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Metabolism and disposition of ritodrine in a pregnant baboon
Volume 152 Number 8
7. Ballard PL. Glucocorticoid receptors in the lung. Fed Proc 1977;36:2660. 8. Gilden C, Sevanian A, Tierney DF, Kaplan SA, Barrett CT. Regulation of fetal lung phosphatidyl choline synthesis by cortisol: role of glycogen and glucose. Pediatr Res 1977;11:845. 9. Farrell PM, Zachman RD. Induction of choline phosphotransferase synthesis in the fetal lung by corticosteroids. Science 1973;179:297. 10. Rooney SA, Gross I, Gassenheimer LN, Motoyama EK. Stimulation of glycerolphosphate phosphatidyltransferase activity in fetal rabbit lung by cortisol administration. Biochim Biophys Acta 1975;398:433. II. Breiher A, Benson BJ, Williams MC, Mason RJ, Ballard PL. Corticosteroid induction of phosphatidic acid phosphatase in fetal rabbit lung. Biochim Biophys Res Commun 1977;77:883.
Glucocorticoids and j3-adrenergic-receptor agonists
12. Ekelund L, Arvidson G, Astedt B: Cortisol-induced accumulation of phospholipids in organ culture of human fetal lung. ScandJ Clin Lab Invest 1975;35:419. 13. Ekelund L, Arvidson G, Emanuelsson H, Myhrberg H, Astedt B. Effect of cortisol on human fetal lung in organ culture. A biochemical electron-microscopic and autoradiographic study. Cell Tissue Res 1975;163:263. 14. Torday JS, Smith BT, Giroud CJP. The rabbit fetal lung as a glucocorticoid target tissue. Endocrinology 1975; 96:1462. 15. Giannopoulos G. Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung. Biochim Biophys Res Commun 1980;95:388. 16. Cheng JB, Goldfien A, Ballard PL, Roberts JM. Glucocorticoids increase pulmonary beta-adrenergic receptors in fetal rabbit. Endocrinology 1980; I 07:1646.
Metabolism and disposition of ritodrine in a pregnant baboon Matana Borrisud, Ph.D., Richard O'Shaughnessy, M.D., MichaelS. Alexander, Ph.D., and Brian D. Andresen, Ph.D. Columbus, Ohio Relatively little is known about the detailed metabolism of ritodrine. The aim of this study was to examine ritodrine metabolism and pharmacokinetics in the maternal and fetal baboon. A fetal-maternal model was made with use of Papio anubis at 144 days of gestation. Tritiated ritodrine was injected as an intravenous bolus into the mother. Maternal and fetal blood samples, amniotic fluid, and maternal urine were collected at timed intervals. Samples were analyzed by a combination of high-pressure liquid chromatography and radiochromatography. Conjugated metabolites were recovered and characterized by cleavage studies with use of j3-glucuronidase and sulfatase. The distribution half-life of ritodrine in the mother was 6 minutes and the elimination half-life was 61 minutes. Metabolites were found in both fetal serum and amniotic fluid. The concentrations of ritodrine and its metabolites in fetal serum were at or above the concentrations in maternal serum at 2 hours after maternal intravenous injection. The principal metabolite identified was the sulfate conjugate. The fetus appears to accumulate metabolites. These data indicate that ritodrine crosses the placenta and, in the baboon, achieves levels in the fetus equal to or higher than those in the mother. (AM
J
0BSTET GYNECOL 1985;152:1067-72.)
Key words: Ritodrine, pharmacokinetics, metabolism (erythro-p-hydroxy-a-[ 1-([p-hydroxyRitodrine phenethyl]-amino)ethyl]benzyl alcohol) (Fig. 1), a new I)-sympathomimetic agent, has been used almost universally' for preterm labor patients.u It was first found to be a potent myometrial inhibitor in 1969. 13 In 1971 ritodrine was first reported to be a well-tolerated uterine relaxant that could arrest preterm labor in most cases.< In pharmacokinetic studies employing tritiated ritodrine, Kleinhout and Veth 5 reported that ritodrine From the Department of Obstetrics and Gynecology, The Ohio State University. Received for publication May 17, 1984; revised March 19, 1985; accepted March 27, 1985. Reprint requests: Richard O'Shaughnessy, M.D., Department of Obstetrics and Gynecology, 561 Means Hall, 1654 Upham Drive, Columbus, OH 43210-1228.
is inactivated by conjugation with glucuronic and sulfuric acids and that the parent compound crossed the placental barrier in a pregnant ewe. In 1980 Gandar et al. 6 developed a radioimmunoassay method for the determination for ritodrine and reported serum levels and half-lives in healthy volunteers. His studies with pregnant women also demonstrated that ritodrine crossed the placenta and entered the fetal circulation. Recently, Nandakumaran et al.' reported a method for the assay for ritodrine by high-pressure liquid chromatography with use of an isolated human placental perfusion model. Their studies also support evidence of ritodrine placental transfer. Ritodrine is currently the only tocolytic drug approved for obstetric use by the United States Food and Drug Administration Advisory Committee on Fertility 1067
1068
Borrisud et al.
