Publications by authors named "Nigel J Waters"

40 Publications

Pharmacokinetic and Pharmacodynamic Considerations for Drugs Binding to Alpha-1-Acid Glycoprotein.

Pharm Res 2018 Dec 28;36(2):30. Epub 2018 Dec 28.

Nonclinical Development, Relay Therapeutics, 215 First Street, Cambridge, Massachusetts, USA.

According to the free drug hypothesis only the unbound drug is available to act at physiological sites of action, and as such the importance of plasma protein binding primarily resides in its impact on pharmacokinetics and pharmacodynamics. Of the major plasma proteins, alpha-1-acid glycoprotein (AAG) represents an intriguing one primarily due to the high affinity, low capacity properties of this protein. In addition, there are marked species and age differences in protein expression, homology and drug binding affinity. As such, a thorough understanding of drug binding to AAG can help aid and improve the translation of pharmacokinetic/pharmacodynamic (PK/PD) relationships from preclinical species to human as well as adults to neonates. This review provides a comprehensive overview of our current understanding of the biochemistry of AAG; endogenous function, impact of disease, utility as a biomarker, and impact on PK/PD. Experimental considerations are discussed as well as recommendations for understanding the potential impact of AAG on PK through drug discovery and early development.
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http://dx.doi.org/10.1007/s11095-018-2551-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089466PMC
December 2018

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

PLoS One 2018 1;13(6):e0197372. Epub 2018 Jun 1.

Epizyme, Inc., Cambridge, Massachusetts, United States of America.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983452PMC
December 2018

The DOT1L inhibitor pinometostat reduces H3K79 methylation and has modest clinical activity in adult acute leukemia.

Blood 2018 06 3;131(24):2661-2669. Epub 2018 May 3.

Memorial Sloan Kettering Cancer Center, New York, NY.

Pinometostat (EPZ-5676) is a first-in-class small-molecule inhibitor of the histone methyltransferase disrupter of telomeric silencing 1-like (DOT1L). In this phase 1 study, pinometostat was evaluated for safety and efficacy in adult patients with advanced acute leukemias, particularly those involving mixed lineage leukemia () gene rearrangements () resulting from 11q23 translocations. Fifty-one patients were enrolled into 6 dose-escalation cohorts (n = 26) and 2 expansion cohorts (n = 25) at pinometostat doses of 54 and 90 mg/m per day by continuous intravenous infusion in 28-day cycles. Because a maximum tolerated dose was not established in the dose-escalation phase, the expansion doses were selected based on safety and clinical response data combined with pharmacodynamic evidence of reduction in H3K79 methylation during dose escalation. Across all dose levels, plasma pinometostat concentrations increased in an approximately dose-proportional fashion, reaching an apparent steady-state by 4-8 hours after infusion, and rapidly decreased following treatment cessation. The most common adverse events, of any cause, were fatigue (39%), nausea (39%), constipation (35%), and febrile neutropenia (35%). Overall, 2 patients, both with t(11;19), experienced complete remission at 54 mg/m per day by continuous intravenous infusion, demonstrating proof of concept for delivering clinically meaningful responses through targeting DOT1L using the single agent pinometostat in leukemia patients. Administration of pinometostat was generally safe, with the maximum tolerated dose not being reached, although efficacy as a single agent was modest. This study demonstrates the therapeutic potential for targeting DOT1L in leukemia and lays the groundwork for future combination approaches in this patient population. This clinical trial is registered at www.clinicaltrials.gov as NCT01684150.
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http://dx.doi.org/10.1182/blood-2017-12-818948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265654PMC
June 2018

Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma.

Sci Rep 2017 12 21;7(1):17993. Epub 2017 Dec 21.

Epizyme, Inc., Cambridge, Massachusetts, USA.

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.
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http://dx.doi.org/10.1038/s41598-017-18446-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740082PMC
December 2017

Mechanisms of Pinometostat (EPZ-5676) Treatment-Emergent Resistance in -Rearranged Leukemia.

