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.

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, a detailed analysis of pressure and flowmetry waveforms could enhance the understanding of toxicological mechanisms and the 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.

The effect of two selective A1 -receptor agonists and the bitopic ligand VCP746 on heart rate and regional vascular conductance in conscious rats.

BACKGROUND AND PURPOSE: Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A1 , A2A , A2B , and A3 ). We have investigated the effect of two A1 -receptor-selective agonists and the novel A1 -receptor bitopic ligand VCP746 on the rat cardiovascular system. EXPERIMENTAL APPROACH: The regional haemodynamic responses of these agonist was investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal, mesenteric arteries and the descending abdominal aorta and the jugular vein and caudal artery catheterized. Cardiovascular responses were measured following intravenous infusion (3 min each dose) of CCPA (120, 400, and 1,200 ng·kg-1 ·min-1 ), capadenoson or adenosine (30, 100, and 300 µg·kg-1 ·min-1 ), or VCP746 (6, 20, and 60 µg·kg-1 ·min-1 ) following pre-dosing with DPCPX (0.1 mg·kg-1 , i.v.) or vehicle. KEY RESULTS: CCPA produced a significant A1 -receptor-mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A1 -receptor-mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A1 -receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A2A - and A2B -receptors. CONCLUSIONS AND IMPLICATIONS: These results suggest VCP746 mediates its cardiovascular effects via activation of A2 rather than A1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A1 allosteric ligand moieties.

Long-term cardiovascular effects of vandetanib and pazopanib in normotensive rats.

Vandetanib and pazopanib are clinically available, multi-targeted inhibitors of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases. Short-term VEGF receptor inhibition is associated with hypertension in 15%-60% of patients, which may limit the use of these anticancer therapies over the longer term. To evaluate the longer-term cardiovascular implications of treatment, we investigated the "on"-treatment (21 days) and "off"-treatment (10 days) effects following daily administration of vandetanib, pazopanib, or vehicle, in conscious rats. Cardiovascular variables were monitored in unrestrained Sprague-Dawley rats instrumented with radiotelemetric devices. In Study 1, rats were randomly assigned to receive either daily intraperitoneal injections of vehicle (volume 0.5 mL; n = 5) or vandetanib 25 mg/kg/day (volume 0.5 mL; n = 6). In Study 2, rats received either vehicle (volume 0.5 mL; n = 4) or pazopanib 30 mg/kg/day (volume 0.5 mL; n = 7), dosed once every 24 hours for 21 days. All solutions were in 2% Tween, 5% propylene glycol in 0.9% saline solution. Vandetanib caused sustained increases in mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) compared to baseline and vehicle. Vandetanib also significantly altered the circadian cycling of MAP, SBP, and DBP. Elevations in SBP were detectable 162 hours after the last dose of vandetanib. Pazopanib also caused increases in MAP, SBP, and DBP. However, compared to vandetanib, these increases were of slower onset and a smaller magnitude. These data suggest that the cardiovascular consequences of vandetanib and pazopanib treatment are sustained, even after prolonged cessation of drug treatment.

The role of evidence in humanitarian assessment: the Seed System Security Assessment and the Emergency Market Mapping and Analysis.

This paper reviews advances in the development and use of two evidence-based assessment toolkits: the Seed System Security Assessment (SSSA) and the Emergency Market Mapping and Analysis (EMMA). Both were created in the past five years and have been employed in a range of acute and chronic stress contexts across Africa, Asia, and parts of the Americas, in periods of civil strife, displacement, and drought, as well as following earthquakes, flooding, and political instability. The aims of this paper are threefold: to review advances with regard to each tool; to compare how each toolkit gathers and uses evidence, while considering possibilities for greater complementarity; and to reflect on the nature of 'evidence' used to guide humanitarian response in sudden-onset and chronic crisis situations. A comparison highlights the importance of triangulation and informed analysis for drawing conclusions from imperfect evidence, understanding the limitations of each assessment methodology, and confronting tacit assumptions.

Predicting in vivo cardiovascular properties of ß-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses.