August 15, 1985 Am J Obstet Gynecol
unidentified
HO-o-~ CH-CH-NH-CH-CH~OH I I 2 2~ OH CH
metabolite
sulfate
3
Fig. 1. Chemical structure of ritodrine.
en u.
serum
CPM 100
o
A
940 CPM/
'------·* o-o
200
fetal
::J <{
600
0
lO
maternal unne
•·· · ··• amruohc
4)-o---o- •• .,_,..--..0
0
______. maleludl s.tuum
10 ul maternal urme
CPM /ML
,,,,a
flu1d
''""""'"'·~-------- .. ---·
400
amniotic fluid
B
maternal
urine . 6 CPM X 10
ritodrine
200
!iO
100 --~ - ...... -u ,.~·-
0""''-~ ...
2!i
--o-----
0
0
0
c
200
100
................... o(J
!iO
30 MIN
Fig. 3. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of the maternal baboon serum after an intravenous injection of 2.18 j.Lmol of tritiated ritodrine in the mother.
.. -·- ...... u-
. .a--·-·
0
0
-
05
------<>-------o
0"".......
"·o..o- ... o--- o-- -.o-..-..-..-.-..--
30
60
.................. .. 120
25 0
180
MIN
Fig. 2. Total radioactivity of ritodrine and its metabolites from the maternal and fetal baboon serum, amniotic fluid (10 j.LI sampling), and maternal urine (total output). Data represent first (A), second (B), and third (C) experimental days.
and Maternal Health Drugs. 2 However, relatively little is known about the detailed metabolism of this drug. The aim of the present study was to examine ritodrine metabolism and pharmacokinetics in both the mother and fetal baboon simultaneously. Material and methods
All reagents used were of analytical grade; solvents were of high-pressure liquid chromatography grade. Both 13-glucuronidase and sulfatase (Sigma Chemical Company) were used in the metabolite cleavage experiments. Ritodrine was obtained as its hydrochloride salt from Astra Pharmaceutical Products. A sample of this material was labeled with tritium by catalytic exchange with use of tritiated water (New England Nuclear, Inc.). The crude tritiated ritodrine was purified by both thin-layer chromatography and high-pressure liquid chromatography" immediately prior to use. Serum, urine, and amniotic fluid samples were obtained from a healthy, fertile, female baboon (Papio
anubis) at a gestational age of 144 days. The baboon was premedicated with ketamine hydrochloride (110 mg) and atropine sulfate (0.6 mg) intramuscularly. The animal was also given hydroxyprogesterone caproate (500 mg) and benzathine penicillin (600,000 U) intramuscularly. The abdomen was shaved, and an intravenous infusion of 5% dextrose in Ringer's lactate was administered in a peripheral arm vein. Electrocardiogram and blood pressure monitoring devices were attached, and the animal was placed in a 15° left lateral position. General anesthesia was administered by endotracheal intubation. The anesthetic consisted of I% halothane and a 50% mixture of nitrous oxide and oxygen, both at a 300 ml/min flow rate. The abdomen was opened and a small incision made in the anterior myometrium over the area of the fetal lower extremities. Excess amniotic fluid was aspirated, kept warm, and returned to the uterine cavity at the end of the operation. The right lower extremity of the fetus was exteriorized and a cutdown was made in the femoral vein; a fine (PV 4) polyvinyl catheter was advanced over an introducer up the vein to a distance of 7 em (the approximate distance of the renal vessels). The fetal groin incision was closed with a fine polyglycolic acid suture. A fetal electrocardiogram electrode was attached to the right buttock, and a large-bore polyethylene catheter was placed within the uterine cavity to monitor intrauterine pressures and for amniotic fluid sampling. The
Metabolism and disposition of ritodrine
Volume 152 Number 8
amniotic fluid removed earlier was returned to the uterine cavity, and the catheters and electrocardiogram device were brought out through the uterine incision, which was closed in layers with polyglycolic acid suture. Another catheter was placed in the maternal femoral vein for maternal blood sampling. All catheters were filled with heparin (I U/cc). A catheter was also placed in the maternal bladder for urine sampling. On the day of operation, the first experiment lasted 1 hour. The catheters were then tunneled into the right flank and coiled in a subcutaneous pouch. Total operation time was 100 minutes. Estimated blood loss was 20 ml (baboon blood volume, 65 to 70 mllkg). 9 Fluid replacement consisted of 500 ml of 5% dextrose in Ringer's lactate. After the operation the animal was returned to the cage and received a normal diet enriched in fruit and vegetables, with water ad libitum. The animal's drinking water contained isoxsuprine at a total dose of 20 mg/day. On the second and fourth postoperative days the animal was returned to surgery after premedication as above. The fetal and intra-amniotic catheters and electrode were exteriorized, and the intra-amniotic catheter and fetal electrode attached to a Corometrics two-channel monitor for evaluation of intrauterine pressure and fetal electrocardiogram. The second experiment was performed on the second postoperative day. The animal's drinking water on the third postoperative day contained ritodrine at a total dose of 50 mg/day as additional insurance against premature labor. The third experiment was performed on the fourth postoperative day. At the end of each experiment, 4 ml of maternal blood was transfused back to the fetus to restore blood volume. At no time during the experiments did the animal have uterine contractions. In the experiments a solution (1 ml, 2.18 11-mol) of ritodrine hydrochloride containing standard tritiated ritodrine (total activity 2.0 X 10 8 cpm) was injected as an intravenous bolus into the mother. Maternal and fetal blood samples as well as amniotic fluid and maternal urine were collected at timed intervals. Blood samples from each experiment were centrifuged immediately. Serum was separated and frozen at -70° C until analysis. The other samples were similarly frozen. An aliquot (10 11-l) of each sample was counted for total radioactivity in a Packard Tri-Carb well-type scintillation counter. All samples were then analyzed by high-pressure liquid chromatography. Serum and amniotic fluid samples were diluted with acetonitrile (I : 3 vol/vol), vortexed thoroughly, and centrifuged. The clear supernatant was separated and taken to dryness under nitrogen at soo C. Each sample
unidentified
1069
metabolite
sulfate
• I
CPM/ML
(/)
600
I
u. :::>
I I I
1.0
<(
,,.•
400
•'•'•' •'••
•'
,!l
N
ci
.
ritodrine
I •'' ~ ~, __ -_,....
