Publications by authors named "Shuguang Ma"

42 Publications

Absorption, metabolism and excretion of pictilisib, a potent pan-class I phosphatidylinositol-3-Kinase (PI3K) inhibitor, in rats, dogs, and humans.

Xenobiotica 2021 Jul 24;51(7):796-810. Epub 2021 May 24.

Clinical Pharmacology, Genentech Inc, South San Francisco, CA, USA.

The absorption, metabolism and excretion of pictilisib, a selective small molecule inhibitor of class 1 A phosphoinositide 3-kinase (PI3K), was characterized following a single oral administration of [C]pictilisib in rats, dogs and humans at the target doses of 30 mg/kg, 5 mg/kg and 60 mg, respectively.Pictilisib was rapidly absorbed with T less than 2 h across species. In systemic circulation, pictilisib represented the predominant total radioactivity greater than 86.6% in all species.Total pictilisib and related radioactivity was recovered from urine and faeces in rats, dogs, and human at 98%, 80% and 95%, respectively, with less than 2% excreted in urine and the rest excreted into faeces.In rat and dog, more than 40% of drug-related radioactivity was excreted into the bile suggesting biliary excretion was the major route of excretion. Unchanged pictilisib was a minor component in rat and dog bile. The major metabolite in bile was -glucuronide of oxidation on indazole moiety (M20, 21% of the dose) in rats and an oxidative piperazinyl ring-opened metabolite M7 (10.8% of the dose) in dogs.Oxidative glutathione (GSH) conjugates (M18, M19) were novel metabolites detected in rat bile, suggesting the potential generation of reactive intermediates from pictilisib. The structure of M18 was further confirmed by NMR to be a -hydroxylated and GSH conjugated metabolite on the moiety of the indazole ring.
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http://dx.doi.org/10.1080/00498254.2021.1923859DOI Listing
July 2021

Preclinical Characterization of the Distribution, Catabolism, and Elimination of a Polatuzumab Vedotin-Piiq (POLIVY) Antibody-Drug Conjugate in Sprague Dawley Rats.

J Clin Med 2021 Mar 23;10(6). Epub 2021 Mar 23.

Preclinical and Translational Pharmacokinetics and Pharmacodynamics, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Polatuzumab vedotin (or POLIVY), an antibody-drug conjugate (ADC) composed of a polatuzumab monoclonal antibody conjugated to monomethyl auristatin E (MMAE) via a cleavable dipeptide linker, has been approved by the United States Food and Drug Administration (FDA) for the treatment of diffuse large B-cell lymphoma (DLBCL). To support the clinical development of polatuzumab vedotin, we characterized the distribution, catabolism/metabolism, and elimination properties of polatuzumab vedotin and its unconjugated MMAE payload in Sprague Dawley rats. Several radiolabeled probes were developed to track the fate of different components of the ADC, with I and In used to label the antibody component and H to label the MMAE payload of the ADC. Following a single intravenous administration of the radiolabeled probes into normal or bile-duct cannulated rats, blood, various tissues, and excreta samples were collected over 7-14 days post-dose and analyzed for radioactivity and to characterize the metabolites/catabolites. The plasma radioactivity of polatuzumab vedotin showed a biphasic elimination profile similar to that of unconjugated polatuzumab but different from unconjugated radiolabeled MMAE, which had a fast clearance. The vast majority of the radiolabeled MMAE in plasma remained associated with antibodies, with a minor fraction as free MMAE and MMAE-containing catabolites. Similar to unconjugated mAb, polatuzumab vedotin showed a nonspecific distribution to multiple highly perfused organs, including the lungs, heart, liver, spleen, and kidneys, where the ADC underwent catabolism to release MMAE and other MMAE-containing catabolites. Both polatuzumab vedotin and unconjugated MMAE were mainly eliminated through the biliary fecal route (>90%) and a small fraction (<10%) was eliminated through renal excretion in the form of catabolites/metabolites, among which, MMAE was identified as the major species, along with several other minor species. These studies provided significant insight into ADC's absorption, distribution, metabolism, and elimination (ADME) properties, which supports the clinical development of POLIVY.
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http://dx.doi.org/10.3390/jcm10061323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004598PMC
March 2021

Characterization of Tissue Distribution, Catabolism, and Elimination of an Anti- THIOMAB Antibody-Antibiotic Conjugate in Rats.

Drug Metab Dispos 2020 11 24;48(11):1161-1168. Epub 2020 Aug 24.

