Ester-type Prodrug Research Articles

Conversion of Olmesartan to Olmesartan Medoxomil, A Prodrug that Improves Intestinal Absorption, Confers Substrate Recognition by OATP2B1.

Olmesartan medoxomil (olmesartan-MX), an ester-type prodrug of the angiotensin II receptor blocker (ARB) olmesartan, is predominantly anionic at intestinal pH. Human organic anion transporting polypeptide 2B1 (OATP2B1) is expressed in the small intestine and is involved in the absorption of various acidic drugs. This study was designed to test the hypothesis that OATP2B1-mediated uptake contributes to the enhanced intestinal absorption of olmesartan-MX, even though olmesartan itself is not a substrate of OATP2B1. Tetracycline-inducible human OATP2B1- and rat Oatp2b1-overexpressing HEK 293 cell lines (hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293, respectively) were established to characterize OATP2B1-mediated uptake. Rat jejunal permeability was measured using Ussing chambers. ARBs were quantified by liquid chromatography-tandem mass spectrometry. Significant olmesartan-MX uptake was observed in hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293 cells, whereas olmesartan uptake was undetectable or much lower than olmesartan-MX uptake, respectively. Furthermore, olmesartan-MX exhibited several-fold higher uptake in Caco-2 cells and greater permeability in rat jejunum compared to olmesartan. Olmesartan-MX uptake in hOATP2B1/T-REx-293 cells and in Caco-2 cells was significantly decreased by OATP2B1 substrates/inhibitors such as 1mM estrone-3-sulfate, 100µM rifamycin SV, and 100µM fluvastatin. Rat Oatp2b1-mediated uptake and rat jejunal permeability of olmesartan-MX were significantly decreased by 50µM naringin, an OATP2B1 inhibitor. Oral administration of olmesartan-MX with 50µM naringin to rats significantly reduced the area under the plasma concentration-time curve of olmesartan to 76.9%. Olmesartan-MX is a substrate for OATP2B1, and the naringin-sensitive transport system contributes to the improved intestinal absorption of olmesartan-MX compared with its parent drug, olmesartan.

Bioconversion and P-gp-Mediated Transport of Depot Fluphenazine Prodrugs after Intramuscular Injection

Fluphenazine (FPZ) decanoate, an ester-type prodrug formulated as a long-acting injection (LAI), is used in the treatment of schizophrenia. FPZ enanthate was also developed as an LAI formulation, but is no longer in use clinically because of the short elimination half-life of FPZ, the parent drug, after intramuscular injection. In the present study, the hydrolysis of FPZ prodrugs was evaluated in human plasma and liver to clarify the reason for this difference in elimination half-lives. FPZ prodrugs were hydrolyzed in human plasma and liver microsomes. The rate of hydrolysis of FPZ enanthate in human plasma and liver microsomes was 15-fold and 6-fold, respectively, faster than that of FPZ decanoate. Butyrylcholinesterase (BChE) and human serum albumin (HSA) present in human plasma, and two carboxylesterase (CES) isozymes, hCE1 and hCE2, expressed in ubiquitous organs including liver, were mainly responsible for the hydrolysis of FPZ prodrugs. FPZ prodrugs may not be bioconverted in human skeletal muscle at the injection site because of lack of expression of BChE and CESs in muscle. Interestingly, although FPZ was a poor substrate for human P-glycoprotein, FPZ caproate was a good substrate. In conclusion, it is suggested that the shorter elimination half-life of FPZ following administration of FPZ enanthate compared with FPZ decanoate can be attributed to the more rapid hydrolysis of FPZ enanthate by BChE, HSA and CESs.

Hydrolase Activity of the Genetic Variants of Human Alpha-1-Acid Glycoprotein.

In human plasma, the main agent of hydrolysis of the ester-type prodrug of levodopa, designated ONO-2160, is alpha-1-acid glycoprotein (AGP), which is a mixture of the F1*S and A variants at molar ratios of 3:1 to 2:1. In this study, the mechanism of AGP esterase-like activity was investigated by evaluating the contribution of the F1*S and A variants to ONO-2160 hydrolysis and identifying the AGP hydrolase active site. We found that although both variants hydrolyzed ONO-2160, their hydrolase activities were different. The intrinsic plasma clearance of the F1*S variant (0.441 mL/h/mg protein) was approximately 30 times higher than that of the A variant (0.0148 mL/h/mg protein), indicating that the F1*S variant contributed the most to AGP esterase-like activity. To identify the hydrolase active site of AGP, we performed inhibition studies of ONO-2160 hydrolysis using 12 AGP-binding drugs with various ligand-binding constants and binding selectivities to the two AGP variants. Inhibition of activity was positively correlated with the constant of ligand binding to the F1*S variant. In addition, compounds with high affinity to the F1*S variant inhibited ONO-2160 hydrolysis the most. Together, our data indicate that ONO-2160 is predominantly hydrolyzed by the F1*S variant at its ligand-binding site.

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.

Evaluation of the Oral Absorption of Ester-type Prodrugs

The first-pass hydrolysis of oral ester-type prodrugs in the liver and intestine is mediated mainly by hCE1 and hCE2 of the respective predominant carboxylesterase (CES) isozymes. In order to provide high blood concentrations of the parent drugs, it is preferable that prodrugs are absorbed as an intact ester in the intestine, then rapidly converted to active parent drugs by hCE1 in the liver. In the present study, we designed a prodrug of fexofenadine (FXD) as a model parent drug that is resistant to hCE2 but hydrolyzed by hCE1, utilizing the differences in catalytic characteristics of hCE1 and hCE2. In order to precisely predict the intestinal absorption of an FXD prodrug candidate, we developed a novel high-throughput system by modifying Caco-2 cells. Further, we evaluated species differences and aging effects in the intestinal and hepatic hydrolysis of prodrugs to improve the estimation of in vivo first-pass hydrolysis of ester-type prodrugs. Consequently, it was possible to design a hepatotropic prodrug utilizing the differences in tissue distribution and substrate specificity of CESs. In addition, we successfully established three useful in vitro systems for predicting the intestinal absorption of hCE1 substrate using Caco-2 cells. However, some factors involved in estimating the bioavailability of prodrugs in human, such as changes in recognition of drug transporters by esterification, and species differences of the first-pass hydrolysis, should be comprehensively considered in prodrug development.

P159 - A novel hydrolytic activation pathway for an ester-type prodrug in human plasma: Contribution of α1-acid glycoprotein variant

P159 - A novel hydrolytic activation pathway for an ester-type prodrug in human plasma: Contribution of α1-acid glycoprotein variant

Unique Hydrolysis of an Ester-Type Prodrug of Levodopa in Human Plasma: Relay-Type Role Sharing between Alpha-1 Acid Glycoprotein and Human Serum Albumin.

ONO-2160 is a newly developed oral ester-type prodrug of levodopa for removing the problems in use of levodopa. It has a structure in which two of the same substituents are bound to levodopa. It is important to understand the pharmacokinetics and metabolic pathway for new drug candidate compounds. The aim of this study was to identify the major enzymes that contribute to the metabolism of ONO-2160 in human plasma. ONO-2160 was hydrolyzed by human serum albumin (HSA) and α1-acid glycoprotein (AGP) in human plasma, although the hydrolysis was not inhibited by various reported esterase inhibitors. The value of the intrinsic clearance per milliliter of plasma of ONO-2160 in AGP solution was greater than that in HSA solution and was comparable to that in human plasma. Therefore, AGP is responsible for the hydrolysis of ONO-2160 in human plasma. ONO-M, which is an intermediate metabolite of ONO-2160, has a structure in which one substituent is removed from ONO-2160 and was mainly generated in AGP solution, but not in human plasma or HSA solution. The hydrolysis of ONO-M by HSA was much greater than by AGP. These results indicate that ONO-M, which is mainly generated from ONO-2160 by AGP, is rapidly hydrolyzed by HSA, and that ONO-2160 generates levodopa via ONO-M in a relay-type reaction through AGP and HSA in human plasma. It has not been reported that AGP has esterase-like activity. These findings could be useful information for drug development of the ester-type prodrug.

