Hiding in Plain Sight: Nebivolol Exhibits Compelling Tocolytic Properties

Abstract Preterm birth before 37 weeks of completed gestation results in numerous health consequences for the foetus. Preterm labour leads to preterm birth in over 50% of cases, and no FDA‐approved treatment can prevent labour or help a foetus remain in the womb until term. Examination of nitric oxide mediated relaxation signaling in the uterine smooth muscle reveals a role for protein S‐nitrosation. The recent discovery of upregulated S‐nitrosoglutathione reductase (GSNOR) in spontaneously preterm labouring women has emphasized the need to explore the function of S‐nitrosation regulation in the maintenance of uterine quiescence. Here we have examined the ability of nebivolol to relax uterine smooth muscle and tested recent claims that nebivolol is a GSNOR inhibitor. In uterine smooth muscle strips from both mouse and human, nebivolol relaxes oxytocin‐induced contractions in a dose dependent manner. Our data indicates that nebivolol has no effect on GSNOR activity, nor does nebivolol inhibit thioredoxin reductase, two of the major protein denitrosylases. The ability of nebivolol to relax uterine smooth muscle is likely the combined effects of increased nitric oxide synthase activity and β3‐adregnegic stimulation.


Spontaneous Preterm Labour (sPTL) leads to Preterm Birth (PTB).
PTB remains the primary cause of neonatal morbidity and hospitalization during pregnancy, 1 and in the United States alone one in eight infants are born prematurely, resulting in 20,000 deaths annually. 2 Currently available drugs are not effective at delaying birth beyond 48-hours. 3 This observation is not surprising when we consider that tocolytics, in general, are borrowed pharmacology. That is to say, nearly every drug used to treat PTL was initially intended to treat maladies in other smooth muscle types, such as vascular, colonic and airway. Even atosiban (Tractocile ® ; Ferring Pharmaceuticals, Parsippany, NJ), a selective oxytocin-vasopressin receptor antagonist designed specifically to mitigate uterine contractions, is not approved for use in the United States and does not reduce the risk of preterm birth beyond 48-hours or improve neonatal outcome. 4 The myometrium is a unique subclass of smooth muscle that exhibits a signalling exception to the dogma that global cGMP accumulation drives nitric oxide (•NO)-mediated relaxation. It is well-established that term human myometrium relaxes to •NO in an cGMP-independent fashion. [5][6][7] •NO is a critical mediator of uterine quiescence; however, •NO's most pervasive role in the myometrium is through protein S-nitrosation (SNO), 8 rather than cGMP accumulation. S-nitrosoglutathione reductase (GSNOR) is an important modulator of •NO and S-nitrosation. 9 GSNOR is upregulated in human sPTL myometrium, and inhibition of GSNOR by N6022 can partially restore native myometrial function. 6 At first glance, nebivolol seems like an unlikely tocolytic. Nebivolol is a third generation, FDA-approved β1-adregneric receptor (β1AR)
Absorbance at 340 nm (A340) was recorded at t = 0, 5, 10 min to ensure stability of the NADH pool prior to the addition of GSNO and/or inhibitors. N6022 (8 nmol L −1 ) (S77589: Selleck Chemicals, Houston, TX), a GSNOR inhibitor, was used to verify negligible NADH conversion to NAD+ in the presence of GSNO.

| SC-TR activity assay
The selenocystamine-thioredoxin reductase (SC-TR) assay was performed as previously described 14

| Analysis
All data analysis were conducted using Prism (version 7.0c for Mac OS 10.11, GraphPad Software, La Jolla, CA). Significance is defined as P < 0.05 using an unpaired, two-tailed, student's t test, unless otherwise stated.

| Nebivolol decreases uterine force and AUC
In order to determine whether nebivolol acts as a negative inotropic agent, we utilized a tissue organ bath with either human or mouse myometrium. Nebivolol was applied at 100 μmol L −1 and 300 μmol L −1 to OT-primed (8nM) term non-labouring human ( Figure 1A, TNL: F I G U R E 2 Nebivolol drives smooth muscle relaxation through multiple pathways. First, nebivolol activates β3ARs on both endothelial and myometrial cells. β3ARs are upregulated in the myometrium during pregnancy, 16 potentially heightening this effect. Nebivolol further enhances endothelial nitric oxide synthase (eNOS) expression and activity. 11 This resulting surge of nitric oxide increases total protein S-nitrosations (SNOs), 12 promoting SNO-mediated myometrial smooth muscle relaxation. 9 Inhibition of S-nitrosoglutathione reductase (GSNOR) also increases nitric oxide availability, which relaxes myometrial tissue 6 ; however, nebivolol does not inhibit the SNO-metabolizing enzymes GSNOR or thioredoxin reductase (TrxR)

| Nebivolol does not inhibit thioredoxin reductase
Another major contributor to •NO metabolism in the myometrium is thioredoxin reductase (TrxR). TrxR depletes •NO from the cell by reducing S-nitrosated thioredoxin. Because S-nitrosations are labile and the NO-moiety can be non-enzymatically transferred between complimentary cysteine thiols, the reduction of S-nitrosated proteins proportionally decreases available •NO in the system. TrxR activity is measured similar to GSNOR using a selenocystamine-thioredoxin reductase assay, except NADPH is substituted for NADH, and the substrate for this assay is selenocystamine, for which TrxR is the only known endogenous metabolizer. We determined that nebivolol (1-100 μmol L −1 ) does not inhibit TrxR activity ( Figure 1D

| DISCUSSION
Our findings indicate that Nebivolol's potential as a tocolytic is most likely the result of enhanced eNOS activity, driven in part by β3AR stimulation (Figure 2). These data compliment our earlier findings that GSNOR is upregulated in some women who undergo sPTL, 6 decreasing the endogenous intracellular •NO pool, and by extension, total protein S-nitrosations. While nebivolol does not inhibit •NO metabolism as others have suggested 12 ( Figure 1B,C), the same functional end state is achieved through nebivolol-mediated •NO generation. We found that nebivolol was able to decrease peak force and AUC in myometrial samples ( Figure 1A) more effectively than N6022, 6 a known GSNOR inhibitor, perhaps in part due to the limited bioavailability of N6022. The use of nebivolol as a tocolytic is further supported by the finding that β3AR is the predominant subtype in human myometrium, is upregulated during pregnancy, 15 and direct stimulation of β3AR has been shown to promote myometrial relaxation. 16 A conspicuous advantage to using nebivolol as a tocolytic agent is that it is already FDA-approved. While other "borrowed" tocolytics have failed to delay PTB, nebivolol is unique in that it promotes relaxation by increasing the •NO pool, which leverages the problem of GSNOR upregulation in sPTL myometrium. 6 That being said, β -blockers use during pregnancy would need to be weighed again potential adverse effects on the foetus, such as small-for-gestationalage infants. 17 While long term use would raise additional issues, 18 these are based upon near continuous administration throughout gestation, while tocolytic use can be invisioned as a shorter treatment course. Further studies are clearly needed to establish safety and efficacy of nibivolol as a tocolytic in pregnant women.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.