Gonadotrophin and testosterone suppression by 7{alpha}-methyl-19-nortestosterone acetate administered by subdermal implant to healthy men

G. Noé1,6, J. Suvisaari2, C. Martin3, A.J. Moo-Young4, K. Sundaram4, S.I. Saleh4, E. Quintero1, H.B. Croxatto1 and P. Lähteenmäki2,5

1 Instituto Chileno de Medicina Reproductiva, Santiago, Chile, 2 Steroid Research Laboratory, Department of Medical Chemistry, Institute of Biomedicine, University of Helsinki, Helsinki, Finland, 3 Center for Reproductive Biology, Edinburgh, UK, 4 The Population Council, Center for Biomedical Research, New York, USA and 5 Väestöliitto, The Family Federation of Finland, Helsinki, Finland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The synthetic androgen 7{alpha}-methyl-19-nortestosterone (MENT) is a potent suppressor of gonadotrophin that has several advantages for long term administration to normal or hypoandrogenic men. The aim of this study was to examine MENT serum concentrations following subdermal insertion of MENT acetate (MENT Ac) implants and their effects on gonadotrophins, testosterone, dihydrotestosterone (DHT), sex hormone-binding globulin, prostate specific antigen and insulin-like growth factor-1 serum concentrations in normal men. A total of 45 healthy men were recruited at three clinics. Each subject received one, two or four implants for 28 days. Serum samples were obtained before insertion and on days 8, 15, 22, 29, 36 and 43 after implant insertion. The average daily dose delivered in vivo by one implant was ~500 µg. One, two or four MENT Ac implants produced dose dependent and sustained serum MENT concentrations for the entire duration of treatment of 0.7 ± 0.1, 1.2 ± 0.1 and 2.0 ± 0.1 nmol/l respectively. This treatment induced a dose dependent decrease in gonadotrophin and androgen serum levels. Two and four implants induced maximal suppression that was maintained throughout treatment and was completely reversed after removal of the implants. The mean decreases were 93 ± 1% for testosterone, 80 ± 3% for DHT, 97 ± 1% for luteinizing hormone and 95 ± 1% for follicle stimulating hormone. No serious adverse reactions were reported by the volunteers and no consistent changes in clinical chemistry and haematology were found. These results indicate that MENT Ac implants are an efficient way of MENT administration and confirm the potent gonadotrophin and androgen suppressive effect of this drug.

Key words: gonadotrophins/implant/male contraception/7{alpha}-methyl-19-nortestosterone/testosterone


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The need for effective and reversible methods of fertility control for men is widely recognized, but continues to be unmet. One approach being considered to suppress spermatogenesis is to inhibit gonadotrophin secretion by administering an androgen alone or combined with a gonadotrophin-releasing hormone analogue or a progestogen. The synthetic androgen 7{alpha}-methyl-19-nortestosterone (MENT) has several advantages for long term administration for male contraception or for androgen substitution therapy: (i) it is a potent suppressor of gonadotrophin secretion in man (Suvisaari et al., 1997Go); (ii) it is ~10 times more androgenic than testosterone (Kumar et al., 1992Go); (iii) it is not metabolized to 5{alpha}-reduced compounds, therefore, in contrast to testosterone, its action is not amplified in the prostate (Kumar et al., 1992Go); (iv) in vitro, it is aromatized to 7{alpha}-methyl oestradiol, which can prevent osteopenia (Anderson et al., 1997Go); (v) MENT exhibits negligible binding to sex hormone-binding globulin (SHBG) (Kumar et al., 1997Go). Presumably, MENT would not accumulate in organs that contain androgen binding protein, such as seminiferous tubules and epididymis (Bordin and Petra 1980Go), and would not mediate SHBG actions through its specific membrane receptors in the prostate (Nakhla and Rosner 1996Go); and (vi) due to its high potency, a low daily dose of MENT will need to be administered, making delivery through an implant system feasible.