200
,
-- --.,
0 0
10
20
30 MIN
Fig. 4. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of fetal baboon serum after an intravenous injection of 2.18 f.Lmol of tritiated ritodrine in the mother.
was reconstituted with 200 11-l of the mobile phase solvent and injected onto the chromatographic column. Urine samples were injected directly onto the column following centrifugation. Analyses were performed by combined high-pressure liquid chromatography and radiochromatography.8 A Beckman Altex Series 322MP gradient liquid chromatograph, equipped with Beckman Model 165 ultraviolet detector, Gilson fraction collector, and a high-performance phenyl-coated column, were used. The absorption maxima for ritodrine at 225 and 275 mm were used for ultraviolet detection. Ritodrine and metabolite levels in serum, urine, and amniotic fluid were measured radiologically. Metabolites were recovered from maternal urine for identification. A urine sample (100 ml) was lyophilized, and the solid residues were triturated in methanol. After centrifugation to remove insoluble solids, the supernatant was applied to a conventional liquid chromatography column (30 by 2 em), packed to a height of 20 em with silica gel 60. The eluting solvent was a mixture of chloroform/methanol/formic acid (10: 5: 1 vollvol), and the sample was eluted under 8 pounds per square inch gauge nitrogen pressure. One hundred 4 ml fractions were collected. An aliquot of each was
1070 Borrisud et al.
August 15, 1985 Am J Obstet Gynecol
unidentified
metabolite
unidentified
sulfate CPMX10 4 /ML 15
metabolite
sulfate
•
12 (/)
LL
::J
CPM/ML 9
tn
I
ritodrine
J
U)
II.
:::1
I"
100
~
~
6
II I I II I I fl Ill I IV I : I I I I
50
3
0
-- I 0
0 30 MIN
Fig. 5. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of maternal baboon urine after an intravenous injection of 2.18 jLmol of tritiated ritodrine in the mother.
counted for radioactivity. Those with significant activity were examined by high-pressure liquid chromatography, and fractions containing the metabolite were combined for analysis. A portion of the combined ritodrine metabolite fractions was analyzed directly as a control. A second portion was taken to dryness and reconstituted with water (1 ml). The solution was adjusted to pH 7 (sodium hydroxide), added to 1000 units of 13-glucuronidase, and stirred in a 3 7° C bath for 3 hours. The reaction was terminated by addition of three volumes of ac;etonitrile and vortexing. After centrifugation and drying of the supernatant under nitrogen, the sample was reconstituted with mobile phase solvent and analyzed by high-pressure liquid chromatography. Fractions (1 ml) were collected and counted for radioactivity. A third portion was also taken to dryness and reconstituted with 0.4 ml of water. This was added to 10 U of sulfatase along with 0.5 ml of 0.2 mol/L of sodium acetate buffer (pH 5) and 0.1 ml of0.2% sodium chloride solution. The mixture was incubated as above for 36 hours and then worked up and analyzed as above.
ci
20
30
MIN
Fig. 6. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of baboon amniotic fluid after an intravenous injection of 2.18 r-t-mol of tritiated ritodrine in the mother.
Results and comment
The total radioactivity plotted against time for the three experiments (A, B, and C in Fig. 2) showed maternal serum total radioactivity fell rapidly in the first 5 minutes and much more slowly after that. The fetal pattern was different: in all three experiments the total radioactivity rose slowly over 30 minutes and remained essentially constant thereafter. In the third experiment (Fig. 2, C) fetal levels exceeded those of the mother after 2 hours. This confirmed that ritodrine and/or its metabolites cross the placenta to the fetal circulation. Kleinhout, in his study of the pregnant ewe, 5 reported that the concentration of ritodrine in the fetus was only 20% of that of the mother 4 hours after a 20-minute infusion of the drug. Our results would seem to indicate that levels in the baboon fetus quickly reach or exceed those in the mother. One may speculate that there are interspecies differences in ritodrine placental transfer. Total urinary radioactivity output from the mother increased over the period of collection in all three experiments. Total activity in the amniotic fluid remained at a constant level, slightly lower than that of fetal serum, over the 3 hours of the third experiment. Hence fetal elimination of ritodrine and its metabolites ap-
Metabolism and disposition of ritodrine
Volume 152 Number 8
.___. ritodrine
80
FETAL I MATERNAL SERUM CONCENTRATION
o-·-·-o sulfate metabolite unidentified
PMOLE I Ml
n---~
FETAL
60
MATERNAl A
1071
80
4
A
60 40
____. ritodrine o··-···o sulfate metabolite
2
o--- -o
unidentified
20 0
80
0 3
8
60 1.6
40
20
0
3
15
c .D
... ..
