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Purpose:Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2–selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL–mediated thrombocytopenia.Experimental Design:We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time.Results:We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2– and Bcl-xL–dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2–selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL–dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models.Conclusions:AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

Figure S6

contains methods.

A physiologically based pharmacokinetic model was developed to describe the tissue distribution kinetics of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) in plasma, liver, spleen, and tumors. Tumor growth data from MV-4-11 tumor-bearing mice were incorporated to investigate the exposure/efficacy relationship. The nanoparticle demonstrated improved antitumor activity compared to the conventional API formulation, owing to the extended released API concentrations at the site of action. Model simulations further enabled the identification of critical parameters that influence API exposure in tumors and downstream efficacy outcomes upon nanoparticle administration. The model was utilized to explore a range of dosing schedules and their effect on tumor growth kinetics, demonstrating the improved antitumor activity of nanoparticles with less frequent dosing compared to the same dose of naked APIs in conventional formulations.

It is common practice to use cannulated rats for pharmacokinetic (PK) in-life studies as it yields high quality PK parameter estimation. While offering many benefits, cannulation requires surgery, post-surgical care, and cannula maintenance. As an alternative approach, the strategy of dosing and bleeding rats via the tail vein in a single experiment is technically feasible and theoretically offers many benefits. Unfortunately, however, as reported by F Tse et al. in 1984 (J Pharm Sci 73: https://doi.org/10.1002/jps.2600731128), parallel tail dosing and bleeding is scientifically flawed and yields inaccurate estimation of PK parameters following intravenous administration. The underlying causality of poor data quality has not been addressed in over 35 years. To overcome the technical flaws associated with parallel tail dosing and bleeding, we have developed a Tail-Dose-Bleed (TDB) method as a substitute for use of cannulated rats. Specifically, the method introduces a flush procedure after dosing, uses separate tail veins for dosing and bleeding, and adjusts dosing and sampling to the proximal and distal portions of the tail, respectively. To demonstrate the proof of principle for this TDB technique, several cassette dosing studies were conducted. The performance of the TDB technique is compared in both stand alone and animal crossover studies employing conventional jugular/femoral bleeding and dosing. The poor data via tail dosing and bleeding previously described by Tse et al. are also recapitulated using their described approach. To ensure broad applicability of the TDB technique, data were generated utilizing compounds of diverse physical chemical properties manifesting a range of clearance and/or volume of distribution characteristics. These data demonstrate that the TDB approach yields comparable PK profiles and parameters as compared to conventional femoral dosing / jugular bleeding. Using this newly described TDB procedure, we demonstrate the ability to overcome documented data quality issues when dosing and bleeding via the tail. The TDB technique has numerous operational advantages of reduced study turnaround time and improved cost effectiveness, but most importantly, addresses key animal welfare concerns relevant to institutional animal care and use committees (IACUC). The notable advantage here is reduced animal stress and discomfort by eliminating the need for surgery and recovery. And by consequence, allows for animals to be group housed and re-used without concern for loss of cannula patency. The tail dose and bleed method is simple and appears readily transferable to other laboratories.

Purpose: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2–selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL–mediated thrombocytopenia. Experimental Design: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. Results: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2– and Bcl-xL–dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2–selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL–dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. Conclusions: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

[Image: see text] Herein we describe our efforts using a late stage functionalization together with more traditional synthetic approaches to generate fluorinated analogues of the clinical candidate AZD9833. The effects of the addition of fluorine on the lipophilicity, permeability, and metabolism are discussed. Many of these changes were tolerated in terms of pharmacology and resulted in high quality molecules which reached advanced stages of profiling in the testing cascade.

Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019 μM–37 μM. The 15 min derivatization incubation time and 1 min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.

Herein, we report the identification and synthesis of a series of tricyclic indazoles as a novel class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to the removal of a reactive metabolite signal in an in vitro glutathione trapping assay. Further optimization, guided by X-ray crystal structures and NMR conformational work, varied the alkyl side chain and pendant aryl group and resulted in compounds with low turnover in human hepatocytes and enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms of pharmacology and demonstrated the desired estrogen receptor α degrader-antagonist profile and demonstrated activity in a xenograft model of breast cancer.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development., Claire Patterson et al. present the design and development of AZD0466, a drug-dendrimer conjugate, and use preclinical and mathematical models to determine the optimal release rate of the drug from the dendrimer carrier for maximal therapeutic index in terms of anti-tumour efficacy and cardiovascular tolerability. This study identifies this promising dual Bcl-2/Bcl-xL inhibitor for progression to clinical development.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xWe used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xWe discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xAZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-x

Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683)., High expression of Mcl-1 promotes tumorigenesis and resistance to anticancer therapies. Here they report a macrocyclic molecule with high selectivity and affinity for Mcl-1 that exhibits potent anti-tumor effects as single agent and in combination with bortezomib or venetoclax in preclinical models of multiple myeloma and acute myeloid leukemia.

