Cardiovascular safety liabilities of VEGFR-2 inhibitors

The identification of the vascular endothelial growth factor (VEGF) as a crucial determinant in neoangiogenesis, as well as the role of its overexpression in tumour growth and dissemination, have provided an attractive target for more specific cancer treatments. In particular, since the clinical approval of the first receptor tyrosine kinase inhibitor (RTKI) in 2001, several agents targeting vascular endothelial growth factor receptor (VEGFR) signalling have been approved for the treatment of a variety of malignancies. However, the improved clinical outcomes associated with these therapeutics have been accompanied by unanticipated cardiovascular toxicities, in particular hypertension, resulting in both acute- and long-term cardiovascular dysfunctions, which still represent a major cause of morbidity and mortality in cancer survivors. As a result, cardiovascular safety of VEGFR inhibitors remains a major challenge in oncology, since unanticipated and poorly controlled cardiovascular toxicities induced by these therapeutics often result in a reduction of therapeutic dosage or treatment interruption, therefore influencing cancer management. To that end, this project aimed to comprehensively characterise the cardiovascular safety liabilities associated with two VEGFR-2 inhibitors, in particular axitinib and lenvatinib, as well as investigate the involvement of endothelin-1 (ET-1) system in the development of RTKI-induced hypertension. Combining diverse in vitro, ex vivo and in vivo approaches, the purpose of this study was also the identification of sensitive approaches to readily detect cardiovascular risk of these novel targeted therapies in preclinical settings. As the incidence of hypertension seems to be correlated to the potency of these therapeutics against VEGFR-2, we first quantified the inhibitory activity of a spectrum of RTKIs on VEGFR-2 mediated responses. The five RTKIs targeting VEGFR-2 tested in this study (axitinib, linifanib, vatalanib, SU-14813 and lenvatinib) showed a potent inhibition of VEGF165a-stimulated nuclear factor of activated T-cell (NFAT) response with IC50 values in nanomolar range, with axitinib and lenvatinib showing highest relative potency for the receptor. These two RTKIs were then selected for the characterisation of their haemodynamic effects in conscious and freely moving rats. Both axitinib and lenvatinib caused a significant hypertensive response, which was associated with an increased vascular tone in hindquarters and mesenteric arteries. Additionally, given the onset time for hypertension in the animal model used (24-48 hours), this in vivo model has proven to be a sensitive and translational approach for the prediction and early detection of haemodynamic effects of these novel targeted anticancer therapies. Second, we investigated the role of dual ETA/ETB receptor blockade with bosentan and selective ETA receptor antagonism with sitaxentan in the prevention of axitinib- and lenvatinib-induced hypertension. In conscious and freely moving rats, sitaxentan completely prevented the hypertensive response to axitinib and lenvatinib. These findings established the contribution of ET-1 to RTKI-induced hypertension. In particular, our results have demonstrated that such increase in BP is purely regulated by ETA receptors. The simultaneous evaluation of the role of endothelin receptors antagonism in preventing the RTKI-mediated alterations of vascular tone in regional vascular beds showed that axitinib- and lenvatinib-induced increase of mesenteric vascular tone could be independent of their hypertensive effect, and it may be mainly mediated by ETB receptors. Third, ex vivo studies were used to investigate the effect of axitinib and lenvatinib on vascular reactivity and endothelial function, while also assessing the role of arterial stiffness in the haemodynamic responses associated with these agents. Experiments on isolated mouse aortic segments showed that the treatment with axitinib and lenvatinib was not associated with endothelial dysfunction, as reported by the unaltered endothelium-dependent relaxation in response to acetylcholine (ACh). Endothelium-independent relaxation in response to nitric oxide (NO) donor was not affected by both axitinib and lenvatinib, suggesting a preserved vascular smooth muscle cells (VSMCs) function. In addition, the rodent oscillatory set-up to study arterial compliance (ROTSAC) was used to investigate the effect of axitinib and lenvatinib on arterial stiffness; no evidence was found that these agents affect arterial compliance in the time period evaluated in this study. Finally, in vivo experiments were performed to assess cardiac functional and structural changes induced by axitinib and lenvatinib in a mouse model. Echocardiographic measurements and pressure-volume loops analysis revealed an impairment of systolic function in response to lenvatinib. The treatment with this RTKI was also associated with structural changes of the left ventricle (LV), including left ventricular wall thinning and cavity enlargement, suggestive of drug-induced dilated cardiomyopathy

