1 Steroid Research Laboratory, Institute of Biomedicine, 2 Department of Obstetrics and Gynecology, University of Helsinki, Finland, 3 The Population Council, Center for Biomedical Research, New York, 4 The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, and 5 Balance Pharmaceuticals Inc., Santa Monica, USA
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Abstract |
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Key words: distribution/lactation/pharmacokinetics/progestin-only contraception/radioimmunoassay
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Introduction |
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Even though lactational contraception with progestin-only contraceptives has been endorsed by international family planning organizations, hormonal methods are often viewed cautiously during lactation. All the contraceptive progestins studied to date can be detected in the milk (Díaz and Croxatto, 1993), thus concerns about the possible ill-effects on the suckling infant remain.
An ideal molecule for lactational hormonal contraceptive would be one with high contraceptive efficacy in the mother, yet it would be without effects on lactation or the nursing infant. Nestorone® (NES, previously known as ST-1435) is an orally inactive progestin, currently in phase II clinical contraceptive trials using implants, vaginal rings, and intracervical or transdermal administration (Kurunmäki et al., 1984; Laurikka-Routti et al., 1990
; Haukkamaa et al., 1991
). NES has high binding affinity to the human progesterone receptor (Lähteenmäki, 1986
), yet due to rapid first-pass metabolism, oral administration of NES is ineffective (Coutinho et al., 1981
; Heikinheimo et al., 1994
). However, parenterally-administered NES is highly effective for contraception, and ovulation is inhibited with very low serum concentrations of NES (Coutinho et al., 1981
; Lähteenmäki et al., 1982
).
NES, therefore, has several key features which may make it an optimal hormonal contraceptive to be used during lactation. While parenteral administration of NES to nursing mothers would guarantee effective contraception, the NES passed to the suckling infant via milk would be rapidly metabolized by the infant's liver.
The purpose of the present study was to examine the distribution as well as the hormonal effects of lactational use of NES in nursing cynomolgus monkeys and their infants.
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Materials and methods |
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Before enrolling the primates into the study, chronic jugular venous catheters were inserted in two of the adult monkeys under ketamine anaesthesia (20 mg/kg i.m.) supplemented with xylazine (1 mg/kg i.m.) to allow serial blood sampling for studies on prolactin secretion. The catheters ended in s.c. ports capped with silicone-coated latex diaphragms which allowed transcutaneous access to the venous system via small (2127)-gauge needles inserted into the ports.
Study protocol
Blood sampling (daily from the mother, every third day from the infant) was initiated at approximately 90 days following delivery. Pre-treatment samples were collected for 2 weeks, after which a 1 cm Nestorone® implant was inserted s.c. The implants were used for a total of 4 weeks, after which the implants were removed, and blood sampling continued for an additional 2 weeks. Based on in-vitro testing, the implants released ~40 µg NES/day. The infants were weighed weekly during the duration of the study.
To study more closely the dynamic variations in prolactin secretion during NES administration, two of the monkeys had indwelling jugular cannulae inserted prior to initiation of the study. Frequent blood sampling (every 15 min between 8:00 and 14:00) was performed at 2 weeks before insertion (pre-treatment), at 2 weeks after insertion (treatment) and at 2 weeks after (post-treatment) removal of the Nestorone® implant. Due to blockade of the system, sample collection was successful in only one of these primates. On the preceding afternoon, the animals were fitted with special vests allowing lactation and mobile steel tethers, which protected the catheters passing from the s.c. port to the back of the cage and into an adjacent room, where the blood samples (2 ml) were drawn.
Animal welfare compliance
This study was approved by the Institutional Animal Care and Use Committee of the Eastern Virginia Medical School. The facilities of the division of Animal Resources are fully accredited by the American Association for the Accreditation of Laboratory Animal Care.
Radioimmunoassays
Serum concentrations of NES were measured as previously described (Lähteenmäki et al., 1981) using 125I-labelled NES as tracer and polyclonal NES antibody, raised against NES-3-(O-carboxymethyl)oximeBSA conjugate in rabbits. The intra- and interassay coefficients of variation (CV) of serum NESradioimmunoassay were 5 and 7% respectively. The practical detection limit was 13 pmol/l.
For the analysis of milk concentrations of NES, the milk samples were extracted with petroleum ether (b.p. 4060°C), evaporated until dry, redissolved into petroleum ether and applied on Sep-Pak® C18 Cartridges (Waters Corp., Milford, MA, USA) as described by the manufacturer. The Sep-Pak® C18 Cartridges were washed with petroleum ether, after which NES was eluted using methanol. Thereafter, the samples were evaporated, and redissolved into PBS buffer used in the NESradioimmunoassay. In the milk assays, 3H-NES was used as a tracer; otherwise the radioimmunoassay was performed as described previously (Lähteenmäki et al., 1981).
Serum oestradiol and progesterone were measured using commercially available radioimmunoassays from ICN Biomedicals Inc. (Costa Mesa, CA, USA). Serum prolactin was measured by specific radioimmunoassays developed for primate work, as described in detail elsewhere (Gordon et al., 1992). CV were calculated from pools of standard serum. Intra-assay and interassay CV were 11 and 18%; 10 and 15%; 10 and 12%; for the oestradiol, progesterone and prolactin at the average hormone concentrations of 195 pmol/l, 4 nmol/l and 10 µg/l respectively. The detection limits were 70 pmol/l, 1 nmol/l and 3 µg/l respectively.