Efficacies of ceftazidime-avibactam (4:1 w/w) and ceftazidime were tested against ceftazidime-susceptible (blaKPC-2-negative), and meropenem- and ceftazidime-resistant (blaKPC-2-positive), Klebsiella pneumoniae in a 52-h, multiple dose, abdominal abscess model in the rat. Efficacies corresponded to minimum inhibitory concentrations (MICs) measured in vitro and were consistent with drug exposures modelled from pharmacokinetics in infected animals. The ceftazidime, ceftazidime-avibactam and meropenem control treatments were effective in the rat abscess model against the susceptible strain, whereas only ceftazidime-avibactam was effective against K. pneumoniae harbouring blaKPC-2.

Phosphorylation of xenobiotics is rare, probably owing to a strong evolutionary pressure against it. This rarity may have attracted more attention recently as a result of intentionally designed kinase-substrate analogs that depend on kinase-catalyzed activation to form phosphorylated active drugs. We report a rare phosphorylated metabolite observed unexpectedly in mouse plasma samples after an oral dose of a Tankyrase inhibitor that was not intended to be a kinase substrate, i.e., (S)-2-(4-(6-(3,4-dimethylpiperazin-1-yl)-4-methylpyridin-3-yl)phenyl)-8-(hydroxymethyl)quinazolin-4(3H)-one (AZ2381). The phosphorylated metabolite was not generated in mouse hepatocytes. In vitro experiments showed that the phosphorylation of AZ2381 occurred in mouse whole blood with heparin as anticoagulant but not in mouse plasma. The phosphorylated metabolite was also produced in rat, dog, and human blood, albeit at lower yields than in mouse. Divalent metal ions are required for the phosphorylation since the reaction is inhibited by the metal chelator EDTA. Further investigations with different cellular fractions of mouse blood revealed that the phosphorylation of AZ2381 was mediated by erythrocytes but did not occur with leukocytes. The levels of 18O incorporation into the phosphorylated metabolite when inorganic 18O4-phosphate and γ-18O4-ATP were added to the mouse blood incubations separately suggested that the phosphoryl transfer was from inorganic phosphate rather than ATP. It remains unclear which enzyme present in red blood cells is responsible for this rare phosphorylation.

Background: Combination therapies have gained momentum in the disease management strategies of various indications. While it is challenging and more time consuming to develop a combined analytical method, the strategy of simultaneous analysis offers significant advantages in terms of efficiency and cost–effectiveness. Results: Due to a significant difference in efficacious dose for ceftazidime and avibactam, the calibration ranges validated in this paper were set to 0.05–50 μg/ml for ceftazidime and 0.005–5.0 μg/ml for avibactam. Interday results of ceftazidime were within 8% for accuracy and 9% for precision and within 9% for both accuracy and precision of avibactam. Conclusion: A sensitive and selective LC–MS/MS method was developed for the simultaneous quantification of ceftazidime and avibactam in rat plasma.

The tumor microenvironment (TME) has multiple mechanisms of immune-suppression, one being recruitment of arginase (ARG) expressing myeloid cells. ARG is an enzyme that catalyzes hydrolysis of L-arginine into urea and L-ornithine. L-arginine is a semi-essential amino acid required for optimal function of T cells and natural killer (NK) cells. Arginine depletion in the TME inhibits T cell and NK cell function. Therefore, inhibition of ARG can reverse immune-suppression and optimize anti-tumor immunity. To determine the dependence to arginine, we cultured human T- and NK cells in arginine concentrations ranging from 0 to 150 µM. Both human CD8+ T cells and NK cells showed decreased proliferation with decreased L-arginine concentration, and a complete proliferation arrest in the absence of L-arginine. L-arginine threshold for optimal proliferation was determined to be ~30 µM for CD8+ T cells and ~ 9 µM for NK cells. Furthermore, both CD8+ T cells and NK cells showed decreases in cytotoxic molecules (e.g. granzyme A, granzyme B, perforin) when cultured under reduced arginine conditions. In addition, NK cells showed a decreased ability to kill target cells in low arginine conditions. We next explored whether arginase inhibition could reverse the immune-suppressive environment in vivo. Administration of arginase inhibitor to tumor bearing mice resulted in a dose dependent increase in both plasma (up to 4 fold) and tumor arginine (up to 12 fold) in all models tested including MC38-ova, CT26, and Lewis Lung. In addition, ARG inhibitor showed signs of in vivo immune cell activation including ~doubling of the number of CD8+ T cells, NK cells, and CD103+ dendritic cells in the tumor microenvironment. Combining with anti PD-L1 further increased the number of CD8+ T cells to ~4-fold of control levels. Arginase inhibitor also increased the activation state of CD8 T cell as determined by percent of granzyme, IFNg, and Ki67 positivity. Moreover, ARG inhibitor resulted in an increase of IFNg and TNFa producing CD8+ T cells in tumor draining lymph nodes. Treatment of tumor bearing mice with ARG inhibitor as monotherapy resulted in modest, but consistent, tumor growth inhibition (TGI) of ~30% in multiple syngeneic models (LL, MC38-ova, CT26). Combining ARG inhibitor with anti-PDL1 significantly improved efficacy reaching ~87% TGI in the MCA38-ova model, with 7/10 mice showing tumor regression. In summary, our pre-clinical data demonstrates that an ARG inhibitor in combination with a checkpoint inhibitor can increase plasma and tumor arginine levels and reverse tumor immune-suppression leading to strong immune activation and anti-tumor responses, suggesting arginase inhibitors could provide an opportunity to increase activity of checkpoint inhibitors clinically. Citation Format: Alwin Schuller, Aatman Doshi, Susan Cantin, Michael Secinaro, Yanjun Wang, Laura Prickett, Sharon Tentarelli, Eric Gangl, Horma Ghadially, Kristina Ilieva, Theresa Proia, Scott Mlynarski, Ray Finlay, Wenlin Shao. Inhibition of arginase in combination with anti-PDL1 leads to increased infiltration and activation of CD8+ T cells, NK cells, and CD103+ dendritic cells in mouse syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4523.