Mol Cancer Ther 2017 08 20;16(8):1669-1679. Epub 2017 Apr 20.

Epizyme Inc., Cambridge, Massachusetts.

DOT1L is a protein methyltransferase involved in the development and maintenance of -rearranged (-r) leukemia through its ectopic methylation of histones associated with well-characterized leukemic genes. Pinometostat (EPZ-5676), a selective inhibitor of DOT1L, is in clinical development in relapsed/refractory acute leukemia patients harboring rearrangements of the gene. The observation of responses and subsequent relapses in the adult trial treating -r patients motivated preclinical investigations into potential mechanisms of pinometostat treatment-emergent resistance (TER) in cell lines confirmed to have -r. TER was achieved in five -r cell lines, KOPN-8, MOLM-13, MV4-11, NOMO-1, and SEM. Two of the cell lines, KOPN-8 and NOMO-1, were thoroughly characterized to understand the mechanisms involved in pinometostat resistance. Unlike many other targeted therapies, resistance does not appear to be achieved through drug-induced selection of mutations of the target itself. Instead, we identified both drug efflux transporter dependent and independent mechanisms of resistance to pinometostat. In KOPN-8 TER cells, increased expression of the drug efflux transporter ABCB1 (P-glycoprotein, MDR1) was the primary mechanism of drug resistance. In contrast, resistance in NOMO-1 cells occurs through a mechanism other than upregulation of a specific efflux pump. RNA-seq analysis performed on both parental and resistant KOPN-8 and NOMO-1 cell lines supported two unique candidate pathway mechanisms that may explain the pinometostat resistance observed in these cell lines. These results are the first demonstration of TER models of the DOT1L inhibitor pinometostat and may provide useful tools for investigating clinical resistance. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0693DOI Listing
August 2017

Implications for Metabolite Quantification by Mass Spectrometry in the Absence of Authentic Standards.

Drug Metab Dispos 2017 05 2;45(5):492-496. Epub 2017 Mar 2.

Drug Metabolism & Pharmacokinetics, Novartis Institutes for Biomedical Research Inc., East Hanover, New Jersey (P.H); Syros Pharmaceuticals, Cambridge, Massachusetts (N.J.W); and Analytical Sciences & Imaging, Novartis Institutes for Biomedical Research Inc., Cambridge, Massachusetts (U.A.A)

Quantification of metabolites by mass spectrometry in the absence of authentic reference standards or without a radiolabel is often called "semiquantitative," which acknowledges that mass spectrometric responses are not truly quantitative. For many researchers, it is tempting to pursue this practice of semiquantification in early drug discovery and even preclinical development, when radiolabeled absorption, distribution, metabolism, and excretion studies are being deferred to later stages of drug development. The caveats of quantifying metabolites based on parent drug response are explored in this investigation. A set of 71 clinically relevant drugs/metabolites encompassing common biotransformation pathways was subjected to flow injection analysis coupled with electrospray ionization (ESI) mass spectrometry. The results revealed a large variation in ESI response even for structurally similar parent drug/metabolite pairs. The ESI response of each metabolite was normalized to that of the parent drug to generate an ESI relative response factor. Overall, relative response factors ranged from 0.014 (>70-fold lower response than parent) to 8.6 (8.6-fold higher response than parent). Various two-dimensional molecular descriptors were calculated that describe physicochemical, topological, and structural properties for each drug/metabolite. The molecular descriptors, along with the ESI response factors, were used in univariate analyses as well as a principal components analysis to ascertain which molecular descriptors best account for the observed discrepancies in drug/metabolite ESI response. This investigation has shown that the practice of using parent drug response to quantify metabolites should be used with caution.
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http://dx.doi.org/10.1124/dmd.117.075259DOI Listing
May 2017

Preclinical Pharmacokinetics and Pharmacodynamics of Pinometostat (EPZ-5676), a First-in-Class, Small Molecule S-Adenosyl Methionine Competitive Inhibitor of DOT1L.