Effect of alcohol on skin permeation and metabolism of an ester-type prodrug in Yucatan micropig skin

We studied the effect that three alcohols, ethanol (EA), propanol (PA), and isopropanol (IPA), have on the skin permeation of p-hydroxy benzoic acid methyl ester (HBM), a model ester-type prodrug. HBM was applied to Yucatan micropig skin in a saturated phosphate buffered solution with or without 10% alcohol, and HBM and related materials in receptor fluid and skin were determined with HPLC. In the absence of alcohol, p-hydroxy benzoic acid (HBA), a metabolite of HBM, permeated the skin the most. The three alcohols enhanced the penetration of HBM at almost the same extent. The addition of 10% EA or PA to the HBM solution led to trans-esterification into the ethyl ester or propyl ester of HBA, and these esters permeated skin as well as HBA and HBM did. In contrast, the addition of 10% IPA promoted very little trans-esterification. Both hydrolysis and trans-esterification in the skin S9 fraction were inhibited by BNPP, an inhibitor of carboxylesterase (CES). Western blot and native PAGE showed the abundant expression of CES in micropig skin. Both hydrolysis and trans-esterification was simultaneously catalyzed by CES during skin permeation. Our data indicate that the alcohol used in dermal drug preparations should be selected not only for its ability to enhance the solubility and permeation of the drug, but also for the effect on metabolism of the drug in the skin.

Down-regulation of carboxylesterases 1 and 2 plays an important role in prodrug metabolism in immunological liver injury rats.

Liver plays a central role in xenobiotics metabolism, thus affecting the in vivo disposition and therapeutic effects of drugs. Carboxylesterases (CESs), with the main isoforms CES1 and CES2, are important in the metabolism of ester-type prodrugs. However, influences of immunological liver injury on the activity of CES remain undefined. In the present study, we demonstrated treatment with lipopolysaccharide (LPS) suppressed the activities of CES1 and CES2. The decreased activities of CES1 and CES2 were preliminarily assessed by the hydrolysis assay for their common substrate p-nitrophenyl acetate (PNPA) with rat hepatic microsomal enzyme. Subsequently, RT-PCR results showed that the levels of CES1 mRNA and mRNA of CES2 (AB010635) and CES2 (AY034877) in the model group were significantly lower than those of the normal control group (P<0.05). Western blot results showed that the expressions of CES1 and CES2 proteins were decreased (P<0.05). To further clarify the effects of LPS on the metabolic activities of CESs, pharmacokinetic studies were performed in rats by utilizing imidapril and irinotecan (CPT-11) as the specific substrates for CES1 and CES2, respectively. After treatment with LPS, AUC0-∞ and Cmax of imidaprilat were decreased from 2084.86±340.66ng·h(-1)·mL(-1) and 234.66±68.85ng·mL(-1) to 983.87±315.34ng·h(-1)·mL(-1) and 113.1±19.69ng·mL(-1) (P<0.05), respectively. Moreover, AUC0-∞ and Cmax of SN-38 were decreased from 8100±918.6ng·h(-1)·mL(-1) and 144.67±20.28ng·mL(-1) to 3270±500.5ng·h(-1)·mL(-1) and 56.19±10.38ng·mL(-1) (P<0.05), respectively. In summary, immunological liver injury remarkably attenuated the expressions and metabolic activities of CES1 and CES2, which may be associated with the regulatory effects of cytokines under inflammation.

