Comparison between Radioanalysis and 19F Nuclear Magnetic Resonance Spectroscopy in the Determination of Mass Balance, Metabolism, and Distribution of Pefloxacin.

Mass balance and metabolism studies using radiolabeled substances are well recognized as an important part of the drug development process. In this study, we directly assessed the use of fluorine nuclear magnetic resonance (19F NMR) to achieve quantitative mass balance, metabolism, and distribution information for fluorinated compounds, without the need for radiolabeled synthesis or study. As a test case, the disposition of pefloxacin, a fluoroquinolone antibiotic, was evaluated in rats using quantitative 19F NMR in parallel with a radiolabeled study. Urine, bile, and feces samples were collected over specific periods after oral administration of either 25 mg/kg [14C]pefloxacin or 25 mg/kg pefloxacin and were subsequently profiled by radioactivity or 19F NMR, respectively. The percentage of dose excreted in each matrix was comparable between the two methods, with the total dose recovered by radioactivity and 19F NMR determined to be 86.8% and 81.8%, respectively. In addition, plasma samples were collected to determine the exposure of pefloxacin and its circulating metabolites. The plasma exposure of pefloxacin determined by 19F NMR was within 5% to that calculated by a validated liquid chromatography-tandem mass spectrometry bioanalytical method. By both methods, pefloxacin was identified as the major circulating entity, with pefloxacin glucuronide as the major circulating metabolite. Quantitative analysis of metabolites in excreta was generally comparable between the two methods. In selected tissues, both methods indicated that the parent drug accounted for most of the drug-related material. In summary, we have demonstrated that 19F NMR can be used as an alternative method to conventional radiolabeled studies for compounds containing fluorine without the need for radiolabeled synthesis/study.

Evaluation and Optimization of Blood Micro-Sampling Methods: Serial Sampling in a Cross-Over Design from an Individual Mouse.

PURPOSE: Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles.  The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice.  In addition, the feasibility of cross-over designs was assessed and recommendations are presented. METHODS: The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice.  The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples.  Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. RESULTS: Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics.  The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective.  Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals. CONCLUSIONS:   Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability.  It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design.  This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse PK studies. 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.

Characterization of the expression and activity of carboxylesterases 1 and 2 from the beagle dog, cynomolgus monkey, and human.

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

Genomic analysis of the carboxylesterases: identification and classification of novel forms.

Large species differences in the expression of carboxylesterases (CE) have been described, but the interrelationships of CEs across species are not well characterized. In the current analyses, sequences with genomic structures similar to human CEs were found in piscine, avian, and mammalian genomes. Analyses of these genes suggest that four CE groups existed prior to mammalian divergence, with another form occurring after eutherian-marsupial divergence, yielding five distinct mammalian CE groups. The CE1 and CE2 groupings appear to have undergone extensive gene duplication in species with herbivorous and omnivorous diets underscoring the potential importance of these two groups in xenobiotic metabolism. However, CE3, CE4, and CE5 have remained at one gene per species in almost all observed cases. In avian and piscine genomes, only two CE groupings each were observed in the currently available sequence data. Finally, this study presents considerations for a broader phylogenetic-based nomenclature that could encompass other serine hydrolases in addition to the CEs.

Transepithelial transport of the group II metabotropic glutamate 2/3 receptor agonist (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740) and its prodrug (1S,2S,5R,6S)-2-[(2'S)-(2'-amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344).

The limited oral bioavailability of the potent and selective group II metabotropic glutamate (mGlu) 2/3 receptor agonist, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), was shown to be improved by its peptidyl prodrug, (1S,2S,5R,6S)-2-[(2'S)-(2'-amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344). The purpose of this study was to elucidate the mechanisms of intestinal absorption of LY354740 and its prodrug LY544344. Transepithelial transport and accumulation studies were performed in Caco-2 cell monolayers; the involvement of the peptide transporter 1 (PEPT1) transporter was also examined. In absorptive transport studies, the peptidyl prodrug LY544344 partially hydrolyzed to release LY354740 intracellularly, and both compounds appeared in the basolateral compartment. The absorptive transport rate of LY544344, basolateral appearance rate of LY354740, and their cellular accumulation after incubation with LY544344 were concentration-dependent. PEPT1 inhibition reduced transepithelial transport and cellular accumulation of LY544344 to 22 and 1.1% of control, respectively. LY354740 showed concentration-independent absorptive transport with negligible cellular accumulation. Efflux and trans-stimulation studies revealed predominantly apical efflux and the existence of specific transporters for LY544344 and intracellularly released LY354740 on the apical and basolateral membranes. LY544344 efflux was also trans-stimulated at the apical side by glycyl-glutamate but not by glycyl-sarcosine. Transport of neither compound was affected by P-glycoprotein-mediated efflux, as shown in transport and uptake inhibition studies in Madin-Darby canine kidney multidrug resistance 1-transfected cell line and inverted membrane vesicles. In conclusion, LY354740 is mainly transported by the paracellular pathway, whereas intestinal absorption of LY544344 is mediated by PEPT1. However, the absorptive transport of LY544344 seems to be modulated by an apical efflux transporter and a rate-limiting transport step across the basolateral membrane.