Preclinical and Translational Pharmacokinetics and Pharmacodynamics (H.C., V.Y., A.V.K., B.-Q.S.), BioAnalytical Sciences (M.V.L., S.W., O.S.), Drug Metabolism and Pharmacokinetics (S.M., S.C.K.), and Safety Assessment (N.L.), Genentech Inc., South San Francisco, California

Invasive infection is a leading cause of infectious disease-related deaths because survives within host phagocytic cells, from which the bacteria are not adequately eliminated using current antibiotic treatments. Anti- THIOMAB antibody-antibiotic conjugate (TAC), an anti- antibody conjugated with antibiotic payload dmDNA31, was designed to deliver antibiotics into phagocytes, thereby killing intracellular Herein, we present the distribution, metabolism/catabolism, and elimination properties for this modality. The tissue distribution of TAC and the release and elimination of its payload dmDNA31 were characterized in rats using multiple approaches. Intravenous injection of unconjugated [C]dmDNA31 to rats resulted in a rapid clearance in both systemic circulation and tissues, with biliary secretion as the major route of elimination. Six major metabolites were identified. When [C]dmDNA31 was conjugated to an antibody as TAC and administered to rat intravenously, a sustained exposure was observed in both systemic circulation and tissues. The dmDNA31 in blood and tissues mainly remained in conjugated form after administering TAC, although minimal deconjugation of dmDNA31 from TAC was also observed. Several TAC catabolites were identified, which were mainly eliminated through the biliary-fecal route, with dmDNA31 and deacetylated dmDNA31 as the most abundant catabolites. In summary, these studies provide a comprehensive characterization of the distribution, metabolism/catabolism, and elimination properties of TAC. These data fully support further clinical development of TAC for the invasive and difficult-to-treat infection. SIGNIFICANCE STATEMENT: The present studies provide a comprehensive investigation of the absorption, distribution, metabolism/catabolism, and elimination of the first antibody-antibiotic conjugate developed for the treatment of an infectious disease. Although many antibody-drug conjugates are in development for various disease indications, only a limited amount of absorption, distribution, metabolism/catabolism, and elimination information is available in the literature. This study demonstrates the use of radiolabeling technology to delineate the absorption, distribution, metabolism/catabolism, and elimination properties of a complex modality and help address the key questions related to clinical pharmacological studies.
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http://dx.doi.org/10.1124/dmd.120.000092DOI Listing
November 2020

Strategies to Mitigate the Bioactivation of Aryl Amines.

Chem Res Toxicol 2020 07 23;33(7):1950-1959. Epub 2020 Jun 23.

Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States.

The bioactivation of xenobiotics to yield reactive metabolites can lead to tolerability and toxicity concerns within a drug discovery program. Development of strategies for mitigating the metabolic liability of commonly encountered toxicophores, such as anilines, relies on an understanding of the relative tendency of these functionalities to undergo bioactivation. In this report, we present the first systematic study of the structure-activity relationships of the bioactivation of aryl amine fragments (molecular weight < 250 Da) using a glutathione (GSH) trapping assay in the presence of human liver microsomes and the reduced form of nicotinamide adenine dinucleotide phosphate. This study demonstrates that conversion of anilines to nitrogen-containing heteroarylamines results in a lower abundance of GSH conjugates in the order phenyl > pyrimidine ≈ pyridine > pyridazine. Introduction of electron-withdrawing functionality on the aromatic ring had a less pronounced effect on the extent of GSH conjugation. Examination of more drug-like compounds sourced from in-house drug discovery programs revealed similar trends in bioactivation between matched pairs containing (hetero)aryl amines. This study provides medicinal chemists with insights and qualitative guidance for the minimization of risks related to aryl amine metabolism.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00138DOI Listing
July 2020

Novel Homodimer Metabolites of GDC-0994 via Cytochrome P450-Catalyzed Radical Coupling.

Drug Metab Dispos 2020 06 31;48(6):521-527. Epub 2020 Mar 31.

Drug Metabolism and Pharmacokinetics (R.H.T., S.B., D.Z., S.M.) and Discovery Chemistry (J.M.G., Y.L., P.B.), Genentech, Inc., South San Francisco, California

Two novel homodimer metabolites were identified in rat samples collected during the in vivo study of GDC-0994. In this study, we investigated the mechanism of the formation of these metabolites. We generated and isolated the dimer metabolites using a biomimetic oxidation system for NMR structure elucidation to identify a symmetric dimer formed via carbon-carbon bond between two pyrazoles and an asymmetric dimer formed via an aminopyrazole-nitrogen to pyrazole-carbon bond. In vitro experiments demonstrated formation of these dimers was catalyzed by cytochrome P450 enzymes (P450s) with CYP3A4/5 being the most efficient. Using density functional theory, we determined these metabolites share a mechanism of formation, initiated by an N-H hydrogen atom abstraction by the catalytically active iron-oxo of P450s. Molecular modeling studies also show these dimer metabolites fit in the CYP3A4 binding site in low energy conformations with minimal protein rearrangement. Collectively, the results of these experiments suggest that formation of these two homodimer metabolites is mediated by CYP3A, likely involving activation of two GDC-0994 molecules by a single P450 enzyme and proceeding through a radical coupling mechanism. SIGNIFICANCE STATEMENT: These studies identified structures and enzymology for two distinct homodimer metabolites and indicate a novel biotransformation reaction mediated by CYP3A. In it, two molecules may bind within the active site and combine through radical coupling. The mechanism of dimerization was elucidated using density functional theory computations and supported by molecular modeling.
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http://dx.doi.org/10.1124/dmd.119.090019DOI Listing
June 2020

Investigation of the absolute bioavailability and human mass balance of navoximod, a novel IDO1 inhibitor.

Br J Clin Pharmacol 2019 08 14;85(8):1751-1760. Epub 2019 Jun 14.

Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.

Aims: Navoximod (GDC-0919, NLG-919) is a small molecule inhibitor of indoleamine-2,3-dioxygenase 1 (IDO1), developed to treat the acquired immune tolerance associated with cancer. The primary objectives of this study were to assess navoximod's absolute bioavailability (aBA), determine the mass balance and routes of elimination of [ C]-navoximod, and characterize navoximod's metabolite profile.

Methods: A phase 1, open-label, two-part study was conducted in healthy volunteers. In Part 1 (aBA), subjects (n = 16) were randomized to receive oral (200 mg tablet) or intravenous (5 mg solution) navoximod in a crossover design with a 5-day washout. In Part 2 (mass balance), subjects (n = 8) were administered [ C]-navoximod (200 mg/600 μCi) as an oral solution.

Results: The aBA of navoximod was estimated to be 55.5%, with a geometric mean (%CV) plasma clearance and volume of distribution of 62.0 L/h (21.0%) and 1120 L (28.4%), respectively. Mean recovery of total radioactivity was 87.8%, with 80.4% detected in urine and the remainder (7.4%) in faeces. Navoximod was extensively metabolized, with unchanged navoximod representing 5.45% of the dose recovered in the urine and faeces. Glucuronidation was identified as the primary route of metabolism, with the major glucuronide metabolite, M28, accounting for 57.5% of the total drug-derived exposure and 59.7% of the administered dose recovered in urine.

Conclusions: Navoximod was well tolerated, quickly absorbed and showed moderate bioavailability, with minimal recovery of the dose as unchanged parent in the urine and faeces. Metabolism was identified as the primary route of clearance and navoximod glucuronide (M28) was the most abundant metabolite in circulation with all other metabolites accounting for <10% of drug-related exposure.
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http://dx.doi.org/10.1111/bcp.13961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624388PMC
August 2019

Unequal Absorption of Radiolabeled and Nonradiolabeled Drug from the Oral Dose Leads to Incorrect Estimates of Drug Absorption and Circulating Metabolites in a Mass Balance Study.

Drug Metab Lett 2019 ;13(1):37-44

Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, United States.

Background: Mass balance studies conducted using radiolabeled material (14C or 3H) definitively characterize the Absorption, Metabolism, and Excretion (AME) of a drug. A critical aspect of these studies is that the radiotracer maintains its proportion to total drug from its administration to its complete elimination from the body. In the study of GDC-0276 in beagle dogs, we observed that the 14C radiotracer proportion (specific activity) varied through the study.

Method: High resolution-accurate mass spectrometric measurements of 12C and 14C isotopes of GDC- 0276 and its metabolites in plasma and excreta samples were used to determine the apparent specific activities, which were higher than the specific activity of the dosing formulation. Drug concentrations were adjusted to the observed specific activities to correct the readouts for GDC-0276 AME and PK.

Results: The enrichment of 14C, which resulted in higher specific activities, was consistent with faster and more extensive absorption of the radiotracer from the dosing formulation. This resulted in overestimating the dose absorbed, the extent of elimination in urine and bile, and the exposures to circulating metabolites. These biases were corrected by the specific activities determined for study samples by mass spectrometry.

Conclusion: Assuming that the radiotracer was proportional to total drug throughout a radiolabeled study was not valid in a 14C study in beagle dogs. This presumably resulted from unequal absorption of the radiotracer and nonradiolabeled test articles from the oral dose due to inequivalent solid forms. We were able to provide a more accurate description of the AME of GDC-0276 in dogs by characterizing the differential absorption of the radiotracer.
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http://dx.doi.org/10.2174/1872312813666181129162237DOI Listing
January 2020

CYP1A1-Mediated Intramolecular Rearrangement of Aminoazepane in GDC-0339.

Drug Metab Dispos 2017 10 8;45(10):1084-1092. Epub 2017 Aug 8.

Departments of Drug Metabolism and Pharmacokinetics (R.H.T., J.W., J.H.C., S.C.K., S.M.), Discovery Chemistry (X.W.), and Small Molecule Pharmaceutical Sciences (N.L.S.), Genentech, Inc., South San Francisco, California.

GDC-0339 is a novel small molecule pan-Pim kinase inhibitor that was discovered as a potential treatment of multiple myeloma. During the in vitro and in vivo metabolite profiling of GDC-0339, a metabolite was detected that had the same elemental composition as the parent but was distinct with respect to its chromatographic separation and mass spectrometric fragmentation pattern. High resolution tandem mass spectrometry data indicated the metabolite was modified at the aminoazepane moiety. The structure was solved by nuclear magnetic resonance analysis of the isolated metabolite and further confirmed by comparing it to a synthetic standard. These results indicated that the metabolite was formed by an intramolecular amine replacement reaction with the primary amine forming a new attachment to pyrazole without any change in stereochemistry. In vitro experiments showed cytochrome P450s catalyzed the reaction and demonstrated high isoform selectivity by CYP1A1. Results from kinetic experiments showed that the CYP1A1-mediated rearrangement of GDC-0339 was an efficient reaction with apparent turnover number (k) and Michaelis constant (K) of 8.4 minutes and 0.6 M, respectively. The binding of GDC-0339 to the cytochrome P450 active site was examined by characterizing the direct inhibition of CYP1A1-mediated phenacetin -deethylation, and GDC-0339 was a potent competitive inhibitor with K of 0.9 M. This high affinity binding was unexpected given a narrow active site for CYP1A1 and GDC-0339 does not conform structurally to known CYP1A1 substrates, which are mostly polyaromatic planar molecules. Further, we explored some of the structural requirements for the rearrangement reaction and identified several analogs to GDC-0339 that undergo this biotransformation.
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http://dx.doi.org/10.1124/dmd.117.076786DOI Listing
October 2017

Mixed Matrix Method Provides A Reliable Metabolite Exposure Comparison for Assessment of Metabolites in Safety Testing (MIST).

