Ayman El-kattan - Academia.edu (original) (raw)

Papers by Ayman El-kattan

Pharmaceutical research, 2000

To test the barrier function of a bio-engineered human skin (BHS) using three model drugs (caffei... more To test the barrier function of a bio-engineered human skin (BHS) using three model drugs (caffeine, hydrocortisone, and tamoxifen) in vitro. To investigate the lipid composition and microscopic structure of the BHS. The human skin substitute was composed of both epidermal and dermal layers, the latter having a bovine collagen matrix. The permeability of the BHS to three model drugs was compared to that obtained in other percutaneous testing models (human cadaver skin, hairless mouse skin, and EpiDerm). Lipid analysis of the BHS was performed by high performance thin layered chromatography. Histological evaluation of the BHS was performed using routine H&E staining. The BHS mimicked human skin in terms of lipid composition, gross ultrastructure, and the formation of a stratum corneum. However, the permeability of the BHS to caffeine, hydrocortisone, and tamoxifen was 3-4 fold higher than that of human cadaver skin. In summary, the results indicate that the BHS may be an acceptable i...

Expert Opinion on Drug Metabolism & Toxicology, 2006

Optimisation of oral bioavailability is a continuing challenge for the pharmaceutical and biotech... more Optimisation of oral bioavailability is a continuing challenge for the pharmaceutical and biotechnology industries. The number of potential drug candidates requiring in vivo evaluation has significantly increased with the advent of combinatorial chemistry. In addition, drug discovery programmes are increasingly forced into more lipophilic and lower solubility chemical space. To aid in the use of in vitro and in silico tools as well as reduce the number of in vivo studies required, a team-based discussion tool is proposed that provides a 'road map' to guide the selection of profiling assays that should be considered when optimising oral bioavailability. This road map divides the factors that contribute to poor oral bioavailability into two interrelated categories: absorption and metabolism. This road map provides an interface for cross discipline discussions and a systematic approach to the experimentation that drives the drug discovery process towards a common goal -acceptable oral bioavailability using minimal resources in an acceptable time frame.

ABSTRACT PURPOSE. To investigate the inter-lot difference in hepatic active uptakes and their cor... more ABSTRACT PURPOSE. To investigate the inter-lot difference in hepatic active uptakes and their correlation to protein expression of hepatic uptake transporters in various lots of cryopreserved human hepatocytes, a higher throughput fast filtration method was developed. In addition, absolute expression of OATP proteins in livers and hepatocytes was determined by using newly developed targeted proteomics LC-MS/MS method. METHODS. The cryopreserved hepatocytes were thawed at 37C and resuspended in Krebs-Henseleit buffer at 2.0x10^6 cells/mL. The viability of hepatocytes used for uptake assays was greater than 70%. The hepatocytes were pre-warmed at 37C for 10 min with or without inhibitors. Uptakes were initiated by adding pre-warmed compounds with or without inhibitors into hepatocyte suspension. The fast filtration was executed through a cell harvester. For quantification of OATPs, membrane fractions of hepatocytes and liver tissue were extracted using Proteo extract membrane kit following the manufacturefs protocol. The membrane fraction samples were then subjected to trypsin digestion and LC-MS/MS analysis. RESULTS. Rosuvastatin and pitavastatin was selected as probe substrates to evaluate the hepatocyte uptake. The hepatic active uptake detected by a cell harvester showed time- and concentration-dependent manner, and was inhibited by rifamycin SV, a prototypical inhibitor for OATPs. The results were consistent with that detected by the literature oil-layer method. Moreover, significant difference in active hepatic uptakes was observed among various lots of cryopreserved hepatocytes. The absolute OATP proteins in hepatocytes and liver tissues were measured by targeted proteomics LC-MS/MS method through monitoring the mass transition of the unique tryptic peptides released from each OATP proteins, e.g. OATP1B1, 1B3 and 2B1. Ongoing process includes to test additional hepatocyte lots and to analysis the correlation between hepatocyte active uptake and protein absolute expression. CONCLUSION. The development of higher throughput hepatic uptake assays enabled to compare the inter-individual difference in hepatocyte uptake. Targeted proteomic LC-MS/MS protein quantification demonstrated as a highly sensitive, selective and reliable method for absolute measurement of OATP proteins. The relationship between active hepatic uptake and protein expression could extend our current understanding on drug hepatic disposition and transporter mediated drug-drug interaction, and further advances system biology models in various translational aspects.

