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ORIGINAL RESEARCH |
From the Pharmacology Research Laboratory, Research and Development, Kissei Pharmaceutical Co. Ltd., Matsumoto City, Japan; and the Departments of Obstetrics and Gynecology and Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan.
Address reprint requests to: Tadashi Matsuda, MD, Hokkaido University School of Medicine, Department of Obstetrics and Gynecology, North 15, West 7, Kitaku, Sapporo 060-8638, Japan; E-mail: choku{at}med.hokudai.ac.jp.
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ABSTRACT |
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METHODS: At 123–125 days’ gestation, ewes (n = 8) were infused with oxytocin (1.0 mU/kg/minute) to induce uterine contractions. One hour later, KUR-1246 was infused for 3 consecutive hours beginning at a dose of 0.001 µg/kg/ minute for 30 minutes and increasing stepwise every 30 minutes to 0.3 µg/kg/minute in the KUR-1246 group (n = 4). The control received saline instead (n = 4). Statistical comparisons of changes with time in the physiologic parameters between the two groups were carried out (analysis of variance).
RESULTS: KUR-1246 suppressed oxytocin-induced uterine contractions more than 90% at doses over 0.03 µg/kg/ minute. Significant differences between the two groups were found at high doses over 0.03 µg/kg/minute for the following parameters: maternal heart rate, diastolic blood pressure, mean blood pressure, base excess, blood K+, blood lactate, plasma glucose, plasma insulin, plasma non-esterified fatty acid levels, and fetal plasma glucose and plasma insulin levels.
CONCLUSION: KUR-1246 significantly inhibited oxytocin-induced uterine contractions at doses over 0.03 µg/kg/ minute and showed reduced cardiovascular and metabolic side effects compared with ritodrine hydrochloride studied earlier in pregnant sheep.
Preterm labor is a major cause of low birth weight, and preventive measures are important for reducing neonatal morbidity and mortality because low birth weight is strongly associated with adverse outcomes in premature infants, such as neonatal death, cardiopulmonary insufficiency, neurodevelopmental delay, and retinopathy of prematurity.1,2 Selective ß2-adrenoceptor agonists, eg, ritodrine hydrochloride and terbutaline sulfate, are representative tocolytic agents currently used to control preterm labor in clinical practice.3,4 Unfortunately, in spite of having high ß2-adrenoceptor selectivity, these drugs are commonly associated with cardiovascular complications at clinical doses due mainly to their effect of concomitantly stimulating ß1-adrenoceptors.3–5 In fact, the use of these drugs to manage persistent preterm labor at doses adequate to arrest uterine contraction has met with difficulty, because the dose is restricted by the occurrence of side effects such as maternal tachycardia and pulmonary edema.3–5
To resolve this problem, we have identified a new selective ß2-adrenoceptor agonist, KUR-1246, which is a compound with a chemical structure similar to those structures of other sympathomimetic amines and characterized by a tetrahydronaphtyl substitution in the terminal amino group of the ethylamine side chain (Figure 1
). This drug showed a 400-fold greater potency in terms of inhibiting uterine contraction and a 100-fold higher selectivity in stimulating ß2-adrenoceptors than did ritodrine hydrochloride in pregnant rats and rabbits in our earlier experiments.7 Accordingly, it is possible that KUR-1246 can arrest preterm labor in pregnant women without the serious cardiovascular side effects induced by ß1-adrenoceptor stimulation.
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The present study was aimed at analyzing simultaneously in pregnant sheep and their fetuses the effects of KUR-1246 on oxytocin-induced uterine contractions, the cardiovascular system, and various metabolic parameters.
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MATERIALS AND METHODS |
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Until completion of the study, arterial and amniotic pressures were continuously measured with a polygraph and recorded on a digital audio tape recorder or a personal computer, or both. All fetal arterial pressure values were corrected for amniotic fluid pressure.
Blood gas data (pH, Po2, Pco2, and base excess) and levels of KUR-1246, K+, glucose, insulin, lactate, and nonesterified fatty acid were measured in heparinized blood samples (5.5 mL) taken from the maternal artery and the fetal abdominal aorta. KUR-1246 levels were also measured in amniotic fluid samples. Using 0.5 mL of the blood samples, we determined blood gas data and K+ and lactate levels with a blood gas analyzer using its optional measuring functions (ion selected electrode and biosensor method).11 Blood gas data were corrected for maternal rectal temperature. The remaining blood samples (5.0 mL) and amniotic fluid samples (3.0 mL) were centrifuged immediately after collection, and the separated plasma and supernatants were stored at -20C until assayed. KUR-1246 levels in plasma and amniotic fluid were determined by modified liquid chromatographytandem mass spectrometry (LC-MS/MS; API-3000, PE-SCIEX, Concord, OT, Canada).12 Plasma glucose, insulin, and nonesterified fatty acid levels were determined, using commercially available kits, by the glucose oxidase method (Glucose B-Test Wako; Wako Pure Chemical Industries, Ltd., Osaka, Japan), radioimmunoassay (Rat Insulin RIA kit; LINCO Research, Inc., St. Charles, MO), and the acyl-CoA oxidase method (NEFA C-Test Wako; Wako Pure Chemical Industries, Ltd.), respectively.13–15
At least 3 days after the surgery, the eight pregnant sheep were randomly divided into two groups, a KUR-1246 group (n = 4) and a control group (n = 4); and the following experiments were conducted: The ewes received infusions of oxytocin at a dose of 1.0 mU/kg/ minute through the right jugular vein until the end of the experiment. After 1 hour of oxytocin infusion, the ewes received infusions of KUR-1246 solution for 3 consecutive hours beginning at a dose of 0.001 µg/kg/minute for 30 minutes and increasing stepwise every 30 minutes: 0.003, 0.010, 0.030, 0.100, and finally 0.300 µg/kg/ minute, in the KUR-1246 group. KUR-1246 was diluted in isotonic saline and infused into the maternal femoral vein at the rate of 60 mL/hour by an infusion pump. In the control group, saline was infused instead in the same manner.