Authors:
Nigel J Waters

Eur J Drug Metab Pharmacokinet 2017 Dec;42(6):891-901

Syros Pharmaceuticals, 620 Memorial Drive, Cambridge, MA, 02139, USA.

Acute leukemias bearing mixed lineage leukemia (MLL) rearrangements are aggressive diseases characterized by a poor overall prognosis despite multi-agent chemotherapy. Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) lead to recruitment of DOT1L, to a multi-protein complex resulting in aberrant methylation of histone H3 lysine 79 at MLL target genes, and ultimately enhanced expression of critical genes for hematopoietic differentiation, including HOXA9 and MEIS1, and as such defines the established mechanism for leukemogenesis in MLL-rearrangement (MLL-r) leukemias. Pinometostat is a first-in-class, small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs, and was the first member of the novel HMT inhibitor class to enter Phase 1 clinical trials in both adult and pediatric MLL-r leukemia patients. In this article, the preclinical pharmacokinetics/pharmacodynamics and drug disposition of pinometostat are reviewed including discussion of how these data were used to inform early clinical studies, and how they translated to the clinical experience.
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http://dx.doi.org/10.1007/s13318-017-0404-3DOI Listing
December 2017

Mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha-1-acid glycoprotein and permeability-limited hepatic uptake.

Xenobiotica 2017 Mar 10;47(3):185-193. Epub 2016 May 10.

a Epizyme Inc , Cambridge , MA , USA and.

1. The plasma clearance of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was shown to be markedly lower in human compared to the preclinical species, mouse, rat and dog. 2. This led to vertical allometry where various interspecies scaling methods were applied to the data, with fold-errors between 4 and 13. We had previously reported the elimination and metabolic pathways of EPZ-5676 were similar across species. Therefore, the aim of this work was to explore the mechanistic basis for the species difference in clearance for EPZ-5676, focusing on other aspects of disposition. 3. The protein binding of EPZ-5676 in human plasma demonstrated a non-linear relationship suggesting saturable binding at physiologically relevant concentrations. Saturation of protein binding was not observed in plasma from preclinical species. Kinetic determinations using purified serum albumin and alpha-1-acid glycoprotein (AAG) confirmed that EPZ-5676 is a high affinity ligand for AAG with a dissociation constant (K) of 0.24 μM. 4. Permeability limited uptake was also considered since hepatocyte CL was much lower in human relative to preclinical species. Passive unbound CL for EPZ-5676 was estimated using a correlation analysis of logD and data previously reported on seven drugs in sandwich cultured human hepatocytes. 5. Incorporation of AAG binding and permeability limited hepatic uptake into the well-stirred liver model gave rise to a predicted clearance for EPZ-5676 within 2-fold of the observed value of 1.4 mL minkg. This analysis suggests that the marked species difference in EPZ-5676 clearance is driven by high affinity binding to human AAG as well as species-specific hepatic uptake invoking the role of transporters.
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http://dx.doi.org/10.3109/00498254.2016.1173265DOI Listing
March 2017

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666.

ACS Med Chem Lett 2016 Feb 2;7(2):162-6. Epub 2015 Dec 2.

Epizyme, Inc. , 400 Technology Square, Cambridge, Massachusetts 02139, United States.

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).
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http://dx.doi.org/10.1021/acsmedchemlett.5b00380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753547PMC
February 2016

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

ACS Med Chem Lett 2016 Feb 27;7(2):134-8. Epub 2015 Aug 27.

Epizyme Inc. , Fourth Floor, 400 Technology Square, Cambridge, Massachusetts 02139, United States.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753551PMC
February 2016

Physiologically Based Pharmacokinetic Modeling in Pediatric Oncology Drug Development.

Drug Metab Dispos 2016 07 2;44(7):934-43. Epub 2016 Mar 2.