Neuropharmacology, 2011

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are sim... more Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Molecular Pharmaceutics, 2012

The Biopharmaceutics Classification System (BCS) is a scientific framework that provides a basis ... more The Biopharmaceutics Classification System (BCS) is a scientific framework that provides a basis for predicting the oral absorption of drugs. These concepts have been extended in the Biopharmaceutics Drug Disposition Classification System (BDDCS) to explain the potential mechanism of drug clearance and understand the effects of uptake and efflux transporters on absorption, distribution, metabolism, and elimination. The objective of present work is to establish criteria for provisional biopharmaceutics classification using pH-dependent passive permeability and aqueous solubility data generated from high throughput screening methodologies in drug discovery settings. The apparent permeability across monolayers of clonal cell line of Madin−Darby canine kidney cells, selected for low endogenous efflux transporter expression, was measured for a set of 105 drugs, with known BCS and BDDCS class. The permeability at apical pH 6.5 for acidic drugs and at pH 7.4 for nonacidic drugs showed a good correlation with the fraction absorbed in human (Fa). Receiver operating characteristic (ROC) curve analysis was utilized to define the permeability class boundary. At permeability ≥5 × 10 −6 cm/s, the accuracy of predicting Fa of ≥0.90 was 87%. Also, this cutoff showed more than 80% sensitivity and specificity in predicting the literature permeability classes (BCS), and the metabolism classes (BDDCS). The equilibrium solubility of a subset of 49 drugs was measured in pH 1.2 medium, pH 6.5 phosphate buffer, and in FaSSIF medium (pH 6.5). Although dose was not considered, good concordance of the measured solubility with BCS and BDDCS solubility class was achieved, when solubility at pH 1.2 was used for acidic compounds and FaSSIF solubility was used for basic, neutral, and zwitterionic compounds. Using a cutoff of 200 μg/mL, the data set suggested a 93% sensitivity and 86% specificity in predicting both the BCS and BDDCS solubility classes. In conclusion, this study identified pH-dependent permeability and solubility criteria that can be used to assign provisional biopharmaceutics class at early stage of the drug discovery process. Additionally, such a classification system will enable discovery scientists to assess the potential limiting factors to oral absorption, as well as help predict the drug disposition mechanisms and potential drug−drug interactions.

Journal of Pharmacokinetics and Pharmacodynamics, 2005

The idea of model-based drug development championed by Lewis Sheiner, in which pharmacostatistica... more The idea of model-based drug development championed by Lewis Sheiner, in which pharmacostatistical models of drug efficacy and safety are developed from preclinical and available clinical data, offers a quantitative approach to improving drug development and development decision-making. Examples are presented that support this paradigm. The first example describes a preclinical model of behavioral activity to predict potency and time-course of response in humans and assess the potential for differentiation between compounds. This example illustrates how modeling procedures expounded by Lewis Sheiner provided the means to differentiate potency and the lag time between drug exposure and response and allow for rapid decision making and dose selection. The second example involves planning a Phase 2a dose-ranging and proof of concept trial in Alzheimer's disease (AD). The issue was how to proceed with the study and what criteria to use for a go/no go decision. The combined knowledge of AD disease progression, and preclinical and clinical information about the drug were used to simulate various clinical trial scenarios to identify an efficient and effective Phase 2 study. A design was selected and carried out resulting in a number of important learning experiences as well as extensive financial savings. The motivation for this case in point was the "Learn-Confirm" paradigm described by Lewis Sheiner. The final example describes the use of Pharmacokinetic and Pharmacodynamic (PK/PD) modeling and simulation to confirm efficacy across doses. In the New Drug Application for gabapentin, data from two adequate and well-controlled clinical trials was submitted to the Food and Drug Administration (FDA) in support of the approval of the indication for the treatment of post-herpetic neuralgia. The clinical trial data was not replicated for each of the sought dose levels in the drug application presenting a regulatory dilemma. Exposure response analysis submitted in the New Drug Application was applied to confirm the evidence of efficacy across these dose levels. Modeling and simulation analyses showed that the two studies corroborate each other with respect to

Journal of Medicinal Chemistry, 2009

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay... more 3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.

Journal of Medicinal Chemistry, 2010

Oral bioavailability (F) is a product of fraction absorbed (Fa), fraction escaping gut-wall elimi... more Oral bioavailability (F) is a product of fraction absorbed (Fa), fraction escaping gut-wall elimination (Fg), and fraction escaping hepatic elimination (Fh). In this study, using a database comprised of Fa, Fg, Fh, and F values for 309 drugs in humans, an analysis of the interrelation of physicochemical properties and the individual parameters was carried out in order to define the physicochemical space for optimum human oral bioavailability. Trend analysis clearly indicated molecular weight (MW), ionization state, lipophilicity, polar descriptors, and free rotatable bonds (RB) influence bioavailability. These trends were due to a combination of effects of the properties on Fa and first-pass elimination (Fg and Fh). Higher MW significantly impacted Fa, while Fg and Fh decreased with increasing lipophilicity. Parabolic trends were observed for bioavailability with polar descriptors. Interestingly, RB has a negative effect on all three parameters, leading to its pronounced effect on bioavailability. In conclusion, physicochemical properties influence bioavailability with typically opposing effects on Fa and first-pass elimination. This analysis may provide a rational judgment on the physicochemical space to optimize oral bioavailability.