The biotransformation of prasugrel, a new thienopyridine prodrug, by the human carboxylesterases 1 and 2.

2-Acetoxy-5-(alpha-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (prasugrel) is a novel thienopyridine prodrug with demonstrated inhibition of platelet aggregation and activation. The biotransformation of prasugrel to its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), requires ester bond hydrolysis, forming the thiolactone 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone(R-95913), followed by cytochrome P450-mediated metabolism to the active metabolite. The presumed role of the human liver- and intestinal-dominant carboxylesterases, hCE1 and hCE2, respectively, in the conversion of prasugrel to R-95913 was determined using expressed and purified enzymes. The hydrolysis of prasugrel is at least 25 times greater with hCE2 than hCE1. Hydrolysis of prasugrel by hCE1 showed Michaelis-Menten kinetics yielding an apparent K(m) of 9.25 microM and an apparent V(max) of 0.725 nmol product/min/microg protein. Hydrolysis of prasugrel by hCE2 showed a mixture of Hill kinetics at low substrate concentrations and substrate inhibition at high concentrations. At low concentrations, prasugrel hydrolysis by hCE2 yielded an apparent K(s) of 11.1 microM, an apparent V(max) of 19.0 nmol/min/microg, and an apparent Hill coefficient of 1.42, whereas at high concentrations, an apparent IC(50) of 76.5 microM was obtained. In humans, no in vivo evidence of inhibition exists. In vitro transport studies using the intestinal Caco-2 epithelial cell model showed a high in vivo absorption potential for prasugrel and rapid conversion to R-95913. In conclusion, the human carboxylesterases efficiently mediate the conversion of prasugrel to R-95913. These data help explain the rapid appearance of R-138727 in human plasma, where maximum concentrations are observed 0.5 h after a prasugrel p.o. dose, and the rapid onset of action of prasugrel.

Effect of buffer components and carrier solvents on in vitro activity of recombinant human carboxylesterases.

INTRODUCTION: The effects of buffer and substrate solvent conditions on in vitro activity of carboxylesterases (CE) have not been previously described. Therefore, it is unknown if the many different assay conditions used by various laboratories have a substantial impact on the activity of CE enzymes. METHODS: Three human CEs were expressed and purified, and the hydrolysis of 4-nitrophenyl butyrate was measured to assess enzyme activity. Four buffers (HEPES, potassium phosphate, sodium phosphate, and Tris) were evaluated for their effects on enzyme activity at concentrations ranging from 5 to 900 mM, as well as phosphate buffered saline. Five commonly used substrate-carrier solvents (acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol) ranging from 0.25 to 6% were also assessed for their effect on enzyme activity. RESULTS: The clearances for the CEs in HEPES, potassium phosphate, sodium phosphate, and Tris up to 100 mM were similar to the CE clearances obtained with phosphate buffered saline. Higher buffer concentrations resulted in differential activity of the CEs. All three CEs tolerated the substrate solvents up to 2% as indicated by little effect of solvent on catalytic activity. At substrate solvent concentrations above 2% the CE activities were found to gradually decrease. In general, CES3 displayed substantially lower activity than CES1 and CES2. DISCUSSION: In conclusion, any of the buffers examined up to 100 mM resulted in clearance values similar to that of phosphate buffered saline for the hydrolysis of 4-nitrophenyl butyrate by the human CEs. With regard to the substrate solvents tested, acetone, acetonitrile, or dimethyl sulfoxide appear to be well tolerated by the CEs up to 2% of the total reaction volume.

Physiologically Based Pharmacokinetic Modelling of Cytochrome P450 2C9-Related Tolbutamide Drug Interactions with Sulfaphenazole and Tasisulam.