Cardiovascular safety liabilities of VEGFR-2 inhibitors

The identification of the vascular endothelial growth factor (VEGF) as a crucial determinant in neoangiogenesis, as well as the role of its overexpression in tumour growth and dissemination, have provided an attractive target for more specific cancer treatments. In particular, since the clinical approval of the first receptor tyrosine kinase inhibitor (RTKI) in 2001, several agents targeting vascular endothelial growth factor receptor (VEGFR) signalling have been approved for the treatment of a variety of malignancies. However, the improved clinical outcomes associated with these therapeutics have been accompanied by unanticipated cardiovascular toxicities, in particular hypertension, resulting in both acute- and long-term cardiovascular dysfunctions, which still represent a major cause of morbidity and mortality in cancer survivors. As a result, cardiovascular safety of VEGFR inhibitors remains a major challenge in oncology, since unanticipated and poorly controlled cardiovascular toxicities induced by these therapeutics often result in a reduction of therapeutic dosage or treatment interruption, therefore influencing cancer management. To that end, this project aimed to comprehensively characterise the cardiovascular safety liabilities associated with two VEGFR-2 inhibitors, in particular axitinib and lenvatinib, as well as investigate the involvement of endothelin-1 (ET-1) system in the development of RTKI-induced hypertension. Combining diverse in vitro, ex vivo and in vivo approaches, the purpose of this study was also the identification of sensitive approaches to readily detect cardiovascular risk of these novel targeted therapies in preclinical settings. As the incidence of hypertension seems to be correlated to the potency of these therapeutics against VEGFR-2, we first quantified the inhibitory activity of a spectrum of RTKIs on VEGFR-2 mediated responses. The five RTKIs targeting VEGFR-2 tested in this study (axitinib, linifanib, vatalanib, SU-14813 and lenvatinib) showed a potent inhibition of VEGF165a-stimulated nuclear factor of activated T-cell (NFAT) response with IC50 values in nanomolar range, with axitinib and lenvatinib showing highest relative potency for the receptor. These two RTKIs were then selected for the characterisation of their haemodynamic effects in conscious and freely moving rats. Both axitinib and lenvatinib caused a significant hypertensive response, which was associated with an increased vascular tone in hindquarters and mesenteric arteries. Additionally, given the onset time for hypertension in the animal model used (24-48 hours), this in vivo model has proven to be a sensitive and translational approach for the prediction and early detection of haemodynamic effects of these novel targeted anticancer therapies. Second, we investigated the role of dual ETA/ETB receptor blockade with bosentan and selective ETA receptor antagonism with sitaxentan in the prevention of axitinib- and lenvatinib-induced hypertension. In conscious and freely moving rats, sitaxentan completely prevented the hypertensive response to axitinib and lenvatinib. These findings established the contribution of ET-1 to RTKI-induced hypertension. In particular, our results have demonstrated that such increase in BP is purely regulated by ETA receptors. The simultaneous evaluation of the role of endothelin receptors antagonism in preventing the RTKI-mediated alterations of vascular tone in regional vascular beds showed that axitinib- and lenvatinib-induced increase of mesenteric vascular tone could be independent of their hypertensive effect, and it may be mainly mediated by ETB receptors. Third, ex vivo studies were used to investigate the effect of axitinib and lenvatinib on vascular reactivity and endothelial function, while also assessing the role of arterial stiffness in the haemodynamic responses associated with these agents. Experiments on isolated mouse aortic segments showed that the treatment with axitinib and lenvatinib was not associated with endothelial dysfunction, as reported by the unaltered endothelium-dependent relaxation in response to acetylcholine (ACh). Endothelium-independent relaxation in response to nitric oxide (NO) donor was not affected by both axitinib and lenvatinib, suggesting a preserved vascular smooth muscle cells (VSMCs) function. In addition, the rodent oscillatory set-up to study arterial compliance (ROTSAC) was used to investigate the effect of axitinib and lenvatinib on arterial stiffness; no evidence was found that these agents affect arterial compliance in the time period evaluated in this study. Finally, in vivo experiments were performed to assess cardiac functional and structural changes induced by axitinib and lenvatinib in a mouse model. Echocardiographic measurements and pressure-volume loops analysis revealed an impairment of systolic function in response to lenvatinib. The treatment with this RTKI was also associated with structural changes of the left ventricle (LV), including left ventricular wall thinning and cavity enlargement, suggestive of drug-induced dilated cardiomyopathy