Journal of Medicinal Chemistry, 2005

As part of a program aimed at generating compounds with affinity for the R 2 -δ subunit of voltag... more As part of a program aimed at generating compounds with affinity for the R 2 -δ subunit of voltage-gated calcium channels, several novel -amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the R 2 -δ subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood-brain barrier. Figure 2. Facile cyclopropanation of 3 with nitroethane. Scheme 1

Journal of Medicinal Chemistry, 2009

Kidney plays an important role in the elimination of drugs, especially with low or negligible hep... more Kidney plays an important role in the elimination of drugs, especially with low or negligible hepatic clearance. An analysis of the interrelation of physicochemical properties and the human renal clearance for a data set of 391 drugs or compounds tested in humans is presented. The data set indicated that lipophilicity shows a negative relationship while polar descriptors show a positive relationship with renal clearance. Analysis of net secreted and net reabsorbed subsets revealed that hydrophilic ionized compounds are probable compounds to show net secretion and a possible drug-drug interaction due to their likely interaction with uptake transporters and inherent low passive reabsorption. The physicochemical space and renal clearance were also statistically analyzed by therapeutic area. In conclusion, ionization state, lipophilicity, and polar descriptors are found to be the physicochemical determinants of renal clearance. These fundamental properties can be valuable in early prediction of human renal clearance and can aid the chemist in structural modifications to optimize drug disposition.

Drug Development and Industrial Pharmacy, 2009

Chitosan glutamate and polyacrylic acid (e.g., carbomer 974P) are known to modulate the tight jun... more Chitosan glutamate and polyacrylic acid (e.g., carbomer 974P) are known to modulate the tight junctions in the intestinal wall and increase permeability and blood exposure of drugs absorbed orally by the paracellular route. To assess the impact of chitosan glutamate and carbomer 974P on the absorption of paracellularly absorbed model drug, acyclovir, in vitro and in rat in vivo. The influence of chitosan glutamate and carbomer 974P (alone and in combination with EDTA-Na2) on the in vitro Caco-2 permeability and oral pharmacokinetic profile in the rat of acyclovir was investigated. In the presence of chitosan glutamate, the apparent permeability of acyclovir across Caco2 monolayer increased 4.1 times relative to control. This increase was accompanied by a significant ( approximately 60%) decrease in transepithelial electrical resistance values indicating opening of the tight junctions in the cell monolayer. In rat, chitosan glutamate doubled oral bioavailability of acyclovir and tripled the amount of acyclovir excreted unchanged into urine. In contrast, the effect of carbomer 974P was not statistically significant at 5% level. In conclusion, chitosan glutamate (1-3%) and chitosan glutamate (1%)/EDTA-Na2 (0.01%) are effective excipients to increase permeability of acyclovir across Caco-2 cell monolayers and the oral absorption in the rat in vivo.

Current Drug Discovery Technologies, 2011

Significant efforts through genomic approaches have been dedicated toward the identification of n... more Significant efforts through genomic approaches have been dedicated toward the identification of novel protein-protein interactions as promising therapeutic targets for indications such as Alzheimer's disease, Parkinson's disease and neuropsychiatric disorders. Additionally, the number of biotherapeutic agents entering the Pharmaceutical sector continues to increase and according to EvaluatePharma's "World Preview 2014" report, "the compounded annual growth rate of biologics is expected to be 8.5 percent from 2008-2014, eight to 10 times greater than the growth rate of small molecules". However, there are limited examples of success in developing biotherapeutic modalities for central nervous system (CNS) diseases in the drug development pipeline. A primary reason for the lack of application of biotherapeutics to neuroscience targets, is that the blood-brain barrier (BBB) isolates and protects CNS structures creating a unique biochemically and immunologically privileged environment, therefore passage of macromolecules across this barrier has additional challenges. An understanding of the anatomical and physiological properties of this barrier with respect to penetration of biotherapeutics is presented in this review document. In this summary, recent advances in biotherapeutic delivery mechanisms across the BBB including transcranial brain drug delivery, focused ultrasound technology, nasal delivery, absorptive endocytosis, and receptor mediated endocytosis are evaluated using an industrial perspective. With acknowledgement that each approach has advantages and disadvantages, this review discusses the opportunities and challenges that are encountered during application of these methods across a variety of therapeutic areas such as, pain, obesity, neuroscience, and oncology. Utilizing an industrial perspective, including consideration of cost of goods and commercial feasibility for these approaches, this review highlights technology features which would enable industry investments toward novel BBB delivery technologies for biologics. Through continued development and improvement of such technology, new therapeutic options to treat and potentially cure central nervous system diseases could eventually evolve.

