Subcutaneous bioabsorbable pellets of norethindrone for contraception in women: Phase I. Clinical study*†‡

Vol. 41, No.5, May 1984

FERTILITY AND STERILITY Copyright © 1984 The American Fertility Society

Printed in U.8A.

Subcutaneous bioabsorbable pellets of norethindrone for contraception in women: Phase I. Clinical study*t*

Gopi N. Gupta, Ph.D.§ Brij B. Saxena, Ph.D., D.Se. Robert Landesman, M.D. William J. Ledger, M.D. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Cornell University Medical College, New York, New York

Ten healthy, normally menstruating female volunteers participated in a 1-year phase I clinical study in which subcutaneous pellet implants of norethindrone (NET) were employed as a low-dose and long-acting potential contraceptive. Two NET pellets were implanted subcutaneously by the aid of a trocar in the forearm of each volunteer on the fifth day after the start of menstrual bleeding. Serum levels of NET, follicle-stimulating hormone, luteinizing hormone, 17rp-estradiol, and progesterone were determined weekly by radioimmunoassay. The daily NET release from the pellets, based on mean values (± standard error of the mean) in five subjects was 79.4 ± 7.6 fLg. The mean serum NET level was initially 1.0 ± 0.34 ng/ml; thereafter, it gradually lowered during the 343 days of the study period to the level of 0.43 ± 0.09 ng/ml. The ovarian response, days of bleeding, and cycle lengths with continuously sustained release of NET from the pellets were similar to those observed in women taking the daily oral "minipill" of 300 WJ NET. The results of the phase I study suggest that NET pellet implants may provide a simple and acceptable approach to long-term contraception in women. Fertil Steril41:726, 1984

Received March 7, 1983; revised and accepted January 25, 1984. *Presented in part at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24, 1982, Las Vegas, Nevada. tSupported by grants from The Rockefeller Foundation, The Robert-Wood Johnson Charitable Trust, New York, New York; and the Program for Applied Research on Fertility Regulation (PARFR), Northwestern University, Chicago, Illinois, subcontract PARFR 330 under contract AIDIDSPE-C-0035. *The contents of this publication do not necessarily reflect the policy of the United States Agency for International Development (AID). §Reprint requests: G. N. Gupta, Ph.D., 103 Lewis Drive; Upper Nyack, New York 10960.

terone) in combined oral pills has ranged from 0.5 to 2 mg/day; the estrogen component has ranged between 30 and 80 j.Lg of ethinyl estradiol. Various dose-dependent side effects have been produced by oral contraceptives. In order to minimize the side effects, estrogen-free, progestin-only "minipills" containing a lower dose of 0.35 mg NET have been commercially available since 1973. Silastic capsules containing lower doses of NET have also been used subcutaneously in women. 2 The contraceptive effect has required, in general, 3 to 12 Silastic capsules 24 mm in length with an outside diameter of 2.4 mm. However, daily NET release from Silastic capsules is not sustained and decreases significantly with time,3 thus rendering them effective only for a limited period; furthermore, the Silastic capsules must be removed on depletion of the medication. NET Silastic rods, containing a high concentration of the

Gupta et al. Norethindrone pellets for contraception

Fertility and Sterility

At present, 80 to 100 million women around the world rely on a daily oral "pill" for contraception. 1 The amount of a commonly used contraceptive, norethindrone (NET; 19-nor-17-I3-ethinyltestos-

726

progestin (80% NET by weight), provide a fairly constant release of NET.4 The release per unit surface area is higher than with Silastic capsules. Within the past few years, totally bioabsorbable pellets of pure NET and of levonorgestrel (LNG), with a small amount of cholesterol, have been prepared by a fusion method (U.S. Patent No. 4,244,949; G. N. Gupta). These pellets release sustained and fairly constant levels of 3H-LNG in rats,5 rabbits,6 and monkeys.7 In humans,s, 9 the fused NET pellets also provide a sustained, constant, and higher release of NET per unit surface area than the Silastic capsules, 2 Silastic rods,4, 10 and various polymeric polylactate NET preparations. 11 , 12 The uses of NET and LNG pellets as potential long-term contraceptive implants have recently been reported. 5, 13 This article describes results of a phase I clinical study with the NET pellets in ten women volunteers for a period of 11 to 12 months. Daily NET release from two pellets implanted subcutaneously in each of the ten women volunteers was determined up to the end of the study period. Serum levels of NET, follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17(3estradiol (E 2), and progesterone (P) were analyzed and correlated with the ovarian response, bleeding patterns, and cycle lengths of women throughout the study period. MATERIALS AND METHODS WOMEN VOLUNTEERS