Epizyme Inc., Cambridge, Massachusetts

Childhood cancer represents more than 100 rare and ultra-rare diseases, with an estimated 12,400 new cases diagnosed each year in the United States. As such, this much smaller patient population has led to pediatric oncology drug development lagging behind that for adult cancers. Developing drugs for pediatric malignancies also brings with it a number of unique trial design considerations, including flexible enrollment approaches, age-appropriate formulation, acceptable sampling schedules, and balancing the need for age-stratified dosing regimens, given the smaller patient populations. The regulatory landscape for pediatric pharmacotherapy has evolved with U.S. Food and Drug Administration (FDA) legislation such as the 2012 FDA Safety and Innovation Act. In parallel, regulatory authorities have recommended the application of physiologically based pharmacokinetic (PBPK) modeling, for example, in the recently issued FDA Strategic Plan for Accelerating the Development of Therapies for Pediatric Rare Diseases. PBPK modeling provides a quantitative and systems-based framework that allows the effects of intrinsic and extrinsic factors on drug exposure to be modeled in a mechanistic fashion. The application of PBPK modeling in drug development for pediatric cancers is relatively nascent, with several retrospective analyses of cytotoxic therapies, and latterly for targeted agents such as obatoclax and imatinib. More recently, we have employed PBPK modeling in a prospective manner to inform the first pediatric trials of pinometostat and tazemetostat in genetically defined populations (mixed lineage leukemia-rearranged and integrase interactor-1-deficient sarcomas, respectively). In this review, we evaluate the application of PBPK modeling in pediatric cancer drug development and discuss the important challenges that lie ahead in this field.
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http://dx.doi.org/10.1124/dmd.115.068031DOI Listing
July 2016

Metabolism and disposition of the DOT1L inhibitor, pinometostat (EPZ-5676), in rat, dog and human.

Cancer Chemother Pharmacol 2016 Jan 8;77(1):43-62. Epub 2015 Dec 8.

Epizyme, Inc., 400 Technology Square, Cambridge, MA, 02139, USA.

Purpose: The metabolism and disposition of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was investigated in rat and dog. Metabolite profiles were compared with those from adult patients in the first-in-man phase 1 study as well as the cross-species metabolism observed in vitro.

Methods: EPZ-5676 was administered to rat and dog as a 24-h IV infusion of [(14)C]-EPZ-5676 for determination of pharmacokinetics, mass balance, metabolite profiling and biodistribution by quantitative whole-body autoradiography (QWBA). Metabolite profiling and identification was performed by radiometric and LC-MS/MS analysis.

Results: Fecal excretion was the major route of elimination, representing 79 and 81% of the total dose in and rat and dog, respectively. QWBA in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 168 h. Fecal recovery of dose-related material in bile duct-cannulated animals as well as higher radioactivity concentrations in the wall of the large intestine relative to liver implicated intestinal secretion as well as biliary elimination. EPZ-5676 underwent extensive oxidative metabolism with the major metabolic pathways being hydroxylation of the t-butyl group (EPZ007769) and N-dealkylation of the central nitrogen. Loss of adenine from parent EPZ-5676 (M7) was observed only in rat and dog feces, suggesting the involvement of gut microbiota. In rat and dog, steady-state plasma levels of total radioactivity and parent EPZ-5676 were attained rapidly and maintained through the infusion period before declining rapidly on cessation of dosing. Unchanged EPZ-5676 was the predominant circulating species in rat, dog and man.

Conclusions: The excretory and metabolic pathways for EPZ-5676 were very similar across species. Renal excretion of both parent EPZ-5676 and EPZ-5676-related material was low, and in preclinical species fecal excretion of parent EPZ-5676 and EPZ007769 accounted for the majority of drug-related elimination.
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http://dx.doi.org/10.1007/s00280-015-2929-yDOI Listing
January 2016

Exploring drug delivery for the DOT1L inhibitor pinometostat (EPZ-5676): Subcutaneous administration as an alternative to continuous IV infusion, in the pursuit of an epigenetic target.

