Prof Michel Azizi 1Faculté de Médecine, The Université

ALDOSTERONE SYNTHASE INHIBITION

Michel Azizi, Paris, France

Chair: Armin Kurtz, Regensburg, Germany

Laszlo Rosivall, Budapest, Hungary

Prof Michel Azizi1Faculté de Médec ine,

The Univers ité P aris Descartes ,

P aris , France

Slide 1

Mr chairman, ladies and gentlemen, dear colleagues, thank you very much for giving me this opportunity to talkabout direct aldosterone synthase inhibitor. I will talk about the history from fadrozole to LCI699.

Slide 2

01/12/12

2/17ndt-educational.org/azizislide2012.htm

This slide shows my conflict of interest very quickly.

Slide 3

So what is today the rationale to investigate aldosterone synthase inhibition? We know that aldosterone is a major hormone which plays a major role in the potassium-sodium regulation and inblood pressure regulation. It has also a pathophysiological role because it has blood pressure independent pro-inflammatory and/or profibrotic effects which at the end gives target organ damage and specifically in the presenceof a high sodium diet. It is involved in cardiovascular and renal disease pathophysiology and prognosis and we know also that it has beenshown that there is an association between serum aldosterone levels and the incidence of hypertension andresistance to therapy. We also know that we have drugs to block the effects of aldosterone. These are the mineralocorticoid receptorblockers such as spironolactone and eplerenone. However, there are some limitations of mineralocorticoid receptorantagonists. We know that there is an increase in renin and aldosterone when we give to patients this type of drugs.Maybe this increase in renin and aldosterone could participate to resistance to treatment. We also know that spironolactone has a non-specific profile and is binding to the progesterone and androgenreceptors, so it has side effects such as sexual side effects: impotency, ginacomastia, menstrual irregularities. So itsuse is limited by this safety profile. On the other side, we have eplerenone which does not display this type of side effects but however, it’s much lesspotent than spironolactone specifically in patients who have a high aldo level like in patients with primary aldosteroneand this is an unmet medical need. We also know that there are both genomic and non-genomic effects of aldosterone and the non-genomic effects ofaldosterone which are partly independent from the MR or are not blocked by MR antagonists.

01/12/12 CME Slides Forum - M. Azizi


A third reason why we should also investigate aldosterone synthase inhibition is that over expression in animalmodels of aldosterone synthase of about 150% in mice is associated with an increased blood pressure on a highsodium diet. There have been associations with the CYP11B2 which is the gene coding for aldosterone synthase polymorphismwith blood pressure. Fadrozole, which is an aromatase inhibitor, has provided the first lead compound allowing to inhibit aldosterone.

Slide 4

This slide is just to remind you how aldosterone is synthesized in the zona glomerulosa of the adrenal cortex fromcholesterol. Aldosterone synthase here will catalyse the last 3 catalytic steps of aldosterone synthases. It catalyses the 11-hydroxylation of 11-deoxycorticosterone to corticosterone. Then 18-hydroxylation of corticosterone to 18-OH-Corticosterone. Finally, an 18-oxidation of this 18-OH corticosterone to aldosterone. So these three steps are completely under the dependence of aldosterone synthase.

Slide 5

It is very interesting to notice immediately that the terminal steps of the aldosterone synthase synthesis are veryclose to the cortisol synthesis. There is another enzyme which is 11-β hydroxylase and is the product of the CYP11 B1 gene which converts 11-deoxycortisol in cortisol in the zona fasciculata of the adrenal gland. So what should we know about these two enzymes? So aldosterone synthase is approximately 93% homologous to11-β-hydroxylase. Which converts 11-deoxycortisol to cortisol.



Secondly these two genes CYP11B1 and CYP11B2 are mapping at the same human chromosome 8q21-22. There isjust very important difference that is the zonation where these enzymes are expressed. CYP11B2 is only expressed in zona glomerulosa and is under the regulation of potassium and angiotensin II.Whereas, CYP11B1 is mainly expressed in zona fasciculata and zona reticularis and is under the dependence mainlyof ACTH.

Slide 6

So the lead compound which allowed to investigate the effects of aldosterone synthase was fadrozole, a structurallyderived inhibitor which is the compound FAD 286A.

Slide 7

So fadrozole is at a low dose 2 mg/day, is a non-steroidal aromatase inhibitor effective in the treatment of advancedbreast cancer. Aromatase is a CYP450 enzyme which is responsible for the conversion of oestrogen in post-menopausal womenwithin the adrenal gland. During the development of fadrozole it was observed that there were some anecdotal cases of hyperkalaemia whichprovoked the investigation of why such hyperkalaemia could occur and whether this fadrozole could inhibitaldosterone synthase. This was in reality the case.

Slide 8



At doses above 4 mg/day it was shown in this very elegant study that it impaired aldosterone secretion in humanswithout impairing glucocorticoid synthesis. So, fadrozole at this dose was administered for 14 days against placebo inhealthy volunteers. These healthy volunteers were challenged by an ACTH test. So these are the aldosterone levels, the first is the pre-treatment day before and after ACTH so you have a rise, a massive rise in aldosterone. After 14 days this is the effect under placebo. So you still have a massive increase in aldosterone after ACTHinjection, whereas you clearly see that this effect is completely blunted by fadrozole at this dose. There was no effect here on the cortisol stimulation and cortisol response and there was an accumulation here of thealdosterone substrate which is 11-deoxycorticosterone, you see here the effects under placebo and you see thiseffect and accumulation of this precursor after inhibition by fadrozole.

Slide 9

Fadrozole has two enantiomers and the dextro-enantiomer of fadrozole is the compound FAD 286A which is muchmore selective for aldosterone synthase than for aromatase inhibition. These are in vitro data, this is the fadrozolecompound and here you have the FAD compound. You see here the IC50 for aromatase is much higher, whereas you haven’t lost here the IC54 inhibition ofaldosterone synthase.

Slide 10



These are the preclinical and in vitro and in vivo data. So these are the IC50 here reported for CYP11B2 andCYP11B1, these are recombinant human CYP11B1. The IC50 is low, 1.6 nanomolars here against CYP11B2 and yousee however, that it is not a very selective compound because the ratio of selectivity is around 6 only because theIC50 here is 9.9 nanomolars. Whereas, the in vivo effects in a different experimental model. First, yes it decreases plasma and urine aldosteronein animal models and increases PRA. It has also a cardioprotective effect here, it prevents death and reduces cardiachypertrophy and proteinuria in dTGR rats to human renin and angiotensinogen. It has also other cardioprotectiveeffects in the rat model of cardio congestive heart failure. Also nephroprotective effects in a model of albuminuriaand azotaemia which is in uninephrectomised rats treated with angiotensin II and high salt model. It has an effectwhich is similar to that of spironolactone.

Slide 11

So the first compound administered to humans is LCI699 and I will show you very quickly the effect of this compoundin humans.

Slide 12



This is the first-in-class alloreactive aldosterone synthase inhibitor. It is structurally derived from the compoundFAD286A. It is rapidly absorbed within 1 hour. It has a short half-life of around 4 hours, no accumulation. Itsexposure is dose-linear and dose-proportional within the dose range of 0.5 to 3 mg. It was well-tolerated after a 2week administration for doses below 3 mg in healthy volunteers on a normal controlled salt diet. On higher doses,up to 10 mg there were clearly signs of hypocortisolism and hypoaldosteronism, so there’s a limitation with this drugof the dose ranges that can be investigated.

Slide 13

This is the effect on plasma aldosterone, just the upper part of the slide, and urinary aldosterone. The control groupwere the placebo group and the active control group was the eplerenone group. So you clearly see that there’s adecrease in plasma aldosterone on day 1 which persists on day 7. You also clearly see that it’s a short acting drug,that after 12 hours the concentration of plasma aldosterone and here after 7 goes up back to the initial levels. In urine excretion data you see this dose dependency of decrease in aldosterone and you see clearly thateplerenone had the reverse effect so it increases, as expected, plasma and urine aldosterone concentrations.

Slide 14



So after this phase I first-in-class study we performed a proof of concept study in patients with primaryaldosteronism.

Slide 15

So this is the study design. This was a single blind placebo controlled sequential and forced-titration study. So 14patients with primary aldosteronism entered the study. They went through a first washout period of 6 weeks to stopall anti-hypertensive drugs interfering with the RAS. Then they received a placebo for 15 days and again from day 1to day 15 they received 0.5 mg of LCI699 bid, force-titrated to 1 mg LCI699 bid for 15 days and again they receiveda placebo. After this we switched the patients to eplerenone 50 mg bid, up titrated to 100 mg bid for one month. These patients received anti-hypertensive drugs to maintain, because we didn’t know when we started the studywhether this LCI would reduce blood pressure, so they received calcium channel blockers and α-blockers.

Slide 16



First, let’s see what’s happening we expect on the classical effects of such types of drugs, whether it will reverse thehypokalaemia observed in patients with primary aldo.So these are the baseline levels, so patients had a low potassium level which after 15 days came even lower that hasa mean value of 3.2 mmol/l. You see that very quickly with 0.5 mg of LCI it increases, it normalizes plasmapotassium. Patients were receiving oral potassium intake here, we stopped oral potassium so this is why it decreasedwith the doubling of the dose. Therefore here patients did not receive any more oral potassium supplementation.When we stopped the drug, within one week you see we have a perfect complete reversal of this effect withhyperkalaemia recurrence. Eplerenone administration here increases as expected plasma potassium levels and indeed this rise in plasmapotassium was significantly higher with the one observed with LCI699 at this dose of 1 mg bid.

Slide 17

So we also expected the blood pressure effect and we were surprised by this very mild effect in terms of bloodpressure lowering. So remember the drug was given for 15 days at 0.5 mg bid and then 1 mg bid for 15 days. Wehad a statistically significant drop in ambulatory blood pressure monitoring of minus 4 mmHg systolic blood pressurewhich was not significant for diastolic blood pressure. You clearly see here that we had a greater efficacy when we gave eplerenone with a decrease of around 9-10 mmHgsystolic blood pressure in terms of ambulatory blood pressure monitoring. This effect was significantly higher thanthis effect observed with LCI699 but it was a sequential study and not a perfectly randomised cross-over study.

Slide 18



So in terms of plasma aldosterone what happened? The things that we expected. So plasma aldosterone hereexpressed in Picomoles/l were high. Then when we gave LCI 0.5 bid, it dropped by around 80% and remainedstable. There was no dose dependency on plasma aldosterone levels whereas we saw this dose dependency in theurinary aldosterone levels. When we stopped the drug, you see that this is a short acting drug very quickly plasmaaldosterone levels came back to their initial levels and when we gave eplerenone, as expected, there was stimulationof aldosterone concentrations which achieved levels which were higher than the initial levels when the patient enteredthe study. Now let’s look at the plasma renin concentration. There was a very mild increase, around a 30% increase in plasmarenin concentrations with this drug. So we were surprised that this indicated that we could not achieve enough sodiumdepletion and clearly this was statistically significant and clearly with eplerenone we can achieve much higher sodiumdepletion than with LCI699.So this may explain why we did not observe perfectly a major drop in blood pressure in these patients who haveprimary aldosteronism.

Slide 19

LCI699 is an aldosterone synthase inhibitor, so we clearly observe a drop in plasma aldosterone levels and also adose-dependent increase here in 11 deoxycorticosterone which is the aldosterone precursor. This effect was veryquickly reversible, so we can immediately see that we achieved aldosterone synthase inhibition in patients withprimary aldo.

Slide 20



What were the effects on the cortisol pathway? So there was no change here in plasma cortisol levels during thewhole duration of the trial but you see clearly that you have accumulation, a dose-dependent accumulation of thecortisol precursor with an increase in plasma 11-deoxycortisol levels which is reversible 1 week after stopping LCI699.

Slide 21

This effect on the glucocorticoid axis was responsible for a dose-dependent increase in plasma ACTH levels. Soplasma ACTH remained normal here, no patient had values above 100 pmol/l. So clearly we can observe here thatthis aldosterone synthase inhibitor is a potent inhibitor of aldosterone synthase but also inhibits partially 11-β-hydroxylase and both effects are reversible after 1 week interruption of LCI699.

Slide 22



Now let’s look at what happened after ACTH stimulation. These are the baseline data on plasma aldosterone. So youhave a massive increase in plasma aldosterone which is completely blunted by LCI699. These are the cortisol data,an increased at baseline which is also completely blunted which also confirms that we have a blockade of theconversion of 11-deoxycortisol to cortisol. So clearly we inhibit also the 11-β-hydroxylase pathway in theglucocorticoid site.

Slide 23

So whereas, the conclusion of the proof of concept study so LCI699 up to 1 mg bid only modestly decreases bloodpressure compared to baseline in patients with primary aldo and this effect was much lower than the one observedwith eplerenone 50 mg bid. It normalised potassium plasma concentration as early as day 8 in all patients withprimary aldo and this effect was slightly lower than the one observed with eplerenone. It was clinically and biologicallywell-titrated, we didn’t observe any signs of hypoaldosteronism or hypocortisolism.

Slide 24



So how can we interpret all these effects? So we fulfilled some expectations. First plasma potassium increased andwe have a decrease in aldosterone production towards normal values, so this is expected. The unexpected result iswhy we didn’t observe an increase in PRA or in renin concentrations and a drop, a more important drop in bloodpressure. First we could think that finally 80% inhibition of aldosterone is not enough despite bid administration. Theproblem is that we cannot increase the dosage of this drug because of this effect on the glucocorticoid side. The second problem we had is we observed a massive increase in plasma 11-deoxycorticosterone by more than700% which s dependent on both sites, first inhibition of CYP11B2 because we inhibited in reality aldosteronesynthase but also because as you have observed, we have an increase in ACTH which will stimulate also thedeoxycorticosterone synthesis inside the zona fasciculata. So there are two reasons of this counteregulation and 11-deoxycorticosterone had a mineralocorticoid effect. So itcan counteract the effects of aldosterone synthase. The third point is the difficulty to define steady states after withdrawal of all the antihypertensive treatments. Inthese patients with primary aldo it is known that when you stop the drugs, even if we take a long wash out ofdifferent drugs, the new steady states in patients with primary aldo is achieved for hormones, for potassium and forrenin but not for blood pressure and maybe because of the sequential nature of this trial this is why we couldn’tobserve an important drop in blood pressure.

Slide 25

So after this study a phase IIa study was performed in patients with essential hypertension.

Slide 26



It’s a typical clinical trial – group study in patients with hypertension which compared 4 doses of LCI699 to placeboand also to a control group which was eplerenone 50 mg bid. So the LCI was given 0.25 mg/day, 0.5 mg/day, 1 mg/day and 0.5 mg/bid. This is the primary endpoint which was adrop in diastolic blood pressure but you have similar data for systolic blood pressure and ambulatory blood pressuremonitoring. So for diastolic blood pressure only the 1 mg dose of LCI699 decreased significantly blood pressure when comparedto placebo. This drop was similar to eplerenone 50 mg/bid. Very interestingly, you have here a lower efficacy of 0.5 mg/bid of eplerenone. We think that this is also due to muchhigher counteregulation achieved when the drug is given twice daily than when only given once daily.

Slide 27

Again, giving LCI699 did not modify plasma cortisol levels but you clearly see that in the black bars here you have adose-dependent increase in plasma ACTH showing that aldosterone synthase inhibitor is not selective for CYP11B2but also inhibits CYP11B1.

Slide 28



So to conclude, LCI699 is a short acting aldosterone synthase inhibitor which partially loses its specificity foraldosterone synthase at doses above 0.5 mg once a day. The use of 0.5 mg once a day of LCI in the treatment ofessential hypertension remains to be discussed in the view of the results we have observed. The expected benefitsof aldosterone synthase inhibition decreasing exposure to aldosterone by comparison to eplerenone, increase inexposure to eplerenone have not yet been investigated in humans. For this compound probably the future is anotherdisease at the last --- Society meeting it was shown that lCI699 at much higher doses, above 10 mg/day because itis massive 11-β-hydroxylase inhibitor is probably a treatment for Cushing syndrome and it normalizes cortisol levelsin patients with Cushing syndrome. So what does the future hold?

Slide 29

First, there are new aldosterone synthase inhibitors derived from metyrapone and these aldosterone synthases whichat the present time have been tested in vitro are much more selective for CYP11B2 you can clearly see very low IC50here and for some compounds very high IC50 for CYP11B1 with a selectivity factor here going from 100 to around200. So we will probably have other compounds which have to be tested in humans that don’t have this problem of non-selectivity against CYP11B1.

Slide30



But however even with this type of compound they will have to be compared to mineralocorticoid receptor antagonistseither the older ones spironolactone and eplerenone

Slide 31

but also newer compounds which are dehydropyridine-based mineralocorticoid receptor antagonists. This compoundhere is derived from a calcium channel blocker but it has no effect on the calcium channel. These are all the IC50, ithas the same efficacy on the mineralocorticoid receptor than spironolactone, it’s much more potent than eplerenonebut it has no effect here on the glucocorticoid receptor, on the androgen receptor or on the progesterone receptor. Sostill tomorrow we will have new investigations performed. Thank you very much for your attention.

Slide 32



Chairman: Doctor Azizi thank you very much for this clear and very interesting presentation and in particular also forthe critical discussion of data. So the presentation is now open for discussion. We have time for a few questions. Somay I start with the first one? You mentioned that the advantage of this aldosterone synthase inhibitor versus thealdosterone receptor antagonist might be that the blockade of the synthase would prevent an exposure to very highaldosterone concentrations. What is the evidence that exposure to high aldosterone concentrations have adetrimental effect if the receptor is blocked?

Prof. Azizi: So, you’re perfectly right the question is as I very quickly mentioned there is no clinical evidence, that Ican assure you but in terms of biology you know that there are both genomic and non-genomic effects so these non-genomic effects don’t depend on the blockade of the receptor. So if we believe that this is still important in terms ofsome biological effects or even noxious biological effects, this is not blocked by aldosterone receptor antagonist. Butthe drawback also could be that by giving aldosterone synthase inhibitors we leave the receptor, the mineralocorticoidreceptor free. So maybe some others specifically in Australia say that the glucocorticoid can still bind to this receptorand may be this is not a good thing that glucocorticoid can bind to the receptor specifically in organs where there isno 11-β– --- which inhibits the effect of glucocorticoid.

Chairman: Thank you Geneviève please a short question.

Question: Yes a very short question. You mentioned that Cushing’s syndrome could be treated with the LCI what wastheir blood pressure level?

Prof. Azizi: It was just presented a few weeks ago I have only the cortisol data. So it normalizes cortisol levels I don’tknow the blood pressure and even in the abstract it’s written that there’s hypokalaemia. I cannot understand why thiscompound could induce hypokalaemia. So I don’t know the blood pressure effects in these patients, I only know thatit normalizes cortisol, that’s all.

Chairman: Last question.

Question: Yes, you mentioned the increase in renin secretion. Did you measure the angiotensin concentration? Thesecond question is have you noticed any kind of difference in efficacy depending on sex and age?

Prof. Azizi: So angiotensin concentration, we did not measure angiotensin concentration because in our patients itwas just a first in-patient study so we had to measure a lot of safety parameters, so we drew a lot of blood so it wasimpossible to measure angiotensin levels so we didn’t measure them. In terms of gender in the patients withprimary aldo because it was a first in-patient study we could only include post-menopausal women so we had onlyone woman but in the hypertension study in – study there were both men and women and there was absolutely nodifferential effect depending on the gender.

Question: And the age?

Prof. Azizi: No the same thing.

Chairman: Ok thank you very much for the discussion.

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Prof Michel Azizi 1Faculté de Médecine, The Université