Canadian Journal of Fisheries and Aquatic Sciences, 1996

ABSTRACT

Bioorganic & Medicinal Chemistry Letters, 2011

The design of drugs with selective tissue distribution can be an effective strategy for enhancing... more The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

Bioorganic & Medicinal Chemistry Letters, 2006

Cyclohexane derivatives Q 0040 Carboxylate Bioisosteres of Gabapentin. -Sulfinate, phosphinate an... more Cyclohexane derivatives Q 0040 Carboxylate Bioisosteres of Gabapentin. -Sulfinate, phosphinate and tetrazole analogues of gabapentin are synthesized and evaluated for their α2-δ binding activity. Tetrazole derivatives (IIa) and (IIb) display comparable affinity for α2-δ as the parent compound gabapentin and similar efficacy against seizures in vivo compared to pregabalin. -(SCHWARZ*, J. B.; et al.; Bioorg. Med. Chem. Lett. 16 (2006) 9, 2333-2336; Pfizer Global Res. Dev., Ann Arbor, MI 48105, USA; Eng.) -M. Schroeter 31-067

Bioorganic & Medicinal Chemistry Letters, 2009

A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several... more A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several were found to exhibit similar potency for the a 2 -d subunit of voltage-gated calcium channels. Oxadiazolone 9 derived from 2 achieved low brain uptake but was nevertheless active in models of osteoarthritis. The high clearance associated with compound 9 was postulated to be a consequence of efflux by OAT and/ or OCT, and was attenuated on co-administration with cimetidine or probenecid.

Bioorganic & Medicinal Chemistry Letters, 2006

Several b-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two co... more Several b-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two convergent, highly efficient routes, and their affinity for the a 2 -d protein examined. Two select compounds with potent affinity for a 2 -d, 8a and 16a, were subsequently tested in vivo in an audiogenic seizure model and found to elicit protective effects.

Arthritis & Rheumatism, 2005

To examine the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage-activated Ca(2+)... more To examine the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage-activated Ca(2+) channels and a compound chemically related to pregabalin and gabapentin, on the development of cartilage structural changes in an experimental dog model of osteoarthritis (OA). The effects of PD-0200347 on the major pathways involved in OA cartilage degradation, including matrix metalloproteinases (MMPs) and the inducible form of nitric oxide synthase (iNOS), were also studied. OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were randomly distributed into 3 groups and treated orally with either 1) placebo, 2) 15 mg/kg/day of PD-0200347, or 3) 90 mg/kg/day of PD-0200347. Dogs were killed 12 weeks after surgery. The severity of the lesions was scored macroscopically and histologically. Cartilage specimens from the femoral condyles and tibial plateaus were processed for RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemistry. Specific probes and antibodies were used to study the messenger RNA and protein levels of iNOS, MMP-1, MMP-3, and MMP-13. No clinical signs of drug toxicity were noted in the treated animals. Treatment with PD-0200347 at both dosages tested (15 and 90 mg/kg/day) reduced the development of cartilage lesions. There was a reduction in the score of lesions, with a statistically significant (P = 0.01) difference when the highest dosage of the drug was administered. The reduction in the score was mainly related to a decrease in the surface size of the lesions. Quantitative RT-PCR showed that PD-0200347 significantly reduced the expression of MMP-13, a key mediator in OA. Immunohistochemical analyses showed that treatment with PD-0200347 significantly reduced the synthesis of all key OA mediators studied. This study demonstrated the efficacy of PD-0200347 in reducing the progression of cartilage structural changes in a dog model of OA. It also showed that this effect is linked to the inhibition of the major pathophysiologic mediators responsible for cartilage degradation.

Expert Opinion on Drug Metabolism & Toxicology, 2012

Drug transporter proteins are expressed on the cell membrane, regulating substrate exposure in sy... more Drug transporter proteins are expressed on the cell membrane, regulating substrate exposure in systemic circulation and/or peripheral tissues. Genetic polymorphism of drug transporter genes encoding these proteins could alter the functional activity and/or protein expression, having effects on absorption, distribution, metabolism and excretion (ADME), efficacy and adverse effects. The authors provide the reader with an overview of the pharmacogenetics (PGx) of 12 membrane transporters. The clinical literature is summarized as to the quantitative significance on pharmacokinetics (PK) and implications on pharmacodynamics (PD) and adverse effects, due to transporter influence on intracellular drug concentrations. Unlike polymorphisms for cytochrome P450s (CYPs) resulting in large magnitude of PK variation, genetic mutations for membrane transporters are typically less than threefold alteration in systemic PK for drugs with a few exceptions. However, substantially greater changes in intracellular drug levels may result. We are aware of 1880 exome variants in 12 of the best-studied transporters to date, and nearly 40% of these change the amino acid. However, the functional consequences of most of these variants remain to be determined, and have only been empirically evaluated for a handful. To the extent that genetic polymorphisms impact ADME, it is a variable that will contribute to ethnic differences due to substantial frequency differences for the known variants.