To study the possibility that NES is metabolized while being passed to milk, a fractionation of diethyl ether extracts of milk and serum samples was carried out using high performance liquid chromatography (HPLC) followed by detection of the immunoreactive NES metabolites with NESradioimmunoassay. The HPLCNESradioimmunoassay system for the studies on the NES metabolism has been described previously (Heikinheimo et al., 1994). The milk samples were processed for HPLC as for radioimmunoassay using ether extraction and Sep-Pak® C18 Cartridges.
Data analysis
A P value 0.05 was considered significant. One way analysis of variance (ANOVA) was used to evaluate the changes in serum concentrations of prolactin.
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Results |
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Concentrations (mean ± SE) of NES in maternal serum, milk and infant serum are shown in Figure 1. The mean (± SD) maternal serum concentrations of NES were 337 (± 90) pmol/l during the use of the implants. The NES concentrations (mean ± SD) in milk were 586 ± 301 pmol/l. The ratio of serum/milk NES was 1.68 ± 0.12 (mean ± SE), and the concentrations of serum and milk NES were significantly correlated (r = 0.75, P < 0.001). NES was not detectable (<13 pmol/l) in any of the infant samples.
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The identity of the measurable material by radioimmunoassay in monkey milk was confirmed by HPLC fractionation of ether-extracted milk samples prior to radioimmunoassay. Practically all material measurable by radioimmunoassay in the milk samples eluted in a single peak with an identical retention time as the NES standard (Figure 2), thus confirming that the radioimmunoassay measured the parent NES in the milk samples.
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Discussion |
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In the present study, the use of 1 cm NES implants resulted in mean NES concentrations of above 270 pmol/l in all of the primates. Previous studies in women have shown that serum NES concentrations of ~100 pmol/l are sufficient for inhibition of ovulation in women (Lähteenmäki et al., 1982; Haukkamaa et al., 1992
). Thus the concentrations of NES seen in this study are well within the clinically-proven effective range. In accordance with the rapid metabolism of NES in women, the serum concentrations of NES decreased rapidly following removal of the implants, the half-life being 1.4 h in the monkey.
Progestins of different chemical structure are passed to human milk differently. The progestins of the prenane class, such as medroxyprogesterone acetate (MPA), are transferred to milk at higher ratios (milk/serum ~0.88) than estrane or gonane progestins; milk/serum ratios of 0.34 and 0.15 have been reported for norethisterone and levonorgestrel (Nilsson et al., 1977; Heikkilä et al., 1982
; Koetsawang et al., 1982
). NES is a pregnane, thus higher milk/serum ratios are to be expected. Previously (Lähteenmäki et al., 1990
) a milk/serum ratio of 0.60 in lactating women using s.c. NES implant was reported. The NES concentrations in primate milk exceeded those measured in the serum, thus the milk/serum ratio was 1.68. Factors thought to increase the progestin content of human milk include the absence of high-affinity binding protein in maternal serum, and an increase in the milk fat content (Nilsson et al., 1977
). In human serum NES is loosely bound by albumin (Lähteenmäki et al., 1983
), which might partly explain the high passage of NES to milk. Moreover, the fat content of the primate milk was not determined in the present study.
Despite the unusually high concentrations of NES in primate milk, no NES could be detected in the infant serum. Similarly in vitro, human fetal liver has been reported rapidly to metabolize 3H-NES (Lähteenmäki, 1986). Also rat liver completely degraded the NES arriving via the portal blood (Heikinheimo et al., 1994
). The fact that infant liver effectively metabolizes the NES in the milk endorses the safety of NES as a lactational contraceptive.
The patterns of prolactin release have been previously evaluated during the use of depot MPA contraception in lactating women (Chaudhury et al., 1977). Increased mean concentrations as well as enhanced prolactin release to suckling stimulus was noted (Chaudhury et al., 1977
). In the present study, a decrease in the pooled concentrations of prolactin was noted between the pre-treatment versus treatment and post-treatment values; however, when analysed at weekly intervals throughout the study, no statistically significant differences were found in the circulating prolactin concentrations (Figure 3
). In addition, the patterns of prolactin release were similar before, during and after the NES implant when analysed in during frequent sampling in one of the primates (Figure 4
).
Progestin contraception during lactation does not affect infant growth or development (WHO, 1994). In the present study, the mean weight increased during the NES treatment. However, a small drop in the mean infant weight was noted at 4 weeks. This was due to weight loss in one of the infants. The reason for this is unknown; however, the effect of repeated blood sampling on the infant growth cannot be ruled out.
In conclusion, use of Nestorone® implants in nursing cynomolgus monkeys results in high concentrations of NES in maternal circulation and in milk. However, NES was not detectable in any of the infants. The weekly maternal serum concentrations of prolactin were unaffected during the study. Furthermore, the infants' weight gain continued during the study. Therefore it appears that NES approaches the ideal lactational contraception an effective contraception in the mother and devoid of effects in the nursing infants.
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Acknowledgments |
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Notes |
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Nestorone® is a registered trademark of The Population Council, New York, NY, USA
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References |
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Submitted on January 27, 1999; accepted on April 21, 1999.