ß-Adrenoceptor antagonists differ in their degree of partial agonism. In vitro assays have provided information on ligand affinity, selectivity, and intrinsic efficacy. However, the extent to which these properties are manifest in vivo is less clear. Conscious freely moving rats, instrumented for measurement of heart rate (ß1; HR) and hindquarters vascular conductance (ß2; HVC) were used to measure receptor selectivity and ligand efficacy in vivo. CGP 20712A caused a dose-dependent decrease in basal HR (P<0.05, ANOVA) at 5 doses between 6.7 and 670 µg/kg (i.v.) and shifted the dose-response curve for isoprenaline to higher agonist concentrations without altering HVC responses. In contrast, at doses of 67 µg/kg (i.v.) and above, ICI 118551 substantially reduced the HVC response to isoprenaline without affecting HR responses. ZD 7114, xamoterol, and bucindolol significantly increased basal HR (ΔHR: +122 ± 12, + 129 ± 11, and + 59 ± 11 beats/min, respectively; n=6), whereas other ß-blockers caused significant reductions (all at 2 mg/kg i.v.). The agonist effects of xamoterol and ZD 7114 were equivalent to that of the highest dose of isoprenaline. Bucindolol, however, significantly antagonized the response to the highest doses isoprenaline. An excellent correlation was obtained between in vivo and in vitro measures of ß1-adrenoceptor efficacy (R(2)=0.93; P<0.0001).

Mesenteric vasoconstriction and hindquarters vasodilatation accompany the pressor actions of exendin-4 in conscious rats.

The hemodynamic effects of the glucagon-like peptide-1 (GLP-1) receptor agonist, exendin-4, and putative underlying mechanisms were assessed in conscious male Sprague-Dawley rats. At a dose of 25 ng kg(-1) i.v., exendin-4 had little effect, but doses of 250 and 2500 ng kg(-1) had significant tachycardic effects (+66 +/- 9 and +95 +/- 16 beats min(-1) at 5 min, respectively) and pressor actions (+10 +/- 2 and +12 +/- 1 mm Hg), accompanied by substantial falls in mesenteric vascular conductance (-38 +/- 3% and -47 +/- 3%) and increases in hindquarters vascular conductance (+82 +/- 14% and +126 +/- 15%). The latter were likely due to adrenaline-mediated activation of beta(2) adrenoceptors since they were abolished by the beta(2) adrenoceptor antagonist, ICI 118551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol) hydrochloride], or propranolol [(RS)-1-[(1-methylethyl)amino]-3-(1-naphthalenyloxy)-2-propanol], and absent in adrenal-demedullated rats. In the presence of beta-adrenoceptor antagonism, the tachycardic effects of exendin-4 were suppressed, but the pressor action was enhanced. Enhancement of the pressor action of exendin-4 was not seen in adrenal-demedullated rats or in animals given phentolamine in addition to propranolol, consistent with a component of the pressor action of exendin-4 being due to an adrenaline-mediated positive inotropic effect mediated by alpha-adrenoceptors. The mesenteric vasoconstrictor effect of exendin-4 was unaffected by antagonism of alpha-adrenoceptors, vasopressin receptors, angiotensin receptors, or GLP-1 receptors, although antagonism of the latter substantially inhibited the hindquarter vasodilator effects of exendin-4. These results are consistent with exendin-4 having cardiovascular effects through GLP-1 receptor-dependent and -independent mechanisms, some of which involve sympathoadrenal activation.

Regional heterogeneity in the haemodynamic responses to urotensin II infusion in relation to UT receptor localisation.