BACKGROUND AND OBJECTIVES: Cytochrome P450 2C9 (CYP2C9) is involved in the biotransformation of many commonly used drugs, and significant drug interactions have been reported for CYP2C9 substrates. Previously published physiologically based pharmacokinetic (PBPK) models of tolbutamide are based on an assumption that its metabolic clearance is exclusively through CYP2C9; however, many studies indicate that CYP2C9 metabolism is only responsible for 80-90% of the total clearance. Therefore, these models are not useful for predicting the magnitude of CYP2C9 drug-drug interactions (DDIs). This paper describes the development and verification of SimCYP®-based PBPK models that accurately describe the human pharmacokinetics of tolbutamide when dosed alone or in combination with the CYP2C9 inhibitors sulfaphenazole and tasisulam. METHODS: A PBPK model was optimized in SimCYP® for tolbutamide as a CYP2C9 substrate, based on published in vitro and clinical data. This model was verified to replicate the magnitude of DDI reported with sulfaphenazole and was further applied to simulate the DDI with tasisulam, a small molecule investigated for the treatment of cancer. A clinical study (CT registration # NCT01185548) was conducted in patients with cancer to assess the pharmacokinetic interaction of tasisulum with tolbutamide. A PBPK model was built for tasisulam, and the clinical study design was replicated using the optimized tolbutamide model. RESULTS: The optimized tolbutamide model accurately predicted the magnitude of tolbutamide AUC increase (5.3-6.2-fold) reported for sulfaphenazole. Furthermore, the PBPK simulations in a healthy volunteer population adequately predicted the increase in plasma exposure of tolbutamide in patients with cancer (predicted AUC ratio = 4.7-5.4; measured mean AUC ratio = 5.7). CONCLUSIONS: This optimized tolbutamide PBPK model was verified with two strong CYP2C9 inhibitors and can be applied to the prediction of CYP2C9 interactions for novel inhibitors. Furthermore, this work highlights the utility of mechanistic models in navigating the challenges in conducting clinical pharmacology studies in cancer patients.

Industry Perspective on Contemporary Protein-Binding Methodologies: Considerations for Regulatory Drug-Drug Interaction and Related Guidelines on Highly Bound Drugs.

Regulatory agencies have recently issued drug-drug interaction guidelines, which require determination of plasma protein binding (PPB). To err on the conservative side, the agencies recommend that a 0.01 lower limit of fraction unbound (fu) be used for highly bound compounds (>99%), irrespective of the actual measured values. While this may avoid false negatives, the recommendation would likely result in a high rate of false positive predictions, resulting in unnecessary clinical studies and more stringent inclusion/exclusion criteria, which may add cost and time in delivery of new medicines to patients. In this perspective, we provide a review of current approaches to measure PPB, and important determinants in enabling the accuracy and precision in these measurements. The ability to measure fu is further illustrated by a cross-company data comparison of PPB for warfarin and itraconazole, demonstrating good concordance of the measured fu values. The data indicate that fu values of ≤0.01 may be determined accurately across laboratories when appropriate methods are used. These data, along with numerous other examples presented in the literature, support the use of experimentally measured fu values for drug-drug interaction predictions, rather than using the arbitrary cutoff value of 0.01 as recommended in current regulatory guidelines.

Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth.

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2-7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drug's half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344-56. ©2016 AACR.

Using dried blood spot sampling to improve data quality and reduce animal use in mouse pharmacokinetic studies.

Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 µL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal-often to a single collection per mouse-thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 µL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study.

Preclinical absorption, distribution, metabolism, and excretion of an oral amide prodrug of gemcitabine designed to deliver prolonged systemic exposure.

Gemcitabine is an intravenously administered nucleoside analog chemotherapeutic agent. The ability to deliver this agent as an oral drug would allow greater flexibility of administration and patient convenience; however, attempts have been fraught with high first-pass metabolism and potential intestinal toxicity. Alternatively, an amide prodrug of gemcitabine (LY2334737) was discovered, which is able to avoid the extensive first-pass metabolism that occurs following administration of gemcitabine. Preclinical in vitro and in vivo experiments were conducted to evaluate the hydrolysis and pharmacokinetics of LY2334737 and its downstream metabolites. In mice, rats, and dogs, the prodrug is absorbed largely intact across the intestinal epithelium and delivers LY2334737 to systemic circulation. The hydrolysis of LY2334737 is relatively slow, resulting in sustained release of gemcitabine in vivo. In vitro experiments identified carboxylesterase 2 (CES2) as a major enzyme involved in the hydrolysis of LY2334737, but with relatively low intrinsic clearance. Following hydrolysis of the prodrug, gemcitabine is cleared predominantly via the formation of its inactive metabolite dFdU. Both biliary and renal excretion was responsible for the elimination of LY2334737 and its metabolites in both mice and dogs.

