Duncan Armstrong

Preservation of cardiomyocytes from the adult heart.

Cardiomyocytes represent one of the most useful models to conduct cardiac research. A single adult heart yields millions of cardiomyocytes, but these cells do not survive for long after isolation. We aimed to determine whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve fully differentiated adult cardiomyocytes. Using inhibitors of the myosin II ATPase, blebbistatin and N-benzyl-p-toluene sulphonamide (BTS), we preserved freshly isolated fully differentiated adult primary cardiomyocytes that were stored at a refrigerated temperature. Specifically, preserved cardiomyocytes stayed viable for a 2-week period with a stable expression of cardiac genes and retained the expression of key markers characteristic of cardiomyocytes. Furthermore, voltage-clamp, action potential, calcium transient and contractility studies confirmed that the preserved cardiomyocytes are comparable to freshly isolated cells. Long-term exposure of preserved cardiomyocytes to four tyrosine kinase inhibitors, sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate, revealed their potential to induce cardiac toxicity that was manifested with a decrease in contractility and induction of cell death, but this toxicity was not observed in acute experiments conducted over the time course amenable to freshly prepared cardiomyocytes. This study introduces the concept that the inhibition of myosin II ATPase safeguards the structure and function of fully differentiated adult cardiomyocytes. The fact that these preserved cardiomyocytes can be used for numerous days after preparation makes them a robust and versatile tool in cardiac research and allows the investigation of long-term exposure to novel drugs on cardiomyocyte function.

Can silicon make an excellent drug even better? An in vitro and in vivo head-to-head comparison between loperamide and its silicon analogue sila-loperamide.

Loperamide (1a), an opioid receptor agonist, is in clinical use as an antidiarrheal agent. Carbon/silicon exchange (sila‐substitution) at the 4‐position of the piperidine ring of 1a (R3COH→R3SiOH) leads to sila‐loperamide (1b). Sila‐loperamide was synthesized in a multistep procedure, starting from triethoxyvinylsilane and taking advantage of the 4‐methoxyphenyl (MOP) unit as a protecting group for silicon. The in vitro and in vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties of the C/Si analogues 1a and 1b were determined and compared. Despite significant differences in the in vitro PK properties of loperamide and sila‐loperamide regarding clearance, permeability, and efflux, both compounds exhibited nearly identical in vivo PK profiles. The increase in metabolic stability of the silicon compound 1b observed in vitro seems to be counterbalanced by an increase in efflux and diminished permeability compared to the parent carbon compound 1a. Overall, sila‐loperamide exhibits high unbound clearance (CLu), leading to a significant decrease in unbound concentration (Cu) and unbound area under the curve (AUCu) after oral exposure, compared to loperamide. In vitro and in vivo metabolic studies showed an altered profile of biotransformation for the silicon compound 1b, leading to the formation of a more polar and quickly cleared metabolite and preventing the formation of the silicon analogue of the neurotoxic metabolite observed for the parent carbon compound 1a. These differences can be correlated with the different chemical properties of the C/Si analogues 1a and 1b. This study provides some of the most detailed insights into the effects of a carbon/silicon switch and how this carbon/silicon exchange affects overall drug properties.

A peripherally restricted P2Y12 receptor antagonist altered rat tumor incidences with no human relevance: Mode of action consistent with dopamine agonism

Background Ticagrelor is an orally available, direct acting and reversible P2Y12 receptor antagonist approved for treatment of acute coronary syndrome. The objectives of these studies were to (1) evaluate the Ticagrelor 2-year rat carcinogenicity bioassay data; (2) investigate potential mode of action (MOA) and (3) interpret human relevance. Methods The following studies were done (1) rat two-year carcinogenicity study in male and female rats, (2) in vitro and in vivo genotoxicity assays, (3) quantitative whole body autoradiography (QWBA; male and female rats), (4) in vitro pharmacological profiling for more than 300 assays, and (5) in vivo ovariectomized rat assay. Results The carcinogenicity study indicated Ticagrelor increased uterine tumor incidence while decreasing mammary and pituitary tumors/hyperplasia incidences in only high dose female rats. However, this altered tumor incidences were not P2Y12 target related since marketed non-reversible P2Y12 receptor antagonists were not associated with alter tumor incidences. MOA studies determined Ticagrelor exposure in the anterior pituitary and Ticagrelor was (1) non-genotoxic, (2) peripherally-restricted, (3) a dopamine transport (DAT) inhibitor with an IC50 lower than systemic free exposure in the rat carcinogenic study and more than a log higher than the free systemic exposure seen in clinical trials and (4) an inhibitor of estradiol-induced prolactin secretion. Discussion Similar to Ticagrelor, centrally active dopamine agonists induce the same altered tumor incidence patterns that according to literature do not translate into the clinical setting, with a MOA involving decreased prolactin secretion. The Ticagrelor MOA data and literature suggest that altered dopamine levels in the hypophyseal part of the hypothalamus–hypophyseal axis (by Ticagrelor) will result in similar altered tumor incidences in rat that do not translate into the clinical setting, based on qualitative species differences. In conclusion Ticagrelor increased uterine tumors in the rat carcinogenesis study by a MOA consistent with reduced dopamine inhibition of prolactin, which is not a patient safety risk.

Biomarkers for safety and toxicology: Drug induced cardiac injury and dysfunction

Abstract Cancer chemotherapeutic drugs, by their very nature of targeting robust and often rapidly dividing cells, inherently carry more toxicological burden and risk than drugs aimed at treating more benign disease. However, clinical oncologists have a continual need to utilise every therapeutic tool in their armoury in their attempts to treat this debilitating and often fatal disease. It is important that the physician has suitable biomarkers not only for efficacy, but also for drug related toxicity, in order to exploit maximally what is often a narrow therapeutic margin between benefit and unacceptable toxicity. Here we present data on a novel anticancer agent intended for combination therapy. The drug caused impaired cardiac functionality in pre- clinical studies at high doses, potentially leading to restrictive exclusion criteria in early clinical trials. To assist in clinical development, we generated biomarker data to be used in a weight of evidence approach to increase confidence in safe clinical progression.