The aim of the study was to measure regional haemodynamic responses to 6 h infusions of human urotensin II (hUII), to identify possible mediators of the effects observed, and to relate the findings to the distribution of urotensin II receptors (UT receptors). Male, Sprague-Dawley rats had pulsed Doppler flow probes and intravascular catheters implanted for measurement of regional haemodynamics in the conscious, freely moving state. Infusions of saline (0.4 ml h(-1)) or hUII (30, 300 and 3,000 pmol kg(-1) h(-1)) were given i.v. for 6 h, and the effects of pretreatment with indomethacin (5 mg kg(-1) h(-1)), N(G)-nitro-L-arginine methyl ester (L-NAME, 3 mg kg(-1) h(-1)) or propranolol (1 mg kg(-1); 0.5 mg kg(-1) h(-1)) on responses to hUII (300 pmol kg(-1) h(-1) for 6 h) were assessed. Cellular localisation of UT receptor-like immunoreactivity was determined in relevant tissues. hUII caused dose-dependent tachycardia and hindquarters vasodilatation, accompanied by a slowly developing rise in blood pressure. Haemodynamic effects of hUII were attenuated by propranolol or L-NAME and abolished by indomethacin. UT receptor-like immunoreactivity was detected in skeletal and vascular smooth muscle. The findings indicate that in conscious rats, infusions of hUII cause vasodilatation, which, of the vascular beds monitored, is selective for the hindquarters and dependent on cyclooxygenase products and nitric oxide. The pressor effect of hUII under these conditions is likely to be due to an increase in cardiac output, possibly due to a positive inotropic effect. UT receptor-like immunoreactivity present in skeletal muscle is consistent with the haemodynamic pattern.

Regional hemodynamic actions of selective corticotropin-releasing factor type 2 receptor ligands in conscious rats.

In conscious male Sprague-Dawley rats, we compared regional hemodynamic actions of the selective corticotropin-releasing factor type 2 (CRF(2)) receptor ligands human and mouse urocortin 2 (hUCN2 and mUCN2, respectively) with those of CRF. Bolus i.v. doses of 3 and 30 pmol kg(-1) hUCN2, mUCN2, or CRF had no significant hemodynamic actions, but at doses of 300 and 3000 pmol kg(-1), all three peptides caused dose-dependent tachycardia and hypotension, with rapid-onset, short-duration, mesenteric vasodilatation and slower-onset, more prolonged hindquarters vasodilatation but little or no change in renal vascular conductance. Pretreatment with the nonselective CRF receptor antagonist astressin or the selective CRF(2) receptor antagonist antisauvagine 30 abolished all the cardiovascular actions of all three peptides. Indomethacin had no effect on responses to hUCN2, and there was no evidence for any involvement of nitric oxide (NO) in the vasodilator actions of hUCN2. There was no evidence that recruitment of angiotensin- and endothelin-mediated vasoconstrictor mechanisms counteracted the vascular actions of hUCN2. The results indicate that the hemodynamic effects of i.v. hUCN2, mUCN2, and CRF depend on activation of CRF(2) receptors and do not involve NO or prostanoids.

Bolus injection of human UII in conscious rats evokes a biphasic haemodynamic response.

A biphasic cardiovascular response to bolus i.v. injection of human urotensin II (hUII, 3 nmol kg(-1)) in conscious, male, Sprague-Dawley (SD) rats was identified and underlying mechanisms were explored. Initially (0-5 min) there was tachycardia, hypotension and mesenteric and hindquarters vasodilatation; later (30-120 min), tachycardia, hindquarters vasodilatation and a modest rise in blood pressure occurred. Pretreatment with indomethacin or N(G) nitro-l-arginine methylester (l-NAME) reduced the mesenteric vasodilator response to hUII, and abolished the late tachycardia and hindquarters vasodilatation. Indomethacin also abolished the hypotension and early hindquarters vasodilatation, and substantially reduced the initial tachycardia. Indomethacin and l-NAME together prevented all haemodynamic responses to hUII. Inhibition of inducible NOS had no effect on responses to hUII, whereas inhibition of neuronal NOS reduced the delayed tachycardic response to hUII but did not significantly affect the vasodilatation. Only the initial tachycardic response to hUII was antagonised by propranolol. In spontaneously hypertensive rats (SHR), the initial haemodynamic responses to hUII were qualitatively similar to those in SD rats, although there was also a modest renal vasodilatation. The secondary response comprised a smaller tachycardia and a small rise in blood pressure, with no significant hindquarters vasodilatation. Haemodynamic responses to hUII were not enhanced by endothelin and angiotensin receptor antagonism in either SD rats or in SHRs. One interpretation of these results is that the primary response to bolus injection of hUII is prostanoid- or prostanoid- and NO-mediated (mesenteric vasodilatation) and that this triggers secondary events, which are dependent on eNOS (hindquarters vasodilatation) and neuronal NOS (tachycardia).