Pharmaceutical research, 2000

To test the barrier function of a bio-engineered human skin (BHS) using three model drugs (caffei... more To test the barrier function of a bio-engineered human skin (BHS) using three model drugs (caffeine, hydrocortisone, and tamoxifen) in vitro. To investigate the lipid composition and microscopic structure of the BHS. The human skin substitute was composed of both epidermal and dermal layers, the latter having a bovine collagen matrix. The permeability of the BHS to three model drugs was compared to that obtained in other percutaneous testing models (human cadaver skin, hairless mouse skin, and EpiDerm). Lipid analysis of the BHS was performed by high performance thin layered chromatography. Histological evaluation of the BHS was performed using routine H&E staining. The BHS mimicked human skin in terms of lipid composition, gross ultrastructure, and the formation of a stratum corneum. However, the permeability of the BHS to caffeine, hydrocortisone, and tamoxifen was 3-4 fold higher than that of human cadaver skin. In summary, the results indicate that the BHS may be an acceptable i...

Expert Opinion on Drug Metabolism & Toxicology, 2006

Optimisation of oral bioavailability is a continuing challenge for the pharmaceutical and biotech... more Optimisation of oral bioavailability is a continuing challenge for the pharmaceutical and biotechnology industries. The number of potential drug candidates requiring in vivo evaluation has significantly increased with the advent of combinatorial chemistry. In addition, drug discovery programmes are increasingly forced into more lipophilic and lower solubility chemical space. To aid in the use of in vitro and in silico tools as well as reduce the number of in vivo studies required, a team-based discussion tool is proposed that provides a 'road map' to guide the selection of profiling assays that should be considered when optimising oral bioavailability. This road map divides the factors that contribute to poor oral bioavailability into two interrelated categories: absorption and metabolism. This road map provides an interface for cross discipline discussions and a systematic approach to the experimentation that drives the drug discovery process towards a common goal -acceptable oral bioavailability using minimal resources in an acceptable time frame.

ABSTRACT PURPOSE. To investigate the inter-lot difference in hepatic active uptakes and their cor... more ABSTRACT PURPOSE. To investigate the inter-lot difference in hepatic active uptakes and their correlation to protein expression of hepatic uptake transporters in various lots of cryopreserved human hepatocytes, a higher throughput fast filtration method was developed. In addition, absolute expression of OATP proteins in livers and hepatocytes was determined by using newly developed targeted proteomics LC-MS/MS method. METHODS. The cryopreserved hepatocytes were thawed at 37C and resuspended in Krebs-Henseleit buffer at 2.0x10^6 cells/mL. The viability of hepatocytes used for uptake assays was greater than 70%. The hepatocytes were pre-warmed at 37C for 10 min with or without inhibitors. Uptakes were initiated by adding pre-warmed compounds with or without inhibitors into hepatocyte suspension. The fast filtration was executed through a cell harvester. For quantification of OATPs, membrane fractions of hepatocytes and liver tissue were extracted using Proteo extract membrane kit following the manufacturefs protocol. The membrane fraction samples were then subjected to trypsin digestion and LC-MS/MS analysis. RESULTS. Rosuvastatin and pitavastatin was selected as probe substrates to evaluate the hepatocyte uptake. The hepatic active uptake detected by a cell harvester showed time- and concentration-dependent manner, and was inhibited by rifamycin SV, a prototypical inhibitor for OATPs. The results were consistent with that detected by the literature oil-layer method. Moreover, significant difference in active hepatic uptakes was observed among various lots of cryopreserved hepatocytes. The absolute OATP proteins in hepatocytes and liver tissues were measured by targeted proteomics LC-MS/MS method through monitoring the mass transition of the unique tryptic peptides released from each OATP proteins, e.g. OATP1B1, 1B3 and 2B1. Ongoing process includes to test additional hepatocyte lots and to analysis the correlation between hepatocyte active uptake and protein absolute expression. CONCLUSION. The development of higher throughput hepatic uptake assays enabled to compare the inter-individual difference in hepatocyte uptake. Targeted proteomic LC-MS/MS protein quantification demonstrated as a highly sensitive, selective and reliable method for absolute measurement of OATP proteins. The relationship between active hepatic uptake and protein expression could extend our current understanding on drug hepatic disposition and transporter mediated drug-drug interaction, and further advances system biology models in various translational aspects.

Neuropharmacology, 2011

Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are sim... more Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents.