The estrogen receptor alpha (ERα) is expressed in >70% of breast cancers and is a clinically validated target in oncology. Anti-hormonal therapies that block ER function directly (e.g. tamoxifen) or therapies that block the production of estrogen itself (e.g., aromatase inhibitors) have proven to be effective treatments of the disease. Further advances in disease control have been made with the development of the Selective Estrogen Receptor Degrader (SERD) fulvestrant, which both antagonizes ERα-driven tumor cell growth and causes degradation of the ERα receptor. The first-generation oral SERDs, including AZD9496, showed full ER degradation in MCF-7 cells but incomplete ER degradation in other cell lines, and partial agonism. Building on that learning, extensive structure-enabled chemical optimization has resulted in the identification of a next generation oral SERD AZD9833, a potent degrader and antagonist of the ERα receptor (both EC50 Citation Format: James S. Scott, Thomas Moss, Steve Stokes, Willem M. Nissink, Christopher J. Morrow, Mandy Lawson, Natalie Cureton, Eric Gangl, Pablo Morentin Gutierrez, Richard Mather, Justin P. Lindemann, Andrew Sykes, David Fisher, Radoslaw Polanski, Danielle Carroll, Aisling Barton Twomey, Teresa Klinowska. Discovery of AZD9833, an oral small molecule selective degrader of the estrogen receptor (SERD) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5674.

