Parenteral administration of progestin Nestorone® to lactating cynomolgus monkeys: an ideal hormonal contraceptive at lactation?

O. Heikinheimo1,2,6, S. Ranta1, A. Moo-Young3, P. Lähteenmäki1 and K. Gordon4,5

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nestorone® (NES) progestin is highly effective for contraception following parenteral administration, but ineffective after oral ingestion due to rapid first-pass metabolism. Thus, NES might be ideal for lactational contraception; possible NES in milk should be metabolized by the nursing infant. We evaluated the distribution of NES, its endocrine effects and infant weight gain in five cynomolgus monkeys and their nursing infants. Nestorone® implants, releasing ~40 µg NES/day in vitro, were placed s.c. in the mothers 3–4 months following delivery, where they remained in situ for 4 weeks. Sampling (blood daily from the mother; milk and blood from the infant at 3 day intervals) was initiated at 2 weeks prior to insertion, and continued for 2 weeks following removal of the implant. NES, oestradiol, progesterone and prolactin were measured by radioimmunoassays and the infants were weighed weekly. The (mean ± SD) maternal serum and milk concentrations of NES were 337 ± 90 and 586 ± 301 pmol/l during the use of the implants. The ratio of milk/serum NES was 1.68 ± 0.12 (mean ± SE), and the serum and milk concentrations were significantly correlated (r = 0.75, P < 0.001). NES was not detectable (<13 pmol/l) in any infant serum samples. Concentrations of prolactin (mean ± SD) were 41.1 ± 32, 26.7 ± 7.6 and 26.3 ± 9.5 ng/ml before, during and after the use of the implants respectively. The (mean ± SE) infant weight increased from 643 ± 54 g 1 week prior to insertion to 713 ± 54 g 1 week following removal. These data confirm that NES in milk is rapidly metabolized by the suckling infant. Therefore, NES appears to be an ideal hormonal contraceptive for use during lactation.

Key words: distribution/lactation/pharmacokinetics/progestin-only contraception/radioimmunoassay


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the worldwide scheme of family planning, extension of the interval between pregnancies would result in major improvement in maternal and child health (Thapa et al., 1988Go). Lactation itself provides a reasonable contraceptive effect initially. However, the duration of lactational amenorrhoea varies greatly (WHO, 1998Go), and even in fully nursing women ovulatory cycles are resumed in 26% at 6 months post-partum (Díaz et al., 1988Go). Pregnancy and childbirth require women to seek medical attention, thus providing an opportunity for contraceptive counselling and introduction. Thus, safe and effective contraceptives which can be initiated during the post-partum period and lactation are important assets in fertility control.

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, 1993Go), 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., 1984Go; Laurikka-Routti et al., 1990Go; Haukkamaa et al., 1991Go). NES has high binding affinity to the human progesterone receptor (Lähteenmäki, 1986Go), yet due to rapid first-pass metabolism, oral administration of NES is ineffective (Coutinho et al., 1981Go; Heikinheimo et al., 1994Go). However, parenterally-administered NES is highly effective for contraception, and ovulation is inhibited with very low serum concentrations of NES (Coutinho et al., 1981Go; Lähteenmäki et al., 1982Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Five nursing cynomolgus monkeys (Macaca fascicularis) and their infants were used for the present study. The mother–infant couples were housed in individual cages under a 12 h:12 h light–dark schedule at 20–23°C; the mother was fed monkey chow (Agway, Elizabeth City, NJ, USA) twice per day with water available ad libitum. Blood samples were drawn by femoral venipuncture under ketamine-induced anaesthesia (10 mg/kg i.m.).

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 (21–27)-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., 1981Go) using 125I-labelled NES as tracer and polyclonal NES antibody, raised against NES-3-(O-carboxymethyl)oxime–BSA conjugate in rabbits. The intra- and interassay coefficients of variation (CV) of serum NES–radioimmunoassay 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. 40–60°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 NES–radioimmunoassay. In the milk assays, 3H-NES was used as a tracer; otherwise the radioimmunoassay was performed as described previously (Lähteenmäki et al., 1981Go).

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., 1992Go). 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 NES–radioimmunoassay. The HPLC–NES–radioimmunoassay system for the studies on the NES metabolism has been described previously (Heikinheimo et al., 1994Go). 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The improved method for the radioimmunoassay measurement of NES in milk was evaluated using blank primate milk spiked with unlabelled NES. The practical detection limit of the milk NES–radioimmunoassay was 52 pmol/l; recovery of the added NES was 68%. The intra-assay CV was 12% and the interassay CV 11–13%, when calculated using primate milk spiked with 135–270 pmol/l of unlabelled NES.

Concentrations (mean ± SE) of NES in maternal serum, milk and infant serum are shown in Figure 1Go. 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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Figure 1. Concentrations (mean ± SE) of Nestorone® (NES) in the mothers' serum ({square}), milk ({blacklozenge}) and infants' serum (•) during lactational use of NES implants. The ratio of milk/serum NES was 1.68 ± 0.12 (mean ± SE); the concentrations of serum and milk NES were significantly correlated (r = 0.75, P < 0.001). NES was not detectable in any of the infant samples studied. The insert shows the disappearance of NES following removal of the implant, the calculated t1/2 (±SD) of NES was 1.4 (±0.4) h.

