Development of a Novel DNA Mono-alkylator Platform for Antibody-Drug Conjugates.

Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing.

Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads.

The Dolaflexin-based Antibody-Drug Conjugate XMT-1536 Targets the Solid Tumor Lineage Antigen SLC34A2/NaPi2b.

Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells.

A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy.

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer.

Mitochondrial DNA deletions in mice in men: substantia nigra is much less affected in the mouse.

Mitochondrial DNA (mtDNA) deletions have been reported to accumulate to high levels in substantia nigra of older humans, and these mutations are suspected of causing age-related degeneration in this area. We have compared levels of mtDNA deletions in humans and mice and report here that levels of deletions in the mouse are very significantly lower than in humans. While human mtDNA from substantia nigra contained more than 5% of deleted molecules, mouse substantia nigra contained less than 0.

Pharmacological inhibition of the hypertensive response to combretastatin A-4 phosphate in rats.

Combretastatin A-4 phosphate (CA4P) is a novel and promising anti-neoplastic agent. However, it is associated with transient hypertension in both animal and human models. In this study, we examined the potential cardiac toxicity and hypertensive effects of CA4P, and defined the most effective pharmacological inhibition of CA4P-induced hypertension in rats.

Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR.

Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process.

Collection of isolated cells for studying mitochondrial DNA mutations within individual cells.

Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. This chapter outlines the methods for collecting individual cells appropriate for analysis of mtDNA mutations by single-molecule PCR.

MicroRNA-1 negatively regulates expression of the hypertrophy-associated calmodulin and Mef2a genes.

Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, the precise control of calmodulin expression is important for the regulation of calcium signaling.

Phosphoinositide 3-kinase Akt signaling pathway interacts with protein kinase Cbeta2 in the regulation of physiologic developmental hypertrophy and heart function.

The phosphoinositide 3-kinase (PI3-kinase)-protein kinase B (Akt) signaling pathway is essential in the induction of physiological cardiac hypertrophy. In contrast, protein kinase C beta2 (PKCbeta2) is implicated in the development of pathological cardiac hypertrophy and heart failure. Thus far, no clear association has been demonstrated between these two pathways.

Elevated levels of placental growth factor represent an adaptive host response in sepsis.

Recently, we demonstrated that circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are increased in sepsis (Yano, K., P.C.

Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways.

Background: Heart failure is the leading cause of death in the United States. By delineating the pathways that regulate cardiomyocyte function, we can better understand the pathogenesis of cardiac disease. Many cardiomyocyte signaling pathways activate protein tyrosine kinases.

Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals.

Observations in hemodialysis patients suggest a survival advantage associated with activated vitamin D therapy. Left ventricular (LV) structural and functional abnormalities are strongly linked with hemodialysis mortality. Here, we investigated whether paricalcitol (PC, 19-nor-1,25(OH)(2)D(2)), an activated vitamin D compound, attenuates the development of LV abnormalities in the Dahl salt-sensitive (DSS) rat and whether humans demonstrate comparable findings.

Uncoupling protein 2 modulates cell viability in adult rat cardiomyocytes.

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane proton carrier that uncouples ATP synthesis. The aim of this study was to determine whether UCP2 plays a role in survival of adult rat cardiac myocytes. We first studied the effects of UCP2 overexpression in vitro.

Response of caspase-independent apoptotic factors to high salt diet-induced heart failure.

The role of caspase-independent apoptotic events in heart failure is largely unknown. The present study examined the response of apoptotic factors, which can function independently of caspase machinery including AIF, EndoG, and HtrA2/Omi to high salt diet-induced pathologic heart failure and exercise-induced physiologic cardiac hypertrophy. Following approximately 4 months of a daily diet containing 6% salt, animals developed clinical evidence of heart failure accompanied by changes in AIF, EndoG, and HtrA2/Omi.

Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure.

An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiomyocytes, the transcription factor Gata4 is required for agonist-induced hypertrophy. We hypothesized that, in the intact organism, Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress.

Overexpression of HAX-1 protects cardiac myocytes from apoptosis through caspase-9 inhibition.

Caspase-9 is a critical regulator of mitochondria-mediated apoptosis. We found that adult cardiac myocytes, but not nonmyocytes, have high caspase-9 expression, and exhibit relative resistance to caspase-9-induced cell death. Thus, we hypothesized that cardiac myocytes possess factors that resist apoptosis.

Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality.

Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies.

Regulation of vascular endothelial growth factor expression and vascularization in the myocardium by insulin receptor and PI3K/Akt pathways in insulin resistance and ischemia.

Objective: This study characterized the role of insulin receptors and resistance on vascular endothelial growth factor (VEGF) expression and myocardial vascularization in physiological conditions and after ischemia.

Methods And Results: Cardiac microvascular density was reduced by 30% in insulin-resistant Zucker fatty rats versus lean controls. This was associated with a parallel 40% inhibition of insulin-stimulated activation of both Akt and VEGF expression in the myocardium and cardiomyocytes.

Genetic expression profiles during physiological and pathological cardiac hypertrophy and heart failure in rats.

Cardiac hypertrophy is a complex and nonhomogenous response to various stimuli. In this study, we used high-density oligonucleotide microarray to examine gene expression profiles during physiological hypertrophy, pathological hypertrophy, and heart failure in Dahl salt-sensitive rats. There were changes in 404/3,160 and 874/3,160 genes between physiological and pathological hypertrophy and the transition from hypertrophy to heart failure, respectively.

Alterations in apoptosis regulatory factors during hypertrophy and heart failure.

Cardiac hypertrophy from pathological stimuli often proceeds to heart failure, whereas cardiac hypertrophy from physiological stimuli does not. In this study, physiological hypertrophy was created by a daily exercise regimen and pathological hypertrophy was created from a high-salt diet in Dahl salt-sensitive rats. The rats continued on a high-salt diet progressed to heart failure associated with an increased rate of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes.

Mutation and intracellular clonal expansion of mitochondrial genomes: two synergistic components of the aging process?

The foundations of the Mitochondrial mutational theory of aging include two assumptions: the high abundance of mitochondrial mutations and their ability to clonally expand within individual cells. The up-to-date data pertinent to these assumptions is reviewed and semi-quantitative estimates of the frequencies of mutants and intracellular expansions are offered. The incidence of mutations in various aged tissues may be on the order of one mutant per mitochondrial genome copy, and most of the cells are likely to be affected by intracellular clonal expansions of mitochondrial genomes.

Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury.

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1.

Gene expression profiling of the aging mouse cardiac myocytes.

Heart disease remains the most frequent cause of death in the general population with increasing incidence in the elderly population. The pathologic failure of the aging heart may be related to structural and functional alterations in cardiac muscle cells. However, the molecular mechanisms underlying the aging-related decline in cardiac muscle function are largely unknown.