AZD9833 is a potent, orally delivered, non-steroidal selective estrogen receptor degrader (SERD) that both antagonizes and degrades ERα. It is currently in clinical testing for the treatment of ER+ metastatic breast cancer (SERENA-1; NCT03616587). While ER is a clinically validated target, sustained inhibition of the target via oral delivery has proven an elusive goal. Despite intensive research, no oral SERD options are currently approved. Poor pharmacokinetic (PK) properties and/or short half-life (t ½) are often underlying features that limit oral SERD candidates from once or twice daily dose regimens. AZD9833 is a clinical stage next generation oral SERD that may overcome these challenges. The pre-clinical PK properties of AZD9833 are consistent with once daily oral dosing in patients and are reported herein. AZD9833 is a low molecular weight ( Citation Format: Eric T. Gangl, Roshini Markandu, Pradeep Sharma, Andy Sykes, Petar Pop-Damkov, Pablo Morentin Gutierrez, James S. Scott, Dermot F. McGinnity, Adrian J. Fretland, Teresa Klinowska. Preclinical pharmacokinetic and metabolic characterization of the next generation oral SERD AZD9833 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1042.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many hematological and solid tumors, but their clinical application has been limited by tolerability issues, including thrombocytopenia. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, showed good efficacy but encountered dose limiting cardiovascular toxicity in preclinical species, and had challenging physicochemical properties which prevented its clinical development. Nanocarriers can provide prolonged circulation time, controlled release, tumor accumulation and retention. Consequently, they have been explored to improve the therapeutic index of small molecules in oncology. This work describes the design and development of AZD0466, a novel drug-dendrimer conjugate, where AZD4320 is chemically conjugated to Starpharma's DEP® dendrimer platform, a 5-generation PEGylated poly-lysine dendrimer via a hydrolytically labile linker. Release of AZD4320 is through hydrolytic cleavage of the linker, which is characterized by a “release half-life”, defined as the time to release 50% of the active moiety. This release half-life can be modified through linker design. Initially, three drug-dendrimer conjugates with a range of AZD4320 release half-lives (from 1.7 h to 217 h) were synthesized and efficacy was investigated in C.B-17 SCID mice bearing RS4;11 tumors while cardiovascular parameters and tolerance were assessed in a telemetered rat model. A mathematical model was developed and used to optimize the desired release rate of the active moiety, AZD4320, from the dendrimer conjugate for maximal therapeutic index in terms of preclinical anti-tumor efficacy and cardiovascular profile. Based on this modeling, AZD0466, with a release half-life of 25.5 h, was synthesized and selected for further in vivo efficacy and tolerability assessment. Efficacy studies in the RS4;11 xenograft model showed similar efficacy to AZD4320, while cardiovascular studies in rat and dog demonstrated that AZD0466 was tolerated at doses of active-moiety that are more than ten-fold higher. In addition, it can be easily formulated for intravenous administration due to significantly enhanced solubility. The AZD4320-dendrimer conjugate, AZD0466, identified in this study has resulted in an improved therapeutic index and enabled progression of this promising Bcl-2/Bcl-xL inhibitor into clinical development. Citation Format: Marianne B. Ashford, Srividya B. Balachander, Lorraine Graham, Iain Grant, Francis D. Gibbons, Kathryn J. Hill, Alexander R. Harmer, Sonya Gales, Sean Redmond, Brian Kelly, William McCoull, Shenghua Wen, Martin Wild, Eric Gangl, David J. Owen, Barry R. Davies. Design and optimization of a dendrimer-conjugated dual Bcl-2/Bcl-xL inhibitor, AZD0466, with improved therapeutic index [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1718.

Objectives: The estrogen receptor alpha (ERα) is highly expressed in breast cancers and is a clinically validated target in oncology. AZD9833 is a novel potent oral selective estrogen receptor degrader (SERD) currently being tested in the clinic [SERENA-1 (NCT03616587)]. We present here the preclinical PK/PD modeling work used to understand the required target modulation and concentration required to see anti-tumor efficacy and therefore help support the dose selection during the early clinical development of AZD9833. Methods: We developed a novel mechanistic mathematical model applied to mouse in both a xenograft ESR1 wild-type (MCF-7) and a PDX model harboring an ESR1 D538G mutation (CTC-174) linking the compound pharmacokinetics with the magnitude of target modulation expressed as relative levels of estrogen (ER) or progesterone receptor (PR) both measured by Western Blotting. These changes at the biomarker level were subsequently linked to inhibition of tumor cell proliferation, via a Hill-Langmuir relationship resulting in macroscopic dynamic effects on tumor size. All model parameters were derived from internal studies; some were estimated using Non-Linear Mixed Effect modeling of individual longitudinal PK, PD biomarker and tumor size data taken from several studies. Results: The model described well the relationship between plasma concentration of AZD9833 and modulation of PR (MCF-7) and ER (CTC-174). In vivo AZD9833 concentration required to see 50% of the maximal target modulation in both animal models (1.6 and 0.4 nM respectively) was in very good agreement with in vitro values when accounting for the different levels of estradiol in both animal models. Population tumor size patterns, for all treatment regimens ranging from 0 to 100% Tumor Growth Inhibition (TGI) across both tumor models were very well described with the model proposed. 75% reduction of ER levels and 45% reduction of PR levels were required to slow the tumor growth rate by one half in the CTC-174 and MCF-7 models respectively. This quantitative relationship was further validated with another SERD (Fulvestrant) in both animal models. Conclusions: This study provides quantitative mechanistic insights into the links between key biomarker modulation (ER and PR) and anti-tumor responses, supporting our understanding of the required high target modulation needed (>70% modulation of PR in MCF-7 and >85% modulation of ER in CTC-174) for maximal anti-tumor effects in these two animal tumor models. This basic mechanistic understanding is valuable to contextualize compound-induced PD modulation in patients, for given doses and schedules and helps to support dose selection decisions. Citation Format: Pablo Morentin Gutierrez, Christopher Morrow, Natalie Cureton, Mandy Lawson, Dawn Trueman, Eric Gangl, Joanne Wilson, Aaron Smith, James Scott, Teresa Klinowska. Preclinical mechanistic PK/PD-efficacy modeling for AZD9833, a novel next generation oral SERD, to support dose selection during early clinical development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4369.