The ten volunteers were healthy, normally menstruating women 19 to 34 years of age, were fully informed about the purpose and methods of the study, and agreed to sign consent forms. All were instructed to use a barrier method for contraception during the study period. STUDY PROTOCOL

Ten-milliliter blood samples were drawn weekly from either arm of each volunteer during one or two control cycles. Two NET pellets were then implanted subcutaneously by means of a trocar in the left ventral forearm of each subject on the fifth day of the menstrual cycle. NET PELLETS

NET ofUSP grade was obtained from Diosynth, Inc., Chicago, IL, and further purified by recrysVol. 41, No.5, May 1984

tallization. NET pellets were prepared individually by a method that melts the progestin and cholesterol and permits a one-step operation for fusion, molding, sterilization, and packaging of the pellet in a Teflon tube. The pellets used were composed of 85% NET and 15% pure cholesterol with an average weight of 34.4 ± 0.6 mg each and were ellipsoidal in shape, measuring 5.5 mm x 3 mm x 2.6 mm with a surface area of 58.0 mm2. Each Teflon tube containing one pellet was stored in a sterile glass vial. The sterility of the pellets was confirmed by the Department of Microbiology, Cornell University Medical College.

IMPLANTATION AND REMOVAL OF PELLETS

A stainless steel trocar consisting of a cannula, a stylet, and a plunger allowed easy and rapid subcutaneous implantation of NET pellets. The surface of the left ventral forearm was cleansed with an antiseptic solution, and a sterile drape was placed over the arm, exposing the site of implantation. One milliliter of 1% lidocaine was injected subcutaneously. After 30 seconds, a 3-mm incision was made into the skin. The skin was pierced by the stylet and the cannula advanced 3 cm under the skin. The stylet was then withdrawn from the cannula. The NET pellets were removed from the Teflon tubes and placed flat in the groove of the cannula with a sterile forceps. The plunger was introduced through the cannula to place the pellets in tandem. The cannula and plunger were withdrawn, and pressure was applied at the implant site for 30 seconds. The incision was closed by approximating the skin and applying a Steri-Strip (Surgical Products Division, St. Paul, MN). For removal, pellets were located by palpation and marked by a sterile dye. Local anesthesia was applied at the implant site as described above. Then, through a minor incision made across the implant site, the pellets were removed along with the intimately surrounding fibrous tissue, and the incision was closed as described above. DETERMINATION OF DAILY ABSORPTION OF NET FROM PELLETS AND NET LEVELS IN SERUM

Pellets were dissected out from the surrounding fibrous tissue and dried overnight at 800 C to a constant weight. The total amount of NET absorbed, determined from the difference in weight between the original and the retrieved pellets, Gupta et aI. Norethindrone pellets for contraception

727

was divided by the number of treatment days to obtain the mean daily absorption rate as micrograms of NET per day. Serum NET levels were measured by a radioimmunoassay (RIA) technique of Walls et al.,14 utilizing a microscintillation counting procedure. 15 3H-NET, labeled at positions 6 and 7 with a specific activity of 55 Cilmmol, was· purch~sed from New England Nuclear Corporation, Boston, MA. A rabbit antiserum against NET-11 a-succinoyl-oxy-bovine serum albumin was obtained from Schering AG, Berlin, West Germany. The NET antiserum was used at a final dilution of 1:32,000. The antiserum had < 0.08% cross-reaction with E2 and P. The intraassay and interassay coefficients of variation were 10% and 15%, respectively. HORMONE ASSAYS

Serum concentrations of FSH and LH were measured by RIA with kits purchased from Cambridge Medical Diagnostics, Inc., Billerica, MA. The sensitivity of the assays for FSH and LH was 3 mlUlml. The intraassay and interassay coefficients of variation were 5% and 11%, respectively. Serum levels of E2 and P were measured by RIA after ether extraction without chromatography. Antisera were supplied by Arnel Products Company, Inc., Brooklyn, NY. 3H-Iabeled E2 and P with a specific activity of 100 Ci/mmol each were purchased from New England Nuclear Corporation. On the basis of serum E2 and P levels, menstrual cycles were classified as anovulatory, mixed, or ovulatory, as described by Landgren and Diczfalusy.16 The first type (anovulatory) exhibited no signs of follicular or luteal activity, as evidenced by the low E2 and P levels; the second type (mixed) showed either normal follicular activity and no luteal function or normal but insufficient luteal activity. The third type (ovulatory) had normal follicular and luteal activity.