MENT acetate (MENT Ac) has been shown by in-vitro studies to be suitable for release from an ethylene vinyl acetate (EVA) copolymer implant delivery system. In-vivo studies have shown it is converted to MENT in the circulation. Therefore the Population Council undertook the development of a MENT Ac subdermal implant to provide adequate androgen replacement for an extended period. Here, MENT serum concentrations following subdermal insertion of one, two or four implants and their effects on gonadotrophins, testosterone, dihydrotestosterone (DHT), SHBG, prostate specific antigen (PSA) and insulin-like growth factor-1 (IGF-1) serum concentrations in normal men are reported.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
The study was carried out in three clinics: the Consultorio de Planificación Familiar, Instituto Chileno de Medicina Reproductiva, Santiago, Chile, the Family Planning Clinic of The Family Federation of Finland (Väestöliitto), Helsinki, Finland and the Center for Reproductive Biology, Edinburgh, Great Britain. A total of 45 healthy men, 15 at each clinic, participated in the study. The average (and range) of the age of Chilean, Finnish and Scottish subjects were 28 (20–42), 32 (22–45) and 31 (23–39) years respectively and their average body mass indexes (and ranges) were 24.3 (20.2–30.1), 24.4 (21.5–30.6) and 25.3 (20.1–31.1). Subjects used no contraceptive other than condoms during the study, but their sexual partner had to be protected by an effective contraceptive method if condoms were not regularly used.

MENT Ac implants
Each implant done by Dr.A.Moo-Young at the Population Council Laboratory was 4.4 cm long, contained 112 ± 4 mg of MENT Ac and released in vitro 396 ± 5 µg of drug per day over a 33 day period (n = 6).

Design of the study
Five subjects were allocated to each dose at each clinic. Each subject received one, two or four implants. The health of the volunteers was evaluated by medical history, general physical examination, routine clinical chemistry (alkaline phosphatase, alanine aminotransferase, gamma glutamyl transferase, lactate dehydrogenase, aspartate amino transferase, total bilirubin, calcium, creatine kinase, creatinine, chloride, iron, glucose, potassium, sodium, albumin, protein total, urea, total cholesterol, cholesterol high density lipoproteins, cholesterol low density lipoproteins, triglycerides) and haematology (haemoglobin, haematocrit, erythrocytes, leukocytes, mean corpuscular volume, platelets). Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), T, DHT, IGF-1, SHBG and PSA were also determined before treatment. After this screening procedure subjects were randomly assigned to one of three groups to receive one, two or four implants.

Insertion was performed under aseptic conditions. After local anaesthesia with 2% lidocaine, the implants were inserted subdermally through a trocar in the medial aspect of the upper non-dominant arm. Removal under local anaesthesia through a 4 mm incision was scheduled to take place on day 29.

Serum samples were obtained at approximately the same time of the day, on days 8, 15, 22, 29, 36 and 43, after implant insertion, for MENT, T, DHT, LH, FSH, and IGF-1 assays. Dihydrotestosterone and IGF-1 were measured in subsets of subjects as indicated in results, while the other hormones were measured in all subjects. At each collection time, three samples were taken at 20 min intervals for LH and FSH. On days 29 and 43 additional samples were drawn for clinical chemistry, haematology, SHBG and PSA analysis.

Semen analysis was not included as an end point of the study, since studies designed to test effects in spermatogenesis require longer exposure that in this case was not allowed by the available preclinical toxicology. However, a further 6 month study is currently underway in which semen analysis is the principal end point.

The study was conducted according to good clinical practice guidelines and the Declaration of Helsinki and as an approved Investigational New Drug (IND) by the Food and Drug Administration, USA. The institutional ethics committees of each clinic approved the study and all subjects gave written consent before enrolment.