Evaluation of Transport Mechanism of Prodrugs and Parent Drugs Formed by Intracellular Metabolism in Caco-2 Cells with Modified Carboxylesterase Activity: Temocapril as a Model Case

The intestinal absorption mechanism of temocapril, an ester-type prodrug of temocaprilat, was evaluated using Caco-2 cell monolayers with or without active carboxylesterase (CES)-mediated hydrolysis. The inhibition of CES-mediated hydrolysis was achieved by pretreatment of the monolayers with bis-p-nitrophenyl phosphate (BNPP), which inhibited 94% of the total hydrolysis of temocapril in the Caco-2 cells. The remaining 6% hydrolysis was due to the presence of serine esterases, other than CES, on the cell membranes. Transport experiments under CES-inhibited conditions showed temocapril not to be a substrate for peptide transporter 1 (PEPT1) or organic anion transporting polypeptides (OATPs), but to be an inhibitor of PEPT1; P-glycoprotein (P-gp) and breast-cancer-resistant protein (BCRP) were responsible for the efflux of temocapril, which was mainly absorbed by passive diffusion at low apical pH. In Caco-2 cell monolayers with CES-mediated hydrolysis intact, temocaprilat derived from temocapril, was 2.5-fold more rapidly transported into the apical compartment than into the basolateral compartment due to the presence of microvilli on the apical membrane. In contrast, temocaprilat at low intracellular concentrations, was preferentially transported across the basolateral membrane under CES-inhibited conditions. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3985–3994, 2011

Oseltamivir in Seasonal, Avian H5N1 and Pandemic 2009 A/H1N1 Influenza

Oseltamivir is the ester-type prodrug of the neuraminidase inhibitor oseltamivir carboxylate. It has been shown to be an effective treatment for both seasonal influenza and the recent pandemic 2009 A/H1N1 influenza, reducing both the duration and severity of the illness. It is also effective when used preventively. This review aims to describe the current knowledge of the pharmacokinetic and pharmacodynamic characteristics of this agent, and to address the issue of possible therapeutic drug monitoring. According to the currently available literature, the pharmacokinetics of oseltamivir carboxylate after oral administration of oseltamivir are characterized by mean ± SD bioavailability of 79 ± 12%, apparent clearance of 25.3 ± 7.0 L/h, an elimination half-life of 7.4 ± 2.5 hours and an apparent terminal volume of distribution of 267 ± 122 L. A maximum plasma concentration of 342 ± 83 μg/L, a time to reach the maximum plasma concentration of 4.2 ± 1.1 hours, a trough plasma concentration of 168 ± 32 μg/L and an area under the plasma concentration-time curve from 0 to 24 hours of 6110 ± 1330 μg · h/L for a 75 mg twice-daily regimen were derived from literature data. The apparent clearance is highly correlated with renal function, hence the dosage needs to be adjusted in proportion to the glomerular filtration rate. Interpatient variability is moderate (28% in apparent clearance and 46% in the apparent central volume of distribution); there is no indication of significant erratic or limited absorption in given patient subgroups. The in vitro pharmacodynamics of oseltamivir carboxylate reveal wide variation in the concentration producing 50% inhibition of influenza A and B strains (range 0.17-44 μg/L). A formal correlation between systemic exposure to oseltamivir carboxylate and clinical antiviral activity or tolerance in influenza patients has not yet been demonstrated; thus no formal therapeutic or toxic range can be proposed. The pharmacokinetic parameters of oseltamivir carboxylate after oseltamivir administration (bioavailability, apparent clearance and the volume of distribution) are fairly predictable in healthy subjects, with little interpatient variability outside the effect of renal function in all patients and bodyweight in children. Thus oseltamivir carboxylate exposure can probably be controlled with sufficient accuracy by thorough dosage adjustment according to patient characteristics. However, there is a lack of clinical study data on naturally infected patients. In addition, the therapeutic margin of oseltamivir carboxylate is poorly defined. The usefulness of systematic therapeutic drug monitoring in patients therefore appears to be questionable; however, studies are still needed to extend the knowledge to particular subgroups of patients or dosage regimens.