J Control Release 2015 Dec 15;220(Pt B):758-65. Epub 2015 Sep 15.

Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139, United States.

Protein methyltransferases are emerging as promising drug targets for therapeutic intervention in human cancers. Pinometostat (EPZ-5676) is a small molecule inhibitor of the DOT1L enzyme, a histone methyltransferase that methylates lysine 79 of histone H3. DOT1L activity is dysregulated in the pathophysiology of rearranged mixed lineage leukemia (MLL-r). Pinometostat is currently in Phase 1 clinical trials in relapsed refractory acute leukemia patients and is administered as a continuous IV infusion (CIV). The studies herein investigated alternatives to CIV administration of pinometostat to improve patient convenience. Various sustained release technologies were considered, and based on the required dose size as well as practical considerations, subcutaneous (SC) bolus administration of a solution formulation was selected for further evaluation in preclinical studies. SC administration offered improved exposure and complete bioavailability of pinometostat relative to CIV and oral administration. These findings warranted further evaluation in rat xenograft models of MLL-r leukemia. SC dosing in xenograft models demonstrated inhibition of MLL-r tumor growth and inhibition of pharmacodynamic markers of DOT1L activity. However, a dosing frequency of thrice daily (t.i.d) was required in these studies to elicit optimal inhibition of DOT1L target genes and tumor growth inhibition. Development of an extended release formulation may prove useful in the further optimization of the SC delivery of pinometostat, moving towards a more convenient dosing paradigm for patients.
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http://dx.doi.org/10.1016/j.jconrel.2015.09.023DOI Listing
December 2015

Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor.

Xenobiotica 2016 ;46(3):268-77

1. Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent. 2. Structure elucidation via high resolution, accurate mass LC-MS(n) revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction. 3. The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes. 4. Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.
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http://dx.doi.org/10.3109/00498254.2015.1072253DOI Listing
October 2016

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

ACS Med Chem Lett 2015 Jun 6;6(6):655-9. Epub 2015 Apr 6.

Epizyme, Inc. , 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02138, United States.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468411PMC
June 2015

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity.

ACS Med Chem Lett 2015 May 4;6(5):491-5. Epub 2015 Mar 4.

Epizyme, Inc. , 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434464PMC
May 2015

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Nat Chem Biol 2015 Jun 27;11(6):432-7. Epub 2015 Apr 27.

Departments of Biology and Molecular Discovery, Epizyme, Inc., Cambridge, Massachusetts, USA.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.
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http://dx.doi.org/10.1038/nchembio.1810DOI Listing
June 2015

Structural and Kinetic Characterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652.

Drug Metab Dispos 2015 Jul 17;43(7):936-43. Epub 2015 Apr 17.

Epizyme, Cambridge, Massachusetts (N.R., L.H.M., S.R., M.P.M., R.A.C., R.C., N.J.W.); RMI Laboratories, North Wales, Pennsylvania (P.T.); and Cyprotex, Macclesfield, Cheshire, United Kingdom (K.P., J.S., K.F.)

Pharmacokinetic and metabolite identification studies were conducted to understand the clearance pathways of EPZ011652 [(2-aminoethyl)(methyl)({3-[4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl}methyl)amine], a potent protein arginine N-methyltransferase inhibitor. Metabolic clearance was the major pathway of EPZ011652 elimination in rats with structural elucidation of metabolites via liquid chromatography - mass spectrometry (LC-MS(n)) accurate mass measurement revealing the formation of a novel aliphatic N-acetylated metabolite (M1) located on the terminal nitrogen of the ethylene-diamine side chain. EPZ015564, a synthetic standard of the N-acetyl product, was prepared and was also generated by human and rat, but not dog hepatocytes. In rat hepatocytes, on incubation with EPZ011652, the concentration of EPZ015564 initially increased before decreasing with incubation time, suggesting that the metabolite is itself a substrate for other metabolizing enzymes, in agreement with the identification of metabolites M2, M3, and M4 in rat bile, all N-acetylated metabolites, undergoing sequential phase I (demethylation, oxidation) or phase II (sulfation) reactions. Reaction phenotyping with recombinant human N-acetyltransferase (NAT) isoforms revealed that both NAT1 and NAT2 are capable of acetylating EPZ011652, although with different catalytic efficiencies. Kinetic profiles of EPZ015564 formation followed classic Michaelis-Menten behavior with apparent Km values of >1000 μM for NAT1 and 165 ± 14.1 µM for NAT2. The in vitro intrinsic clearance for EPZ011652 by NAT2 (110 μL/min/mg) was 500-fold greater than by NAT1. In summary, we report the unusual N-acetylation of an aliphatic amine and discuss the implications for drug discovery and clinical development.
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http://dx.doi.org/10.1124/dmd.115.064014DOI Listing
July 2015