Drug Metab Lett 2017 11;11(1):21-28

Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Background: The regulatory guidances on metabolites in safety testing (MIST) by US Food and Drug Administration (FDA) and International Conference on Harmonisation (ICH) describe the necessity to assess exposures of major circulating metabolites in humans at steady state relative to exposures achieved in nonclinical safety studies prior to the initiation of large scale clinical trials. This comparison can be accomplished by measuring metabolite concentrations in animals and humans with validated bioanalytical methods. However, bioanalysis of metabolites in multiple species and multiple studies is resource intensive and may impact the timelines of clinical studies.

Method: A simple, reliable and accurate method has been developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volume of human plasma with blank plasma of animal species and vice versa followed by an analysis using LC-SRM or LC-HRMS. Here, we explored the reliability and accuracy of this method in several development projects at Genentech and compared the results to those obtained from validated bioanalytical methods.

Results: The mixed-matrix method provided comparable accuracy (within ±20%) to those obtained from validated bioanalysis but does not require authentic standards or radiolabeled compounds, which could translate to time and resource savings in drug development.

Conclusion: Quantitative assessment of metabolite coverage in safety species can be made using mixed matrix method with similar accuracy and scientific rigor to those obtained from validated bioanalytical methods. Moving forward, we are encouraging the industry and regulators to consider accepting the mixed matrix method for assessing metabolite exposure comparisons between humans and animal species used in toxicology studies.
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http://dx.doi.org/10.2174/1872312811666170710193229DOI Listing
November 2017

Novel Mechanism of Decyanation of GDC-0425 by Cytochrome P450.

Drug Metab Dispos 2017 05 10;45(5):430-440. Epub 2017 Feb 10.

Departments of Drug Metabolism and Pharmacokinetics (R.H.T., J.S.H., Y.C., C.E.C.A.H., S.C.K., S.M.), and Discovery Chemistry (M.S.), Genentech, Inc., 1 DNA Way, South San Francisco, California.

GDC-0425 [5-((1-ethylpiperidin-4-yl)oxy)-9H-pyrrolo[2,3-b:5,4-c']dipyridine-6-carbonitrile] is an orally bioavailable small-molecule inhibitor of checkpoint kinase 1 that was investigated as a novel cotherapy to potentiate chemotherapeutic drugs, such as gemcitabine. In a radiolabeled absorption, distribution, metabolism, and excretion study in Sprague-Dawley rats, trace-level but long-lived C-labeled thiocyanate was observed in circulation. This thiocyanate originated from metabolic decyanation of GDC-0425 and rapid conversion of cyanide to thiocyanate. Excretion studies indicated decyanation was a minor metabolic pathway, but placing C at nitrile magnified its observation. Cytochrome P450s catalyzed the oxidative decyanation reaction in vitro when tested with liver microsomes, and in the presence of O, one atom of O was incorporated into the decyanated product. To translate this finding to a clinical risk assessment, the total circulating levels of thiocyanate (endogenous plus drug-derived) were measured following repeated administration of GDC-0425 to rats and cynomolgus monkeys. No overt increases were observed with thiocyanate concentrations of 121-154 M in rats and 71-110 M in monkeys receiving vehicle and all tested doses of GDC-0425. These findings were consistent with results from the radiolabel rat study where decyanation accounted for conversion of <1% of the administered GDC-0425 and contributed less than 1 M thiocyanate to systemic levels. Further, in vitro studies showed only trace oxidative decyanation for humans. These data indicated that, although cyanide was metabolically released from GDC-0425 and formed low levels of thiocyanate, this pathway was a minor route of metabolism, and GDC-0425-related increases in systemic thiocyanate were unlikely to pose safety concerns for subjects of clinical studies.
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http://dx.doi.org/10.1124/dmd.116.074336DOI Listing
May 2017

Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice.

ACS Med Chem Lett 2016 Oct 26;7(10):896-901. Epub 2016 Aug 26.

Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.

A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 μM lactate IC in a MiaPaca2 cell line. Optimization of the series led to , a potent cell active molecule (MiaPaca2 IC = 0.67 μM) that also possessed good exposure when dosed orally to mice.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066143PMC
October 2016

Non-cytochrome P450-mediated bioactivation and its toxicological relevance.

Drug Metab Rev 2016 11 20;48(4):473-501. Epub 2016 Sep 20.

b Department of Drug Metabolism & Pharmacokinetics, Genentech , South San Francisco , CA , USA.