ChemInform Abstract: A Short-Step Synthesis of Orally Active Carbapenem Antibiotic CS-834.

An orally bioavailable carbapenem CS-834, which is a pivaloyloxymethyl (POM) ester-type prodrug and has (R)-5-oxopyrrolidin-3-ylthio moiety at the C-2 position of the 1 beta-methylcarbapenem skeleton, is currently under clinical trial. We accomplished a short-step synthesis of CS-834 by using phosphorus ylide from the intramolecular Wittig-type reaction in the key step for cyclization to the bicyclic carbapenem system. The POM ester group was found to be suitable for the cyclization conditions.

Abstract C98: Blocking glycolysis with 2-deoxy-D-glucose prodrugs to target brain tumors: A novel concept

Abstract Glycolysis is the major energy producing pathway for fast growing, glycolytically dependent tumors, such as gliomas and ependymomas. Blocking glycolysis is, therefore, an important therapeutic strategy when used alone or as a combination therapy to enhance the effects of chemotherapy in energy-starved tumors. In our previous studies, we examined D-glucose-based antimetabolites, including 2-deoxy-D-glucose (2-DG), and confirmed their ability to block glycolysis, discovered that they can induce autophagic cell death in vitro, and established their antitumor activity in vivo in an orthotopic glioma model. However, during our evaluation of 2-DG, we determined that these compounds do not possess sufficient drug-like properties (reasonable biostability, pharmacokinetic characteristics, or distribution to the target) to warrant further investigations into their clinical effectiveness. In an attempt to improve the pharmacokinetic profile of 2-DG, we have designed synthesized, and tested a series of ester-type prodrugs that enhance biostability, biodistribution, and delivery of 2-DG to its CNS target. We will present the synthesis and preliminary evaluation of these compounds, leading to the selection of WP1122 as our lead compound for further drug development. We will also present the subsequent evaluation of WP1122 in vivo in orthotopic brain tumor models and demonstrate its superior pharmacokinetic profile and enhanced CNS uptake. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C98.

Development of a Novel System for Estimating Human Intestinal Absorption Using Caco-2 Cells in the Absence of Esterase Activity

Both mRNA and protein levels of the carboxylesterase (CES) isozymes, hCE1 and hCE2, in Caco-2 cells increase in a time-dependent manner, but hCE1 levels are always higher than those of hCE2. In human small intestine, however, the picture is reversed, with hCE2 being the predominant isozyme. Drugs hydrolyzed by hCE1 but not by hCE2 can be hydrolyzed in Caco-2 cells, but they are barely hydrolyzed in human small intestine. The results in Caco-2 cells can be misleading as a predictor of what will happen in human small intestine. In the present study, we proposed a novel method for predicting the absorption of prodrugs in the absence of CES-mediated hydrolysis in Caco-2 cells. The specific inhibition against CES was achieved using bis-p-nitrophenyl phosphate (BNPP). The optimal concentration of BNPP was determined at 200 microM by measuring the transport and hydrolysis of O-butyryl-propranolol (butyryl-PL) as a probe. BNPP concentrations of more than 200 microM inhibited 86% of hydrolysis of butyryl-PL, resulting in an increase in its apparent permeability. Treatment with 200 microM BNPP did not affect paracellular transport, passive diffusion, or carrier-mediated transport. Furthermore, the proposed evaluation system was tested for ethyl fexofenadine (ethyl-FXD), which is a superior substrate for hCE1 but a poor one for hCE2. CES-mediated hydrolysis of ethyl-FXD was 94% inhibited by 200 microM BNPP, and ethyl-FXD was passively transported as an intact prodrug. From the above observations, the novel evaluation system is effective for the prediction of human intestinal absorption of ester-type prodrugs.