Assays
The concentrations of MENT, T, DHT, FSH, LH, IGF-1 and SHBG were measured in the Steroid Research Laboratory, Department of Medical Chemistry, University of Helsinki. The intra- and inter-assay coefficient of variation and limit of detection for each assay are summarized in Table IGo. MENT concentrations were measured by radioimmunoassay as described previously (Kumar et al., 1990Go). The cross-reactivity of T in the MENT radioimmunoassay was 1.1% and this is probably responsible for the high limit of detection of MENT, 1 nmol/l (Table IGo). Testosterone concentrations were determined by conventional RIA according to a standard operating procedure of the World Health Organization (WHO) (Sufi et al., 1993Go). The cross-reactivity of MENT in the testosterone radioimmunoassay was 1.2%. Serum DHT levels were measured using the TRK 600 assay kit from Biotrak (Amersham Life Science, Bucks., UK). The concentrations of FSH, LH and SHBG were measured by time-resolved fluoroimmunoassay using commercially available kits (DELFIA®, Wallac OY, Turku, Finland). For IGF-1 determination an immunoradiometric kit from Immunotech (Paris, France) was used. All hormones and SHBG measurements were made in duplicate, and duplicates with a CV exceeding 15% were re-assayed.


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Table I. Properties of the hormone and SHBG assays
 
Clinical chemistry, haematology and PSA assays were carried out in the local laboratories of each clinic.

Assessment of in-vitro and in-vivo release rate
Twenty-five implants recovered intact were used to determine the in-vitro release rate of MENT Ac for a 19 day period. Each one was placed in an individual amber-capped bottle, containing a known volume of 1:750 aqueous solution of Zephiran® (benzalkonium chloride) and was incubated in a water-bath maintained at 37°C while shaking at a rate of 100 strokes/min. The incubation medium was changed daily and the MENT Ac was determined spectrophotometrically at 244 nm against a Zephiran® solution reference. Appropriate standard solutions of MENT Ac were also read with the unknown samples. In order to determine the in-vivo release rate of MENT Ac, the steroid content of 100 recovered implants was assayed. Each implant was cut into small pieces, ~1 mm in thickness. The pieces were placed in a Soxhlet apparatus and extracted with absolute ethanol for 24 h. The ethanol extracts were transferred quantitatively to volumetric flasks and made to mark with absolute ethanol. These served as unknown working solutions. Appropriate standards were prepared in duplicate. Standard and unknown solutions were measured spectrophotometrically at 240 nm, against an absolute ethanol reference on a Perkin-Elmer Lambda 2 spectrophotometer.

Statistical analysis
The concentration of LH and FSH was determined in each of the three serum samples taken at 20 min intervals and expressed as the mean of the three samples.

Statistical analysis was performed using Statistical Analysis System (NC: SAS Institute Inc., 1987) release 6.12. To meet the assumption of normality of T, FSH and SHBG values comparisons were made using logarithm transformation. A P value < 0.05 was considered significant. Variance analysis of independent measurement, including clinic effect, were performed for comparison between doses by each sampling day. The interaction clinic-dose was not included in the model because it was not significant. The mean comparisons were done by Tukey's test. Variance analysis for repeated measurements was done for comparisons throughout time. The recovery of hormone levels after implant removal with respect to baseline concentration were analysed by t-test for paired data.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Luteinizing hormone, FSH, testosterone, SHBG and PSA basal concentrations were similar in the three clinics and since no significant dosexclinic interactions were detected for any of these hormones, SHBG, PSA and MENT, the results are expressed as the mean ± SE of the 15 subjects per dose group.

The use of one, two, or four MENT implants for 28 days caused a clear dose dependent biological response. All three doses were effective in lowering gonadotrophins and testosterone. One implant had a moderate effect, giving mean maximal decreases of 61 ± 7% for testosterone; 40 ± 9% for LH and 31 ± 11% for FSH (baseline = 100%). Two and four implants had near maximum effect. There was no significant difference between the response to two and four implants. Gonadotrophins and testosterone had fallen to the lowest concentrations in the first sample taken 8 days after implant insertion and remained so for the duration of treatment. The mean maximal decreases were 93 ± 1% for testosterone; 97 ± 1% for LH and 95 ± 1% for FSH. Two weeks after removal of the implants, all hormone concentrations had returned to pre-treatment values. In the group that used four implants, a rebound increase in LH and FSH was detected 1 week after implant removal (Figure 1Go).



