Concentration-Response Study

For all drugs, cumulative treatments inhibited spontaneous pregnant human myometrial contractions ex vivo in a concentration-dependent manner. In Figs. 1, 2, 3, 4, 5 and 6, panel A shows representative traces of contractile activity before and after drug treatment. Each drug was assessed against spontaneously contractile tissue strips from n = 8–10 women. The CRCs for each drug generated from the corresponding amplitude and AUC measurements are shown in Figs. 1, 2, 3, 4, 5 and 6, panel B, while panel C shows the effect of each drug’s cumulative treatment on contraction frequency. During the treatment courses (2.5–3.5 h), the administration of volume-matched cumulative doses of drug vehicles (DMSO: up to a maximum of 0.42%; Milli-Q water; KREBS) did not affect the contraction amplitude, frequency, or AUC (Supplementary Figures S3 and S4, and Figs. 1, 2, 3, 4, 5 and 6, panel B). The mean IC25 and IC50 concentrations for each drug, based on their inhibitory effects on contraction amplitude or AUC, are summarized in Table 1. In terms of inhibiting spontaneous pregnant human myometrial contractility ex vivo, when amplitude alone was used as an index, the order of potency of the tocolytics (from highest to lowest) was NIF > GH > ROL > 2-APB > IND > AMP. However, when AUC was used as an index, the order of potency was NIF > ROL > GH > 2-APB > IND > AMP. The order of potency of the tocolytics therefore changed, depending on whether effects on AUC or amplitude alone were used to calculate IC25 and IC50.

Fig. 1

Concentration-response analysis of aminophylline. (A) A representative trace for the effect of cumulative treatment of aminophylline (n = 8) on spontaneous contractions. Dotted lines indicate the points at which treatments were added to the organ bath. (B) The plotted concentration-response curves for the effect of aminophylline on spontaneous pregnant human myometrial contractions ex vivo. Contractility was measured as AUC or amplitude and expressed relative to the contraction baseline (100%). (C) The effect of cumulative drug treatments on contraction frequency. The mean percentages of contraction frequency for the cumulative doses are expressed relative to the frequency baselines (100%). Data were checked for normality using the Shapiro-Wilk normality test and then analyzed by 1-way ANOVA with multiple comparisons (Dunnett’s relative to Baseline in all panels). Data are mean ± SEM. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; NS, non-significant

Fig. 2

Concentration-response analysis of rolipram. (A) A representative trace for the effect of cumulative treatment of rolipram (n = 8) on spontaneous contractions. Dotted lines indicate the points at which treatments were added to the organ bath. (B) The plotted concentration-response curves for the effect of rolipram on spontaneous pregnant human myometrial contractions ex vivo. Contractility was measured as AUC or amplitude and expressed relative to the contraction baseline (100%). (C) The effect of cumulative drug treatments on contraction frequency. The mean percentages of contraction frequency for the cumulative doses are expressed relative to the frequency baselines (100%). Data were checked for normality using the Shapiro-Wilk normality test and then analyzed by 1-way ANOVA with multiple comparisons (Dunnett’s relative to Baseline in all panels). Data are mean ± SEM. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; NS, non-significant

Fig. 3

Concentration-response analysis of indomethacin. (A) A representative trace for the effect of cumulative treatment of indomethacin (n = 10) on spontaneous contractions. Dotted lines indicate the points at which treatments were added to the organ bath. (B) The plotted concentration-response curves for the effect of indomethacin on spontaneous pregnant human myometrial contractions ex vivo. Contractility was measured as AUC or amplitude and expressed relative to the contraction baseline (100%). (C) The effect of cumulative drug treatments on contraction frequency. The mean percentages of contraction frequency for the cumulative doses are expressed relative to the frequency baselines (100%). Data were checked for normality using the Shapiro-Wilk normality test and then analyzed by 1-way ANOVA with multiple comparisons (Dunnett’s relative to Baseline in all panels). Data are mean ± SEM. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; NS, non-significant