Oseltamivir (Tamiflu) Is a Substrate of Peptide Transporter 1

Oseltamivir, an ester-type prodrug of the neuraminidase inhibitor [3R,4R,5S]-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate phosphate (Ro 64-0802), has been developed for the treatment of A and B strains of the influenza virus but has neuropsychiatric and other side effects. In this study, we characterized the transport across intestinal epithelial cells and the absorption of oseltamivir in rats. Uptake by Caco-2 cells (human carcinoma cell line) and HeLa cells transfected with peptide transporter 1 (HeLa/PEPT1) was time- and temperature-dependent and was inhibited by typical PEPT1 inhibitors such as glycyl-sarcosine (Gly-Sar). The uptake by Caco-2 cells and HeLa/PEPT1 was saturable, with similar K(m) values. Oseltamivir absorption in adult rats was greatly reduced by simultaneous administration of milk, casein, or Gly-Sar. Furthermore, the plasma and brain concentrations of oseltamivir were higher in fasting than in nonfasting rats after oral administration. These results suggest that oseltamivir is a substrate of PEPT1 and that PEPT1 is involved in its intestinal absorption.

Integrated pharmacokinetic assessment and strategy for orally effective prodrugs overcoming luminal degradation and biological membrane barriers

Because no kinetic principles have been proposed for designing orally effective prodrugs, the author recently reported a kinetic model for membrane transport of prodrugs (Chem-Bio Informatics Journal, 8, 25-32 (2008)), and proposed the kinetic classification and criteria for effective membrane-permeable prodrugs (KCCEMP). The present study addressed more practical conditions, where a prodrug is metabolized/degraded to a drug in the luminal tract after oral administration. Primary factors in orally effective prodrugs are luminal degradation/metabolism and absorption clearance (permeability), which includes the mechanism of membrane transport and metabolism in intestinal cells. The fraction of absorbed prodrug is expressed by the functions of these parameters. Based on the required improvement ratio of the absorption clearance, the kinetic classification and criteria of orally effective prodrugs (KCCOEP) are proposed as a decision tree with conditional equations for guiding kinetic assessment and strategy for the rational development of prodrugs. The assessment of lenampicillin, which was selected as an example of successful prodrugs, according to the procedure indicated a significant impact of luminal degradation/metabolism on the absorbed fraction, and suggests that most ester-type prodrugs on the market degrade in the luminal tract. Thus, a comprehensive study on the fraction of luminal degradation/metabolism and the absorption clearance (permeability) should be conducted to develop orally effective prodrugs, in particular, quantitative assessment of the fraction of contribution (fc,dd) of the drug formed from the prodrug in the luminal tract to the absorption following oral administration of the prodrug is emphasized.

Faropenem Medoxomil

Replidyne is developing faropenem medoxomil, the ester-type prodrug of faropenem, for the treatment of bacterial infections and respiratory tract infections, including acute exacerbations of chronic bronchitis (AECB), acute bacterial sinusitis (ABS) and community-acquired pneumonia (CAP). Faropenem medoxomil is also being developed for the treatment of tonsillitis, pharyngitis and otitis media in children. Faronpenem medoxomil was discovered by scientists at Suntory Institute for Biomedical Research (now Asubio Pharma). The compound has significantly improved oral bioavailability and is dehydropeptidase-I stable. Following absorption, faropenem medoxomil is rapidly hydrolysed to the active drug faropenem. An NDA was filed in the US but was deemed not approvable by the US authorities. Following the termination of the license agreement between Replidyne and Forest Laboratories, Replidyne is now exploring other partnering opportunities for faropenem medoxomil. Daiichi Suntory Pharma (now Asubio Pharma) has granted Replidyne the exclusive rights to faropenem medoxomil for the US and Canada and an exclusive option to develop and commercialize the compound in the rest of the world, excluding Japan. Replidyne has rights to the preclinical and clinical data generated up to the time of the agreement (August 2004) and will complete clinical development of the drug. Replidyne is also developing a paediatric formulation for the treatment of common bacterial infections. In February 2006, Replidyne sublicensed development, commercialization and distribution rights of faropenem medoxomil in the US to Forest Laboratories Holdings (Forest Laboratories). However, the agreement was terminated in February 2007, following the US FDA's non-approvable letter for the product. Replidyne re-acquired all US adult and paediatric rights previously granted to Forest. Bayer AG previously licensed exclusive worldwide rights to develop faropenem medoxomil from Suntory (now Asubio Pharma) and conducted a number of phase III clinical trials. This agreement appears to have been superseded by the agreement with Replidyne in 2004. In April 2007, Daiichi Asubio Pharma was renamed as Asubio Pharma Co., Ltd. Daiichi Asubio Pharma was the name used by Daiichi Suntory Pharma after it became a wholly owned subsidiary of Daiichi Pharmaceutical in September 2005. Daiichi Suntory Pharma was the joint venture company owned by Daiichi Pharmaceutical and Suntory. In April 2006, Daiichi Pharmaceutical merged with Sankyo to form Daiichi Sankyo Inc. The FDA issued a non-approvable letter in October 2006 for faropenem medoxomil in the treatment of ABS, CAP, AECB and uncomplicated skin and skin structure infections. Consequently, drug development has reverted back to phase III in the US. The agency has indicated that four phase III trials in three adult respiratory indications, ABS, CAP and AECB, will be required for a US marketing application. According to this advice, Replidyne may be required to conduct one superiority study (versus placebo) each for the ABS and AECB indications and two non-inferiority, active-controlled studies for the treatment of CAP. The required dose of faropenem medoxomil in future trials will be 600 mg, administered twice daily, and trials will involve approximately 1500 patients to ensure an acceptable database of safety information for review. Replidyne is continuing to work with the FDA on further trial details. Replidyne first filed the NDA seeking approval for faropenem medoxomil in December 2005. This submission marked the first marketing approval application for faropenem medoxomil worldwide. The NDA, which was accepted in February 2006, was primarily based on data from 11 phase III trials in patients with respiratory tract and skin infections; the safety data included information from more than 5000 patients treated with the drug. The proposed commercial name for faropenem medoxomil, Orapemtrade mark, was not approved by the FDA due to its similarity to another commercially approved drug. Replidyne and the FDA are working together to identify a suitable alternative. Two phase III trials have been conducted that demonstrated faropenem medoxomil was non-inferio o azithromycin and clarithromycin in the treatment of AECB. Replidyne's phase II trial evaluating an oral liquid formulation of faropenem medoxomil (7.5-40 mg/kg) in paediatric patients with acute otitis media (AOM), met its primary endpoint. The trial was completed in March 2007 and enrolled approximately 310 patients in Costa Rica and Israel. Replidyne intends to meet with the US authorities to discuss the design of the planned phase III trial in paediatric AOM. In addition to 5 years of Hatch-Waxman exclusivity granted upon approval, faropenem medoxomil is protected by an issued US composition of matter patent, which expires in 2015. Extension of exclusivity under Hatch-Waxman legislation is expected.

Oseltamivir (Tamiflu) Efflux Transport at the Blood-Brain Barrier via P-Glycoprotein

Oseltamivir (Tamiflu, Roche, Nutley, NJ), an ester-type prodrug of the anti-influenza drug Ro 64-0802 (oseltamivir carboxylate), has been reported to be associated with neuropsychiatric side effects, which are likely to be caused by distribution of oseltamivir and/or its metabolite into the central nervous system. Enhanced toxicity and brain distribution of oseltamivir in unweaned rats led us to hypothesize that the low level of distribution of oseltamivir and/or Ro 64-0802 in adult brain was caused by the presence of a specific efflux transporter at the blood-brain barrier. We examined the possible role of P-glycoprotein (P-gp) as the determinant of brain distribution of oseltamivir and Ro 64-0802 both in vitro using LLC-GA5-COL150 cells, which overexpress human multidrug resistance protein 1 P-gp on the apical membrane, and in vivo using mdr1a/1b knockout mice. The permeability of oseltamivir in the basal-to-apical direction was significantly greater than that in the opposite direction. The directional transport disappeared on addition of cyclosporin A, a P-gp inhibitor. The brain distribution of oseltamivir was increased in mdr1a/1b knockout mice compared with wild-type mice. In contrast, negligible transport of Ro 64-0802 by P-gp was observed in both in vitro and in vivo studies. These results show that oseltamivir, but not Ro 64-0802, is a substrate of P-gp. Accordingly, low levels of P-gp activity or drug-drug interactions at P-gp may lead to enhanced brain accumulation of oseltamivir, and this may in turn account for the central nervous system effects of oseltamivir observed in some patients.

Lack of Improvement of Oral Absorption of ME3277 by Prodrug Formation Is Ascribed to the Intestinal Efflux Mediated by Breast Cancer Resistant Protein (BCRP/ABCG2)

ME3229, an ester-type prodrug of a hydrophilic glycoprotein IIb/IIIa antagonist (ME3277), failed to show improved oral absorption. Okudaira et al. (J. Pharmacol. Exp. Ther. 294. 580-587, 2000) provided a piece of evidence that this is ascribed to an efflux system, distinct from P-gp and MRP2, that extrudes ME3277 formed from ME3229 in the intestinal epithelial cells. The aim of the present study is to examine the involvement of breast cancer resistant protein (BCRP/ABCG2) as a cause of low oral absorption of ME3229. The transport activity of ME3277 in the presence and absence of ATP was determined using a rapid filtration method with the membrane vesicles prepared from LLC-PK1 cells expressing BCRP. The plasma concentrations of ME3229 and its metabolites were compared between Bcrp1(-/-) mice and wild-type mice after a single-pass perfusion of small intestine with ME3229. The ATP-dependent uptake of ME3277 was greater in BCRP-expressing membrane vesicles than that in the control vesicles. Furthermore, it was found that after intestinal perfusion with ME3229 for 60 min, the plasma concentrations of ME3277 and PM-5, a metabolite of ME3229, increased 2-fold and 3-fold, respectively, in Bcrp1 knockout mice. It is possible that BCRP acts synergistically with intestinal carboxylesterases. These results suggest that Bcrp1 plays an important role in the intestinal efflux of ME3277 and, probably, PM-10 and PM-11, metabolites of ME3229, and limits its BA after oral administration of ME3229.

Pharmacokinetics and bioavailability of oral cephalosporins, KR-984055 and its prodrugs, KR-999001 and KR-999002, in the rat.

KR-984055 is a new oral cephalosporin antibiotic with activity against both gram-positive and gram-negative bacteria. Lipophilic ester-type prodrugs of KR-984055, i.e., KR-999001 and KR-999002, have been synthesized in an attempt to increase the oral bioavailability of this broad-spectrum antibiotic agent. In this study we determined the oral bioavailability of KR-984055 and its prodrugs in the rat, and evaluated the pharmacokinetic model that best describes the plasma concentration behavior following single intravenous (i.v.) and oral single dose. In addition, concentrations in plasma as well as biliary and urinary recovery of KR-984055 were determined. Also, protein binding of KR-984055 in plasma was examined in vitro. The degree of protein binding of KR-984055 was in the range of 92.09-94.77%. KR-984055 exhibited poor oral bioavailability (7.02 +/- 1.58%). The observed oral bioavailabilities of KR-984055 from KR-999001 and KR-999002 were 38.77 +/- 2.81% and 39.81 +/- 5.25%, respectively. These data were calculated from the levels of free KR-984055 in plasma. Oral KR-999001 and KR-999002 were not recovered from plasma, suggesting that it was readily cleaved to free KR-984055. KR-999001 and KR-999002 appear to be an efficient oral prodrug of KR-984055 that deserved further clinical evaluation in human.