period, and it fell to the level of 0.43 ± 0.09 ng/ml during the 343 days of the study period. After implantation of the pellets, the midcycle preovulatory FSH and LH surge, as well as the follicular and luteal phase levels of FSH and LH, were suppressed during the initial two to three cycles. OVARIAN RESPONSE DURING THE TREATMENT PERIOD

The ovarian resporise to the continuous low dose of NET was reflected in serum E2 and P levels in all ten subjects. The control cycles during pretreatment periods were ovulatory in all the subjects. As shown in Table 1, the percentage of distributions of ovulatory, mixed, and anovulatory patterns in all the subjects during initial postimplant cycles were 40%, 20%, and 40%, respectively, and subsequently changed to 50%, 10%, and 40% in later cycles. BLEEDING. SPOTTING. AND CYCLE LENGTHS

The average number of days of bleeding and spotting calculated per 28-day interval during the treatment period of up to 372 days in ten women is summarized in Table 2. The average number of days of bleeding and spotting was 3.85 ± 0.58. There were minor variations in cycle length during treatment periods. HISTOPATHOLOGIC EXAMINATIONS OF TISSUES SURROUNDING THE PELLETS

There was no infection or scarring in any subject at the site of the implant. The histopathologic examination of the tissue surrounding the retrieved pellets revealed formation of a thin fibrous capsule associated with a light chronic inflammation. Different degrees of inflammatory cell reaction were shown involving mainly mononuclear cells and foreign body giant cells.

DISCUSSION RESULTS DAILY ABSORPTION OF NET FROM PELLETS AND SERUM LEVELS OF NET AND HORMONES

As determined in five subjects, the mean daily NET absorption from two pellets with a total length of 1.1 cm per subject was 79.4 ± 7.6 f.J.g/day with a range of 59.1 to 101.7 f.J.g/day. As shown in Figure 1, the mean serum NET level in ten women was 1.0 ± 0.34 ng/ml during the early 14-day 728

Gupta et al. Norethindrone pellets for contraception

The results of the present phase I study have shown that the mean daily absorption of NET over a period of 11 to 12 months in women who received two NET pellets was 79.4 ± 7.6 f.J.g/day. The daily level of NET in the serum was sustained, which was consistent with earlier results observed in animals6 and in humans. 9 The release rate of NET per unit surface area from the pellets was more uniform and significantly higher than with other subcutaneous delivery systems, Fertility and Sterility