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Figure 1. Concentration of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone and 7{alpha}-methyl-19-nortestosterone (MENT) before, during and after administration of one ({square}), two (•) or four ({blacktriangleup}) MENT acetate (MENT Ac) implants for 28 days in 14 healthy men and for 38 days in one subject corresponding to the group of four implants. In that case, days 39, 46 and 53 were considered equivalent to days 29, 36 and 43 for the purpose of analysis. Implants were inserted on day 1 and removed on day 29. Each value represents the mean ± SE of 15 subjects, except for days 36 and 43 in the group of four implants in which n = 14. MENT could not be detected after treatment (see Results). aSignificantly different from baseline, bsignificantly different from one implant and csignificantly different from two implants.

 
MENT was measured in samples before, during and after treatment. During the use of the implants, the levels of MENT were dose dependent, reaching concentrations of 0.69 ± 0.1, 1.18 ± 0.1 and 2.03 ± 0.2 nmol/l for one, two and four implants respectively (Figure 1Go). Serum MENT concentrations were not detected in samples obtained during the recovery period.

SHBG concentrations decreased significantly in the groups that used two and four implants (P = 0.043 and 0.001 respectively) (Figure 2Go). Treatment with MENT had no effect on PSA concentrations (Figure 2Go).



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Figure 2. Concentration of serum hormone binding globulin (SHBG) and prostate specific antigen (PSA) before, during and after administration of one ({square}), two (•) or four ({blacktriangleup}) MENT Ac implants for 28 days in 14 healthy men and for 38 days in one subject corresponding to the group of four implants. In that case, days 39, 46 and 53 were considered equivalent to days 29, 36 and 43 for the purpose of analysis. Each value represents the mean ± SE of 15 subjects, except for day 43 in the group of four implants in which n = 14. *Significantly different from baseline.

 
Dihydrotestosterone was determined in subgroups of eight subjects for doses of one and two implants and in seven subjects for the dose of four implants. The concentration of DHT in serum showed a dose-dependent fall, parallel to testosterone decrease (not shown). The maximal declines observed in the one, two and four implant groups were 60 ± 3, 76 ± 1 and 77 ± 2% respectively.

In the Finnish clinic, IGF-1 was measured in three, three and four subjects in the one, two and four implant groups respectively. IGF-1 serum levels remained constant throughout the study (data not shown).

There was one case of local infection, in the group of four implants, which responded to antibiotic therapy without necessitating removal of the implants.

Symptoms reported by the volunteers were: pain at the site of implant insertion (n = 7), mood change (n = 4), androgenic effects (n = 7), sweating and insomnia (n = 2) and muscle cramps, diarrhoea and headache (n = 1). Subjects in Helsinki complained that the location of the implants interfered with their physical activity. No subject reported decreased libido.

Five subjects had intercurrent infectious diseases during the use of the implants, which affected the airways in four and the middle ear in one. None of them was considered to be related to MENT administration.

No consistent changes in blood cell count or clinical chemistry assays indicating adverse effects of MENT were detected (Table IIGo).


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Table II. Clinical chemistry and haematology
 
Two volunteers kept the implants in place for more than 28 days; one who had one of the four implants for 6 weeks due to problems of removal and another who could not attend the scheduled visit on day 29 for implant removal and had the implants in situ for an additional 10 days. In the first case, samples of days 35 and 43 were not used for the purpose of the analysis because those samples, which corresponded to the recovery period, were drawn during the use of one implant. In the second case, days 39, 46 and 53 were considered equivalent to days 29, 36 and 43, assuming that the use of the implants for an additional 10 days was unlikely to change the pharmacodynamic effects of the drug.