Efficacies of ceftazidime-avibactam (4:1 w/w) and ceftazidime were tested against ceftazidime-susceptible (bla

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

The estrogen receptor alpha (ERα) is expressed in >70% of breast cancers and is a clinically validated target in oncology.1 Anti-hormonal therapies that directly block ER function (e.g., tamoxifen) or therapies that block the production of estrogen itself (e.g., aromatase inhibitors) have proven to be effective treatments of the disease. Further advances have been made with the development of SERDs (Selective Estrogen Receptor Degraders) such as fulvestrant which both antagonise ERα-driven tumor cell growth and cause degradation of the ERα receptor.2 We previously disclosed the identification of a tricyclic indole scaffold that led to the orally active clinical candidate AZD9496.3 Additional work to identify novel chemotypes including phenol 14 and indazole 25 was also disclosed together. The work previously disclosed relied on the presence of the acrylic acid for efficient degradation of the estrogen receptor. In this poster we will discuss the replacement of the acrylic acid on the tricyclic indole scaffold with amines (e.g. compound 3). Compound 3 is a degrader of the estrogen receptor in the MCF-7 cell line (pIC50 8.4). Further optimisation of lead structure 3 led to more potent selective degraders of the estrogen receptor in multiple cell lines (e.g. pIC50 >10 in MCF-7) with suitable properties for oral absorption (e.g. oral AUC 44 μM.h in mice at 20 mg/kg). We will discuss some of the medicinal chemistry challenges that were faced along the way and the optimisation strategy (use of NMR derived solution phase conformations, understanding of the binding mode in the estrogen receptor by X-ray crystallography). ol> Citation Format: Bernard Barlaam, Jason Breed, Rodrigo J Carbajo, Eric Gangl, Samantha Hughes, Christopher J Morrow, Thomas A Moss, Radoslaw Polanski, Willem M Nissink, Daniel O'Donovan, Jeffrey Varnes, Bin Yang, James S Scott. Small Molecule Degraders of the Estrogen Receptor (SERDs): Optimization of the tricyclic indole scaffold beyond AZD9496 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A107. doi:10.1158/1535-7163.TARG-19-A107

The sedative clomethiazole (CMZ) has been used in Europe since the mid-1960s to treat insomnia and alcoholism. It has been previously demonstrated in clinical studies to reversibly inhibit human CYP2E1 in vitro and decrease CYP2E1-mediated elimination of chlorzoxazone. We have investigated the selectivity of CMZ inhibition of CYP2E1 in pooled human liver microsomes (HLMs). In a reversible inhibition assay of the major drug-metabolizing cytochrome P450 (P450) isoforms, CYP2A6 and CYP2E1 exhibited IC50 values of 24 µ M and 42 µ M, respectively with all other isoforms exhibiting values >300 µ M. When CMZ was preincubated with NADPH and liver microsomal protein for 30 minutes before being combined with probe substrates, however, more potent inhibition was observed for CYP2E1 and CYP2B6 but not CYP2A6 or other P450 isoforms. The substantial increase in potency of CYP2E1 inhibition upon preincubation enables the use of CMZ to investigate the role of human CYP2E1 in xenobiotic metabolism and provides advantages over other chemical inhibitors of CYP2E1. The K I and k inact values obtained with HLM-catalyzed 6-hydroxylation of chlorzoxazone were 40 µ M and 0.35 minute−1, respectively, and similar to values obtained with recombinant CYP2E1 (41 µ M, 0.32 minute−1). The K I and k inact values, along with other parameters, were used in a mechanistic static model to explain earlier observations of a profound decrease in the rate of chlorzoxazone elimination in volunteers despite the absence of detectable CMZ in blood.

Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

Approximately 70% of breast cancers express estrogen receptor alpha (ERα) and anti-hormonal therapies which either block the production of estrogen (e.g. anastrozole) or directly block ERα function (e.g. tamoxifen) remain the mainstay of treatment for these patients. While these therapies are highly effective resistance can occur. A common resistance mechanism to anti-hormonal agents is the mutation of ESR1, the gene that encodes ERα, which leads to ligand independent activity. Evidence is emerging that the selective estrogen receptor degrader (SERD) fulvestrant is effective in patients with ESR1 mutations. However, given the low bioavailability of fulvestrant and the still detectable levels of ER in clinical samples after treatment with fulvestrant it is hypothesised that SERDs with improved pharmacokinetic properties which are able to drive greater degradation of ERα may provide additional clinical benefit. We have previously described the discovery and characterisation of the orally bioavailable SERD AZD9496, which is currently in phase I clinical trials in ER+ breast cancer patients. Here we report the preclinical activity of AZD9496 in cell line and patient derived xenograft models expressing clinically relevant ESR1 mutations. We have engineered MCF-7 cells to express Y537S ESR1 which confers the ability to proliferate in the absence of estradiol, consistent with ligand independent activation of ER signalling. AZD9496 is able to inhibit proliferation of these cells and downregulate progesterone receptor protein expression at low nanomolar concentrations. Furthermore, when MCF-7 Y537S ESR1 cells are implanted as xenografts they grow in the absence of exogenous estradiol, are as sensitive to AZD9496 as parental MCF7 xenografts and demonstrate downregulation of ER dependent biomarkers. AZD9496 also has anti-tumour and pharmacodynamic efficacy in patient derived xenograft models expressing D538G ESR1. Taken together, these data strengthen the body of data suggesting that SERDs may be active in patients with tumours containing ESR1 mutations and supports the inclusion of this patient population in AZD9496 clinical trials. Citation Format: Morrow CJ, Lawson M, Ladd B, de Almeida C, James N, Curwen JO, Hanson L, Bell K, Goeppert A, Fisher D, Gangl E, De Savi C, Scott JS, D'Cruz C, Klinowska T, Weir HM. Utility of the orally bioavailable selective estrogen receptor degrader AZD9496 in ESR1 mutant preclinical models of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-10.