Evaluation of drug-drug interactions for oncology therapies: in vitro-in vivo extrapolation model-based risk assessment.

Authors:
Nigel J Waters

Br J Clin Pharmacol 2015 Jun;79(6):946-58

Epizyme, Inc., 400 Technology Square, Cambridge, MA, USA.

Aims: Understanding drug-drug interactions (DDI) is a critical part of the drug development process as polypharmacy has become commonplace in many therapeutic areas including the cancer patient population. The objectives of this study were to investigate cytochrome P450 (CYP)-mediated DDI profiles available for therapies used in the oncology setting and evaluate how models based on in vitro-in vivo extrapolation performed in predicting CYP-mediated DDI risk.

Methods: A dataset of 125 oncology therapies was collated using drug label and approval history information, incorporating in vitro and clinical PK data. The predictive accuracy of the basic and net effect mechanistic static models was assessed using this oncology drug dataset, for both victim and perpetrator potential of CYP3A-mediated DDI.

Results: The incidence of CYP3A-mediated interaction potential was 47%, 22% and 11% for substrates, inhibitors and inducers, respectively. The basic models for precipitants gave conservative predictions with no false negatives, whilst the mechanistic static models provided reasonable quantitative predictions (2.3-3-fold error). Further analysis revealed that incorporating DDI at the level of the intestine was in most cases over-predicting interaction magnitude due to overestimates of the rate and extent of oral absorption of the precipitant. Quantifying victim DDI potential was also demonstrated using fmCYP3A estimates from ketoconazole clinical DDI studies to predict the magnitude of interaction on co-administration with the CYP3A inducer, rifampicin (1.6-3.3 fold error).

Conclusions: This work illustrates the utility and limitations of current DDI risk assessment approaches applied to a range of contemporary anti-cancer agents, and discusses the implications for therapeutic combination strategies.
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http://dx.doi.org/10.1111/bcp.12563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456127PMC
June 2015

Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma.

Mol Cancer Ther 2014 Apr 21;13(4):842-54. Epub 2014 Feb 21.

Authors' Affiliations: Epizyme Inc., Cambridge; Eisai Inc., Andover, Massachusetts; and Eisai Co. Ltd., Tsukuba-shi, Ibaraki, Japan.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0773DOI Listing
April 2014

Consideration of the unbound drug concentration in enzyme kinetics.

Methods Mol Biol 2014 ;1113:119-45

Drug Metabolism and Pharmacokinetics, Epizyme Inc., Cambridge, MA, USA.