Molecular Pharmaceutics, 2012

The Biopharmaceutics Classification System (BCS) is a scientific framework that provides a basis ... more The Biopharmaceutics Classification System (BCS) is a scientific framework that provides a basis for predicting the oral absorption of drugs. These concepts have been extended in the Biopharmaceutics Drug Disposition Classification System (BDDCS) to explain the potential mechanism of drug clearance and understand the effects of uptake and efflux transporters on absorption, distribution, metabolism, and elimination. The objective of present work is to establish criteria for provisional biopharmaceutics classification using pH-dependent passive permeability and aqueous solubility data generated from high throughput screening methodologies in drug discovery settings. The apparent permeability across monolayers of clonal cell line of Madin−Darby canine kidney cells, selected for low endogenous efflux transporter expression, was measured for a set of 105 drugs, with known BCS and BDDCS class. The permeability at apical pH 6.5 for acidic drugs and at pH 7.4 for nonacidic drugs showed a good correlation with the fraction absorbed in human (Fa). Receiver operating characteristic (ROC) curve analysis was utilized to define the permeability class boundary. At permeability ≥5 × 10 −6 cm/s, the accuracy of predicting Fa of ≥0.90 was 87%. Also, this cutoff showed more than 80% sensitivity and specificity in predicting the literature permeability classes (BCS), and the metabolism classes (BDDCS). The equilibrium solubility of a subset of 49 drugs was measured in pH 1.2 medium, pH 6.5 phosphate buffer, and in FaSSIF medium (pH 6.5). Although dose was not considered, good concordance of the measured solubility with BCS and BDDCS solubility class was achieved, when solubility at pH 1.2 was used for acidic compounds and FaSSIF solubility was used for basic, neutral, and zwitterionic compounds. Using a cutoff of 200 μg/mL, the data set suggested a 93% sensitivity and 86% specificity in predicting both the BCS and BDDCS solubility classes. In conclusion, this study identified pH-dependent permeability and solubility criteria that can be used to assign provisional biopharmaceutics class at early stage of the drug discovery process. Additionally, such a classification system will enable discovery scientists to assess the potential limiting factors to oral absorption, as well as help predict the drug disposition mechanisms and potential drug−drug interactions.

Journal of Pharmacokinetics and Pharmacodynamics, 2005

The idea of model-based drug development championed by Lewis Sheiner, in which pharmacostatistica... more The idea of model-based drug development championed by Lewis Sheiner, in which pharmacostatistical models of drug efficacy and safety are developed from preclinical and available clinical data, offers a quantitative approach to improving drug development and development decision-making. Examples are presented that support this paradigm. The first example describes a preclinical model of behavioral activity to predict potency and time-course of response in humans and assess the potential for differentiation between compounds. This example illustrates how modeling procedures expounded by Lewis Sheiner provided the means to differentiate potency and the lag time between drug exposure and response and allow for rapid decision making and dose selection. The second example involves planning a Phase 2a dose-ranging and proof of concept trial in Alzheimer's disease (AD). The issue was how to proceed with the study and what criteria to use for a go/no go decision. The combined knowledge of AD disease progression, and preclinical and clinical information about the drug were used to simulate various clinical trial scenarios to identify an efficient and effective Phase 2 study. A design was selected and carried out resulting in a number of important learning experiences as well as extensive financial savings. The motivation for this case in point was the "Learn-Confirm" paradigm described by Lewis Sheiner. The final example describes the use of Pharmacokinetic and Pharmacodynamic (PK/PD) modeling and simulation to confirm efficacy across doses. In the New Drug Application for gabapentin, data from two adequate and well-controlled clinical trials was submitted to the Food and Drug Administration (FDA) in support of the approval of the indication for the treatment of post-herpetic neuralgia. The clinical trial data was not replicated for each of the sought dose levels in the drug application presenting a regulatory dilemma. Exposure response analysis submitted in the New Drug Application was applied to confirm the evidence of efficacy across these dose levels. Modeling and simulation analyses showed that the two studies corroborate each other with respect to

Journal of Medicinal Chemistry, 2009

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay... more 3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.

Journal of Medicinal Chemistry, 2010

Oral bioavailability (F) is a product of fraction absorbed (Fa), fraction escaping gut-wall elimi... more Oral bioavailability (F) is a product of fraction absorbed (Fa), fraction escaping gut-wall elimination (Fg), and fraction escaping hepatic elimination (Fh). In this study, using a database comprised of Fa, Fg, Fh, and F values for 309 drugs in humans, an analysis of the interrelation of physicochemical properties and the individual parameters was carried out in order to define the physicochemical space for optimum human oral bioavailability. Trend analysis clearly indicated molecular weight (MW), ionization state, lipophilicity, polar descriptors, and free rotatable bonds (RB) influence bioavailability. These trends were due to a combination of effects of the properties on Fa and first-pass elimination (Fg and Fh). Higher MW significantly impacted Fa, while Fg and Fh decreased with increasing lipophilicity. Parabolic trends were observed for bioavailability with polar descriptors. Interestingly, RB has a negative effect on all three parameters, leading to its pronounced effect on bioavailability. In conclusion, physicochemical properties influence bioavailability with typically opposing effects on Fa and first-pass elimination. This analysis may provide a rational judgment on the physicochemical space to optimize oral bioavailability.

