Brad Hirakawa

Effect of the Multitargeted Tyrosine Kinase Inhibitors Imatinib, Dasatinib, Sunitinib, and Sorafenib on Mitochondrial Function in Isolated Rat Heart Mitochondria and H9c2 Cells

Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.

pan-FGFR inhibition leads to blockade of FGF23 signaling, soft tissue mineralization, and cardiovascular dysfunction

The fibroblast growth factor receptors (FGFR) play a major role in angiogenesis and are desirable targets for the development of therapeutics. Groups of Wistar Han rats were dosed orally once daily for 4 days with a small molecule pan-FGFR inhibitor (5mg/kg) or once daily for 6 days with a small molecule MEK inhibitor (3mg/kg). Serum phosphorous and FGF23 levels increased in all rats during the course of the study. Histologically, rats dosed with either drug exhibited multifocal, multiorgan soft tissue mineralization. Expression levels of the sodium phosphate transporter Npt2a and the vitamin D-metabolizing enzymes Cyp24a1 and Cyp27b1 were modulated in kidneys of animals dosed with the pan-FGFR inhibitor. Both inhibitors decreased ERK phosphorylation in the kidneys and inhibited FGF23-induced ERK phosphorylation in vitro in a dose-dependent manner. A separate cardiovascular outcome study was performed to monitor hemodynamics and cardiac structure and function of telemetered rats dosed with either the pan-FGFR inhibitor or MEK inhibitor for 3 days. Both compounds increased blood pressure (~+ 17 mmHg), decreased heart rate (~-75 bpm), and modulated echocardiography parameters. Our data suggest that inhibition of FGFR signaling following administration of either pan-FGFR inhibitor or MEK inhibitor interferes with the FGF23 pathway, predisposing animals to hyperphosphatemia and a tumoral calcinosis-like syndrome in rodents.

miR-208a as a Biomarker of Isoproterenol-induced Cardiac Injury in Sod2+/− and C57BL/6J Wild-type Mice

This investigation examined microRNA-208a (miR-208a) as a potential biomarker of isoproterenol (ISO)-induced cardiac injury in superoxide dismutase-2 (Sod2+/− ) and the wild-type mice, and the potential sensitivity of Sod2+/− mice to ISO-induced toxicity. A single intraperitoneal injection of ISO was administered to age-matched wild-type and Sod2+/− mice at 0, 80, or 160 mg/kg. Plasma miR-208a, cardiac troponin I (cTnI), and ISO systemic exposure were measured at various time points postdose. Hearts were collected for histopathology examination and for tissue expression of miR-208a and myosin heavy chain 7. ISO administration caused increases in cTnI and miR-208a plasma levels that correlated with myocardial damage; however, the magnitude of increase differed according to the types of mice. At similar ISO systemic exposure, the magnitude of cTnI was greater in wild-type mice compared to Sod2+/ − mice; however, the magnitude of miR-208a was greater in Sod2+/− mice than that of the wild-type mice. Myocardial degeneration occurred at ≥3 hr in the wild-type and ≥6 hr in Sod2+/ − mice. At ≥24 hr after ISO administration, miR-208a appeared superior to cTnI in indicating myocardial injury in both wild-type and Sod2+/− mice. Sod2+/− mice were not more sensitive than wild-type mice to ISO-induced toxicity.

RTP801 Gene Expression Is Differentially Upregulated in Retinopathy and Is Silenced by PF-04523655, a 19-Mer siRNA Directed Against RTP801

PURPOSE The intraocular pharmacodynamics of PF-04523655, a small-interfering RNA (siRNA) directed against RTP801, was characterized using rat models of retinopathy. METHODS Rat models of streptozotocin-induced diabetes and wet AMD were used to determine the onset, extent, and duration of siRNA inhibition of retinal RTP801 expression by PF-04523655, and this inhibition was characterized by pharmacokinetic/pharmacodynamic (PK/PD) modeling. A rat model of wet AMD was also used to examine PF-04523655 dose-dependent effects on the incidence of clinical grade 3 or 4 choroidal neovascularization lesions. Whole homogenate versus laser-capture microdissected (LCM) retinal samples were analyzed by quantitative PCR for RTP801 expression. RESULTS RTP801 expression in RPE/choroid (RPE/C) increased in diabetic rats by up to 70% above nondiabetic rat levels. Inhibition of retinal RTP801 expression by PF-04523655 began 1 day after intravitreous injection and was observed through day 7 in the neurosensory retina and through day 14 or longer in RPE/C. PF-04523655 inhibition of RTP801 expression was maintained well after clearance of PF-04523655 from the eye and was best characterized by an effect compartment PK/PD model. Moreover, PF-04523655 administration decreased the incidence of clinical grade 3 or 4 lesions by approximately 60% (P = 0.053), and dose-dependently inhibited retinal RTP801 expression (P < 0.01). RTP801 expression was enriched in the outer nuclear layer in LCM samples. CONCLUSIONS In rodent retinopathy models, administration of the siRNA, PF-04523655, reduced RTP801 expression in the retina, consistent with the RNA-induced silencing complex (RISC) mechanism of action. The pharmacodynamic profile from the animal models could be useful to elucidate dose and exposure dependency of RTP801 expression inhibition by siRNA.

A tyrosine kinase inhibitor-induced myocardial degeneration in rats through off-target phosphodiesterase inhibition.