 
Following insertion high serum concentrations of NES were measured at 1 h; at 2 h the mean concentration of NES was 957 pmol/l, thus NES was rapidly distributed from the site of implantation. Following removal of the implants, serum concentrations of NES rapidly decreased; the calculated half-life (± SD) was 1.4 (± 0.4) h (Figure 1Go, insert).

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 2Go), thus confirming that the radioimmunoassay measured the parent NES in the milk samples.



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Figure 2. The identity of the NES in primate milk was ascertained by HPLC + radioimmunoassay analysis of the milk samples. The behaviour of NES in milk ({blacklozenge}) was identical to that of a NES-standard ({square}), thus parent NES was passed to and measured in the milk.

 
Serum concentrations of prolactin (mean ± SE) are depicted in Figure 3Go. The mean (±SD) concentrations of prolactin in the daily blood sample were 43 (±33), 26 (±17) and 29 (±26) µg/l in the samples collected prior to, during and after the use of the NES implants respectively. Comparison of the pooled prolactin values using Student's t-test revealed statistically significant differences when the pre-implant concentrations were compared with those measured during (P < 0.001) and after (P < 0.05) the use of the Nestorone® implants. One way ANOVA, performed with prolactin concentrations at weekly intervals over the study period, indicated no statistically significant differences at different time points of the study.



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Figure 3. Serum concentrations of prolactin (mean ± SE) in the nursing female monkeys during the use of NES implant.

 
Figure 4Go shows the individual concentrations of prolactin concentrations during frequent blood sampling in one of the monkeys before, during and after the use of Nestorone® implant. The mean (±SD) concentrations of prolactin over the frequent sampling period in this individual monkey in the pre-implant, implant and post-implant samples were 61.1 (±37.2), 70.0 (±26.7) and 66.8 (±6.0) µg/l respectively.



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Figure 4. Prolactin concentrations during frequent blood sampling in one of the monkeys before ({square}), during ({blacklozenge}) and after ({circ}) the use of NES implant. Samples were collected via indwelling jugular cannule between 08:00 and 14:00 h at 2 weeks before insertion (pre-implant), 2 weeks after insertion (implant) and 2 weeks following removal of the implant (post-implant).

 
Measurable concentrations of oestradiol were detected in some of the primates prior to implant use. However, during the use of the NES implants, serum oestradiol remained below the detection concentration of the currently used oestradiol radioimmunoassay (Figure 5Go). Similarly, progesterone was not detectable in any of the maternal serum samples (data not shown).



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Figure 5. Maternal serum concentrations of oestradiol during the use of Nestorone® implants. The oestradiol concentrations appeared to be suppressed during the first 2 weeks of the implant use. However, some follicular activity appeared towards the end of the implant use, and specially thereafter. Progesterone was not detectable in any of the samples.

 
The infant weight gain throughout the study period is shown in Figure 6Go. The mean weight of 620 g at the beginning of the study increased to 734 g, measured at 2 weeks after implant removal. A notch in the mean weight at 4 weeks was due to weight loss in one of the five infant primates.



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Figure 6. Infant weight (mean ± SD) during the use of NES implants.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
High contraceptive efficacy and rapid first-pass metabolism, and thus lack of effect following oral administration, make Nestorone® progestin a strong candidate for lactational contraception. We studied the endocrine effects and distribution of parenterally administered NES in nursing cynomolgus monkeys and their infants.

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., 1982Go; Haukkamaa et al., 1992Go). 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., 1977Go; Heikkilä et al., 1982Go; Koetsawang et al., 1982Go). NES is a pregnane, thus higher milk/serum ratios are to be expected. Previously (Lähteenmäki et al., 1990Go) 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., 1977Go). In human serum NES is loosely bound by albumin (Lähteenmäki et al., 1983Go), 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, 1986Go). Also rat liver completely degraded the NES arriving via the portal blood (Heikinheimo et al., 1994Go). 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., 1977Go). Increased mean concentrations as well as enhanced prolactin release to suckling stimulus was noted (Chaudhury et al., 1977Go). 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 3Go). 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 4Go).

Progestin contraception during lactation does not affect infant growth or development (WHO, 1994Go). 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.


    Acknowledgments
 
We wish to thank Mrs Eeva Harju of the Steroid Research Laboratory for her expert editorial help. This work was supported in part by the contraceptive development programme sponsored by the International Committee for Contraception Research of The Population Council Inc., New York, NY, USA. Also grants from The Jalmari and Rauha Ahokas and the Sigrid Jusélius Foundation, Helsinki, Finland (O.Heikinheimo) as well as The Andrew Mellon Foundation, New York, NY, USA (K.Gordon) are gratefully acknowledged.


    Notes
 
6 To whom correspondence should be addressed at: Steroid Research Laboratory, Institute of Biomedicine, PO Box 8, FIN-00014, University of Helsinki, Finland Back

Nestorone® is a registered trademark of The Population Council, New York, NY, USA Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 27, 1999; accepted on April 21, 1999.





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