The maternal and fetal mean blood pressure and heart rate values at each time point were calculated as averages of values obtained every 5 minutes. The changes of intrauterine pressure over time were estimated by integration of values between intrauterine pressure monitoring curve versus baseline as averages of values obtained every 10 minutes (HyperWave, Kissei Comtec, Co., Ltd., Tokyo, Japan). For sampling, maternal arterial and fetal abdominal aortic blood and amniotic fluid were collected just before oxytocin infusion and at 30-minute intervals from just before the beginning of KUR-1246 infusion to 7 hours after the start of infusion. After each fetal blood sampling, an equivalent volume of maternal heparinized blood was infused into the fetus through the venous catheter.
All values were expressed as means ± standard errors. Repeated-measures analysis of variance was performed for statistical comparison of changes over time in physiologic parameters between the two groups. In brief, the interactions of time and group were judged by the F test of variance ratio (unbiased variance of group and time divided by that of sheep and times within groups). If significant changes were suggested, Dunnett test was performed to test for significant changes from the KUR-1246 preinfusion value. P < .05 was considered significant. For this, SAS system 6.12 (SAS Institute Inc., Cary, NC) was used.
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RESULTS |
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Figure 2 displays changes in KUR-1246 levels in the maternal plasma in the KUR-1246 group. These levels increased exponentially during the infusion, reaching 23.0 ± 5.1 ng/mL at the end of the infusion, and then immediately decreased after the infusion was stopped. Fetal plasma and amniotic fluid KUR-1246 levels in all of the samples were below the limit of detection (0.10 ng/mL) for the assay (data not shown).
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shows changes with time in the intrauterine pressure in the two groups. The intrauterine pressure values before KUR-1246 infusion were 3454 ± 942 cm H2O • minute in the KUR-1246 group and 2711 ± 1396 cm H2O • minute in the control group; and in terms of Montevideo units, these values were equivalent to 219 ± 15 in the KUR-1246 group and 199 ± 25 in the control group. No significant difference was seen between the two groups at this point. However, a significant difference between the two groups was found in changes with time of the intrauterine pressure (P < .01). In the KUR-1246 group, the intrauterine pressure decreased significantly from the end of infusion at 0.03 µg/kg/minute to that at 0.3 µg/kg/minute, as compared with the preinfusion value (P < .05).
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). Significant differences were found in changes over time between the two groups in maternal heart rate (P < .01), diastolic blood pressure (P < .01), and mean blood pressure (P < .01). On the other hand, there were no significant differences with time between the two groups in maternal systolic blood pressure or in fetal heart rate and blood pressure (data not shown). The maternal heart rate in the KUR-1246 group increased significantly from the end of the 0.01 µg/kg/minute to 1 hour after the end of the 0.3 µg/kg/minute infusion of KUR-1246, as compared with the preinfusion value (P < .05); and increments were between 13.9% and 33.8% of the preinfusion value (Figure 4A). On continuous monitoring of the electrocardiogram there were neither arrhythmia, except for maternal sinus tachycardia, nor apparent flattening of Twave in either group. Maternal diastolic blood pressure in the KUR-1246 group decreased significantly at the cessation of the 0.3 µg/kg/minute infusion of KUR-1246 (Figure 4B), whereas there was no significant change in the maternal mean blood pressure (Figure 4C), compared with preinfusion values.
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). There were no significant differences with time in the two groups in terms of maternal pH, Pco2, and Po2 and fetal pH, Pco2, Po2, and base excess (data not shown). No physical findings related to maternal pulmonary edema, such as cough, wheezing, tachypnea, and retraction, were evident in either group.
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and 7. Significant changes versus the preinfusion value (P < .05) were found in the following four parameters in the KUR-1246 group: 1) Maternal plasma glucose levels were significantly increased from the end of the 0.1 µg/kg/minute to 2.5 hours after that of the 0.3 µg/kg/minute infusion of KUR-1246 (Figure 6C), 2) maternal plasma nonesterified fatty acid levels were significantly increased from the cessation of the 0.01 µg/kg/minute to 0.5 hours after that of the 0.3 µg/kg/minute infusion of the drug (Figure 6D), 3) fetal plasma glucose levels were significantly increased from the end of the 0.1 µg/kg/minute to 3 hours after that of the 0.3 µg/kg/minute infusion of KUR-1246 (Figure 7A), and 4) fetal plasma insulin levels were significantly increased from 0.5 to 2.5 hours after the end of the 0.3 µg/kg/minute infusion (Figure 7B).