PF‐04254644 is a selective kinase inhibitor of mesenchymal epithelial transition factor/hepatocyte growth factor receptor with known off‐target inhibitory activity against the phosphodiesterase (PDE) family. Rats given repeated oral doses of PF‐04254644 developed a mild to moderate myocardial degeneration accompanied by sustained increase in heart rate and contractility. Investigative studies were conducted to delineate the mechanisms of toxicity. Microarray analysis of Sprague–Dawley rat hearts in a 6 day repeat dose study with PF‐04254644 or milrinone, a selective PDE3 inhibitor, revealed similar perturbation of the cyclic adenosine monophosphate (c‐AMP) pathway. PDE inhibition and activation of c‐AMP were further substantiated using PDE3B immunofluorescence staining and through a c‐AMP response element reporter gene assay. The intracellular calcium and oxidative stress signaling pathways were more perturbed by treatment with PF‐04254644 than milrinone. The rat cardiomyocytes calcium assay found a dose‐dependent increase in intracellular calcium with PF‐04254644 treatment. These data suggest that cardiotoxicity of PF‐04254644 was probably due to activation of c‐AMP signaling, and possibly subsequent disruption of intracellular calcium and oxidative stress signaling pathways. The greater response with PF‐04254644 as compared with milrinone in gene expression and micro‐ and ultrastructural changes is probably due to the broader panel of PDEs inhibition. Copyright

Toxicogenomic Analysis of Cardiotoxicity in Rats

Evidence of cardiotoxicity in the preclinical testing of drugs will often lead to compound attrition. The standard method for identifying cardiotoxic compounds involves histopathological analysis of tissue sections, a resource intensive process. In an effort to reduce attrition and capture safety endpoints early within the drug discovery paradigm, a more rapid assessment of target organ effects is desired. Here we describe the results of a preliminary study in which a group of common genes were affected by in vivo exposure to compounds known to cause dose-dependant cardiotoxicity. Adult male Sprague-Dawley rats were treated intraperitoneally with a single dose of digoxin (20 mg/kg), doxorubicin (30 mg/kg), isoproterenol (70 mg/kg), lipopolysaccharide (10 mg/kg) or carbon tetrachloride (800 mg/kg) and euthanized either 6 or 24 hours post-dose. Digoxin, doxorubicin, isoproterenol, and lipopolysaccharide were chosen for this study based on their diverse mechanisms of cardiotoxicity. Carbon tetrachloride, a known liver toxicant, was chosen as a non-cardiotoxic negative control. Genes commonly affected by all four cardiotoxic compounds were grouped together as a list of potential biomarkers. Gene expression changes were subsequently quantified using quantitative PCR. These genes were compared to those affected by novel experimental compounds previously shown to cause cardiotoxicity in rats. These compounds also affected over half of the genes on the biomarker list, whereas the non-cardiotoxic control compound did not affect any genes on the biomarkers list. These data indicate that measuring changes in gene expression could aid in the prioritization of compounds before they are tested in more resource intensive studies.

Abstract LB-203: Development of a nonclinical model for the assessment of oncology therapy-induced cardiomyopathy

With the increasing number of people surviving cancer, patients and doctors are becoming equally focused on the quality of life in survivorship as they are on fighting cancer. Consequently, cardio-oncology has emerged as a new clinical discipline with focus on both the stratification of patients at risk and therapies that pose a cardiovascular risk, eg molecularly targeted therapies including tyrosine kinase inhibitors (TKI). Therefore, nonclinical research should strive not only to detect preclinical drug-induced changes in cardiac structure or function, but also to understand the relevance of these cardiotoxic effects to humans. As is typical for new disciplines, there is an unmet need for early and predictive tools to identify both of these factors. Currently, preclinical assessment of cardiovascular toxicity consists of repeat-dose in vivo toxicity studies focused primarily on histopathological endpoints; additional measures, such as cardiac function and biomarkers of cardiac injury, are not routinely assessed in these studies. Furthermore, analysis of these studies suggests that using young and healthy, drug-naive animals do not adequately translate the TKI-induced cardiomyopathy phenotype; therefore, new nonclinical models are needed to address this gap. The developed model would ideally be used to assess the preclinical cardiotoxicity of proposed oncology therapies, but may also have utility in assessing cardioprotective therapies for use in cardio-oncology. This poster will propose a new model for studying cardio-oncology nonclinically that includes myocardial transcriptional signatures, strain echocardiography imaging, and pharmacologic (dobutamine) stress challenge. In general, rats with radiotelemetry were treated with vehicle or low dose doxorubicin (2 mg/kg/week IP) for 1 month. Cardiac structure and function at rest and during stress were assessed using strain echocardiography in concert with a weekly dobutamine infusion. Circulating blood biomarkers of cardiac toxicity (eg, NTproBNP, NTproANP, cTropin, and miR-208) were collected weekly and at necropsy. At the end of the study, samples of cardiac tissue were taken from the ventricular apex and were submitted for transcriptional profiling along with standard histopathology. These data as well as recent publications give high confidence in developing a rodent model predictive of drug-induced cardiomyopathy. Citation Format: Michelle Hemkens, Samantha Cardenas, Jessica Frey, Brad Hirakawa, Petra Koza-Taylor, Jianying Wang, Paul Butler. Development of a nonclinical model for the assessment of oncology therapy-induced cardiomyopathy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-203.