Mcl-1 is a member of the Bcl/Mcl family of proteins that promotes cell survival by preventing induction of apoptosis in a broad range of cancers. High expression of Mcl-1 has been linked to tumor development and resistance to anticancer therapies, underscoring the potential of Mcl-1 inhibitors as anticancer drugs. We have previously shown that AZD5991, a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1 and high selectivity, induces rapid and irreversible commitment to apoptosis in Mcl-1-dependent cancer cells in a manner dependent on proapoptotic BAK. Here, we demonstrate that AZD5991 exhibits cytotoxic activity (GI50 Citation Format: Adriana E. Tron, Matthew A. Belmonte, Steven Criscione, Edwin A. Clark, Eric Gangl, Francis D. Gibbons, Jeffrey W. Tyner, Stephen E. Kurtz, Qing Ye, Alexander W. Hird, Alwin Schuller, J. Paul Secrist. Selective Mcl-1 inhibition by AZD5991 induces on-target cell death and achieves antitumor activity in multiple myeloma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 302.

The proteins Bcl2 and Bcl-xL are often up-regulated in cancer, and hold in check the apoptosis that would normally be initiated by accumulation of the BH3-only proteins Bax and Bak in response to genomic dysregulation. AZD4320 potently disrupts that interaction, initiating the apoptotic cascade in Bcl-2 or Bcl-xL-dependent tumors. Because platelets are known to be dependent on Bcl-xL, thrombocytopenia is an expected on-target effect. AZD4320 was administered at dose levels 0.5-10 mg/kg, both intravenously and extravascularly, to immune-compromised mice inoculated subcutaneously with the RS4;11 model of acute lymphocytic leukemia (ALL). Drug concentrations were measured by liquid chromatography-mass spectrometry (LC-MS) in plasma and tumor. A cleaved-caspase-3 ELISA was used to assess apoptotic activity in the tumor. Parallel efficacy studies, were used to assess tumor growth compared to vehicle, following tumors from initial regression at tolerated doses to regrowth. Tumors were measured using calipers, and tumor volumes computed using an ellipsoid approximation. We present a mini-physiologically based pharmacokinetic/pharmacodynamic (mPBPK/PD) model that links drug concentrations in plasma and tumor to observed caspase activity and efficacy in an integrated manner, across multiple dose levels and schedules. The tumor is modeled as a pool of sensitive cells which can be triggered rapidly by AZD4320 to apoptose, from which point they transition gradually to death, reducing tumor volume. Cleaved caspase-3 is used as a marker of apoptosis, and modeled using a sigmoidal response function with steep slope parameter. In this way, we effectively capture the transient nature of the response, despite AZD4320's long residence in the tumor. Thrombocytopenia is described not as an effect on the megakaryocyte precursors, but as a linear concentration-dependent effect on circulating platelets. Feedback from the circulation to megakaryocytes drives increased production to fill the deficit. Parameters are well-estimated throughout. Together, these components provide a comprehensive means to investigate the effects of dose and schedule with a dual Bcl2/BCL-xL inhibitor. Citation Format: Francis D. Gibbons, Ammar Adam, Marie-Eve Beaudoin, Eric Gangl, Paul Secrist. Target engagement, thrombocytopenia, and efficacy induced by the dual Bcl2/xL inhibitor AZD4320 are quantitatively linked by a PK/PD model in leukemia xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3975.