.. .a-
-:.--~: ..... -·0'"'"
120
0 30 60
120
180
Fig. 7. Fetal and maternal baboon serum levels of ritodrine and its metabolites after an intravenous injection of 2.18 ,...mol of tritiated ritodrine in a pregnant baboon, from first (A), second (B), and third (C) experimental days.
pears to be very slow in comparison to that of the mother. When the samples were individually examined by high-pressure liquid chromatography, three separate peaks of radioactivity were found, which corresponded to ritodrine and two metabolites. There is a quantitative similarity in the maternal and fetal serum samples (Figs. 3 and 4) and in the maternal urine and amniotic fluid (Figs. 5 and 6), with the latter showing much higher relative ratios of parent drug than the former. This would seem to be due to urinary output of the fetus into the amniotic fluid. The identity of the early-eluting metabolite has not yet been determined, although its extremely short retention time indicates a highly polar compound; there is no firm evidence for its structure at this time, although in other experiments in this laboratory it has been demonstrated that ritodrine is capable of undergoing phase 1 metabolism in the rat with cleavage of the secondary amino group. The second metabolite was positively identified as the sulfate conjugate of ritodrine. Fig. 7 summarizes the results of those experiments. From a semilog graph of maternal ritodrine serum
-----·-
............... ..o
180 MIN
Fig. 8. Ratio of the concentration of ritodrine and its metabolites in the fetal serum to maternal serum, from first (A), second (B), and third (C) experimental days.
concentration versus time, a distribution half-life of 6 ± 1 minute and an elimination half-life of 6I ± I minute were determined. The elimination half-life was about half that found by Gandar6 in nonpregnant human female volunteers receiving a I-hour constant-rate ritodrine infusion. In the fetus the serum concentration pattern of ritodrine varies among the three experiments. In the first two experiments the ritodrine levels in the serum increased up to I hour and then declined, but in the third the levels decreased continuously from 10 minutes to 3 hours. This was obviously due to the ritodrine the mother had been receiving in her drinking water on the day previous to the experiment. Fig. 8 summarizes the calculated fetal/maternal serum concentration ratios for the three experiments. The maternal serum showed a very rapid rise of both metabolites within the first 10 minutes (Fig. 7), with the sulfate co~ugate as the predominant compound. In the second and third experiments the rate of elimination of all three compounds appeared approximately equal. Both metabolites were also found in the fetal serum, although they did not show the initial surge seen in the mother. Rather, their concentrations increased slowly, achieving levels comparable to those of the mother after 60 minutes. Their continued slow build-up, as seen in the third experiment, suggests that the fetus is at a
1072
Borrisud et al.
disadvantage relative to the mother in disposing of these compounds and that they can accumulate in the fetal circulation even though the parent drug is cleared, perhaps because of a poor recrossing of the placental barrier. The enzymatic degradation studies of the metabolite isolated from the maternal urine showed that it was inert toward ~-glucuronidase but was converted in high yield to the parent drug by sulfatase. Thus the only isolable conjugate of ritodrine in the baboon was the sulfate ester. Ritodrine administered to the pregnant baboon can cross the placenta and achieve levels in the fetal circulation equal to or higher than levels in the mother. The fetus appears to accumulate the metabolites to some extent. It would seem reasonable to assume that any extrauterine effects of ritodrine caused by its sympathomimetic activity should therefore be expected to be seen in the fetus as well as the mother. REFERENCES 1. Coutinho EM, de Sousa F, Wilson KH, Landesman R. Inhibitory action of a new sympathomimetic amine on the
August 15, 1985 Am J Obstet Gynecol
nongravid uterus. AM 1 0BSTET GYNECOL 1969;104:1053. 2. Barden TP, Peter 1B, Mer katz IR. Ritodrine hydrochloride: a betamimetic agent for use in preterm labor. Obstet Gynecol 1980;56: 1. 3. Gamissans 0, Esteban-Altirriba1, Maiques V. Inhibition of human myometrial activity by a new beta adrenergic drug. 1 Obstet Gynecol Br Commonw 1969;76:656. 4. Wesselius-de Casparis A, Thiery M, Yo le Sian A, et a!. Results of double-blind multicenter study with ritodrine in premature labor. Br Med 1 1971 ;3: 144. 5. Kleinhout 1, Veth A. Placental transfer of ritodrine and its effect on the fetal cardiovascular system. In: Eskes TKAB, de Haan1, Van Kessel H, Stolk1G, eds. Aspects of obstetrics today. Amsterdam: Excerpta Medica, 1975:385. 6. Gandar R, de Zoeten LW, van der Schoot VB. Serum level of ritodrine in man. Eur 1 Clin Pharmacal 1980; 17:117. 7. Nandakumaran M, Gardey C, Rey E, et al. Transfer of ritodrine and norepinephrine in human placenta: in vitro study. Dev Pharmacal Ther 1982;4:71. 8. Borrisud M. Metabolic study of ritodrine with high performance liquid chromatography [Dissertation]. Columbus, Ohio: The Ohio State University, 1983. 9. Horwitz DL, Ballantine TV, Herman CM. Acute effects of septic shock on plasma and red cell volumes in baboons. 1 Appl Physiol 1972;33:320.