The bioactivation of drugs is often associated with toxicological outcomes; however, for most cases, the causal relationship between bioactivation and toxicity is not well established despite extensive research that attempts to elucidate the mechanisms leading to the formation of chemically reactive species, presumably the initial step towards adverse reactions. Due to rapid advancement in the research of cytochrome P450s (CYPs) and the prevalence of CYP involvement in the metabolic clearance of pharmaceuticals, CYP-mediated bioactivation is widely investigated and reviewed, while non-CYP-mediated bioactivation has not been emphasized. The widespread use of metabolic stability screening in drug discovery, however, has led to the identification of new chemical entities that rely on non-CYP enzymes for clearance, and the number of drugs that undergo metabolism via these enzymes has increased. Non-CYP enzymes can be divided into four general categories according to their enzymatic function, namely, oxidative, reductive, conjugative and hydrolytic. The aim of this review is to complement the existing literature on CYP-mediated metabolism by focusing on bioactivation mediated non-CYP enzymes and provide representative examples in each category.
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http://dx.doi.org/10.1080/03602532.2016.1225756DOI Listing
November 2016

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition.

Nat Chem Biol 2016 10 1;12(10):779-86. Epub 2016 Aug 1.

Translational Oncology, Genentech, South San Francisco, California, USA.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
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http://dx.doi.org/10.1038/nchembio.2143DOI Listing
October 2016

Elucidating the Mechanism of Tofacitinib Oxidative Decyanation.

Drug Metab Lett 2016 ;10(2):136-43

Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way MS 412a, South San Francisco, CA 94080 USA.

Background: Tofacitinib is known to generate two metabolites M2 (alcohol) and M4 (acid), which are formed as the result of oxidation and loss of the nitrile [1].

Method: Systematic in vitro investigation into generation of M2 and M4 from tofacitinib.

Results: In vitro using human liver microsomes, we found a new geminal diol metabolite of tofacitinib (MX) that lost the nitrile. MX was further reduced or oxidized to M2 (alcohol) and M4 (acid), respectively by enzymes such as aldo-keto reductase 1C1, aldehyde oxidase and possibly CYP3A4. Stable label studies using H2 18O and D2O suggested the source of oxygen was from water in the media. This was due to rapid water exchange with MX in the media prior to reduction to M2. In case of deuterium, one was incorporated in M2 and this was mainly as a result of tofacitinib rapid exchange of two deuterium atoms from D2O onto methylene position. After formation of MX, there was one deuterium that no longer exchanged with water and therefore retained in M2 for further reduction.

Conclusion: The proposed mechanism involved the initial oxidation by P450 at the α-carbon to the nitrile group generating an unstable cyanohydrin intermediate; followed by the loss of the nitrile group to form a new geminal diol metabolite (MX).
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http://dx.doi.org/10.2174/1872312810666160427104954DOI Listing
October 2017

Absorption, metabolism and excretion of cobimetinib, an oral MEK inhibitor, in rats and dogs.

Xenobiotica 2017 Jan 8;47(1):50-65. Epub 2016 Apr 8.

a Department of Drug Metabolism and Pharmacokinetics , Genentech, Inc. , South San Francisco , CA , USA.

1. The absorption, metabolism and excretion of cobimetinib, an allosteric inhibitor of MEK1/2, was characterized in mass balance studies following single oral administration of radiolabeled (C) cobimetinib to Sprague-Dawley rats (30 mg/kg) and Beagle dogs (5 mg/kg). 2. The oral dose of cobimetinib was well absorbed (81% and 71% in rats and dogs, respectively). The maximal plasma concentrations for cobimetinib and total radioactivity were reached at 2-3 h post-dose. Drug-derived radioactivity was fully recovered (∼90% of the administered dose) with the majority eliminated in feces via biliary excretion (78% of the dose for rats and 65% for dogs). The recoveries were nearly complete after the first 48 h following dosing. 3. The metabolic profiles indicated extensive metabolism of cobimetinib prior to its elimination. For rats, the predominant metabolic pathway was hydroxylation at the aromatic core. Lower exposures for cobimetinib and total radioactivity were observed in male rats compared with female rats, which was consistent to in vitro higher clearance of cobimetinib for male rats. For dogs, sequential oxidative reactions occurred at the aliphatic portion of the molecule. Though rat metabolism was well-predicted in vitro with liver microsomes, dog metabolism was not. 4. Rats and dogs were exposed to the two major human circulating Phase II metabolites, which provided relevant metabolite safety assessment. In general, the extensive sequential oxidative metabolism in dogs, and not the aromatic hydroxylation in rats, was more indicative of the metabolism of cobimetinib in humans.
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http://dx.doi.org/10.3109/00498254.2016.1157645DOI Listing
January 2017

The use of stable isotope-labeled drug as microtracers with conventional LC-MS/MS to support human absolute bioavailability studies: are we there yet?

Bioanalysis 2016 Apr 23;8(8):731-3. Epub 2016 Mar 23.

Department of Drug Metabolism & Pharmacokinetics, Millennium Pharmaceuticals, Inc., A Wholly Owned Subsidiary of Takeda Pharmaceutical Company Limited, 40 Landsdowne Street, Cambridge, MA 02139, USA.