Fig. 4

Concentration-response analysis of nifedipine. (A) A representative trace for the effect of cumulative treatment of nifedipine (n = 10) on spontaneous contractions. Dotted lines indicate the points at which treatments were added to the organ bath. (B) The plotted concentration-response curves for the effect of nifedipine on spontaneous pregnant human myometrial contractions ex vivo. Contractility was measured as AUC or amplitude and expressed relative to the contraction baseline (100%). (C) The effect of cumulative drug treatments on contraction frequency. The mean percentages of contraction frequency for the cumulative doses are expressed relative to the frequency baselines (100%). Data were checked for normality using the Shapiro-Wilk normality test and then analyzed by 1-way ANOVA with multiple comparisons (Dunnett’s relative to Baseline in all panels). Data are mean ± SEM. ****p ≤ 0.000; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; NS, non-significant

Fig. 5

Concentration-response analysis of glycyl-11-52. (A) A representative trace for the effect of cumulative treatment of glycyl-H-11-52 (n = 8) on spontaneous contractions. Dotted lines indicate the points at which treatments were added to the organ bath. (B) The plotted concentration-response curves for the effect of glycyl-11-52 on spontaneous pregnant human myometrial contractions ex vivo. Contractility was measured as AUC or amplitude and expressed relative to the contraction baseline (100%). (C) The effect of cumulative drug treatments on contraction frequency. The mean percentages of contraction frequency for the cumulative doses are expressed relative to the frequency baselines (100%). Data were checked for normality using the Shapiro-Wilk normality test and then analyzed by 1-way ANOVA with multiple comparisons (Dunnett’s relative to Baseline in all panels). Data are mean ± SEM. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; NS, non-significant

Fig. 6

Concentration-response analysis of 2-aminoethoxydiphenyl borate. (A) A representative trace for the effect of cumulative treatment of 2-aminoethoxydiphenyl borate (n = 8) on spontaneous contractions. Dotted lines indicate the points at which treatments were added to the organ bath. (B) The plotted concentration-response curves for the effect of 2-aminoethoxydiphenyl borate on spontaneous pregnant human myometrial contractions ex vivo. Contractility was measured as AUC or amplitude and expressed relative to the contraction baseline (100%). (C) The effect of cumulative drug treatments on contraction frequency. The mean percentages of contraction frequency for the cumulative doses are expressed relative to the frequency baselines (100%). Data were checked for normality using the Shapiro-Wilk normality test and then analyzed by 1-way ANOVA with multiple comparisons (Dunnett’s relative to Baseline in all panels). Data are mean ± SEM. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; NS, non-significant

Table 1 Summary of concentration-response curve parameters for each tocolyticAssessing AUC Versus Amplitude: Effect on IC25 and IC50

Of the tocolytics analyzed, AMP, ROL, and IND revealed the most marked variations in CRCs when assayed according to amplitude alone versus AUC (Figs. 1, 2 and 3, panels B).

For AMP treatment, the CRCs in Fig. 1, panel B, show that the difference between contraction amplitude and AUC inhibition was evident across the full concentration range. AMP had a more negligible effect on the amplitude and a relatively more prominent effect on contraction AUC (Fig. 1, panel A), which ultimately translated into significant differences in the IC25 and IC50 concentrations calculated from either AUC or amplitude-based assessment of the contraction traces. For AMP treatment, the IC25(AUC) and IC25(Amplitude) concentrations were determined to be 175 µM and 277 µM, respectively (p = 0.04), while the IC50(AUC) and IC50(Amplitude) were determined to be 318.5 µM and 450 µM, respectively (p = 0.03) (Tables 1 and 2).

