Measurement of Anti-Cancer Agent Methoxyamine in Plasma by Tandem Mass Spectrometry with On-Line Sample Extraction

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis® HLB extraction column ( mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00–1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1–94.7 and 90.1–96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20 mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse

Development and Validation of An LC–MS/MS Method for Pharmacokinetic Study of Methoxyamine in Phase I Clinical Trial

Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. This work described the development and validation of an LC–MS/MS method for quantitative determination of MX in human plasma. In this method, MX and its stable isotope methoxyl-d3-amine (MX-d3 as internal standard) were directly derivatized in human plasma with 4-(N,N-diethylamino)benzaldehyde. The derivatized MX and IS were extracted by methyl-tert-butyl ether, and separated isocratically on a Xterra C18 column (2.1 mm × 100 mm) using an aqueous mobile phase containing 45% acetonitrile and 0.4% formic acid at a flow rate of 0.200 ml/min. Quantitation of MX was carried out by multiple-reaction-monitoring (MRM) mode of positive turbo-ion-spray tandem mass spectrometry. This method has been validated according to FDA guidelines for bioanalytical method. The linear calibration range for MX was 1.25–500 ng/ml in human plasma with a correlation coefficient ≥ 0.9993. The intra- and inter-assay precision (%CV) at three concentration levels (3.50, 45.0 and 450 ng/ml) ranged 0.9–1% and 0.8–3%, respectively. The stability studies showed that MX met the acceptable criteria under all tested conditions. The method developed had been applied to the determination of plasma MX concentrations in the first-in-human phase I clinical trial, and PK data were presented

Development and Validation of An LC–MS/MS Method for Pharmacokinetic Study of Methoxyamine in Phase I Clinical Trial

Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. This work described the development and validation of an LC–MS/MS method for quantitative determination of MX in human plasma. In this method, MX and its stable isotope methoxyl-d3-amine (MX-d3 as internal standard) were directly derivatized in human plasma with 4-(N,N-diethylamino)benzaldehyde. The derivatized MX and IS were extracted by methyl-tert-butyl ether, and separated isocratically on a Xterra C18 column (2.1 mm × 100 mm) using an aqueous mobile phase containing 45% acetonitrile and 0.4% formic acid at a flow rate of 0.200 ml/min. Quantitation of MX was carried out by multiple-reaction-monitoring (MRM) mode of positive turbo-ion-spray tandem mass spectrometry. This method has been validated according to FDA guidelines for bioanalytical method. The linear calibration range for MX was 1.25–500 ng/ml in human plasma with a correlation coefficient ≥ 0.9993. The intra- and inter-assay precision (%CV) at three concentration levels (3.50, 45.0 and 450 ng/ml) ranged 0.9–1% and 0.8–3%, respectively. The stability studies showed that MX met the acceptable criteria under all tested conditions. The method developed had been applied to the determination of plasma MX concentrations in the first-in-human phase I clinical trial, and PK data were presented

MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors

AbstractThe DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin−Sca-1+c-kit+ (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT+GFP+) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect

MMR Deficiency Does Not Sensitize or Compromise the Function of Hematopoietic Stem Cells to Low and High LET Radiation

One of the major health concerns on long‐duration space missions will be radiation exposure to the astronauts. Outside the earth’s magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation‐sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56Fe radiation on HSC function in Mlh1+/+ and Mlh1‐/‐ marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. Stem Cells Translational Medicine 2018;7:513–520The hematopoietic system is essential for life, and normally has the capacity to sustain function for the duration of our lifetimes in spite of natural declination, which is associated with loss of DNA repair (including as DNA mismatch repair). Astronauts are exposed to ionizing radiation sources that are not commonly found on earth (such as HZE ions) and thus may display unforseen risks that need accounting in NASA risk models.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145333/1/sct312310.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145333/2/sct312310_am.pd

Spontaneous Autologous Graft-versus-Host Disease in Plasma Cell Myeloma Autograft Recipients: Flow Cytometric Analysis of Hematopoietic Progenitor Cell Grafts

Nine plasma cell myeloma patients spontaneously developed histologically proven autologous graft-versus-host disease (GVHD) limited predominantly to the gastrointestinal tract within 1 month of initial autologous hematopoietic cell transplantation (AHCT) using high-dose melphalan conditioning. All recipients responded promptly to systemic and nonabsorbable oral corticosteroid therapy. All patients previously received systemic therapy with thalidomide, lenalidomide, or bortezomib before AHCT. Using enzymatic amplification staining-enhanced flow cytometry, we evaluated expression of selected transcription regulators, pathway molecules, and surface receptors on samples of the infused hematopoietic cell grafts. We demonstrated significantly enhanced expression of GATA-2, CD130, and CXCR4 on CD34+ hematopoietic progenitor cells of affected patients compared with 42 unaffected AHCT controls. These 3 overexpressed markers have not been previously implicated in autologous GVHD. Although we did not specifically evaluate T cells, we postulate that exposure over time to the various immunomodulating therapies used for induction treatment affected not only the CD34+ cells but also T cells or relevant T cell subpopulations capable of mediating GVHD. After infusion, the affected hematopoietic progenitor cells then encounter a host that has been further altered by the high-dose melphalan preparative regimen; such a situation leads to the syndrome. These surface markers could be used to develop a model to predict development of this syndrome. Autologous GVHD potentially is a serious complication of AHCT and should be considered in plasma cell myeloma patients with otherwise unexplained gastrointestinal symptoms in the immediate post-AHCT period. Prompt recognition of this condition and protracted treatment with nonabsorbable or systemic corticosteroids or the combination may lead to resolution