Haemodynamic effects of the selective phosphodiesterase 5 inhibitor, UK-357,903, in conscious SHR.

1. Regional haemodynamic responses to a continuous, 4-day infusion of the selective phosphodiesterase type 5 inhibitor, UK-357,903 (0.133 or 1.33 mg x kg(-1) h(-1)) were measured in conscious spontaneously hypertensive rats, and compared with those of enalapril (1 mg x kg(-1) h(-1)). 2. Both doses of UK-357,903 caused modest reductions in mean blood pressure that were not dose-dependent and only significantly different from the vehicle effects on Day 1 of the study (mean -11.8 and -15.3 mmHg for low and high doses, respectively). UK-357,903 had mesenteric and hindquarters vasodilator effects, which were, again, similar for both dose levels and only significantly different from vehicle on Day 1. Neither dose of UK-357,903 affected renal vascular conductance or heart rate. 3. Although the haemodynamic effects of UK-357,903 were not clearly dose-related and some appeared to wane with time, geometric mean plasma levels of UK-357,903 increased in proportion to dose, and were sustained throughout the infusion period. Furthermore, plasma cyclic guanosine monophosphate, a biomarker of phosphodiesterase 5 inhibition, was persistently elevated, and increased with increasing dose. 4. Enalapril caused a fall in mean blood pressure on day 1 (-14.1 mmHg) that was associated with dilatation in renal, mesenteric and hindquarters vascular beds. The haemodynamic effects of enalapril were sustained or increased over the 4-day infusion, although plasma free drug levels were stable. 5. In conclusion, we have shown regional and temporal changes in the haemodynamic effects of UK-357,903, which may be due to activation of compensatory mechanisms, but there were no signs of functional compensation to the cardiovascular effects of enalapril.

Comparative regional haemodynamic effects of the nitric oxide synthase inhibitors, S-methyl-L-thiocitrulline and L-NAME, in conscious rats.

1. The regional haemodynamic effects of the putative nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC), were compared with those of the nonselective NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), in conscious, male Sprague-Dawley rats. 2. SMTC (0.3 mg kg(-1) bolus) produced a significant, short-lived, pressor effect associated with renal, mesenteric and hindquarters vasoconstriction; the same dose of L-NAME did not affect mean blood pressure (BP), although it caused bradycardia and mesenteric vasoconstriction. 3. At the highest dose tested (10 mg kg(-1)), L-NAME produced a significantly greater bradycardia and fall in mesenteric vascular conductance than SMTC, although the initial pressor response to SMTC was greater, but less sustained, than that to L-NAME. 4. Infusion of SMTC or L-NAME (3 mg kg(-1) h(-1)) induced rises in BP and falls in renal, mesenteric and hindquarters vascular conductances, but the effects of L-NAME were greater than those of SMTC, and L-NAME also caused bradycardia. 5. The renal vasodilator response to acetylcholine was markedly attenuated by infusion of L-NAME, but unaffected by SMTC. The hindquarters vasodilatation induced by salbutamol was attenuated by L-NAME, but not by SMTC. The mesenteric vasodilator response to bradykinin was modestly enhanced by SMTC, but not by L-NAME. The depressor and renal, mesenteric and hindquarters vasodilator responses to sodium nitroprusside were enhanced by L-NAME, whereas SMTC modestly enhanced the hypotensive and renal vasodilator effects of sodium nitroprusside, but attenuated the accompanying tachycardia. 6. The results are consistent with the cardiovascular effects of low doses of SMTC being attributable to nNOS inhibition.