The implants were returned to the Population Council laboratory for assessment of the daily in-vitro and in-vivo release rate of MENT Ac. Among these, 25 implants, which were intact (i.e. without damage to the integrity of the implant), were used to assess the in-vitro release rate of the steroid. The mean daily release rate was estimated to be 360 ± 3 µg of MENT Ac. This compared favourably to the release rate of 396 ± 5 µg/day noted in the unused control implants. The amount of MENT Ac lost by each of the 100 implants was calculated by subtracting the drug content assessed after implant use from the known drug load of each implant. The average daily in-vivo release rate was estimated by dividing the steroid loss by the days of implant use. Sets of one, two and four implants resulted in average daily in-vivo release of 477 ± l9, 103l ± 58 and l944 ± 82 µg MENT Ac respectively (Figure 3Go).



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Figure 3. The average in-vivo daily release rates of MENT Ac from 100 implants recovered following continuous use for 4 weeks by men. The rates were obtained by dividing the amount of steroid lost by the number of days of implant use. Each column and bar represent the mean and SE, respectively of sets of one, two or four implants obtained in each clinic and the mean of the three clinics. The numbers within the column represent the sample size.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
One, two or four MENT Ac implants produced dose dependent and sustained serum MENT levels for the entire duration of treatment. It was not possible to determine the decrease of MENT after implant removal, probably because MENT was cleared during the week that elapsed from the end of treatment to the next blood sample.

The average daily dose delivered, calculated by in-vivo release rate was directly proportional to the number of implants used, i.e. four and two implants delivered four and two times more steroid respectively than one implant. The differences in the release rate estimated in vivo and in vitro were probably due to loss of drug during manipulation of the implants or to a higher release rate during the first few days after insertion. It should be noted that, although some implants were damaged during removal, the insertion and removal of the implants were not difficult procedures. The damage to the integrity of the implants resulted from grasping the implants with serrated or toothed forceps during the removal procedure. This resulted in punctures mostly to the end of the implant. In the opinion of the investigators, this does not pose a risk to the subject.

MENT induced a dose dependent decrease in testosterone, DHT, LH and FSH serum levels. Two and four implants induced near maximum gonadotrophin, testosterone and DHT suppression that was maintained throughout treatment and was completely reversed after removal of the implants. There were no significant differences in gonadotrophin and testosterone concentrations between the groups that used two and four implants. These results confirm the gonadotrophin and T suppressive effect of MENT observed after six consecutive daily i.m. injections of MENT (Suvisaari et al., 1997Go).

The reciprocal cross-reactivity of MENT and testosterone in their respective radioimmunoassays of ~1% limits the accuracy of the assays, since each one may show higher than actual concentrations when the cross-reactive steroid is increased. Thus, actual concentrations of testosterone and MENT could be somewhat lower than those shown in Figure 1Go.

It has been reported (Handelsman et al., 1992Go) that gonadotrophin suppression between 4 and 16 weeks after implantation of six pellets containing in total 1200 mg of fused crystalline testosterone and releasing 9 mg/day. This dose of testosterone was ~10 fold higher than the dose of MENT Ac used in the present study and both induced the same effect, showing the higher potency of MENT in comparison with testosterone. In addition, the mean total and free testosterone serum concentrations increased during the use of testosterone pellets and were one order of magnitude higher than the MENT concentration, pointing again to the high potency of MENT in comparison with testosterone. Later, similar suppression of gonadotrophins was reported by these authors using a lower dose of 800 mg testosterone, (6 mg/day) plus 300 mg of depot medroxyprogesterone acetate (DMPA). With this regimen, testosterone concentrations were maintained in the normal range (Handelsman et al., 1996Go).

The decrease in SHBG serum concentration after 28 days of use of two and four implants may reflect a specific effect of MENT on secretion of this protein rather than unspecific toxicity. This is supported by the fact that none of the subjects showed an alteration of the lipid profile or liver function tests. A similar decrease in SHBG was observed after 1 month of use of 800 mg testosterone implants supplemented with 300 mg DMPA. Since 800 mg testosterone alone did not change SHBG concentrations significantly, it was proposed that DMPA could be responsible for the SHBG decline (Handelsman et al., 1996Go).