Table 2 Summary of statistical differences between AUC-based and amplitude-based IC values for each tocolytic

Consistent findings were observed for ROL treatment on spontaneous contractions, as the IC values were significantly affected according to whether AUC or amplitude was assessed (Fig. 2, panel B). For ROL treatment, the IC25(AUC) and IC25(Amplitude) were determined to be 515 µM and 15 µM, respectively (p = 0.01), while the IC50(AUC) and IC50(Amplitude) were determined to be 4.3 µM and 55 µM, respectively (p = 0.009) (Tables 1 and 2).

Similarly, the IC values of IND differed according to whether AUC or amplitude was assessed (Fig. 3, panel B). IND inhibited both amplitude and AUC in a dose-dependent manner. The inhibitory effect of IND on contraction amplitude was less pronounced at lower doses, but both amplitude and AUC were affected at higher doses. For IND treatment, the IC25(AUC) and IC25(Amplitude) were determined to be 28 µM and 42 µM, respectively (p = 0.025), while the IC50(AUC) and IC50(Amplitude) were determined to be 59.5 µM and 75 µM, respectively (p = 0.03) (Tables 1 and 2).

Slight differences were also found between AUC and amplitude-based assessment of the contraction traces for NIF and GH (Figs. 4 and 5, panels A and B). Scrutiny of the CRCs for NIF revealed that contraction AUC and amplitude were almost equally affected across the lower doses (1st and 2nd dose), however, as drug concentration increased, the AUC declined at a shallower slope, due to contraction frequency increasing, which partially offset the loss of amplitude across each 30 min window. However, as the contractions progressively diminished in amplitude, AUC still decreased overall despite the increase in frequency (Fig. 4, panel B). Despite slight differences in the CRC slopes, assessment of NIF treatment contraction traces based on amplitude alone versus AUC did not significantly affect the calculated mean IC25 and IC50 concentrations. For NIF treatment, the IC25(AUC) and IC25(Amplitude) were calculated to be 3.4 and 3.6 nM, respectively (p = 0.09), while the IC50(AUC) and IC50(Amplitude) were 10 nM and 10 nM, respectively (p = 0.1) (Tables 1 and 2).

Consistent findings were observed for GH treatment (Fig. 5, panel B), in that the IC25 and IC50 concentrations determined for GH treatment were not significantly affected by whether AUC or amplitude alone was assessed. For GH treatment, the IC25(AUC) and IC25(Amplitude) were calculated to be 4.6 µM and 3.6 µM, respectively (p = 0.2), while the IC50 (AUC) and IC50(Amplitude) were calculated to be 18.2 µM and 15 µM, respectively (p = 0.08) (Tables 1 and 2).

The effect of 2-APB on both contraction AUC and amplitude is shown in Fig. 6, panel B. The inhibitory effect on contraction AUC and amplitude was slightly different across the concentration range. At the lower treatment concentrations (1 and 10 µM), 2-APB had little effect on amplitude, however, amplitude declined rapidly at concentrations of 50 µM and above (Fig. 6, panel A), producing the steepest hill slope of all the agents analyzed (Table 1). The shallower hill slope observed for AUC-based assessment of 2-APB’s effects was attributable to 2-APB increasing contraction frequency, in particular at 50 and 100 µM (3rd and 4th doses), which partially offset the rapid loss of contraction amplitude, thus producing a shallower hill slope. Analysis of the CRCs of 2-APB revealed a significant difference between IC25(AUC) and IC25(Amplitude), which were 27 and 40 µM, respectively (p = 0.01), however, there was no significant difference between IC50(AUC) and IC50(Amplitude), which were 53 and 57 µM, respectively (p = 0.3) (Tables 1 and 2).

The IC25 and IC50 concentrations determined for each tocolytic by assessment of either AUC or amplitude are summarized in Table 1 and the differences between AUC and amplitude-based IC values are summarized in Table 2.