7. Ballard PL. Glucocorticoid receptors in the lung. Fed Proc 1977;36:2660. 8. Gilden C, Sevanian A, Tierney DF, Kaplan SA, Barrett CT. Regulation of fetal lung phosphatidyl choline synthesis by cortisol: role of glycogen and glucose. Pediatr Res 1977;11:845. 9. Farrell PM, Zachman RD. Induction of choline phosphotransferase synthesis in the fetal lung by corticosteroids. Science 1973;179:297. 10. Rooney SA, Gross I, Gassenheimer LN, Motoyama EK. Stimulation of glycerolphosphate phosphatidyltransferase activity in fetal rabbit lung by cortisol administration. Biochim Biophys Acta 1975;398:433. II. Breiher A, Benson BJ, Williams MC, Mason RJ, Ballard PL. Corticosteroid induction of phosphatidic acid phosphatase in fetal rabbit lung. Biochim Biophys Res Commun 1977;77:883.
Glucocorticoids and j3-adrenergic-receptor agonists
12. Ekelund L, Arvidson G, Astedt B: Cortisol-induced accumulation of phospholipids in organ culture of human fetal lung. ScandJ Clin Lab Invest 1975;35:419. 13. Ekelund L, Arvidson G, Emanuelsson H, Myhrberg H, Astedt B. Effect of cortisol on human fetal lung in organ culture. A biochemical electron-microscopic and autoradiographic study. Cell Tissue Res 1975;163:263. 14. Torday JS, Smith BT, Giroud CJP. The rabbit fetal lung as a glucocorticoid target tissue. Endocrinology 1975; 96:1462. 15. Giannopoulos G. Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung. Biochim Biophys Res Commun 1980;95:388. 16. Cheng JB, Goldfien A, Ballard PL, Roberts JM. Glucocorticoids increase pulmonary beta-adrenergic receptors in fetal rabbit. Endocrinology 1980; I 07:1646.
Metabolism and disposition of ritodrine in a pregnant baboon Matana Borrisud, Ph.D., Richard O'Shaughnessy, M.D., MichaelS. Alexander, Ph.D., and Brian D. Andresen, Ph.D. Columbus, Ohio Relatively little is known about the detailed metabolism of ritodrine. The aim of this study was to examine ritodrine metabolism and pharmacokinetics in the maternal and fetal baboon. A fetal-maternal model was made with use of Papio anubis at 144 days of gestation. Tritiated ritodrine was injected as an intravenous bolus into the mother. Maternal and fetal blood samples, amniotic fluid, and maternal urine were collected at timed intervals. Samples were analyzed by a combination of high-pressure liquid chromatography and radiochromatography. Conjugated metabolites were recovered and characterized by cleavage studies with use of j3-glucuronidase and sulfatase. The distribution half-life of ritodrine in the mother was 6 minutes and the elimination half-life was 61 minutes. Metabolites were found in both fetal serum and amniotic fluid. The concentrations of ritodrine and its metabolites in fetal serum were at or above the concentrations in maternal serum at 2 hours after maternal intravenous injection. The principal metabolite identified was the sulfate conjugate. The fetus appears to accumulate metabolites. These data indicate that ritodrine crosses the placenta and, in the baboon, achieves levels in the fetus equal to or higher than those in the mother. (AM
J
0BSTET GYNECOL 1985;152:1067-72.)
Key words: Ritodrine, pharmacokinetics, metabolism (erythro-p-hydroxy-a-[ 1-([p-hydroxyRitodrine phenethyl]-amino)ethyl]benzyl alcohol) (Fig. 1), a new I)-sympathomimetic agent, has been used almost universally' for preterm labor patients.u It was first found to be a potent myometrial inhibitor in 1969. 13 In 1971 ritodrine was first reported to be a well-tolerated uterine relaxant that could arrest preterm labor in most cases.< In pharmacokinetic studies employing tritiated ritodrine, Kleinhout and Veth 5 reported that ritodrine From the Department of Obstetrics and Gynecology, The Ohio State University. Received for publication May 17, 1984; revised March 19, 1985; accepted March 27, 1985. Reprint requests: Richard O'Shaughnessy, M.D., Department of Obstetrics and Gynecology, 561 Means Hall, 1654 Upham Drive, Columbus, OH 43210-1228.
is inactivated by conjugation with glucuronic and sulfuric acids and that the parent compound crossed the placental barrier in a pregnant ewe. In 1980 Gandar et al. 6 developed a radioimmunoassay method for the determination for ritodrine and reported serum levels and half-lives in healthy volunteers. His studies with pregnant women also demonstrated that ritodrine crossed the placenta and entered the fetal circulation. Recently, Nandakumaran et al.' reported a method for the assay for ritodrine by high-pressure liquid chromatography with use of an isolated human placental perfusion model. Their studies also support evidence of ritodrine placental transfer. Ritodrine is currently the only tocolytic drug approved for obstetric use by the United States Food and Drug Administration Advisory Committee on Fertility 1067
1068
Borrisud et al.
