Angiotensin II type 2 receptor agonists – where should they be applied?

Abstract

It is now widely accepted that the renin–angiotensin system (RAS) not only contributes to pathological mechanisms involved, e.g. in hypertension or hypertensive and diabetic end-organ damage, but also harbors a “protective arm” represented mainly by two receptors, the AT2 (angiotensin type 2) receptor and the Mas receptor, both mediating tissue-protective and pro-regenerative actions. Several compounds are currently in preclinical and clinical development, which aim at targeting the “protective RAS” by agonism on the AT2 or the Mas receptor. In a recent issue of Expert Opinion on Investigational Drugs Koen Verdonk and co-authors review the physiology and patho-physiology of the AT2 receptor and discuss potential future clinical indications and putative adverse effects of AT2 receptor agonists. This article comments the review by Verdonk et al., suggests some additional possible indications, and particularly re-reviews whether there is preclinical in vivo evidence for adverse effects of AT2 receptor agonists.

Keywords::

• angiotensin
• AT2R
• cardiology
• renin–angiotensin system

The renin–angiotensin system (RAS) is still mainly associated with blood pressure and volume regulation, hypertension and cardiovascular disease effects, which are all mediated via the AT1 receptor (AT1R). Indications for approved drugs targeting the RAS (ACE inhibitors, AT1R blockers, and renin inhibitors) are also in the cardiovascular field. All of these drugs aim at preventing angiotensin II (Ang II) exerting its effects via the AT1R.

In contrast to the AT1R, the angiotensin AT2 receptor (AT2R) has long been a “neglected child” within the RAS. As reviewed in detail by Koen Verdonk and co-authors in a recent issue of Expert Opinion of Investigational Drugs [1], the AT2R in most cases seems to counteract the AT1R thus acting tissue-protective. This initially unexpected nature of the AT2R has attracted the academic interest of several research groups in the past. Interest in this receptor has further grown substantially with the synthesis of a first drug-like, non-peptide agonist for the AT2R named Compound 21 (C21), which has changed the conception of the AT2R from yet another receptor of academic interest to a pharmacological target for a putative new drug class [2].

Consequently, Verdonk et al. pose the question, where AT2R agonists should be applied? – in other words, what may be future clinical indications?

They suggest that AT2R agonists may be suitable for the treatment of stroke, aneurysm formation, inflammation, myocardial fibrosis, or myocardial infarction. This expert opinion is based on favorable effects of AT2R stimulation in experimental disease models. However, it is not really clear on which basis the authors have made this selection, because not for all of these indications a treatment effect of an AT2R agonist has already been shown [3] in contrast to other indications such as kidney disease, hypertension-induced vascular remodeling, cognitive decline/Alzheimer's disease [4-8], which are not mentioned.

With respect to aortic aneurysm, evidence that AT2R stimulation may delay disease progression is only indirect and based on the observation that a favorable effect of AT1R blockers (ARB) could be blocked by an AT2R antagonist thus suggesting that the effect of the ARB was due to indirect AT2R stimulation by reactively elevated plasma levels of angiotensin II [3]. Still, this is an interesting observation and it is hoped that an AT2R agonist will soon be tested in a model of aortic aneurysm to confirm this indirect evidence.

Regarding stroke, AT2R agonists have so far only been shown to delay onset of symptoms or reduce infarct size and neurological deficits when applied before stroke induction/occurrence, thus in prevention protocols [5,9]. However, the more clinically relevant situation would be applying the drug after the event of stroke. Such studies are currently undertaken by several groups and results are to be awaited.