Effect of Tocolytics on Contraction Frequency

Panel C in Figs. 1, 2, 3, 4, 5 and 6 shows the effects of the tocolytics on contraction frequency. Treatment with AMP and ROL significantly reduced the frequency of spontaneous pregnant human myometrial contractions ex vivo in a concentration-dependent manner (Figs. 1 and 2, panel C), whereas IND did not significantly affect contraction frequency across the concentration range, with the exception of abolishing contractions at the maximum inhibitory concentration (5th dose) (Fig. 3, panel C). In contrast, NIF, GH, and 2-APB significantly increased contraction frequency as drug concentration increased (Figs. 4, 5 and 6, panel C), highlighting that the tocolytics’ different biochemical mechanisms of action can manifest as markedly different physiological effects on pregnant human uterine contractility.

AUC Vs. Amplitude: Assessment of Experimentally Determined IC25 and IC50 Concentrations

Having determined the IC25 and IC50 for each drug based upon assessment of both AUC (IC25(AUC), IC25(AUC)) and amplitude (IC25(Amplitude), IC50(Amplitude)), follow-up studies were conducted to assess the effects of a single dose of the IC25/50(AUC) concentrations against both AUC and amplitude. This was done to assess/confirm that the IC25/50(AUC) concentrations reduced AUC by 25/50% and to determine whether the AUC-based IC25/50 concentrations also reduced amplitude by 25/50%. Similarly, the effects of a single dose of the IC25/50(Amplitude) concentrations were assessed against both AUC and amplitude. This was done to assess/confirm that the IC25/50(Amplitude) concentrations reduced amplitude by 25/50% and to determine whether the amplitude-based IC concentrations also reduced AUC by 25/50%. The study layout for assessing/confirming the effects of the determined IC25/50 concentrations is illustrated in Fig. 7.

Fig. 7

Overview of study layout. The effects of the IC25 concentrations were assessed/confirmed for each tocolytic via AUC and amplitude-based assessment. The above experimental layout was repeated for IC50 concentrations determined for each of the tocolytics

Effect of IC25/50(AUC) Concentrations on Contraction AUC and Amplitude

Looking at the results presented in Fig. 8, when each of the tocolytics was applied as a single dose at the IC25(AUC) concentration, AUC was consistently reduced (1 h pre-treatment vs. 1 h post-treatment) by approximately 25% [AMP = 23.1 ± 1% (n = 10); ROL = 23 ± 2.5% (n = 5); IND = 23.6% ± 1% (n = 10); NIF = 24.8 ± 0.5% (n = 10); GH = 24.4 ± 0.5% (n = 10); 2-APB = 23 ± 0.8% (n = 10)] (Fig. 8, panel G), thus confirming accuracy of the IC25(AUC) concentrations determined during prior concentration-response studies. However, when the effects of the IC25(AUC) were assessed against amplitude, a 25% reduction in amplitude was observed for only two of the six tocolytics. That is, whilst contraction amplitude was indeed reduced by approximately 25% by NIF (23.8 ± 0.7% (n = 10)) and GH (24.8 ± 1.1% (n = 10)), IND and 2-APB reduced amplitude by 19.8% ± 0.8% (n = 10) and 18.8 ± 1.2% (n = 10), respectively, whilst AMP and ROL reduced amplitude by just 10.2 ± 1.9% (n = 10) and 5.05 ± 0.9% (n = 5), respectively (Fig. 8, panel H).

Fig. 8

Assessment of the experimentally determined IC25(AUC) concentrations. Representative traces showing the extent of contraction inhibition following treatment of spontaneously contracting pregnant human myometrial strips with the experimentally determined AUC-based IC25 concentrations of (A) AMP (n = 10), (B) ROL (n = 5), (C) IND (n = 10), (D) NIF (n = 10), (E) GH (n = 10), and (F) 2-APB (n = 10). Dotted lines indicate the points at which the treatment was added to the organ bath. (G) The mean percentage inhibition of AUC (relative to the contraction AUC baselines, 100%) induced by each drug when applied at the IC25(AUC) for 60 min. (H) The mean percentage inhibition of amplitude (relative to the contraction amplitude baselines, 100%) induced by each drug when applied at the IC25(AUC) for 60 min. IC, inhibitory concentration; AUC, area under the curve; AMP, aminophylline; ROL, rolipram; IND, indomethacin; NIF, nifedipine; GH, glycyl-h-1152; 2-APB, 2-aminoethoxydiphenyl borate