August 15, 1985 Am J Obstet Gynecol
unidentified
HO-o-~ CH-CH-NH-CH-CH~OH I I 2 2~ OH CH
metabolite
sulfate
3
Fig. 1. Chemical structure of ritodrine.
en u.
serum
CPM 100
o
A
940 CPM/
'------·* o-o
200
fetal
::J <{
600
0
lO
maternal unne
•·· · ··• amruohc
4)-o---o- •• .,_,..--..0
0
______. maleludl s.tuum
10 ul maternal urme
CPM /ML
,,,,a
flu1d
''""""'"'·~-------- .. ---·
400
amniotic fluid
B
maternal
urine . 6 CPM X 10
ritodrine
200
!iO
100 --~ - ...... -u ,.~·-
0""''-~ ...
2!i
--o-----
0
0
0
c
200
100
................... o(J
!iO
30 MIN
Fig. 3. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of the maternal baboon serum after an intravenous injection of 2.18 j.Lmol of tritiated ritodrine in the mother.
.. -·- ...... u-
. .a--·-·
0
0
-
05
------<>-------o
0"".......
"·o..o- ... o--- o-- -.o-..-..-..-.-..--
30
60
.................. .. 120
25 0
180
MIN
Fig. 2. Total radioactivity of ritodrine and its metabolites from the maternal and fetal baboon serum, amniotic fluid (10 j.LI sampling), and maternal urine (total output). Data represent first (A), second (B), and third (C) experimental days.
and Maternal Health Drugs. 2 However, relatively little is known about the detailed metabolism of this drug. The aim of the present study was to examine ritodrine metabolism and pharmacokinetics in both the mother and fetal baboon simultaneously. Material and methods
All reagents used were of analytical grade; solvents were of high-pressure liquid chromatography grade. Both 13-glucuronidase and sulfatase (Sigma Chemical Company) were used in the metabolite cleavage experiments. Ritodrine was obtained as its hydrochloride salt from Astra Pharmaceutical Products. A sample of this material was labeled with tritium by catalytic exchange with use of tritiated water (New England Nuclear, Inc.). The crude tritiated ritodrine was purified by both thin-layer chromatography and high-pressure liquid chromatography" immediately prior to use. Serum, urine, and amniotic fluid samples were obtained from a healthy, fertile, female baboon (Papio
anubis) at a gestational age of 144 days. The baboon was premedicated with ketamine hydrochloride (110 mg) and atropine sulfate (0.6 mg) intramuscularly. The animal was also given hydroxyprogesterone caproate (500 mg) and benzathine penicillin (600,000 U) intramuscularly. The abdomen was shaved, and an intravenous infusion of 5% dextrose in Ringer's lactate was administered in a peripheral arm vein. Electrocardiogram and blood pressure monitoring devices were attached, and the animal was placed in a 15° left lateral position. General anesthesia was administered by endotracheal intubation. The anesthetic consisted of I% halothane and a 50% mixture of nitrous oxide and oxygen, both at a 300 ml/min flow rate. The abdomen was opened and a small incision made in the anterior myometrium over the area of the fetal lower extremities. Excess amniotic fluid was aspirated, kept warm, and returned to the uterine cavity at the end of the operation. The right lower extremity of the fetus was exteriorized and a cutdown was made in the femoral vein; a fine (PV 4) polyvinyl catheter was advanced over an introducer up the vein to a distance of 7 em (the approximate distance of the renal vessels). The fetal groin incision was closed with a fine polyglycolic acid suture. A fetal electrocardiogram electrode was attached to the right buttock, and a large-bore polyethylene catheter was placed within the uterine cavity to monitor intrauterine pressures and for amniotic fluid sampling. The
Metabolism and disposition of ritodrine
Volume 152 Number 8
amniotic fluid removed earlier was returned to the uterine cavity, and the catheters and electrocardiogram device were brought out through the uterine incision, which was closed in layers with polyglycolic acid suture. Another catheter was placed in the maternal femoral vein for maternal blood sampling. All catheters were filled with heparin (I U/cc). A catheter was also placed in the maternal bladder for urine sampling. On the day of operation, the first experiment lasted 1 hour. The catheters were then tunneled into the right flank and coiled in a subcutaneous pouch. Total operation time was 100 minutes. Estimated blood loss was 20 ml (baboon blood volume, 65 to 70 mllkg). 9 Fluid replacement consisted of 500 ml of 5% dextrose in Ringer's lactate. After the operation the animal was returned to the cage and received a normal diet enriched in fruit and vegetables, with water ad libitum. The animal's drinking water contained isoxsuprine at a total dose of 20 mg/day. On the second and fourth postoperative days the animal was returned to surgery after premedication as above. The fetal and intra-amniotic catheters and electrode were exteriorized, and the intra-amniotic catheter and fetal electrode attached to a Corometrics two-channel monitor for evaluation of intrauterine pressure and fetal electrocardiogram. The second experiment was performed on the second postoperative day. The animal's drinking water on the third postoperative day contained ritodrine at a total dose of 50 mg/day as additional insurance against premature labor. The third experiment was performed on the fourth postoperative day. At the end of each experiment, 4 ml of maternal blood was transfused back to the fetus to restore blood volume. At no time during the experiments did the animal have uterine contractions. In the experiments a solution (1 ml, 2.18 11-mol) of ritodrine hydrochloride containing standard tritiated ritodrine (total activity 2.0 X 10 8 cpm) was injected as an intravenous bolus into the mother. Maternal and fetal blood samples as well as amniotic fluid and maternal urine were collected at timed intervals. Blood samples from each experiment were centrifuged immediately. Serum was separated and frozen at -70° C until analysis. The other samples were similarly frozen. An aliquot (10 11-l) of each sample was counted for total radioactivity in a Packard Tri-Carb well-type scintillation counter. All samples were then analyzed by high-pressure liquid chromatography. Serum and amniotic fluid samples were diluted with acetonitrile (I : 3 vol/vol), vortexed thoroughly, and centrifuged. The clear supernatant was separated and taken to dryness under nitrogen at soo C. Each sample
unidentified
1069
metabolite
sulfate
• I
CPM/ML
(/)
600
I
u. :::>
I I I
1.0
<(
,,.•
400
•'•'•' •'••
•'
,!l
N
ci
.
ritodrine
I •'' ~ ~, __ -_,....
