imidaprilat metabolized from imidapril by CES1 was too low to be detected in rat intestinal S9 fractions, while there was little and even no expression of CES1 mRNA in intestinal segments. In contrast, Vmax values for CPT-11 diminished gradually from proximal to distal segments within the rat intestine which was consistent with the mRNA expression level of CES2. These results indicated that CES2 represents the major CESs isoform in the rat complete intestine and decreased from duodenum to colon, whereas the expression of CES1 was too low to influence the metabolism of ester prodrugs. The expression of PXR and CAR decreased slightly along the entire intestine on both mRNA and protein levels which indicated that PXR and CAR may be one of the major factors which contribute to the expression of CES1 and CES2. Thus, the knowledge about the characteristic and site-specific expression of CES1 and CES2 in rat intestine will help to predict the oral bioavailability of ester prodrugs.