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http://dx.doi.org/10.4155/bio.16.25DOI Listing
April 2016

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans.

Drug Metab Dispos 2016 Jan 8;44(1):28-39. Epub 2015 Oct 8.

Departments of Drug Metabolism and Pharmacokinetics (R.H.T., E.F.C., S.M., S.W., J.H., Y.D., C.E.C.A.H., S.C.K.,), Product Development Oncology (I.R.), Early Clinical Development (M.G.), and Clinical Pharmacology (M.J.D, L.M.), Genentech, South San Francisco, California.

The pharmacokinetics, metabolism, and excretion of cobimetinib, a MEK inhibitor, were characterized in healthy male subjects (n = 6) following a single 20 mg (200 μCi) oral dose. Unchanged cobimetinib and M16 (glycine conjugate of hydrolyzed cobimetinib) were the major circulating species, accounting for 20.5% and 18.3% of the drug-related material in plasma up to 48 hours postdose, respectively. Other circulating metabolites were minor, accounting for less than 10% of drug-related material in plasma. The total recovery of the administered radioactivity was 94.3% (±1.6%, S.D.) with 76.5% (±2.3%) in feces and 17.8% (±2.5%) in urine. Metabolite profiling indicated that cobimetinib had been extensively metabolized with only 1.6% and 6.6% of the dose remaining as unchanged drug in urine and feces, respectively. In vitro phenotyping experiments indicated that CYP3A4 was predominantly responsible for metabolizing cobimetinib. From this study, we concluded that cobimetinib had been well absorbed (fraction absorbed, Fa = 0.88). Given this good absorption and the previously determined low hepatic clearance, the systemic exposures were lower than expected (bioavailability, F = 0.28). We hypothesized that intestinal metabolism had strongly attenuated the oral bioavailability of cobimetinib. Supporting this hypothesis, the fraction escaping gut wall elimination (Fg) was estimated to be 0.37 based on F and Fa from this study and the fraction escaping hepatic elimination (Fh) from the absolute bioavailability study (F = Fa × Fh × Fg). Physiologically based pharmacokinetics modeling also showed that intestinal clearance had to be included to adequately describe the oral profile. These collective data suggested that cobimetinib was well absorbed following oral administration and extensively metabolized with intestinal first-pass metabolism contributing to its disposition.
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http://dx.doi.org/10.1124/dmd.115.066282DOI Listing
January 2016

Elucidating the Mechanisms of Formation for Two Unusual Cytochrome P450-Mediated Fused Ring Metabolites of GDC-0623, a MAPK/ERK Kinase Inhibitor.

Drug Metab Dispos 2015 Dec 5;43(12):1929-33. Epub 2015 Oct 5.

Departments of Drug Metabolism and Pharmacokinetics (R.H.T., S.M., Q.Y., E.F.C., S.C.K.) and Small Molecule Pharmaceutical Sciences (S.J.R.), Genentech, Inc., South San Francisco, California.

Two isomeric metabolites of GDC-0623 [5-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide], a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) kinase inhibitor, were identified in radiolabeled mass balance studies in rats and dogs (approximately 5% in excreta) and were also observed in human circulation (nonradiolabeled). Mass spectrometric data indicated that both metabolites had formed a new ring structure fused to the imidazopyridine core. Given their unusual structures, we conducted experiments to elucidate their chemical structures and understand the mechanisms for their formation. For the first metabolite, M14, a pyrazol-3-ol ring was generated by N-N bond formation between the aniline and hydroxamate. For the second metabolite, M13, an imidazol-2-one was generated by a Hofmann-type rearrangement that involved C-C bond cleavage and C-N bond formation. Both reactions were catalyzed by CYP2C9 and CYP2C19. M14 was generated directly from GDC-0623 and we speculate that its formation was via oxidative activation of the hydroxamic ester by cytochrome P450 (P450) and intramolecular nucleophilic displacement of the ester side chain. M13 (the rearranged metabolite) formed from the N-reduced hydroxamate (amide) and not from GDC-0623 directly. We propose for M13 that a P450-mediated reaction formed a cationic amide intermediate, which enabled the molecular rearrangement of the imidazopyridine core migrating from the amide carbon to the nitrogen and subsequent cyclization reaction. Each of these metabolic pathways constitutes a novel biotransformation mediated by P450 enzymes.
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http://dx.doi.org/10.1124/dmd.115.067181DOI Listing
December 2015

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase.

Bioorg Med Chem Lett 2015 Jan 10;25(1):75-82. Epub 2014 Nov 10.

Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (4–10 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.
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http://dx.doi.org/10.1016/j.bmcl.2014.11.008DOI Listing
January 2015

Measuring NAD(+) levels in mouse blood and tissue samples via a surrogate matrix approach using LC-MS/MS.

Bioanalysis 2014 Jun;6(11):1445-57

Drug Metabolism & Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, USA.

Background: NAD(+) is an endogenous analyte and is unstable during blood sample collection, both of which present obstacles for quantitation. Moreover, current procedures for NAD(+) sample collection require onsite treatment with strong acid to stabilize the NAD(+) in mouse blood cells.

Results: NAD(+) can be stabilized by addition of acid before the frozen mouse blood sample was thawed. A simple sample collection procedure was proposed to facilitate the analysis of NAD(+) in mouse blood and tissue samples. A LC-MS/MS method was developed for quantifying NAD(+) in mouse blood and various tissue samples. The described method was used to measure endogenous NAD(+) levels in mouse blood following oral administration of the nicotinamide phosphoribosyltransferase inhibitor GNE-617.

Conclusion: This study presents a suitable assay and sample collection procedure for high throughput screening of NAD(+) samples in preclinical discovery studies.
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http://dx.doi.org/10.4155/bio.14.8DOI Listing
June 2014

Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase.

Bioorg Med Chem Lett 2014 Aug 1;24(16):3764-71. Epub 2014 Jul 1.

WuXi AppTec Co., Ltd, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, PR China.

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).
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http://dx.doi.org/10.1016/j.bmcl.2014.06.076DOI Listing
August 2014

Dose-dependent exposure and metabolism of GNE-892, a β-secretase inhibitor, in monkeys: contributions by P450, AO, and P-gp.

Eur J Drug Metab Pharmacokinet 2015 Jun 3;40(2):171-85. Epub 2014 Apr 3.

Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA,

(R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of β-secretase 1 (β-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study. With cryopreserved monkey hepatocytes, concentration-dependency for substrate turnover and the relative contribution of P450- versus AO-mediated metabolism were observed. Characterization of the kinetics of these metabolic pathways demonstrated differences in the affinities of P450 and AO for GNE-892, which supported the metabolic profiles that had been obtained. To test if this metabolic shift occurred in vivo, mass balance studies in monkeys were conducted at doses of 0.085 and 15 mg/kg. Plasma exposure of GNE-892 following oral administration was more than 20-fold greater than dose proportional at the high-dose. P-gp-mediated efflux was unable to explain the discrepancy. The profiles of metabolites in circulation and excreta were indicative that oxidative metabolism limited the exposure to unchanged GNE-892 at the low dose. Further, the in vivo data supported the concentration-dependent metabolic shift between P450 and AO. In conclusion, microdosing of GNE-892 was not predictive of pharmacokinetics at a more pharmacologically relevant dose due to saturable absorption and metabolism. Therefore, it is important to consider ADME liabilities and their potential concentration-dependency when deciding upon a clinical microdosing strategy.
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http://dx.doi.org/10.1007/s13318-014-0198-5DOI Listing
June 2015

A tiered approach to address regulatory drug metabolite-related issues in drug development.

Bioanalysis 2014 Mar;6(5):587-90

Department of Drug Metabolism & Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

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http://dx.doi.org/10.4155/bio.14.40DOI Listing
March 2014

Elucidating the mechanism of cytochrome P450-mediated pyrimidine ring conversion to pyrazole metabolites with the BACE1 inhibitor GNE-892 in rats.

Drug Metab Dispos 2014 May 4;42(5):890-8. Epub 2014 Mar 4.

Departments of Drug Metabolism and Pharmacokinetics (R.T., S.M., Q.Y., C.E.H., X.L., S.C.K), Small Molecule Pharmaceutical Sciences (A.D.), and Discovery Chemistry (M.S.), Genentech, Inc., South San Francisco, California; XenoBiotic Laboratories, Inc., Plainsboro, New Jersey (H.K., Y.Y.); and Array BioPharma, Boulder, Colorado (K.W.H., N.C.K.).

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one), a β-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of cytochrome P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation, followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. After hydrolysis of the formamide, a carbon is lost as formic acid, together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.
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http://dx.doi.org/10.1124/dmd.114.057141DOI Listing
May 2014

Investigations into the mechanisms of pyridine ring cleavage in vismodegib.

Drug Metab Dispos 2014 Mar 3;42(3):343-51. Epub 2014 Jan 3.

Department of Drug Metabolism and Pharmacokinetics (S.C.K., Q.Y., S.M., J.Z.C., T.M., R.T., J.L., C.E.C.A., H.W.), Department of Discovery Chemistry (G.C., J.L.), gRED Non Clinical Operations (K.M.), Small Molecule Pharmaceutical Sciences (W.J.), and Small Molecule Clinical Pharmacology (L.L.), Genentech Inc., South San Francisco, California.

Vismodegib (Erivedge, GDC-0449) is a first-in-class, orally administered small-molecule Hedgehog pathway inhibitor that is approved for the treatment of advanced basal cell carcinoma. Previously, we reported results from preclinical and clinical radiolabeled mass balance studies in which we determined that metabolism is the main route of vismodegib elimination. The metabolites of vismodegib are primarily the result of oxidation followed by glucuronidation. The focus of the current work is to probe the mechanisms of formation of three pyridine ring-cleaved metabolites of vismodegib, mainly M9, M13, and M18, using in vitro, ex vivo liver perfusion and in vivo rat studies. The use of stable-labeled ((13)C2,(15)N)vismodegib on the pyridine ring exhibited that the loss of carbon observed in both M9 and M13 was from the C-6 position of pyridine. Interestingly, the source of the nitrogen atom in the amide of M9 was from the pyridine. Evidence for the formation of aldehyde intermediates was observed using trapping agents as well as (18)O-water. Finally, we conclude that cytochrome P450 is involved in the formation of M9, M13, and M18 and that M3 (the major mono-oxidative metabolite) is not the precursor for the formation of these cleaved products; rather, M18 is the primary cleaved metabolite.
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http://dx.doi.org/10.1124/dmd.113.055715DOI Listing
March 2014

Data acquisition and data mining techniques for metabolite identification using LC coupled to high-resolution MS.

Bioanalysis 2013 May;5(10):1285-97

Department of Drug Metabolism & Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Metabolite identification plays a pivotal role through all stages of drug discovery and development. The task of detecting and characterizing drug metabolites in complex biological matrices is very challenging, due in part to the co-existence of drug-related material with a large excess of endogenous material. Deciphering information on drug metabolites in these complex biological systems requires not only sophisticated LC-MS systems, but also software that can help differentiate drug-related compounds from endogenous material in the MS data. Fortunately, there have been considerable advances in high-resolution MS technologies with improved mass accuracy. The high resolution and mass accuracy capabilities have necessitated and augmented the development of integrated data acquisition methods, which have significantly facilitated metabolite detection and identification. In this review, we discuss various data-dependent and -independent acquisition methods in combination with accurate mass-based data mining tools for metabolite identification in drug discovery and development.
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http://dx.doi.org/10.4155/bio.13.103DOI Listing
May 2013

Application of LC-high-resolution MS with 'intelligent' data mining tools for screening reactive drug metabolites.

Bioanalysis 2012 Mar;4(5):501-10

Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, CA, USA.

Biotransformation of chemically stable compounds to reactive metabolites that can bind covalently to macromolecules (such as proteins and DNA) is considered an undesirable property of drug candidates. Due to the possible link, which has not yet been conclusively demonstrated, between reactive metabolites and adverse drug reactions, screening for metabolic activation of lead compounds through in vitro chemical trapping experiments has become an integral part of the drug discovery process in many laboratories. In this review, we provide an overview of the recent advances in the application of high-resolution MS. These advances facilitated the development of accurate-mass-based data mining tools for high-throughput screening of reactive drug metabolites in drug discovery.
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http://dx.doi.org/10.4155/bio.12.5DOI Listing
March 2012

Analytical strategies for assessment of human metabolites in preclinical safety testing.

Anal Chem 2011 Jul 20;83(13):5028-36. Epub 2011 Apr 20.

Merck Research Laboratories, Kenilworth, NJ 07033, USA.

Strategies for the detection, identification, and quantification of in vivo drug metabolites from non-radiolabeled studies are reviewed with an emphasis on the utilization of accurate-mass-based data mining tools. New approaches to the determination of coverage of human drug metabolites in preclinical species without using radiolabeled drugs or synthetic standards are also discussed.
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http://dx.doi.org/10.1021/ac200349gDOI Listing
July 2011

Determination of exposure multiples of human metabolites for MIST assessment in preclinical safety species without using reference standards or radiolabeled compounds.

Chem Res Toxicol 2010 Dec;23(12):1871-3

Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA.

A simple, reliable, and accurate method was developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volumes of human plasma with blank plasma of animal species and vice versa followed by an analysis using high-resolution full-scan accurate mass spectrometry. This approach provided comparable results (within (±15%) to those obtained from regulated bioanalysis and did not require synthetic standards or radiolabeled compounds. In addition, both qualitative and quantitative data were obtained from a single LC-MS analysis on all metabolites and, therefore, the coverage of any metabolite of interest can be obtained.
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http://dx.doi.org/10.1021/tx100363kDOI Listing
December 2010

Side population of a murine mantle cell lymphoma model contains tumour-initiating cells responsible for lymphoma maintenance and dissemination.

J Cell Mol Med 2010 Jun 28;14(6B):1532-45. Epub 2009 Jul 28.

Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

'Cancer stem cells' or 'tumour initiating cells' in B-cell non-Hodgkin lymphomas have not been demonstrated, although some studies focused on other cancer types suggest that such populations exist and represent tumour cells resistant to therapy and involved in relapse. These cells may also represent a putative neoplastic 'cell of origin' in lymphomas, but there is little substantive data to support this suggestion. Using cell lines derived from a recently established murine IL-14alphax c-Myc double transgenic/mantle cell lymphoma-blastoid variant model, heretofore referred to as DTG cell lines, we identified a subset of cells within the side population (SP) with features of 'tumour-initiating cells'. These features include higher expression of ABCG2 and BCL-2, longer telomere length, greater self-renewal ability and higher in vitro clonogenic and in vivo tumorigenic capacities compared with non-SP. In addition, in vitro viability studies demonstrated that the non-SP lymphoma subpopulation has a limited lifespan in comparison with the SP fraction. Syngenic transplant studies showed that non-SP derived tumours, in comparison to the SP-derived tumours, exhibit greater necrosis/apoptosis and less systemic dissemination capability. In conclusion, our data support the interpretation that the DTG SP fraction contains a cell population highly capable of tumour maintenance and systemic dissemination and lends support to the concept that 'tumour-initiating cells' occur in lymphomas.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00865.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829019PMC
June 2010
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