200
,
-- --.,
0 0
10
20
30 MIN
Fig. 4. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of fetal baboon serum after an intravenous injection of 2.18 f.Lmol of tritiated ritodrine in the mother.
was reconstituted with 200 11-l of the mobile phase solvent and injected onto the chromatographic column. Urine samples were injected directly onto the column following centrifugation. Analyses were performed by combined high-pressure liquid chromatography and radiochromatography.8 A Beckman Altex Series 322MP gradient liquid chromatograph, equipped with Beckman Model 165 ultraviolet detector, Gilson fraction collector, and a high-performance phenyl-coated column, were used. The absorption maxima for ritodrine at 225 and 275 mm were used for ultraviolet detection. Ritodrine and metabolite levels in serum, urine, and amniotic fluid were measured radiologically. Metabolites were recovered from maternal urine for identification. A urine sample (100 ml) was lyophilized, and the solid residues were triturated in methanol. After centrifugation to remove insoluble solids, the supernatant was applied to a conventional liquid chromatography column (30 by 2 em), packed to a height of 20 em with silica gel 60. The eluting solvent was a mixture of chloroform/methanol/formic acid (10: 5: 1 vollvol), and the sample was eluted under 8 pounds per square inch gauge nitrogen pressure. One hundred 4 ml fractions were collected. An aliquot of each was
1070 Borrisud et al.
August 15, 1985 Am J Obstet Gynecol
unidentified
metabolite
unidentified
sulfate CPMX10 4 /ML 15
metabolite
sulfate
•
12 (/)
LL
::J
CPM/ML 9
tn
I
ritodrine
J
U)
II.
:::1
I"
100
~
~
6
II I I II I I fl Ill I IV I : I I I I
50
3
0
-- I 0
0 30 MIN
Fig. 5. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of maternal baboon urine after an intravenous injection of 2.18 jLmol of tritiated ritodrine in the mother.
counted for radioactivity. Those with significant activity were examined by high-pressure liquid chromatography, and fractions containing the metabolite were combined for analysis. A portion of the combined ritodrine metabolite fractions was analyzed directly as a control. A second portion was taken to dryness and reconstituted with water (1 ml). The solution was adjusted to pH 7 (sodium hydroxide), added to 1000 units of 13-glucuronidase, and stirred in a 3 7° C bath for 3 hours. The reaction was terminated by addition of three volumes of ac;etonitrile and vortexing. After centrifugation and drying of the supernatant under nitrogen, the sample was reconstituted with mobile phase solvent and analyzed by high-pressure liquid chromatography. Fractions (1 ml) were collected and counted for radioactivity. A third portion was also taken to dryness and reconstituted with 0.4 ml of water. This was added to 10 U of sulfatase along with 0.5 ml of 0.2 mol/L of sodium acetate buffer (pH 5) and 0.1 ml of0.2% sodium chloride solution. The mixture was incubated as above for 36 hours and then worked up and analyzed as above.
ci
20
30
MIN
Fig. 6. Ultraviolet and radiochromatogram obtained by highpressure liquid chromatography (solvent programming) of baboon amniotic fluid after an intravenous injection of 2.18 r-t-mol of tritiated ritodrine in the mother.
Results and comment
The total radioactivity plotted against time for the three experiments (A, B, and C in Fig. 2) showed maternal serum total radioactivity fell rapidly in the first 5 minutes and much more slowly after that. The fetal pattern was different: in all three experiments the total radioactivity rose slowly over 30 minutes and remained essentially constant thereafter. In the third experiment (Fig. 2, C) fetal levels exceeded those of the mother after 2 hours. This confirmed that ritodrine and/or its metabolites cross the placenta to the fetal circulation. Kleinhout, in his study of the pregnant ewe, 5 reported that the concentration of ritodrine in the fetus was only 20% of that of the mother 4 hours after a 20-minute infusion of the drug. Our results would seem to indicate that levels in the baboon fetus quickly reach or exceed those in the mother. One may speculate that there are interspecies differences in ritodrine placental transfer. Total urinary radioactivity output from the mother increased over the period of collection in all three experiments. Total activity in the amniotic fluid remained at a constant level, slightly lower than that of fetal serum, over the 3 hours of the third experiment. Hence fetal elimination of ritodrine and its metabolites ap-
Metabolism and disposition of ritodrine
Volume 152 Number 8
.___. ritodrine
80
FETAL I MATERNAL SERUM CONCENTRATION
o-·-·-o sulfate metabolite unidentified
PMOLE I Ml
n---~
FETAL
60
MATERNAl A
1071
80
4
A
60 40
____. ritodrine o··-···o sulfate metabolite
2
o--- -o
unidentified
20 0
80
0 3
8
60 1.6
40
20
0
3
15
c .D
... ..