The study of enzyme kinetics in drug metabolism involves assessment of rates of metabolism and inhibitory potencies over a suitable concentration range. In all but the very simplest in vitro system, these drug concentrations can be influenced by a variety of nonspecific binding reservoirs that can reduce the available concentration to the enzyme system under investigation. As a consequence, the apparent kinetic parameters that are derived, such as K m or K i, can deviate from the true values. There are a number of sources of these nonspecific binding depots or barriers, including membrane permeation and partitioning, plasma or serum protein binding, and incubational binding. In the latter case, this includes binding to the assay apparatus, as well as biological depots, depending on the characteristics of the in vitro matrix being used. Given the wide array of subcellular, cellular, and recombinant enzyme systems utilized in drug metabolism, each of these has different components that can influence the free drug concentration. The physicochemical properties of the test compound are also paramount in determining the influential factors in any deviation between true and apparent kinetic behavior. This chapter describes the underlying mechanisms determining the free drug concentration in vitro and how these factors can be accounted for in drug metabolism studies, illustrated with case studies from the literature.
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http://dx.doi.org/10.1007/978-1-62703-758-7_7DOI Listing
September 2014

Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor.

Biopharm Drug Dispos 2014 May 14;35(4):237-52. Epub 2014 Feb 14.

Epizyme Inc., 400 Technology Square, Cambridge, MA, USA.

(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).
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http://dx.doi.org/10.1002/bdd.1889DOI Listing
May 2014

Structure-efficiency relationship of [1,2,4]triazol-3-ylamines as novel nicotinamide isosteres that inhibit tankyrases.

J Med Chem 2013 Sep 14;56(17):7049-59. Epub 2013 Aug 14.

Novartis Institutes for Biomedical Research Incorporated , 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

Tankyrases 1 and 2 are members of the poly(ADP-ribose) polymerase (PARP) family of enzymes that modulate Wnt pathway signaling. While amide- and lactam-based nicotinamide mimetics that inhibit tankyrase activity, such as XAV939, are well-known, herein we report the discovery and evaluation of a novel nicotinamide isostere that demonstrates selectivity over other PARP family members. We demonstrate the utilization of lipophilic efficiency-based structure-efficiency relationships (SER) to rapidly drive the evaluation of this series. These efforts led to a series of selective, cell-active compounds with solubility, physicochemical, and in vitro properties suitable for further optimization.
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http://dx.doi.org/10.1021/jm400826jDOI Listing
September 2013

Identification of NVP-TNKS656: the use of structure-efficiency relationships to generate a highly potent, selective, and orally active tankyrase inhibitor.

J Med Chem 2013 Aug 13;56(16):6495-511. Epub 2013 Aug 13.

Novartis Institutes for Biomedical Research, Inc., 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.
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http://dx.doi.org/10.1021/jm400807nDOI Listing
August 2013

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia.

Blood 2013 Aug 25;122(6):1017-25. Epub 2013 Jun 25.

Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139, USA.

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.
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http://dx.doi.org/10.1182/blood-2013-04-497644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739029PMC
August 2013

Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 2: clearance.

J Clin Pharmacol 2013 Feb 24;53(2):178-91. Epub 2013 Jan 24.

Metabolism and Pharmacokinetics Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

A comprehensive analysis on the prediction of human clearance based on intravenous pharmacokinetic data from rat, dog, and monkey for approximately 400 compounds was undertaken. This data set has been carefully compiled from literature reports and expanded with some in-house determinations for plasma protein binding and rat clearance. To the authors- knowledge, this is the largest publicly available data set. The present examination offers a comparison of 37 different methods for prediction of human clearance across compounds of diverse physicochemical properties. Furthermore, this work demonstrates the application of each prediction method to each charge class of the compounds, thus presenting an additional dimension to prediction of human pharmacokinetics. In general, the observations suggest that methods employing monkey clearance values and a method incorporating differences in plasma protein binding between rat and human yield the best overall predictions as suggested by approximately 60% compounds within 2-fold geometric mean-fold error. Other single-species scaling or proportionality methods incorporating the fraction unbound in the corresponding preclinical species for prediction of free clearance in human were generally unsuccessful.
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http://dx.doi.org/10.1177/0091270012440282DOI Listing
February 2013

Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 1: volume of distribution at steady state.

J Clin Pharmacol 2013 Feb 24;53(2):167-77. Epub 2013 Jan 24.