The results were largely consistent when each tocolytic was applied as a single treatment but at their respective IC50(AUC) concentrations (Fig. 9). That is, for each tocolytic, the IC50(AUC) consistently reduced AUC by approximately 50% [AMP = 47 ± 1.3% (n = 10); ROL = 48 ± 3.5% (n = 5); 48.7% ± 1% for IND (n = 10); NIF = 50 ± 1% (n = 10); GH = 48 ± 0.9% (n = 10); 2-APB = 49 ± 1% (n = 10)] (Fig. 9, panel G); however, when a single dose of the IC50(AUC) was assessed against amplitude, amplitude was reduced by approximately 50% by NIF (47.8 ± 1.9% (n = 10)), GH (50.8 ± 1% (n = 10)), and 2-APB (47.4 ± 1.9% (n = 10)), but IND reduced amplitude by 40.2% ± 1.4% (n = 10), whilst AMP and ROL reduced amplitude by only 17.8 ± 3% (n = 10) and 13.8 ± 0.9% (n = 5), respectively (Fig. 9, panel H).

Fig. 9

Assessment of the experimentally determined IC50(AUC) concentrations. Representative traces showing the extent of contraction inhibition following treatment of pregnant human myometrial strips with the experimentally determined AUC-based IC50 concentrations for (A) AMP (n = 10), (B) ROL (n = 5), (C) IND (n = 10), (D) NIF (n = 10), (E) GH (n = 10) and (F) 2-APB (n = 10). Dotted lines indicate the points at which the treatment was added to the organ bath. (G) The mean percentage inhibition of AUC (relative to the contraction AUC baselines, 100%) induced by each drug when applied at the IC50(AUC) for 60 min. (H) The mean percentage inhibition of amplitude (relative to the contraction amplitude baselines, 100%) induced by each drug when applied at the IC50(AUC) for 60 min. IC, inhibitory concentration; AUC, area under the curve; AMP, aminophylline; ROL, rolipram; IND, indomethacin; NIF, nifedipine; GH, glycyl-h-1152; 2-APB, 2-aminoethoxydiphenyl borate

Effect of IC25/50(Amplitude) Concentrations on Contraction Amplitude and AUC

Just as single treatments of the IC25(AUC) and IC50(AUC) concentrations of each tocolytic were assessed against AUC and amplitude, single treatments of the IC25(Amplitude) and IC50(Amplitude) concentrations were also assessed against AUC and amplitude, as indicated in Fig. 7.

The representative contraction traces in Fig. 10, panels A – F, show the inhibitory effects of a single dose of each tocolytic applied at IC25(Amplitude) concentration (1 h pre-treatment vs. 1 h post-treatment). Amplitude analysis (Fig. 10, panel G) revealed that the IC25(Amplitude) concentration of AMP, ROL, IND, NIF, and GH reduced contraction amplitude by ~ 25% [AMP = 22 ± 1.2% (n = 5); ROL = 25.2 ± 1.8% (n = 5); IND = 25.3% ± 0.6% (n = 5); NIF = 24.2 ± 0.3% (n = 5); GH = 24 ± 0.4% (n = 5)], as expected, however the IC25(Amplitude) for 2-APB reduced amplitude by 36 ± 1.2% (n = 5). Moreover, when the inhibitory effects of the IC25(Amplitude) concentrations were assessed against AUC, the reduction in AUC was not consistently ~ 25%. That is, the observed reduction in AUC was ~ 25% for NIF (25.2 ± 0.5% (n = 5)) and GH (26.7 ± 0.4% (n = 5)), but the reduction in AUC for the other drugs was greater than 25% [AMP = 35.3 ± 2% (n = 5); ROL = 81.1 ± 2.9% (n = 5); IND = 32.4% ± 1% (n = 5); 2-APB = 41.7 ± 1.4% (n = 5)] (Fig. 10, panel H).