The authors mention “inflammation” as a potential indication, but this is of course an endlessly wide field. Apart from classical (chronic) inflammatory diseases, inflammation also contributes to the pathogenesis of basically all cardiovascular or renal diseases – though usually in a more secondary way. Nevertheless, anti-inflammation seems an important way of action of AT2R agonists and could be identified to be involved in favorable treatment effects in almost all studies published so far including myocardial infarction, acute and chronic renal disease, and vascular stiffening [4,5,7,10]. However, the proportional impact of the anti-inflammatory effect on the overall amelioration of disease is not yet clear. It has also not yet been clarified whether the anti-inflammatory effect of AT2R agonists is sufficiently strong for the treatment of classical inflammatory diseases such as autoimmune disease (e.g. rheumatoid arthritis, multiple sclerosis). The thought of treating an autoimmune disease by targeting the RAS seems unusual at first sight, but is less so considering that stimulation of the AT2R inhibits NF-κB activity as shown independently by two groups and that the resulting inhibition of cytokine transcription seems to be in the same range as inhibition by a weaker glucocorticoid (hydrocortisone) [10,11]. Again, animal studies are on their way to answer the question whether AT2R agonism may have the potential as a future treatment strategy for chronic inflammatory diseases or whether the anti-inflammatory effect is “only” part of a combination of actions which are effective in (cardiovascular/renal) diseases, in which inflammation contributes to the pathology.

A major proportion of the review by Verdonk et al. is dedicated to the discussion of potential side-effects of AT2 agonists. This is a valid point, because in particular with regard to cardiac hypertrophy and vasoconstriction/vasodilation/blood pressure effects, data about AT2R function are controversial.

With regard to cardiac hypertrophy, it has recently been reviewed in detail that an almost equal number of studies support either pro-hypertrophic, anti-hypertrophic or neutral effects of the AT2R [12]. Notably, and as Verdonk et al. state as well, all studies observing a pro-hypertrophic effect have been performed in genetically altered animals or cells, but not in wild-type animals [12]. The transferability of these results into wild-type organisms is questionable, and this concern is supported by the fact that i) cardiac hypertrophy was only observed in one of two existing AT2R knockout strains and ii) as shown by one research group in two publications, pro- or anti-hypertrophic effects of the AT2R on the myocardium seem to depend on the number of transfected AT2 receptors, with a pro-hypertrophic response only occurring in case of an artificially high number of receptors [12]. Studies looking at cardiac hypertrophy by direct AT2R stimulation may clarify this controversy, but these are still missing. However, although no publication has been addressing cardiac hypertrophy as a main focus, there are data from two independent studies in hypertensive rats (SHR-SP and L-NAME model), which developed left ventricular hypertrophy and in which treatment with C21 over 6 weeks had no effect on heart weight (although treatment was effective in terms of vascular remodeling) [6,7]. Although it is desirable to get more detailed data (histology) about the effect of C21 on cardiac hypertrophy in the future, these two studies rather contradict the concern of Verdonk et al. that cardiac hypertrophy may be a potential side effect of AT2R stimulation.

The second major concern of the authors regarding potential side-effects is that under certain pathological conditions AT2R stimulation may result in vasoconstriction and thus hypertension. Here, the situation is more complex:

First, it should be noted that most studies looking at the effect of AT2Rs on vascular tone and blood pressure (BP) have been performed in isolated vessels ex vivo using Langendorff preparations or Mulvany myographs [1]. In several of these studies, the authors conclude that their ex vivo findings translate into the in vivo situation, i.e. ex vivo vasodilation would result in a fall and vasoconstriction in a rise in BP. This apparently obvious and valid conclusion may not hold true for the AT2R.

For example, while most ex vivo studies on isolated vessels report vasodilation in response to AT2R stimulation, the vast majority of in vivo studies demonstrate that the AT2R is BP neutral [4-8,13]. The recent article by Sanja Bosnyak et al. is one of the rare publications in which ex vivo and in vivo hemodynamic effects of AT2R stimulation are reported in parallel [13]. The authors show that C21 causes vasodilation in mouse aorta, mesenteric artery from normotensive rats, and in aortic rings from SHR. In contrast, C21 did not decrease BP in vivo, neither in normotensive nor in hypertensive rats. The latter results are in concordance with all nine studies published so far which measured BP in response to C21 and in which there was also no BP-lowering effect [4-8,13-15]. The only exceptions are a publication by Gao et al., in which C21 was applied intracerebroventricularly [16], and the original publication about synthesis and design of C21, in which BP was lowered in anesthetized SHR [2]. It is not clear yet why in this single study a reduction of BP was recorded, but it has been speculated that barbiturate anesthesia may have hampered homeostatic reflex mechanisms [13].