Journal of Medicinal Chemistry, 2005

As part of a program aimed at generating compounds with affinity for the R 2 -δ subunit of voltag... more As part of a program aimed at generating compounds with affinity for the R 2 -δ subunit of voltage-gated calcium channels, several novel -amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the R 2 -δ subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood-brain barrier. Figure 2. Facile cyclopropanation of 3 with nitroethane. Scheme 1

Journal of Medicinal Chemistry, 2009

Kidney plays an important role in the elimination of drugs, especially with low or negligible hep... more Kidney plays an important role in the elimination of drugs, especially with low or negligible hepatic clearance. An analysis of the interrelation of physicochemical properties and the human renal clearance for a data set of 391 drugs or compounds tested in humans is presented. The data set indicated that lipophilicity shows a negative relationship while polar descriptors show a positive relationship with renal clearance. Analysis of net secreted and net reabsorbed subsets revealed that hydrophilic ionized compounds are probable compounds to show net secretion and a possible drug-drug interaction due to their likely interaction with uptake transporters and inherent low passive reabsorption. The physicochemical space and renal clearance were also statistically analyzed by therapeutic area. In conclusion, ionization state, lipophilicity, and polar descriptors are found to be the physicochemical determinants of renal clearance. These fundamental properties can be valuable in early prediction of human renal clearance and can aid the chemist in structural modifications to optimize drug disposition.

Drug Development and Industrial Pharmacy, 2009

Chitosan glutamate and polyacrylic acid (e.g., carbomer 974P) are known to modulate the tight jun... more Chitosan glutamate and polyacrylic acid (e.g., carbomer 974P) are known to modulate the tight junctions in the intestinal wall and increase permeability and blood exposure of drugs absorbed orally by the paracellular route. To assess the impact of chitosan glutamate and carbomer 974P on the absorption of paracellularly absorbed model drug, acyclovir, in vitro and in rat in vivo. The influence of chitosan glutamate and carbomer 974P (alone and in combination with EDTA-Na2) on the in vitro Caco-2 permeability and oral pharmacokinetic profile in the rat of acyclovir was investigated. In the presence of chitosan glutamate, the apparent permeability of acyclovir across Caco2 monolayer increased 4.1 times relative to control. This increase was accompanied by a significant ( approximately 60%) decrease in transepithelial electrical resistance values indicating opening of the tight junctions in the cell monolayer. In rat, chitosan glutamate doubled oral bioavailability of acyclovir and tripled the amount of acyclovir excreted unchanged into urine. In contrast, the effect of carbomer 974P was not statistically significant at 5% level. In conclusion, chitosan glutamate (1-3%) and chitosan glutamate (1%)/EDTA-Na2 (0.01%) are effective excipients to increase permeability of acyclovir across Caco-2 cell monolayers and the oral absorption in the rat in vivo.

Current Drug Discovery Technologies, 2011

Significant efforts through genomic approaches have been dedicated toward the identification of n... more Significant efforts through genomic approaches have been dedicated toward the identification of novel protein-protein interactions as promising therapeutic targets for indications such as Alzheimer's disease, Parkinson's disease and neuropsychiatric disorders. Additionally, the number of biotherapeutic agents entering the Pharmaceutical sector continues to increase and according to EvaluatePharma's "World Preview 2014" report, "the compounded annual growth rate of biologics is expected to be 8.5 percent from 2008-2014, eight to 10 times greater than the growth rate of small molecules". However, there are limited examples of success in developing biotherapeutic modalities for central nervous system (CNS) diseases in the drug development pipeline. A primary reason for the lack of application of biotherapeutics to neuroscience targets, is that the blood-brain barrier (BBB) isolates and protects CNS structures creating a unique biochemically and immunologically privileged environment, therefore passage of macromolecules across this barrier has additional challenges. An understanding of the anatomical and physiological properties of this barrier with respect to penetration of biotherapeutics is presented in this review document. In this summary, recent advances in biotherapeutic delivery mechanisms across the BBB including transcranial brain drug delivery, focused ultrasound technology, nasal delivery, absorptive endocytosis, and receptor mediated endocytosis are evaluated using an industrial perspective. With acknowledgement that each approach has advantages and disadvantages, this review discusses the opportunities and challenges that are encountered during application of these methods across a variety of therapeutic areas such as, pain, obesity, neuroscience, and oncology. Utilizing an industrial perspective, including consideration of cost of goods and commercial feasibility for these approaches, this review highlights technology features which would enable industry investments toward novel BBB delivery technologies for biologics. Through continued development and improvement of such technology, new therapeutic options to treat and potentially cure central nervous system diseases could eventually evolve.