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DISCUSSION |
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First, KUR-1246 possessed a more powerful tocolytic action compared with ritodrine hydrochloride under the experimental conditions used. Table 1 summarizes the suppressive effect of KUR-1246 on oxytocin-induced uterine contractions compared with that of ritodrine hydrochloride shown in our previous study.9 The present results indicate that 0.01 µg/kg/minute of infusion of KUR-1246 had a potency equal to that of 3 µg/kg/minute infusion of ritodrine hydrochloride with respect to inhibition of oxytocin-induced uterine contractions in pregnant sheep. Strikingly, whereas the 0.3 µg/kg/minute infusion of KUR-1246 inhibited the contractions by 96.3%, a 100-fold higher dose of ritodrine hydrochloride did so by only 79.4%. Taking doses of 50% inhibition of uterine contraction into consideration, KUR-1246 possessed about a 300 to 600-fold higher potency of tocolytic action than ritodrine hydrochloride under this experimental condition of pregnant sheep.
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It is well known that ß-adrenoceptor stimulation causes vasodilation, raises plasma glucose, lactate, and nonesterified fatty acid levels, and induces changes in plasma electrolyte levels such as hypokalemia.16,17 In the present results, decreases in maternal diastolic blood pressure (Figure 4B
) and blood K+ level (Figure 6A), and increases in blood lactate, plasma glucose, and plasma nonesterified fatty acid levels (Figure 6B–D) were a consequence of the KUR-1246 infusion. Significant changes versus the preinfusion value in maternal diastolic blood pressure and plasma glucose levels were surely observed over 0.1 µg/kg/minute infusion of KUR-1246, but it would be not meaningful in clinical practice because the dose was more than 10 times higher than that required to inhibit the uterine contraction effectively. Even in such high doses of KUR-1246 infusion, moreover, maternal diastolic hypotension did not induce acute pulmonary edema from standpoints of both physical findings and blood gas analysis, and no abnormal electrocardiogram except for sinus tachycardia was found. The significant decrease in the maternal base excess may have resulted from the elevation of the maternal blood lactate level, though it was not so severe as to cause acidemia. On the other hand, the maternal plasma nonesterified fatty acid levels significantly increased at the cessation of the 0.01 µg/kg/minute infusion of KUR-1246, which dose was within the range that effectively inhibited the uterine contractions. Therefore, the plasma nonesterified fatty acid levels must be monitored carefully in administering KUR-1246 to pregnant women, especially those with diabetic lipidemia.
Third, the data made clear that KUR-1246, administered intravenously, was minimally transferred to fetuses across the placenta. During infusion of the maximal dose of KUR-1246 (0.30 µg/kg/minute), plasma KUR-1246 levels in fetuses were no more than one two-hundredth of those in maternal sheep. Placental transfer of KUR-1246 was apparently low compared with that of ritodrine hydrochloride; because in our previous study, in which ritodrine hydrochloride was administered to pregnant sheep (maximal dose of 30 µg/kg/minute), the plasma ritodrine hydrochloride levels in fetuses were one twenty-fifth of those in maternal sheep.8 It has been reported that the epitheliochorial placenta of the sheep generally constitutes a significant barrier to hydrophilic substances with molecular weights of more than 300.18 The difference in molecular weight between these two compounds is likely to be among the important reasons for the difference in placental transfer because the molecular weights of ritodrine hydrochloride and KUR-1246, dissolved in blood, are 287.4 and 428.5, respectively, and both compounds are hydrophilic.
Intravenous administration of KUR-1246 in maternal sheep increased plasma glucose and insulin levels in the fetuses (Figure 7). Based on the placental transfer of KUR-1246, it was not plausible that KUR-1246 in the fetal circulation acted directly on ß-adrenoceptors to cause these changes in fetuses. The increase in the fetal plasma glucose levels appeared simultaneously with that in the maternal level, and the change with time in fetus resembled closely that of the maternal sheep. Because plasma glucose moves across the placenta readily in pregnant sheep,19 it is quite likely that the changes observed in fetal plasma glucose resulted from transplacental transfer of maternal plasma glucose. As for insulin, this molecule showed minimal transfer across the sheep placenta,19 and thus the increase in the fetal plasma insulin level can probably be attributed to the increased fetal plasma glucose levels.
Based on these considerations, we reasonably conclude that KUR-1246 is a potentially beneficial tocolytic agent, as compared with ritodrine hydrochloride, in view of its lack of serious cardiovascular and metabolic side effects when infused at the minimal dose that effectively inhibits uterine contractions in pregnant ewes.
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Footnotes |
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Received November 1, 2001. Received in revised form February 22, 2002. Accepted March 21, 2002.
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REFERENCES |
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