Involvement of β-adrenoceptors in the cardiovascular responses induced by selective adenosine A2A and A2B receptor agonists

A2A and A2B adenosine receptors produce regionally selective regulation of vascular tone and elicit differing effects on mean arterial pressure (MAP), whilst inducing tachycardia. The tachycardia induced by the stimulation of A2A or A2B receptors has been suggested to be mediated by a reflex increase in sympathetic activity. Here, we have investigated the role of β1- and β2-adrenoceptors in mediating the different cardiovascular responses to selective A2A and A2B receptor stimulation. Hemodynamic variables were measured in conscious male Sprague-Dawley rats (350–450 g) via pulsed Doppler flowmetry. The effect of intravenous infusion (3 min per dose) of the A2A-selective agonist CGS 21680 (0.1, 0.3, 1.0 µg.kg−1.min−1) or the A2B-selective agonist BAY 60–6583 (4.0, 13.3, 40.0 µg.kg−1.min−1) in the absence or following pre-treatment with the non-selective β-antagonist propranolol (1.0 mg.kg−1), the selective β1-antagonist CGP 20712A (200 µg.kg−1), or the selective β2-antagonist ICI 118,551 (2.0 mg.kg−1) was investigated (maintenance doses also administered). CGP 20712A and propranolol significantly reduced the tachycardic response to CGS 21680, with no change in the effect on MAP. ICI 118,551 increased BAY 60–6583-mediated renal and mesenteric flows, but did not affect the heart rate response. CGP 20712A attenuated the BAY 60–6583-induced tachycardia. These data imply a direct stimulation of the sympathetic activity via cardiac β1-adrenoceptors as a mechanism for the A2A- and A2B-induced tachycardia. However, the regionally selective effects of A2B agonists on vascular conductance were independent of sympathetic activity and may be exploitable for the treatment of acute kidney injury and mesenteric ischemia

Regionally selective cardiovascular responses to adenosine A2A and A2B receptor activation

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein-coupled receptors (A1, A2A, A2B, A3). Here, we have investigated the effect of A2A and A2B-selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague-Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A2A-selective agonist CGS 21680 (0.1, 0.3, 1 µg kg−1 min−1) or the A2B-selective agonist BAY 60-6583 (4,13.3, 40 µg kg−1 min−1) following predosing with the A2A-selective antagonist SCH 58261 (0.1 or 1 mg kg−1 min−1), the A2B/A2A antagonist PSB 1115 (10 mg kg−1 min−1) or vehicle. The A2A-selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B-selective agonist BAY 60-6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury

COVID-19-Induced Myocarditis: Pathophysiological Roles of ACE2 and Toll-like Receptors