Efficacy of low-dose oral metronomic dosing of the prodrug of gemcitabine, LY2334737, in human tumor xenografts.

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.

Effect of gastric pH on the pharmacokinetics of a BCS class II compound in dogs: utilization of an artificial stomach and duodenum dissolution model and GastroPlus,™ simulations to predict absorption.

Dogs are one of the most commonly used non-rodent species in toxicology studies and are known to have basal stomach pH ranging from 2 to 7 in the fasted state. Thus absorption and resulting plasma exposure of weakly basic compounds administered as crystalline suspensions to dogs are often variable. LY2157299 is a potent and selective transforming growth factor (TGF)-beta receptor type 1 kinase (TGF-ßRI) inhibitor that displayed variable absorption in early dog studies. This molecule is a weakly basic Biopharmaceutics Classification System (BCS)Class II compound, and depends on the rate and extent of dissolution to drive oral absorption. An artificial stomach and duodenum (ASD) dissolution model was utilized to evaluate potential effect of gastric pH on the absorption of suspension and buffered solution formulations. GastroPlus™ was also employed to predict the magnitude of gastric pH changes on LY2157299 absorption. The ASD experiments demonstrated that administration of a buffered acidic solution could improve the potential for absorption by normalizing gastric pH and enabling supersaturation in the duodenum. GastroPlus™ modeling suggested that direct modulation of gastric pH could lead to marked changes in bioavailability. Pharmacokinetic experiments were conducted in dogs to evaluate the effect of gastric pH modification on plasma exposure. The data were qualitatively consistent with the predictions.

Dried blood spot sampling: coupling bioanalytical feasibility, blood-plasma partitioning and transferability to in vivo preclinical studies.

BACKGROUND: The adoption of dried blood spot (DBS) sampling and analysis to support drug discovery and development requires the understanding of its bioanalytical feasibility as well as the distribution of the analyte in blood. RESULTS: Demonstrated the feasibility of adopting DBS for four test analytes representing diverse physico-chemical as well as pharmacokinetic parameters. The key findings include the use of a single extraction procedure across all four analytes, assay range of 1 to 5000 ng/ml, stability in whole blood as well as on-card, and the non-impact of blood volume. In vivo data were used to calculate the blood-to-plasma ratio (using both AUC and average of individual time points), which was then used to predict plasma concentration from DBS data. The predicted data showed an excellent correlation with actual plasma data. CONCLUSION: Transition from plasma to DBS can be supported for preclinical studies by conducting a few well-defined bioanalytical experiments followed by an in vivo bridging study. Blood:plasma ratio derived from the bridging study can be used to predict plasma concentrations from DBS data.

The intestinal absorption of a prodrug of the mGlu2/3 receptor agonist LY354740 is mediated by PEPT1: in situ rat intestinal perfusion studies.

LY354740 is a potent mGlu2/3 agonist with a limited oral bioavailability. Its alanyl prodrug, LY544344, showed high affinity to the intestinal peptide transporter PEPT1, and improved the oral bioavailability of LY354740 in various animal models. The aim of the present study was to investigate the mechanism of in vivo absorption of the dipeptidic prodrug LY544344. The permeabilities of LY544344 and LY354740 were examined in the rat in situ single-pass intestinal perfusion model. The intestinal absorptive flux of LY354740 was shown to be very low in comparison with LY544344. The absorptive flux of LY544344 could best be described by a Michaelis-Menten process in parallel with a linear process. The estimated parameters were: J(max) = 26.7 x 10(-5) micromol/(cm(2)-s), K(m) = 2.6 mM. The absorptive permeability of LY544344 was reduced to approximately 5% of control in the presence of excess Gly-Sar, a known PEPT1 substrate. Intracellular accumulation of LY354740 and LY544344, estimated postperfusion, showed high levels of LY354740 over LY544344 at all perfusate concentrations studied. However, there was a decline in the intracellular ratio of LY354740 to LY544344 at higher concentrations, suggesting that the metabolic activation to release LY354740 is saturable.

Synthesis, crystallization, and biological evaluation of an orally active prodrug of gemcitabine.

The design, synthesis, and biological characterization of an orally active prodrug (3) of gemcitabine are described. Additionally, the identification of a novel co-crystal solid form of the compound is presented. Valproate amide 3 is orally bioavailable and releases gemcitabine into the systemic circulation after passing through the intestinal mucosa. The compound has entered clinical trials and is being evaluated as a potential new anticancer agent.

Pharmacokinetics, metabolism, and excretion of the intestinal peptide transporter 1 (SLC15A1)-targeted prodrug (1S,2S,5R,6S)-2-[(2'S)-(2-amino)propionyl]aminobicyclo[3.1.0.]hexen-2,6-dicarboxylic acid (LY544344) in rats and dogs: assessment of first-pass bioactivation and dose linearity.

The peptidyl prodrug (1S,2S,5R,6S)-2-[(2'S)-(2-Amino)propionyl]a-minobicyclo[3.1.0.]hexen-2,6-dicarboxylic acid, also known as LY544344, was discovered to improve the oral bioavailability of the parent drug (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), a potent group II metabotropic glutamate receptor agonist. This prodrug has been shown to deliver high plasma concentrations of the active drug via intestinal peptide transporter 1 (SLC15A1) (PepT1)-mediated intestinal transport and presystemic hydrolysis in preclinical species. The current data describe the pharmacokinetic behavior of LY544344 and LY354740, with a specific focus on the first-pass activation processes and dose linearity in rats and dogs. The PepT1 transporter makes an attractive prodrug target because of its high capacity and relatively broad substrate specificity. This was demonstrated by the wide dose proportionality observed in both species (up to 1000 mg/kg in rats and 140 mg/kg in dogs). After oral administration of LY544344, absorption and bioactivation were extensive and rapid, with greater than 97% of prodrug hydrolysis occurring before its appearance in the hepatic portal vein. Systemic activation was likewise extensive, with 100% conversion of a 7-mg/kg intravenous dose in dogs. Radiolabeled studies confirmed that hydrolysis to LY354740 was the only metabolic pathway and that the excretion pattern of the active drug was not altered by administration of the prodrug. These results demonstrate the nearly ideal prodrug properties of LY544344 and further validate the utility of the peptide transporter-directed approach to prodrug design.

Improved bioavailability of the mGlu2/3 receptor agonist LY354740 using a prodrug strategy: in vivo pharmacology of LY544344.

Numerous studies have indicated that selective agonists of group II metabotropic glutamate (mGlu) receptors, such as LY354740 [(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate] and LY379268 [(-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate], may be useful in the treatment of many psychiatric disorders, including psychosis, anxiety, and drug withdrawal. Although animal and human studies demonstrate potential therapeutic utility, poor oral bioavailability is a limiting factor in the clinical development of these compounds. Therefore, a novel prodrug approach is being pursued to increase exposure levels of active compound after oral administration. Here, we demonstrate a 10-fold increase in brain, plasma, and cerebrospinal fluid levels of LY354740 after oral prodrug administration. Furthermore, we compare the oral efficacy of the mGlu2/3 receptor agonist LY354740 and its prodrug LY544344 [(1S,2S,5R,6S)-2-[(2'S)-(2'-amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride] in rodent models of psychosis and anxiety. Phencyclidine (PCP)-induced hyperlocomotion was dose dependently inhibited in rats receiving oral administration of 30 or 100 mg/kg LY544344, whereas LY354740 did not significantly reverse PCP-mediated behaviors at doses up to 100 mg/kg. Orally administered LY544344 (30 mg/kg) and subcutaneously administered LY354740 (10 mg/kg) attenuated stress-induced hyperthermia in DBA/2 mice, with the prodrug producing anxiolytic effects at lower oral doses than the parent compound. Although oral administration of LY354740 did not significantly affect fear-induced suppression of operant responding in rats, subcutaneously administered LY354740 (10 or 20 mg/kg) and orally administered LY544344 (10 or 30 mg/kg) produced significant anxiolytic effects in this model. The present data confirm that mGlu2/3 receptor agonists produce antipsychotic and anxiolytic effects in animal behavioral models and demonstrate that oral bioavailability of LY354740 was substantially increased using a prodrug strategy.