.. .a-
-:.--~: ..... -·0'"'"
120
0 30 60
120
180
Fig. 7. Fetal and maternal baboon serum levels of ritodrine and its metabolites after an intravenous injection of 2.18 ,...mol of tritiated ritodrine in a pregnant baboon, from first (A), second (B), and third (C) experimental days.
pears to be very slow in comparison to that of the mother. When the samples were individually examined by high-pressure liquid chromatography, three separate peaks of radioactivity were found, which corresponded to ritodrine and two metabolites. There is a quantitative similarity in the maternal and fetal serum samples (Figs. 3 and 4) and in the maternal urine and amniotic fluid (Figs. 5 and 6), with the latter showing much higher relative ratios of parent drug than the former. This would seem to be due to urinary output of the fetus into the amniotic fluid. The identity of the early-eluting metabolite has not yet been determined, although its extremely short retention time indicates a highly polar compound; there is no firm evidence for its structure at this time, although in other experiments in this laboratory it has been demonstrated that ritodrine is capable of undergoing phase 1 metabolism in the rat with cleavage of the secondary amino group. The second metabolite was positively identified as the sulfate conjugate of ritodrine. Fig. 7 summarizes the results of those experiments. From a semilog graph of maternal ritodrine serum
-----·-
............... ..o
180 MIN
Fig. 8. Ratio of the concentration of ritodrine and its metabolites in the fetal serum to maternal serum, from first (A), second (B), and third (C) experimental days.
concentration versus time, a distribution half-life of 6 ± 1 minute and an elimination half-life of 6I ± I minute were determined. The elimination half-life was about half that found by Gandar6 in nonpregnant human female volunteers receiving a I-hour constant-rate ritodrine infusion. In the fetus the serum concentration pattern of ritodrine varies among the three experiments. In the first two experiments the ritodrine levels in the serum increased up to I hour and then declined, but in the third the levels decreased continuously from 10 minutes to 3 hours. This was obviously due to the ritodrine the mother had been receiving in her drinking water on the day previous to the experiment. Fig. 8 summarizes the calculated fetal/maternal serum concentration ratios for the three experiments. The maternal serum showed a very rapid rise of both metabolites within the first 10 minutes (Fig. 7), with the sulfate co~ugate as the predominant compound. In the second and third experiments the rate of elimination of all three compounds appeared approximately equal. Both metabolites were also found in the fetal serum, although they did not show the initial surge seen in the mother. Rather, their concentrations increased slowly, achieving levels comparable to those of the mother after 60 minutes. Their continued slow build-up, as seen in the third experiment, suggests that the fetus is at a
1072
Borrisud et al.
disadvantage relative to the mother in disposing of these compounds and that they can accumulate in the fetal circulation even though the parent drug is cleared, perhaps because of a poor recrossing of the placental barrier. The enzymatic degradation studies of the metabolite isolated from the maternal urine showed that it was inert toward ~-glucuronidase but was converted in high yield to the parent drug by sulfatase. Thus the only isolable conjugate of ritodrine in the baboon was the sulfate ester. Ritodrine administered to the pregnant baboon can cross the placenta and achieve levels in the fetal circulation equal to or higher than levels in the mother. The fetus appears to accumulate the metabolites to some extent. It would seem reasonable to assume that any extrauterine effects of ritodrine caused by its sympathomimetic activity should therefore be expected to be seen in the fetus as well as the mother. REFERENCES 1. Coutinho EM, de Sousa F, Wilson KH, Landesman R. Inhibitory action of a new sympathomimetic amine on the
August 15, 1985 Am J Obstet Gynecol
nongravid uterus. AM 1 0BSTET GYNECOL 1969;104:1053. 2. Barden TP, Peter 1B, Mer katz IR. Ritodrine hydrochloride: a betamimetic agent for use in preterm labor. Obstet Gynecol 1980;56: 1. 3. Gamissans 0, Esteban-Altirriba1, Maiques V. Inhibition of human myometrial activity by a new beta adrenergic drug. 1 Obstet Gynecol Br Commonw 1969;76:656. 4. Wesselius-de Casparis A, Thiery M, Yo le Sian A, et a!. Results of double-blind multicenter study with ritodrine in premature labor. Br Med 1 1971 ;3: 144. 5. Kleinhout 1, Veth A. Placental transfer of ritodrine and its effect on the fetal cardiovascular system. In: Eskes TKAB, de Haan1, Van Kessel H, Stolk1G, eds. Aspects of obstetrics today. Amsterdam: Excerpta Medica, 1975:385. 6. Gandar R, de Zoeten LW, van der Schoot VB. Serum level of ritodrine in man. Eur 1 Clin Pharmacal 1980; 17:117. 7. Nandakumaran M, Gardey C, Rey E, et al. Transfer of ritodrine and norepinephrine in human placenta: in vitro study. Dev Pharmacal Ther 1982;4:71. 8. Borrisud M. Metabolic study of ritodrine with high performance liquid chromatography [Dissertation]. Columbus, Ohio: The Ohio State University, 1983. 9. Horwitz DL, Ballantine TV, Herman CM. Acute effects of septic shock on plasma and red cell volumes in baboons. 1 Appl Physiol 1972;33:320.