The increase in PSA is the marker of prostate hyperplasia most frequently examined. In the present study MENT had no effect on serum PSA levels over the entire period of examination. It has been recently reported that there is a strong correlation between serum IGF-1 levels and the risk of developing prostate cancer (Chang et al., 1998Go). Administration of testosterone enanthate to hypogonadal men increased IGF-1 serum levels (Ip et al., 1995Go); the same was observed in normal subjects (Hobbs et al., 1993Go). No change in IGF-1 was detected in the present study.

Serum DHT concentrations changed in parallel with serum testosterone concentrations. This behaviour was consistently observed with every dose and it is not surprising, since most of the circulating DHT in adult males is derived from the 5{alpha}-reduction of testicular testosterone. The inhibition of 5{alpha}-reductase with finasteride has been shown to increase the incidence of cancer in patients with a high risk of prostate cancer (Cote et al., 1998Go). However, the participation of low DHT levels on this effect remains to be assessed. A recent study in which rat prostate gene expression was compared in normal, castrated and finasteride-treated animals suggests that testosterone and DHT regulate common as well as different genes in this organ (Avila et al., 1998Go). Thus, MENT reduces both testosterone and DHT but would not simulate the pattern of gene expression caused by finasteride alone. The effect of long-term administration of MENT in the prostate has still to be investigated.

Minor adverse reactions with no outstanding symptoms were noted by volunteers. The androgenic effects reported by seven volunteers were: increased libido, increased sex activity, pricking in testes, acne, weight gain and increased hairiness. The short period of MENT administration, 28 days, makes it difficult to evaluate the significance of these symptoms. Not a single volunteer reported ejaculatory dysfunction. As expected, MENT substituted testosterone in the maintenance of libido, since no subject complained of decreased libido. In a separate study, it was shown that MENT Ac implants restored libido and sexual function in hypogonadal men (Anderson, 1999Go). Transitory local symptoms at the implant insertion site were the most frequent complaint.

The use of EVA implants appears to be an efficient way to administer MENT Ac because it maintained low and stable circulating levels of MENT throughout treatment. The data also suggest that different doses of MENT Ac can be delivered by subdermal implants depending on the indication, whether for androgen replacement therapy or for contraception.

These results warrant extended phase I/II clinical trials designed to observe effects on spermatogenesis. It will be of interest to determine if the gonadotrophin and T suppressive effect of MENT is sufficient to produce azoospermia. Other androgens used for male contraception show high variability in individual responsiveness to hormonal suppression (Handelsman et al., 1992Go; Behre et al., 1995Go; Meriggiola et al., 1997Go, 1998Go) Whether or not MENT is more efficacious and its effects more predictable than these other androgens, remains to be seen.


    Acknowledgments
 
This study undertaken by the International Committee for Contraception Research of the Population Council is part of the Contraceptive Development Program at the Population Council's Center for Biomedical Research, New York. We wish to acknowledge the generous support received from the US Agency for International Development (Cooperative Agreement CCP-A-0094–00013–04); the United Nations Population Fund (UNFPA); The Andrew W.Mellon Foundation; The George Hecht Family Trust, The Educational Foundation of America; The Lita Annenberg Hazen Foundation and Brigham Trust. The study in Edinburgh was supported in part by the Department for International Development and Medical Research Council (Grant 9523250) to the contraceptive Development Network, Centre for Reproductive Biology, Edinburgh. Our thanks are also due to Dr Kaisa Elomaa who took care of the insertion and removal of the implants in Helsinki.


    Notes
 
6 To whom correspondence should be addressed at: Instituto Chileno de Medicina Reproductiva, Correo 22, Casilla 96, Santiago, Chile Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 22, 1999; accepted on June 11, 1999.