While AT2R agonists alone do not seem to lower BP in conscious rats, inhibition of a permanent “angiotensinergic” vasoconstrictive tone in vivo by an AT1R blocker is apparently able to unmask the BP-lowering effect of the AT2R [13]. This effect becomes particularly apparent when the AT1R blocker is used at a low dose that itself has only a minor effect on BP.

However, as stated above, it is not only AT2R-mediated ex vivo vasodilation which does not seem to translate into a lowering of BP in vivo, but also ex vivo vasoconstriction in vessels of SHR. Verdonk et al. based a major concern regarding potential side effects (specifically a rise in BP) of AT2R agonists in future clinical use on AT2R-mediated vasoconstriction that has been reported to occur in SHR and old rats [1]. Again, all of the studies cited by the authors to support their concern are ex vivo studies. In vivo data about AT2R stimulation in SHR can again be found in the study by Bosnyak et al., in which C21 led to a dilation (and not constriction) of aortic rings from SHR and also lowered (not elevated) BP in SHR under concomitant AT1R blockade [13]. However, when infusing C21 at a very high dose (1000 ng/kg/min), they observed a rise in BP. This is not really surprising, because C21 – although very specific for the AT2R with a Ki of 0.4 nM for the AT2R and > 10,000 nM for the AT1R [2] – starts binding and activating the AT1R once plasma/tissue concentrations reach the Ki for the AT1R. This obviously was the case when SHR were infused with 1000 ng/kg/min C21, because the rise in BP could be prevented by blockade of the AT1R with Candesartan [13]. Thus, in the only study published so far which looked at the effect of C21 on BP in SHR, there was definitely no AT2R-mediated rise in BP.

Apart from this single study in SHR, there are two more studies which used stroke-prone SHR (SP-SHR). Both studies report no rise in BP except for a short and transient rise in BP in the study by Rehman et al. in 7-week-old SHR-SP after 1 week of treatment with 1 mg/kg C21 p.o., which was not present any more after 2 weeks and during the remaining 6-week study course [5,7]. It is difficult to decide at present whether this was a valid effect, because Gelosa et al. reported no effect of C21 on BP in SHR-SP of the same age, which however were on a high-salt diet and therefore presented with generally higher BP levels than the rats in the study by Rehman et al. There was also no effect on BP by C21 in hypertension models of two-kidney-one-clip-induced hypertension and L-NAME-induced hypertension [4,6]. In Sprague–Dawley rats, Hilliard et al. reported an ∼ 4 mmHg rise in MAP in response to a graded infusion of 100 – 300 ng/mg/min C21, but this was not statistically significant when compared with controls, in which MAP rose 2 – 3 mmHg as well, but only when compared to animals receiving C21 + PD123319 (AT2R antagonist) [14].

Expert opinion

Since the synthesis of a first drug-like, non-peptide AT2R agonist, the AT2R has become a potential drug target for future clinical use. A clinical Phase I study is planned for C21 in late 2012/early 2013. Entry of Phase I will of course depend on a favorable outcome of an extensive regulatory toxicity program which is currently being implemented by a commercial, certified lab. A Phase I study will then look at dosage, pharmacokinetics and potential intolerances in humans, and only if successful, a Phase II study will follow. Not until this stage, a decision about a first indication for AT2R agonists will be needed. Such a decision will be based on successful tests of C21 in animal models (so far achieved for: myocardial infarction, vascular remodeling, stroke prevention, inflammatory kidney disease, and cognitive decline), on potential problems/adverse effects in animal models (not evident so far), but these are not the only criteria. Since a successful Phase II study will lead the way to a Phase III study which will then eventually decide about approval of the drug, several other determinants have to be kept in mind such as duration/size/costs of Phase II and III studies or the putative future market situation. AT2R agonists are still only at the onset of this developmental process.

Declaration of interest

T Unger has modest financial interest in Vicore Pharma. The authors have no other competing interests to declare.