Fig. 10

Assessment of the experimentally determined IC25(Amplitude) concentrations. Representative traces showing the extent of contraction inhibition following treatment of pregnant human myometrial strips with the experimentally determined amplitude-based IC25 concentrations for (A) AMP (n = 5), (B) ROL (n = 5), (C) IND (n = 5), (D) NIF (n = 5), (E) GH (n = 5) and (F) 2-APB (n = 5). Dotted lines indicate the points at which the treatment was added to the organ bath. (G) The mean percentage inhibition of amplitude (relative to the contraction amplitude baselines, 100%) induced by each drug when applied at the IC25(Amplitude) for 60 min. (H) The mean percentage inhibition of AUC (relative to the contraction AUC baselines, 100%) induced by each drug when applied at the IC25(Amplitude) for 60 min. IC, inhibitory concentration; AUC, area under the curve; AMP, aminophylline; ROL, rolipram; IND, indomethacin; NIF, nifedipine; GH, glycyl-h-1152; 2-APB, 2-aminoethoxydiphenyl borate

Mostly consistent results were found when each of the tocolytics was applied as a single dose but at their respective IC50(Amplitude) concentrations (Fig. 11). That is, while the IC50(Amplitude) of AMP (47.2 ± 2.5% (n = 5)), IND (47.5% ± 2% (n = 5)), NIF (49 ± 1.5% (n = 5)), and GH (48 ± 1% (n = 10)) reduced contraction amplitude by approximately 50%, as expected, ROL and 2-APB reduced amplitude by 100% (n = 5) and 62 ± 2% (n = 5), respectively (Fig. 11, panel G). Similarly, when the effects of the IC50(Amplitude) were assessed against AUC, a ~ 50% reduction in AUC was observed for NIF (50.2 ± 1% (n = 5)), GH (52 ± 1% (n = 5)), and 2-APB (50 ± 1.2% (n = 5), however, IND and AMP reduced amplitude by ≥60% [IND = 60% ± 1.2% (n = 5); AMP = 66.3 ± 2.2% (n = 5)], while ROL reduced AUC by 100% (n = 5) (Fig. 11, panel H).

Fig. 11

Assessment of the experimentally determined IC50(Amplitude) concentrations. Representative traces showing the extent of contraction inhibition following treatment of pregnant human myometrial strips with the experimentally determined amplitude-based IC50 concentrations for (A) AMP (n = 5), (B) ROL (n = 5), (C) IND (n = 5), (D) NIF (n = 5), (E) GH (n = 5) and (F) 2-APB (n = 5). Dotted lines indicate the points at which the treatment was added to the organ bath. (G) The mean percentage inhibition of amplitude (relative to the contraction amplitude baselines, 100%) induced by each drug when applied at the IC50(Amplitude) for 60 min. (H) The mean percentage inhibition of AUC (relative to the contraction AUC baselines, 100%) induced by each drug when applied at the IC50(Amplitude) for 60 min. IC, inhibitory concentration; AUC, area under the curve; AMP, aminophylline; ROL, rolipram; IND, indomethacin; NIF, nifedipine; GH, glycyl-h-1152; 2-APB, 2-aminoethoxydiphenyl borate

The overall observed percent reductions in contraction AUC and amplitude induced by single treatments of the IC25(AUC) and IC25(Amplitude) are summarized in Table 3, while observed percent reductions induced by the IC50(AUC) and IC50(Amplitude) are summarized in Table 4.

Table 3 Summary of the percentage reductions of AUC and amplitude induced by the IC25(AUC) and IC25(Amplitude) of each tocolyticTable 4 Summary of the percentage reductions of AUC and amplitude induced by the IC50(AUC) and IC50(Amplitude) of each tocolytic