The clinical manifestations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection responsible for coronavirus disease 2019 (COVID-19) commonly include dyspnoea and fatigue, and they primarily involve the lungs. However, extra-pulmonary organ dysfunctions, particularly affecting the cardiovascular system, have also been observed following COVID-19 infection. In this context, several cardiac complications have been reported, including hypertension, thromboembolism, arrythmia and heart failure, with myocardial injury and myocarditis being the most frequent. These secondary myocardial inflammatory responses appear to be associated with a poorer disease course and increased mortality in patients with severe COVID-19. In addition, numerous episodes of myocarditis have been reported as a complication of COVID-19 mRNA vaccinations, especially in young adult males. Changes in the cell surface expression of angiotensin-converting enzyme 2 (ACE2) and direct injury to cardiomyocytes resulting from exaggerated immune responses to COVID-19 are just some of the mechanisms that may explain the pathogenesis of COVID-19-induced myocarditis. Here, we review the pathophysiological mechanisms underlying myocarditis associated with COVID-19 infection, with a particular focus on the involvement of ACE2 and Toll-like receptors (TLRs)

Monitoring haemodynamic changes in rodent models to better inform safety pharmacology: Novel insights from in vivo studies and waveform analysis

Animal models are essential for assessing cardiovascular responses to novel therapeutics. Cardiovascular safety liabilities represent a leading cause of drug attrition and better preclinical measurements are essential to predict drug-related toxicities. Presently, radiotelemetric approaches recording blood pressure are routinely used in preclinical in vivo haemodynamic assessments, providing valuable information on therapy-associated cardiovascular effects. Nonetheless, this technique is chiefly limited to the monitoring of blood pressure and heart rate alone. Alongside these measurements,Doppler flowmetry can provide additional information on the vasculature by simultaneously measuring changes in blood flow in multiple different regional vascular beds. However, due to the time-consuming and expensive nature of this approach, it is not widely used in the industry. Currently, analysis of waveform data obtained from telemetry and Doppler flowmetry typically examines averages or peak values of waveforms. Subtle changes in the morphology and variability of physiological waveforms have previously been shown to be early markers of toxicity and pathology. Therefore, adetailed analysis of pressure and flowmetry waveforms could enhance the understanding of toxicological mechanisms andthe ability to translate these preclinical observations to clinical outcomes. In this review, we give an overview of the different approaches to monitor the effects of drugs on cardiovascular parameters (particularly regional blood flow, heart rate and blood pressure) and suggest that further development of waveform analysis could enhance our understanding of safety pharmacology, providing valuable information without increasing the number of in vivo studies needed

Role of endothelin ETA receptors in the hypertension induced by the VEGFR-2 kinase inhibitors axitinib and lenvatinib in conscious freely-moving rats

Receptor tyrosine kinase inhibitors (RTKIs) suppress tumour growth by targeting vascular endothelial growth factor receptor 2 (VEGFR-2) which is an important mediator of angiogenesis. Here, we demonstrate that two potent RTKIs, axitinib and lenvatinib, are associated with hypertensive side effects. Doppler flowmetry was used to evaluate regional haemodynamic profiles of axitinib and lenvatinib. Male Sprague Dawley rats (350-500 g) were instrumented with Doppler flow probes (renal and mesenteric arteries and descending abdominal aorta) and catheters (jugular vein and distal abdominal aorta, via the caudal artery). Rats were dosed daily with axitinib (3 or 6 mg.kg-1) or lenvatinib (1 or 3 mg.kg-1) and regional haemodynamics were recorded over a maximum of 4 days. Both RTKIs caused significant (p<0.05) increases in mean arterial pressure (MAP), which was accompanied by significant (p<0.05) vasoconstriction in both the mesenteric and hindquarters vascular beds. To gain insight into the involvement of endothelin-1 (ET-1) in RTKI-mediated hypertension, we also monitored heart rate (HR) and MAP in response to axitinib or lenvatinib in animals treated with the ETA receptor selective antagonist sitaxentan (5 mg.kg-1) or the mixed ETA/ETB receptor antagonist bosentan (15 mg.kg-1) over two days. Co-treatment with bosentan or sitaxentan markedly reduced the MAP effects mediated by both RTKIs (p<0.05). Bosentan, but not sitaxentan, also attenuated ET-1 mediated increases in HR. These data suggest that selective antagonists of ETA receptors may be appropriate to alleviate the hypertensive effects of axitinib and lenvatinib