Canadian Journal of Fisheries and Aquatic Sciences, 1996

ABSTRACT

Bioorganic & Medicinal Chemistry Letters, 2011

The design of drugs with selective tissue distribution can be an effective strategy for enhancing... more The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

Bioorganic & Medicinal Chemistry Letters, 2006

Cyclohexane derivatives Q 0040 Carboxylate Bioisosteres of Gabapentin. -Sulfinate, phosphinate an... more Cyclohexane derivatives Q 0040 Carboxylate Bioisosteres of Gabapentin. -Sulfinate, phosphinate and tetrazole analogues of gabapentin are synthesized and evaluated for their α2-δ binding activity. Tetrazole derivatives (IIa) and (IIb) display comparable affinity for α2-δ as the parent compound gabapentin and similar efficacy against seizures in vivo compared to pregabalin. -(SCHWARZ*, J. B.; et al.; Bioorg. Med. Chem. Lett. 16 (2006) 9, 2333-2336; Pfizer Global Res. Dev., Ann Arbor, MI 48105, USA; Eng.) -M. Schroeter 31-067

Bioorganic & Medicinal Chemistry Letters, 2009

A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several... more A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several were found to exhibit similar potency for the a 2 -d subunit of voltage-gated calcium channels. Oxadiazolone 9 derived from 2 achieved low brain uptake but was nevertheless active in models of osteoarthritis. The high clearance associated with compound 9 was postulated to be a consequence of efflux by OAT and/ or OCT, and was attenuated on co-administration with cimetidine or probenecid.

Bioorganic & Medicinal Chemistry Letters, 2006

Several b-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two co... more Several b-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two convergent, highly efficient routes, and their affinity for the a 2 -d protein examined. Two select compounds with potent affinity for a 2 -d, 8a and 16a, were subsequently tested in vivo in an audiogenic seizure model and found to elicit protective effects.

Arthritis & Rheumatism, 2005

To examine the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage-activated Ca(2+)... more To examine the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage-activated Ca(2+) channels and a compound chemically related to pregabalin and gabapentin, on the development of cartilage structural changes in an experimental dog model of osteoarthritis (OA). The effects of PD-0200347 on the major pathways involved in OA cartilage degradation, including matrix metalloproteinases (MMPs) and the inducible form of nitric oxide synthase (iNOS), were also studied. OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were randomly distributed into 3 groups and treated orally with either 1) placebo, 2) 15 mg/kg/day of PD-0200347, or 3) 90 mg/kg/day of PD-0200347. Dogs were killed 12 weeks after surgery. The severity of the lesions was scored macroscopically and histologically. Cartilage specimens from the femoral condyles and tibial plateaus were processed for RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemistry. Specific probes and antibodies were used to study the messenger RNA and protein levels of iNOS, MMP-1, MMP-3, and MMP-13. No clinical signs of drug toxicity were noted in the treated animals. Treatment with PD-0200347 at both dosages tested (15 and 90 mg/kg/day) reduced the development of cartilage lesions. There was a reduction in the score of lesions, with a statistically significant (P = 0.01) difference when the highest dosage of the drug was administered. The reduction in the score was mainly related to a decrease in the surface size of the lesions. Quantitative RT-PCR showed that PD-0200347 significantly reduced the expression of MMP-13, a key mediator in OA. Immunohistochemical analyses showed that treatment with PD-0200347 significantly reduced the synthesis of all key OA mediators studied. This study demonstrated the efficacy of PD-0200347 in reducing the progression of cartilage structural changes in a dog model of OA. It also showed that this effect is linked to the inhibition of the major pathophysiologic mediators responsible for cartilage degradation.

Expert Opinion on Drug Metabolism & Toxicology, 2012

Drug transporter proteins are expressed on the cell membrane, regulating substrate exposure in sy... more Drug transporter proteins are expressed on the cell membrane, regulating substrate exposure in systemic circulation and/or peripheral tissues. Genetic polymorphism of drug transporter genes encoding these proteins could alter the functional activity and/or protein expression, having effects on absorption, distribution, metabolism and excretion (ADME), efficacy and adverse effects. The authors provide the reader with an overview of the pharmacogenetics (PGx) of 12 membrane transporters. The clinical literature is summarized as to the quantitative significance on pharmacokinetics (PK) and implications on pharmacodynamics (PD) and adverse effects, due to transporter influence on intracellular drug concentrations. Unlike polymorphisms for cytochrome P450s (CYPs) resulting in large magnitude of PK variation, genetic mutations for membrane transporters are typically less than threefold alteration in systemic PK for drugs with a few exceptions. However, substantially greater changes in intracellular drug levels may result. We are aware of 1880 exome variants in 12 of the best-studied transporters to date, and nearly 40% of these change the amino acid. However, the functional consequences of most of these variants remain to be determined, and have only been empirically evaluated for a handful. To the extent that genetic polymorphisms impact ADME, it is a variable that will contribute to ethnic differences due to substantial frequency differences for the known variants.