CYP11B2, the aldosterone synthase, and CYP11B1, the cortisol synthase, are two highly homologous enzymes implicated in a range of cardiovascular and metabolic diseases. We have previously reported the discovery of LCI699, a dual CYP11B2 and CYP11B1 inhibitor that has provided clinical validation for the lowering of plasma aldosterone as a viable approach to modulate blood pressure in humans, as well normalization of urinary cortisol in Cushing’s disease patients. We now report novel series of aldosterone synthase inhibitors with single-digit nanomolar cellular potency and excellent physicochemical properties. Structure–activity relationships and optimization of their oral bioavailability are presented. An illustration of the impact of the age of preclinical models on pharmacokinetic properties is also highlighted. Similar biochemical potency was generally observed against CYP11B2 and CYP11B1, although emerging structure–selectivity relationships were noted leading to more CYP11B1-selective analogs.

The structural elucidation of metabolites of ritonavir and indinavir, HIV-protease inhibitor drugs, by liquid chromatography-electrospray ionization mass spectrometry is described. Ritonavir and indinavir were biotransformed separately by incubation with transplant quality human liver microsomes. The incubation mixture was then analyzed by HPLC coupled to ion trap (ITMS) and triple quadrupole mass analyzers. The metabolites retained most of the structural features of the parent molecules. Baseline chromatographic resolution of isobaric species by gradient elution HPLC permitted rapid structural identification of these metabolites. Both drugs were biotransformed primarily by oxidative and hydrolytic pathways to numerous metabolites that retained many of the features of the parent molecules. Triple quadrupole and ion trap mass spectrometry were applied jointly to thoroughly detect and thoroughly characterize these metabolites. Furthermore, retention-time and data-dependent scanning assured acquisition of detailed MS-MS spectra for rapid detection of metabolic pathways of ritonavir and indinavir. Comparison of the ITMS and triple quadrupole data showed qualitative and quantitative differences in the mass spectral patterns, suggesting that these instruments should be used in parallel to ensure comprehensive metabolite detection and characterization by LC-MS.

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vitro and in vivo sources. Constant neutral loss (CNL) and selective reaction monitoring (SRM) techniques with a triple-quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts in vitro and in animals. Detection of 1 adduct in 10(4) unmodified bases is achieved using CNL scanning detection, while the lower detection limits using SRM approach 1 adduct in 10(7) unmodified bases using 300 microg of DNA. The DNA adducts N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4, 5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N(2)-IQ) were detected in kidney tissues of chronically treated cynomolgus monkeys at levels and in proportions consistent with previously published (32)P-postlabeling data [Turesky, R. J., et al. (1996) Chem. Res. Toxicol. 9, 403-408]. Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

Current diagnosis of drug-induced kidney injury (DIKI) primarily relies on detection of elevated plasma creatinine (Cr) or blood urea nitrogen (BUN) levels; however, both are indices of overall kidney function and changes are delayed with respect to onset of nephron injury. Our aim was to investigate whether early changes in new urinary DIKI biomarkers predict plasma Cr, BUN, renal hemodynamic and kidney morphological changes associated with kidney injury following a single dose of cisplatin (CDDP) using an integrated platform in rodent. Conscious surgically prepared male Han Wistar rats were given a single intraperitoneal dose of CDDP (15 mg/kg). Glomerular filtration rate (GFR), effective renal plasma flow (ERPF), urinalysis, DIKI biomarkers, CDDP pharmacokinetics, blood pressures, heart rate, body temperature and electroencephalogram (EEG) were measured in the same vehicle- or CDDP-treated animals over 72 h. Plasma chemistry (including Cr and BUN) and renal tissues were examined at study termination. Cisplatin caused progressive reductions of GFR, ERPF, heart rate and body temperature from day 1 (0–24 h). DIKI biomarkers including alpha-glutathione S-transferase (α-GST) significantly increased as early as 6 h post-dose, which preceded significant declines of GFR and ERPF (24 h), increased plasma Cr and BUN (72 h), and associated with renal acute tubular necrosis at 72 h post-dose. The present study adds to the current understanding of CDDP action by demonstrating that early increases in urinary excretion of α-GST predict DIKI risk following acute exposure to CDDP in rats, before changes in traditional DIKI markers are evident.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.

Synthetic studies of the antimicrobial secondary metabolite thiomuracin A (1) were initiated to improve chemical stability and physicochemical properties. Functional group modifications of 1 included removing the C2–C7 side chain, derivatizing the C84 epoxide region, and altering the C44 hydroxyphenylalanine motif. The resulting derivatives simplified and stabilized the chemical structure and were evaluated for antibacterial activity relative to 1. The simplified structure and improved organic solubility of the derivatives facilitated isolation yields from fermentation broths and simplified the procedures involved for the process. These advancements increased material supply for continued medicinal chemistry optimization and culminated in the identification of 2, a structurally simplified and chemically stable analogue of 1 which retained potent antibiotic activity.