.~ iE 2.0

~

~~~'\ 1.5

o

14

28

42

56

Figure 1 Mean ± standard error of the mean serum NET levels in ten women over a period of 343 days.

70 OAYS

factory antifertility effect. While the vaginal ring contained more NET and released 200 fJ-g NET/ day, it was twice as potent as administration of 300 fJ-g orally by the minipills; yet the higher dose of NET caused increased days of bleeding and spotting. In our study, the mean serum NET level in ten women initially was 1.0 ± 0.34 ng/ml (range, 0.4 to 1.1 ng/ml) and remained at a sustained level of 0.43 ± 0.09 ng/ml (range, 0.34 to 0.52 ng/ml) up to 11 to 12 months. It is noteworthy that in spite of the individual variations, the low serum NET levels obtained from the pellets were effective in changing the menstrual patterns of the women initially into 40% ovulatory, 20% mixed, and 40% anovulatory cycles, which subsequently changed to 50% ovulatory, 10% mixed, and 40% anovulatory cycles. In the present study the mean days of bleeding and spotting per interval were 3.85 days, slightly lower than occurred during the pretreatment period of 4.9 to 5.4 days. This finding also concurs with the recent observation of the low-dose effect of NET by Landgren et al. 20 Furthermore, Moghissi et al. 21 and Gutierrez-Najar et al. 22 reported earlier that the low-dose progestins caused breakthrough bleeding only when ovulation was apparently suppressed. The mechanism of action of NET appears to be dose-related, and the NET may have multiple loci in target organs. It has also been reported that

namely, the Silastic capsules, Silastic rods, and various polymeric polylactate-NET preparations. Furthermore, the pellets produced no initial surge or "burst effect" of NET in the blood, which usually occurs with the Silastic capsules and polymeric polylactate-NET preparations. The very early studies of Shimkin et al. 17 and Bishop and Folley 18 also noted increased uniformity in the absorption rate of sex steroid hormones from the fused pellets. It is interesting to note that a daily absorption of79.4 ± 7.6 fJ-g NET/day from two pellets, which is - 25% of the NET provided by the daily oral minipill, produced 40% ovulatory, 20% mixed, and 40% anovulatory cycles, similar to the pattern obtained from the oral mini pill. These findings confirm the earlier observation of Chang and Kincl 19 that the bioavailability and effectiveness ofprogestins through the subcutaneous route was several times higher than through the oral route. The probable reason is that a drug through the oral route is affected by the first-pass effect and thus a large amount of the drug is metabolized in the liver. Recently, Landgren et al. 20 reported comparative effects of the continuous release of NET from vaginal rings and daily oral NET minipills. In their study, the release of 50 fJ-g NET/day from the vaginal ring produced a marginal effect on the ovary and was insufficient to achieve a satis-

Table 1. Ovarian Response as Reflected by Serum E2 and P Levels During Exposure to a Continuous Low Dose of NET Up to a Period of 11 to 12 Months a Treatment cycles Subject M.e. B. K. G.M. M.A. B.E. S. F. D. B. E.H. L.A. B. J.

Pretreatment cycles

Initial cycles

F

L

%

F

+ + + + + + + + + +

+ + + + + + + + + +

20

+ + + + +

40

+ 40

Later cycles L - (M) - (M) + (Ov) + (Ov) + (Ov) - (Anov) + (Ov) - (Anov) - (Anov) - (Anov)

%

F

L

+ + +

+ (Ov)

10

+ +

+ (Ov) + (Ov)

50 40

+

- (M)

+ (Ov) - (Anov) - (Anov) - (Anov) + (Ov) - (Anov)

aF, follicular; L, luteal; +, normal; -, suppressed or absent; M, mixed; Ov, ovulatory; Anov, anovulatory. Vol. 41, No.5, May 1984

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Table 2. Mean ± Standard Error of the Mean Days of Bleeding and Spotting in Ten Women During the Treatment Period Up to 372 Days Subjects

Treatment period

Total bleed· ing and spotting

Bleeding and spotting/2Sday intervala

M.C. B. K. G.M. M.A. B.E.

325 247 325 372 357 327 291 201 319 307

31 31 65 74 68 7 44 8 59 52

2.7 3.5 5.6 5.6 5.3 0.6 4.2 1.1 5.2 4.7

S. F. D.B. E. H. L.A.

B. J.

aMean ± standard error of the mean

=

3.85 ± 0.58.

the suppression of ovulation is a dose-dependent phenomenon. 23 For example, higher doses may affect the feedback mechanism at the hypothalamus-pituitary level, thus causing anovulation. The lower doses may affect the steroid receptor content in the endometrium, thereby interfering with implantation. The effect oflow levels of NET may also be visualized as changing the biochemical properties and consistency of the cervical mucus, which may interfere with sperm migration. Moghissi et al.,21 as early as 1973, indicated that a safe and effective contraceptive device should utilize the minimum dose of a progestin to achieve a maximum effect on cervical mucus and endometrium without inhibition of ovulation. The present study suggests that the subcutaneous NET pellet implant may prove a useful approach in that direction. It may be mentioned that during our studies ~ 50% of the subjects voluntarily did not use other contraception. One of the ten volunteers conceived during the eighth month of the study and delivered a normal female infant. This fact may indicate a needed increase in the daily serum NET levels to allow for individual variations in order to render NET pellets an effective contraceptive for use by a larger number of women over a longer period of time. In conclusion, the two NET pellets in the phase I clinical study have provided a sustained and fairly constant serum NET level over a period of 11 to 12 months. There was a high degree of acceptability of the NET pellets by all volunteers. The smaller size of the pellets also has the advantages of ease and speed in the implantation and retrieval of the pellets. These factors have further enhanced the potential of NET pellet implants as 730