Identification of 6-Benzylthioinosine as a Myeloid Leukemia Differentiation-Inducing Compound

As the pathophysiology of acute myelogenous leukemia (AML) involves a block of myeloid maturation, a desirable therapeutic strategy is to induce leukemic cell maturation to increase the efficacy and to avoid the side effects of traditional chemotherapeutics. Through a compound library screen, 6-benzylthioinosine (6BT) was identified as a promising differentiation-inducing agent. 6BT induces monocytic differentiation of myeloid leukemia cell lines such as HL-60 and OCI-AML3, as well as primary patient samples as evidenced by morphology, immunophenotyping, and nitroblue tetrazolium reduction. Not only can 6BT induce differentiation but a subset of AML cell lines such as MV4-11 and HNT34 instead undergo 6BT-mediated cell death. Despite inducing cell death in some leukemic cells, 6BT exhibits extremely low toxicity on several nonmalignant cells such as fibroblasts, normal bone marrow, and endothelial cells. This toxicity profile may relate to the function of 6BT as an inhibitor of the nucleoside transporter, ent1, which is thought to prevent it from entering many cell types. In contrast, 6BT likely enters at least some leukemic cell lines as shown by its requirement for phosphorylation for its differentiation activity. 6BT is also able to synergize with currently used myeloid differentiation agents such as ATRA and decitabine. Early studies indicate that the mechanism of action of this compound may involve ATP depletion that leads to growth inhibition and subsequent differentiation. Besides in vitro activity, 6BT also shows the ability to impair HL-60 and MV4-11 tumor growth in nude mice. 6BT is a promising new monocytic differentiation agent with apparent leukemic cell–specific activity

A Phase 1 Study of TRC102, An Inhibitor of Base Excision Repair, and Pemetrexed in Patients with Advanced Solid Tumors

Introduction TRC102 potentiates the activity of cancer therapies that induce base excision repair (BER) including antimetabolite and alkylating agents. TRC102 rapidly and covalently binds to apurinic/apyrimidinic (AP) sites generated during BER, and TRC102-bound DNA causes topoisomerase II-dependent irreversible strand breaks and apoptosis. This study assessed the safety, maximum-tolerated dose (MTD), pharmacokinetics and pharmacodynamics of TRC102 alone and in combination with pemetrexed. Purpose Patients with advanced solid tumors received oral TRC102 daily for 4 days. Two weeks later, patients began standard-dose pemetrexed on day 1 in combination with oral TRC102 on days 1 to 4. The pemetrexed-TRC102 combination was repeated every 3 weeks until disease progression. Methods Twenty-eight patients were treated with TRC102 at 15, 30, 60 or 100 mg/m2/d. The MTD was exceeded at 100 mg/m2/d due to grade 3 anemia in 50 % of patients. TRC102 exposure increased in proportion to dose with a mean t1/2 of 28 h. A pharmacodynamic assay confirmed that TRC102 binds to pemetrexed-induced AP sites at all doses studied. Stable disease or better was achieved in 15 of 25 patients evaluable for response (60 %), including one patient with recurrent metastatic oropharyngeal carcinoma that expressed high levels of thymidylate synthase, who achieved a partial response and was progression free for 14 months. Conclusions When administered with pemetrexed, the maximum tolerated dose of oral TRC102 is 60 mg/m2/d for 4 days. Randomized controlled studies are planned to evaluate the clinical benefit of adding TRC102 to pemetrexed and other agents that induce BER. © 2012 Springer Science+Business Media, LLC

A second-generation 15-PGDH inhibitor promotes bone marrow transplant recovery independently of age, transplant dose and granulocyte colony-stimulating factor support

Hematopoietic stem cell transplantation following myeloablative chemotherapy is a curative treatment for many hematopoietic malignancies. However, profound granulocytopenia during the interval between transplantation and marrow recovery exposes recipients to risks of fatal infection, a significant source of transplant-associated morbidity and mortality. We have previously described the discovery of a small molecule, SW033291, that potently inhibits the prostaglandin degrading enzyme 15-PGDH, increases bone marrow prostaglandin E2, and accelerates hematopoietic recovery following murine transplant. Here we describe the efficacy of (+)-SW209415, a second-generation 15-PGDH inhibitor, in an expanded range of models relevant to human transplantation. (+)-SW209415 is 10,000-fold more soluble, providing the potential for intravenous delivery, while maintaining potency in inhibiting 15-PGDH, increasing in vivo prostaglandin E2, and accelerating hematopoietic regeneration following transplantation. In additional models, (+)-SW209415: (i) demonstrated synergy with granulocyte colony-stimulating factor, the current standard of care; (ii) maintained efficacy as transplant cell dose was escalated; (iii) maintained efficacy when transplant donors and recipients were aged; and (iv) potentiated homing in xenotransplants using human hematopoietic stem cells. (+)-SW209415 showed no adverse effects, no potentiation of in vivo growth of human myeloma and leukemia xenografts, and, on chronic high-dose administration, no toxicity as assessed by weight, blood counts and serum chemistry. These studies provide independent chemical confirmation of the activity of 15-PGDH inhibitors in potentiating hematopoietic recovery, extend the range of models in which inhibiting 15-PGDH demonstrates activity, allay concerns regarding potential for adverse effects from increasing prostaglandin E2, and thereby, advance 15-PGDH as a therapeutic target for potentiating hematopoietic stem cell transplantation

Matricellular protein CCN3 mitigates abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease