Why did the NHE inhibitor clinical trials fail?

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<ul><li><p>fa</p><p>tral</p><p>causing a rise in [Na+]i which then stimulates a rise in cytosolic Ca2+</p><p>ain aree risehat it does. I propose that wethe following questions.</p><p>rise in [ +</p><p>during ischemia. This interpretation was questioned based on thefollowing points. Firstly, the low extracellular pH during ischemia hasbeen suggested to inhibit NHE. Secondly, amiloride was shown to also</p><p>result in [Na ]i entry during ischemia. Thirdly, although amilorideinhibited the rise in [Na+]i during ischemia, it did not increase the fallin pH during ischemia as might be expected with inhibition of NHE.</p><p>Journal of Molecular and Cellular Cardiology 46 (2009) 137141</p><p>Contents lists available at ScienceDirect</p><p>Journal of Molecular an</p><p>.e lperfused hearts [611]. Some earlier studies by Allen and coworkers,using rat hearts paced at 2 Hz and at 35 C, found little or no increasein [Na+]i during ischemia [2,5,11]. The smaller rise in [Na+]i in thesestudies could be related to the lower rate of beating and lowerischemic temperature. Consistent with the effect of metabolic rate on[Na+]i during ischemia, a recent study byWilliams et al. with ischemiaat 37 C and pacing at 5 Hz reported a 10mM increase (a doubling) in[Na+]i during ischemia [6]. This increase is still somewhat lower than</p><p>+ 23</p><p>occur immediately and therefore NHE will operate during thebeginning of ischemia leading to a rise in [Na+]i. The second pointregarding the inhibition of persistent Na+ channels by amiloride andrelated compounds is an issue that was not fully recognized in ouroriginal paper, in which we attributed all of the amiloride dependentinhibition of the rise in [Na+]i during ischemia to inhibition of NHE. Itis clear that amiloride can inhibit persistent Na+ channels andtherefore some of the amiloride dependent increase in [Na+]i duringthe typically 2030 mM increase in [Na ]i o[7]. InterestinglyWilliams et al. used female raImahashi et al. [12] found that females had alower rise in [Na+]i during ischemia. Taken</p><p> Corresponding author.E-mail address: murphy1@mail.nih.gov (E. Murphy).</p><p>0022-2828/$ see front matter. Published by Elsevier Idoi:10.1016/j.yjmcc.2008.09.715Na ]i during ischemia in as inhibition. More importantly, the fall in extracellular pH does not1. Does [Na+]i rise during ischemia?</p><p>Most data in the literature show anumber of recent clinical trials. The mto be whether NHE contributes to thwould argue that the data suggest tdiscuss the controversy by focusing onextracellular pH will reduce the rate of NHE activity, some low levelof activity can still occur [13]. Slowing the rate of a NHE is not the sameWe have responded to these issues as follows. Although lowa of disagreement seemsin Nai during ischemia. I</p><p>block persistent Na+ channels, and these persistent Na+ channels will+via the NaCa exchange. This controversy has implications for aPoint/Counterpoint</p><p>Why did the NHE inhibitor clinical trials</p><p>Elizabeth Murphy a,, David G. Allen b</p><p>a National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20892, USAb School of Medical Sciences and Bosch Institute F13, University of Sydney, NSW 2006, Aus</p><p>a r t i c l e i n f o</p><p>Article history:Received 11 July 2008Received in revised form 16 September 2008Accepted 18 September 2008Available online 5 November 2008</p><p>Murphy</p><p>Karmazyn [1] rst showed that the NaH exchange (NHE) inhibitoramiloride improved the contractile recovery of the isolated rat heartfollowing ischemia. This result has been repeatedly conrmed in arange of animal models and with various NHE1 inhibitors and hasnaturally triggered interest in whether ischemic damage to humanhearts would show a similar benet.</p><p>There has been some disagreement regarding the involvement ofthe plasma membrane NHE in the rise in intracellular Na+ ([Na+]i)during ischemia [26]. Fig. 1 illustrates the proposed role of NHE in</p><p>j ourna l homepage: wwwbserved with Na NMRts, and a recent study byslightly but signicantlytogether, although there</p><p>nc.il?</p><p>ia</p><p>may be some difference in the extent of the rise in [Na+]i duringischemia depending on pacing rate, temperature, and gender, thereappears to be agreement that in beating perfused hearts paced at 5 Hzat 37 C there is an increase in [Na+]i during ischemia.</p><p>2. Is NHE responsible for the rise in [Na+]i during ischemia?</p><p>A number of studies have reported a rise in [Na+]i during ischemiathat was blocked by NHE inhibitors such as amiloride [7,8]. Weinterpreted these data as showing a role for NHE in [Na+]i loading</p><p>d Cellular Cardiology</p><p>sev ie r.com/ locate /y jmccischemia is likely due to amiloride mediated inhibition of persistentNa+ channels; we have acknowledged this point in reviews over thepast 10 years [4]. Inhibition of persistent Na+ channels by TTX andlidocaine has been shown to reduce the rise in [Na+]i during ischemia[6,9]. However addition of lidocaine reduced but did not block the risein [Na+]i during ischemia [9], suggesting that other mechanisms areinvolved. Furthermore a role for Na+ channels in the rise in [Na+]i</p></li><li><p>cha</p><p>138 E. Murphy, D.G. Allen / Journal of Molecular and Cellular Cardiology 46 (2009) 137141during ischemia does not preclude a role for NHE. Indeed recentstudies with more specic NHE inhibitors show that these morespecic inhibitors also reduce the rise in [Na+]i during ischemia[1416]. Also, consistent with NHE-dependent effects during ische-mia, mice lacking NHE were shown to be resistant to ischemiareperfusion injury with better preservation of ATP during ischemia anda reduced ischemic contracture [17]. These benecial effects areconsistent with an effect of NHE during ischemia. Furthermore theredoes not appear to be a clear relationship between inhibition ofpersistent Na+ channels and inhibition of the rise in [Na+]i duringischemia. Williams et al. [6] report that zoniporide reduced persistent</p><p>+</p><p>Fig.1. [Na+]i has been shown to rise during ischemia concomitant with a fall in pH. The meto activation of NHE, activation of persistent Na channels or a combination of both.Na channels to 41% (a 60% reduction), but yet had no effect on the risein [Na+]i during ischemia. In contrast, Baetz et al. [18] report thatcariporide reduced the Na+ current by 24%, but the rise in [Na+]i duringischemia by over 50%. If the rise in [Na+]i during ischemia is due onlyto the inhibition of the persistent Na+ channels, these results aredifcult to interpret. Regarding the third point, the NHE is not the onlypH regulatory transporter and although inhibition of NHE would beexpected to change the rate at which pH reaches its set point, it wouldnot necessarily alter the nal pH. Also inhibition of NHE duringischemia would reduce the Ca2+ load during ischemia and this wouldreduce Ca2+ activation of the sarcoplasmic reticulum ATPase andthereby reduce metabolism and generation of metabolic acid. Thereduction in Ca observed with NHE inhibitors would also be expectedto indirectly alter pHi by reducing [Ca2+]i release of H+ from commonbinding sites [19]. Thus, I would suggest that both NHE and persistentNa+ channels contribute to the rise in [Na+]i during ischemia.</p><p>3. Does [Na+]i rise during reperfusion?</p><p>In perfused heart studies using 23Na NMR, there is little or nomeasurable increase in bulk [Na+]i on reperfusion [10,20]. However,this lack of rise in [Na+]i on reperfusion does not mean that NHE doesnot result in Na+ entry during ischemia. Indeed if the NaK ATPase isinhibited during reperfusion there is a measurable rise in [Na+]i onreperfusion, which is reduced in the presence of NHE inhibitors [10].Taken together most data suggest that NHE is active on reperfusionand results in Na+ entry into the myocytes; however the Na+ thatenters via NHE is rapidly removed from the myocyte via NaKATPaseand the NaCa exchanger. In contrast to the studies showing little orno rise in [Na+]i during reperfusion, Williams et al. report a very largerise on reperfusion to levels of 40 mM [6]. The reasons for this largerise in [Na+]i on reperfusion are unclear and I would be interested inDr. Allen's thoughts on this issue.</p><p>4. Is NHE responsible for the rise in [Na+]i during reperfusion?</p><p>I think there is generally an agreement that NHE is active duringreperfusion. Inhibitors of NHE slow the rate at which intracellular pHreturns to normal [20]. Inhibitors of NHE will also reduce the rise in</p><p>+</p><p>nism for the rise in Na during ischemia is somewhat controversial. It has been attributed[Na ]i during reperfusion in hearts in which the NaK ATPase isinhibited [10].</p><p>Allen</p><p>We both agree that [Na+]i rises during ischemia and that the criticalquestion is the mechanism by which NHE inhibitors achieve thereduction in [Na+]i during ischemia. We also agree that duringreperfusion there is additional Na+ entry which often causes a furthertransient rise in [Na+]i e.g. Fig. 2A. Murphy et al. [7] showed thatamiloride reduced the ischemic rise of [Na+]i and this result has beenconrmed by many groups e.g. Fig. 2B. Murphy et al. proposed thatNHE1 had been activated by the large intracellular acidosis of ischemiaandwas removing H+ in exchange for Na+ entry. You now concede thatthis interpretation is challenged by (i) the large extracellular acidosiswould be expected to inhibit NHE1 [21], (ii) the fact that all NHE1inhibitors so far tested (amiloride, EIPA, cariporide and zoniporide)appear to block the persistent Na+ channels [6,18,22,23] and (iii)although NHE inhibitors reduce the rise of [Na+]i they do not increasethe magnitude of the acidosis. A further challenge to this interpreta-tion is the nding that metabolic inhibition [24,25], hypoxia [11] andischemia [11] have all been shown to inhibit NHE1 although themechanism remains uncertain [26].</p><p>An alternative (or additional) hypothesis to explain the ischemicrise in [Na+]i is that it arises through Na+ channels. During ischemiaconventional (transient) Na+ channels are inactivated by depolariza-tion [27] but the activity of persistent Na+ channels are both enhancedby hypoxia and unaffected by depolarization [28]. A number of studies</p></li><li><p>d at</p><p>139E. Murphy, D.G. Allen / Journal of Molecular and Cellular Cardiology 46 (2009) 137141have shown that the rise of [Na+]i during ischemia can be blocked bydrugs which block the persistent Na+ channels [2,23,29] and anexample is shown in Fig. 2C.</p><p>You argue above that the problem of persistent Na+ channelinhibition by NHE inhibitors may be minimal on the grounds thatmore specic NHE inhibitors also block the rise of [Na+]i. RecentNHE1 inhibitors are certainly more potent and more selective againstNHE1 compared to the other NHE subtypes [30] but whether they aremore specic, in the sense of relatively greater NHE1 inhibition v. sideeffects such as blocking persistent Na+ channels, remains to bedetermined. For example, we recently tested zoniporide, which is apotent inhibitor of NHE1 [30] and found it to have substantially lesspersistent Na+ channel blockade than amiloride, but, as shown in Fig.2D, it had no effect on the rise of [Na+]i during ischemia in contrast toyour assertion.</p><p>Thus many studies support the idea that persistent Na+ channels</p><p>Fig. 2. [Na+]i measurements from isolated rat hearts (37 C, 5 Hz stimulation rate, perfuseas indicated by lower bar. Modied from Williams et al. [6].contribute to the rise of [Na+]i during ischemia, and the quantitativerole of NHE1 has yet to be reassessed following the discovery thatmany NHE1 inhibitors also block persistent Na+ channels. In contrastthere is general agreement in the eld that the Na+ entry duringreperfusion is principally through the NHE1 and is consequentlyblocked by either amiloride or zoniporide (Figs. 2B, D) but not by a lowconcentration of tetrodotoxin (Fig. 2C).</p><p>An interesting feature of the literature is that in most studies e.g.[7] [Na+]i falls monotonically on reperfusion whereas in some studies[Na+]i rises transiently on reperfusion (e.g. Fig. 2A). Presumably [Na+]irises if Na+ inux through NHE1 and other sources is greater than Na+</p><p>extrusion by the Na+ pump and the Na+/Ca2+ exchanger. An interestinginsight into this variability is provided by a study of cardiac ischemiainwhich Na+ pump activity and [Na+]i weremeasured in hearts duringlow ow ischemia [31]. When glucose was absent from the perfusate,the rise in ischemic [Na+]i was large and the Na+ pump was inhibited,and when these hearts were reperfused the [Na+]i fell monotonically,suggesting that reactivation of the Na+ pump and efux on the Na+/Ca2+ exchanger overwhelmed any Na+ inux associated with NHE1activity. Conversely, when glucose was present, the Na+ pump showedcontinued activity during ischemia and the [Na+]i rose minimally, andon reperfusion, [Na+]i showed a transient rise. Thus aspects of themetabolic state dependent on the degree of ischemia inuence therise of [Na+]i during ischemia and reperfusion and may well inuencethe response to NHE1 inhibitors.These issues are important because they inform how and when toadminister an NHE1 inhibitor or a persistent Na+ channel blocker toobtain maximal protection during an ischemic episode. It is generallyagreed that the level to which [Na+]i rises is inversely related to therecovery of function after a period of ischemia/reperfusion; thus theaim of treatment is to minimize the rise in [Na+]i. Obviously since Na+</p><p>appears to enter during both ischemia and reperfusion it would belogical to block both paths if feasible; thus optimal therapy wouldinclude a persistent Na+ channel blocker and an NHE1 inhibitor duringthe ischemic period and a NHE1 inhibitor during the reperfusionperiod. Thus it may yet turn out to be a clinically valuable feature thatmost NHE1 inhibitors also block persistent Na+ channels.</p><p>Murphy</p><p>Given the very strong experimental support for cardioprotection</p><p>a constant ow rate) subjected to 30 min global ischemia (ow stopped). Drugs appliedbyNHE inhibitors in animalmodels of cardiac ischemia, a key questionis understanding why the clinical trials with NHE inhibitors failed toshow protection.</p><p>Clinical trials to test the benecial effects of NHE inhibitors in thesetting of myocardial infarction have largely failed to show a benet[32]. A post hoc analysis, of a study in which the NHE inhibitorcariporide was administered to patients undergoing coronary arterybypass (CABG), showed a benecial effect [33]. In this study, a one-hour intravenous infusion was given before surgery and administeredevery 8 h for 2 to 7 days. This benecial effect in CABG patients wasattributed, at least in part, to the administration of cariporide to theCABG cohort prior to surgery (ischemia). A follow up trial to test theeffect of cariporide in CABG used a higher dose of cariporide and thetrial was stopped due to an increased incidence of stroke. Interestinglyin this prospective trial the NHE inhibitor cariporide given prior toCABG signicantly reduced myocardial infarction (MI) and MI deathscompared to the placebo group. However, this benecial effect wasoffset by an overall increase in mortality from 1.5% in the placebogroup to 2,2% in the cariporide group; this increase in mortality wasdue to an in...</p></li></ul>