4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for their activity against Gram positive bacterial infections. Optimization efforts focused on improving the physicochemical properties (e.g., aqueous solubility and chemical stability) of the 4-aminothiazolyl natural product template while improving the in vitro and in vivo antibacterial activity. Structure-activity relationships were defined, and the solubility and efficacy profiles were improved over those of previous analogues and 1. These studies identified novel, potent, soluble, and efficacious elongation factor-Tu inhibitors, which bear cycloalkylcarboxylic acid side chains, and culminated in the selection of development candidates amide 48 and urethane 58.

Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50)10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.

Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the in vitro and in vivo activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC 90 ≤ 0.25 μg/ml) but weaker against the streptococci (MIC 90 ≥ 4 μg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the tuf gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal S. aureus infections with 50% effective doses (ED 50 ) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic E. faecalis infections with ED 50 of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active in vitro and in vivo activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.

A series of 2-[(2,6)-dimethylphenyl]benzimidazole analogs displayed strong potential for mutagenicity following metabolic activation in either TA98 or TA100 Salmonella typhimurium strains. The number of revertants was significantly reduced by replacing the 2,6-dimethylphenyl group with a 2,6-dichlorophenyl moiety. Time-dependent CYP3A4 inhibition was also observed with a compound containing a 2-[(2,6)-dimethylphenyl] benzimidazole ring, implying risk for this scaffold to generate reactive metabolites.

Electrospray ionization mass spectrometry is a valuable tool in the identification and quantification of drug metabolites in biological fluids. However, there are many instances where matrix components present in these fluids interfere with analyte detection and prevent the acquisition of accurate or complete results. In some instances, the matrix can suppress ionization to such an extent that analytes are completely undetectable by MS. In this work, we investigate how ionization and ion-transfer efficiencies are affected by drastically reducing the flow into the MS. A postcolumn concentric flow-splitting device was constructed to allow the measurement of analyte signal and ionization suppression across a range of flow rates (0.1-200 microL/min). Using this device, the effects of flow rate on signal intensity and ionization suppression were measured in analytical experiments that included flow injection analysis MS, postcolumn addition LC-MS, and on-line LC-MS analysis of metabolites generated from rat liver microsomes. The device used to deliver 0.1 microL/min flows is referred to as a nanosplitter because it achieved high split ratios (2000:1), producing flow rates comparable to those observed in nanoelectrospray. The nanosplitter maintained chromatographic integrity with high fidelity and allowed the direct comparison of analyte signal across a range of flow rates (0.1-200 microL/min). A significant improvement in concentration and mass sensitivity as well as a reduction in signal suppression is observed when the performance at 200 versus 0.1 microL/min flow rate is compared. Using this specially designed concentric splitting device, the advantages of ultralow flow ESI were easily exploited for applications employing large bore chromatography.

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vivo sources. Adjustments were made to a previously described methodology such that analyte detection could be improved by nearly 2 orders of magnitude. These adjustments included changing the electrospray ionization sprayer configuration, increasing the sample injection volume, improving the solid-phase extraction procedure, and increasing peak efficiency by modifying chromatographic conditions. While this scheme for improving analyte detection was targeted for DNA adducts, it could be applied to almost any LC/MS methodology where sensitive analysis is the primary objective. Selective reaction monitoring) techniques with a triple quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts, with detection limits approaching 1 adduct in 10(9) unmodified bases using approximately 500 microg of DNA. The DNA adducts N-(2'-deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline and 5-(2'-deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline were detected in pancreas tissue of a cynomolgus monkey sacrificed 24 h after a single administration of 10 mg/kg carcinogen. The LC/MS results were consistent with previously published 32P-postlabeling data (Turesky et al. Chem Res. Toxicol. 1996, 9, 403-408). Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

A modular subatmospheric electrospray interface with fiber optic UV detection close to the electrospray tip was developed for coupling of microcolumn separation techniques with mass spectrometry. The interface was based on a liquid junction with a removable microelectrospray tip. The electrospray tip was enclosed in a subatmospheric chamber attached in front of the sampling orifice of the mass spectrometer. The inlet of the liquid junction was maintained at atmospheric pressure, and thus no pressure drop developed across the separation column. The flow rate of the electrosprayed liquid from the liquid junction reservoir was adjusted by the pressure in the electrospray chamber. In this approach, a continuous and stable electrospray could be achieved without the use of an external pump. Since the electrospray did not depend on fluid delivery from the separation column, coated capillaries without electroosmotic flow as well as capillaries with electroosmotic flow could be used for capillary electrophoresis. In addition, the interface was found to be effective with capillary liquid chromatography. The use of a fiber optic UV detector placed close to the exit of the separation column provided additional detection information and a simple means of troubleshooting. The interface did not significantly influence the quality of the separation, even with columns generating several hundred thousand theoretical plates. Peptide samples in the submicromolar concentration range were detected, corresponding to a limit of detection in the attomole range.