Gupta et aI. Norethindrone pellets for contraception

long-acting, low-dose, and safe contraceptive agents. Acknowledgment. The authors gratefully acknowledge the excellent technical assistance of Mrs. Bokja Park.

REFERENCES 1. Hatcher RA, Stewart GK, Stewart F, Guest F, Stratton P, Wright AH: Hormone contraceptives: an overview. In Contraceptive Technology 1978-1979. New York, Irvington Publisher, 1978, p 35 2. Croxatto HB, Diaz S, Quinteros E, Simoneti L, Kaplan E, Rencoret R, Leixeland P, Martinez C: Clinical assessment of subdermal implant of megestrol acetate, d-norgestrel, and norethindrone as a long-term contraception in women. Contraception 12:615, 1975 3. Wiese J, Marker IL, Holma P, Vartiainen E, Osler M, Pyoriilii T,Johansson E, Luukkainen T: Long-term contraception with norethindrone subcutaneous capsules. Ann Clin Res 8:93, 1976 4. Gupta GN: Prolonged release of testosterone propionate form new solid steroid-saturated Silastic implants in rats. Presented at the Fifty-Seventh Annual Meeting of the Endocrine Society, June 13 to 20, 1975, New York. Abstract 387 5. Gupta GN: Sustained absorption of 3H-norgestrel from S.C. fused pellets in rats: a potential totally bioabsorbable implant for human contraception. Fed Proc (Abstr 3833) 36:977, 1977 6. Gupta GN: Development ofbio-erodable contraceptive implants. Springfield, Virginia, U.S. National Technical Information Service, 1978, PB 292-383-A-S 7. Maitra A, Joshi UM, Kholkute SD, Munshi SR, Gupta GN, Tejuja S: Release of norethindrone from a bioaborbable implant in female Bonnet monkeys (Macaca radiata). Contraception 21:165, 1980 8. Odlind V, Moo-young AJ, Gupta GN, Weiner E, Johansson EDB: Subdermal norethindrone pellets-a method for contraception. Contraception 19:639, 1979 9. Toddywala VS, Mehta S, Virker KD, Saxena BN: Release of 19-nortestosterone type of contraceptive steroids through different drug delivery systems into breast milk oflactating women. Contraception 21:217, 1980 10. Odlind V, Weiner E, Johansson EDB: Plasma levels of norethindrone and effect upon ovarian function during treatment with Silastic implants containing norethindrone. Contraception 19:197, 1979 11. Beck LR, Cowsar DR, Lewis DR, Gibson JW, Flowers CE: New long-acting injectable microcapsule COjtraceptive system. Am J Obstet Gynecol 135:419, 1979 12. Beck LR, Ramos RA, Flowers CE, Lopez GZ, Lewis DH, Cowsar DR: Clinical evaluation of injectable biodegradable contraceptive system. Am J Obstet Gynecol 140:799, 1981 13. Gupta GN, Saxena BB, Landesman R, Ledger WJ: Bioabsorbable long-acting norethindrone (NET) pellets for contraception: sustained NET serum concentrations and levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17[3-estradiol (E 2 ), and progesterone (P) in women. Fertil Steril (Abstr) 37:317, 1982

Fertility and Sterility

14. Walls C, Vose CW, Horth CE, Palmer RF: Radioimmunoassay of plasma norethindrone afterethynodiol diacetate administration. J Steroid Biochem 8:167, 1977 15. Gupta GN: New method of micro-liquid scintillation counting in plastic mini-bags for 3H, 14C36 and 32p samples. In Organic Scintillators and Liquid Scintillation Counting, Edited by DL Horrocks. New York, Academic Press, 1971, p 747 16. Landgren BM, Diczfalusy E: Hormonal effects of the 300 ILg norethisterone (NET) minipill. I. Daily steroid levels in 43 subjects during a pretreatment cycle and during the second month of NET .administration. Contraception 21:87,1980 17. Shimkin MB, Lorenz E, Wyman R, Norton SG: Absorption of subcutaneously implanted hormone pellets. Endocrinology 35:283, 1944 18. Bishop PMF, Folley SH: Absorption of hormone implants in man. Lancet 2:229,.1951 19. Chang CC, Kincl FA: Sustained release hormonal preparation. 4. Biologic effectiveness. of steroid hormones. Fertil Steril 21:134, 1970

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20. Landgren BM, Oriowo MA, Diczfalusy E: Pharmacokinetics and .pharmacodynamics studies with vaginal devices releasing norethisterone at a constant, near zero order. Contraception 24:29, 1981 21. Moghissi KS, Syner FN, McBride LC: Contraceptive mechanism of microdose norethindrone. Obstet Gynecol 41:585, 1973 22. Gutierrez-Najar A, Marquez-Monter H, Cortes-Gallegos V, Giner-Velazquez J, Martinez-Manautou J: Presence of corpus luteum as evidence of ovulation in women treated with low doses of chloromadinone acetate. Am J Obstet Gyneco1102:1018, 1968 23. Nilsson CG, LahteenmaId P, Robertson DN, Luukkainen T: Plasma concentrations of levonorgestrel as a function of the release rate oflevonorgestrel from medicated intrauterine devices. Acta Endocrinol (Copenh) 93:380, 1980

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