Metabolism and Pharmacokinetics Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

The authors present a comprehensive analysis on the estimation of volume of distribution at steady state (VD(ss) ) in human based on rat, dog, and monkey data on nearly 400 compounds for which there are also associated human data. This data set, to the authors- knowledge, is the largest publicly available, has been carefully compiled from literature reports, and was expanded with some in-house determinations such as plasma protein binding data. This work offers a good statistical basis for the evaluation of applicable prediction methods, their accuracy, and some methods-dependent diagnostic tools. The authors also grouped the compounds according to their charge classes and show the applicability of each method considered to each class, offering further insight into the probability of a successful prediction. Furthermore, they found that the use of fraction unbound in plasma, to obtain unbound volume of distribution, is generally detrimental to accuracy of several methods, and they discuss possible reasons. Overall, the approach using dog and monkey data in the íie-Tozer equation offers the highest probability of success, with an intrinsic diagnostic tool based on aberrant values (<0 or >1) for the calculated fraction unbound in tissue. Alternatively, methods based on dog data (single-species scaling) and rat and dog data (íie-Tozer equation with 2 species or multiple regression methods) may be considered reasonable approaches while not requiring data in nonhuman primates.
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http://dx.doi.org/10.1177/0091270012440281DOI Listing
February 2013

In silico prediction of total human plasma clearance.

J Chem Inf Model 2012 Aug 18;52(8):2069-78. Epub 2012 Jul 18.

Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusettes 02139, USA.

The prediction of the total human plasma clearance of novel chemical entities continues to be of paramount importance in drug design and optimization, because it impacts both dose size and dose regimen. Although many in vivo and in vitro methods have been proposed, a well-constructed, well-validated, and less resource-intensive computational tool would still be very useful in an iterative compound design cycle. A new completely in silico linear PLS (partial least-squares) model to predict the human plasma clearance was built on the basis of a large data set of 754 compounds using physicochemical descriptors and structural fragments, the latter able to better represent biotransformation processes. The model has been validated using the "ELASTICO" approach (Enhanced Leave Analog-Structural, Therapeutic, Ionization Class Out) based on ten therapeutic/structural analog classes. The model yields a geometric mean fold error (GMFE) of 2.1 and a percentage of compounds predicted within 2- and 3-fold error of 59% and 80%, respectively, showing an improved performance when compared with previous published works in predicting clearance of neutral compounds, and a very good performance with ionized molecules at pH 7.5, able to compare favorably with fairly accurate in vivo methods.
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http://dx.doi.org/10.1021/ci300155yDOI Listing
August 2012

Protein kinase C inhibitor sotrastaurin selectively inhibits the growth of CD79 mutant diffuse large B-cell lymphomas.

Cancer Res 2011 Apr 15;71(7):2643-53. Epub 2011 Feb 15.

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-2525DOI Listing
April 2011

The role of metabonomics at the interface between drug metabolism and safety assessment.

Authors:
Nigel J Waters

Curr Drug Metab 2010 Oct;11(8):686-92

Metabolism & Pharmacokinetics, Novartis Institutes for Biomedical Research, 250 Massachusetts Ave, Cambridge, MA 02139, USA.

Safety assessment of candidate drugs is a key stage in drug development and one which represents a significant attritional hurdle. There is also a focused effort in drug metabolism studies to assess bioactivation potential based on the notion this could lead to the risk of macromolecule adduct formation and subsequent toxicological consequences. However, characterization of the molecular mechanisms of drug toxicity still remains an enormous challenge. Recent advancements in 'omics sciences, and in particular metabonomics, has enabled some elucidation or insights into toxicological sequelae. Metabonomics is a global metabolic profiling framework which utilizes high resolution analytics (typically NMR and mass spectrometry) together with chemometric statistical tools (such as principal component analysis and partial least squares) to derive an integrated picture of both endogenous and xenobiotic metabolism. This review details some of the current progress in the application of metabonomics in drug safety and metabolism.
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http://dx.doi.org/10.2174/138920010794233512DOI Listing
October 2010