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Contraceptive steroid administration by subdermal implants: Serum concentrations of R-2323, estrogen and progesterone in rhesus monkeys
CONTRACEPTION
CONTRACEPTIVE
STEROID ADMINISTRATION
SERUM CONCENTRATIONS AND PROGESTERONE
BY SUBDERMAL
OF R-2323,
IMPLANTS:
ESTROGEN
IN RHESUS MONKEYS"
M. Rasheed Bahgat**
and Linda E. Atkinson
The Population Council, Biomedical Division The Rockefeller University, New York, N.Y. 10021
ABSTRACT Silasti.I@ capsules containing R-2323 (13-ethyl-17ðinyl-17_hydroxygona-4,9,11-trien-3-one) were implanted subdermally in six normal female rhesus monkeys (Macaca mulatta) for one year. Serum concentrations of R-2323, estrogenspprogesterone were estimated by radioimmunoassay and the R-2323 remaining in the capsule reservoir was measured. The -in situ diffusion rate of R-2323 from the capsules averaged 14.0 + 1.2,&/cm2/day (mean + S.E.). Serum R-2323 was found one hour after insertion of the capsules and reached maximum values of 0.50-1.41 ng/ml within three days. Thereafter, the R-2323 serum concentrations declined, stabilizing after three months within a range of 0.23-0.54 ng/ml. Serum R-2323 values could not be predicted by the number of capsules inserted but reflected the differences in release of steroid from each implant. Ovulation and subsequent corpus luteum formation, as appraised by estrogen and progesterone blood levels, were inhibited for variable periods which were dependent on the serum R-2323 concentration attained.
Accepted
for
publication
January
13,
1977
*This work was undertaken as part of the contraceptive development research program sponsored and coordinated by the International Committee for Contraception Research of the Population Council. **Postdoctoral Fellow of The Ford Foundation Present address: Department of Biochemistry, Cairo University, Cairo,Egypt.
MARCH 1977 VOL. 15 NO. 3
Faculty of Medicine,
335
CONTRACEPTION
INTRODUCTION The utility of subdermal Silasti.@ devices as reservoirs of contraceptive steroids has been envisioned and tested by investigators for several years (l-5). Potentially, these devices offer a constant controlled release of steroid into circulation that may provide fertility regulation by ovulation suppression or modification of other reproductive functions in women for a year or longer. Clinical testing of subdermal capsules containing megestrol acetate (174-acetoxy-6-methyl-pregna-4,6-diene-3,20dione), norgestrienone (13-methyl-1~-ethinyl-l7-hydroxy-gona-4,9,ll-trien3-one) or R-2323 have shown that at initially effective doses, pregnancy protection decreases with time -in situ (6-9). It has not been established if this effect is the result of depletion of the steroid reservoir, or a progressively declining diffusion rate of steroid from the device or variations in biological response to the available steroid. R-2323 is a totally synthesized contraceptive steroid. In animal studies it exhibits very low estrogenic, androgenic and progestomimetic activities, some anti-estrogenic and pituitary inhibitory activities and a striking antideciduogenic, antiprogestomimetic, antiprogestational and anti-implantation activities showing that it is a strong progesterone antagonist (10). The availability of a specific radioimmunoassay (RIA) for R-2323 (13ethyl-17LY-ethinyl-l7-hydroxy-gona-4,9,1l-trien-3-one) enabled us to directly measure the blood level and fluctuations of steroid achieved by diffusion from subdermal Silasti 8 capsules implanted in monkeys during a 12-month period. In addition, the concomitant biological effects of these concentrations of R-2323 on serum estrogens, progesterone and menstruation in rhesus monkeys were observed.
MATERIALS
AND METHODS
Animals. Six rhesus monkeys (Macaca mulatta) having 24- to 31-day -___ The last menstrual cycle was judged length menstrual cycles were selected. to be ovulatory by finding serum progesterone levels above two ng/ml durMonkey body weights ranged from 6 to ing the second half of the cycle. 10.7 kg. The monkeys were caged individually in a light and temperature regulated environment, provided with water ad libidum and fed a commercial monkey diet supplemented with fresh fruit. Their vaginas were swabbed daily with a cotton-tipped stick to detect uterine bleeding. contained 30.3 f0.2 mg (mean f S.E.) Implants. The Silasti'&capsules of R-2323 in a filled length of 31 mm. The tubing had an outer diameter The capsules were manufactured of 2.4 mm and an inner diameter of 1.6mm. by Laboratorios Gutfol, S.A., Mexico City, for the International Committee The capsules were sterilized by radiafor Contraception Research (ICCR). tion (Cobalt 60). Crystalline ~-2323 was obtained from Roussel, UCLAF, Paris, France. SilastiWis a registered land, Michigan, U.S.A.
336
trademark
of the Dow-Corning
Corporation,
Mid-
MARCH 1977 VOL. 15 NO. 3
CONTRACEPTION
Experimental Procedure. The Silastl.c@ capsules were implanted on days 5-9 of the menstrual cycle. Monkeys 195 and 242 received one R-2323 Monkeys 71,94 and 480 were given two capsules and monkey 381 implant. The capsules were inserted in the had three R-2323 capsules implanted. subcutaneous fat about 3 cm on either side of the umbilicus through separate incisions. Blood samples were collected at 1, 2, 4 and 24 hours postimplantation and then on days 7-15 of the menstrual cycle and every other day thereIn addition, after for 90 days and then every week for about one year. blood samples were obtained from monkeys 94 and 381 daily during the last two weeks of the experiment. Blood was drawn from the saphenous vein using a vacutainer syringe and the serum was separated by centrifugation and stored at -2O'C until analysis. Steroid Radioimmunoassays. Serum samples were analyzed for estrogen, progesterone and R-2323 by RIA using the charcoal-dextran method as previously described (11). Crystalline R-2323, 6,7-3H-R-2323 (S.A.) 45 Ci/mmole) as well as the antiserum to R-2323 were a gift from Roussel LJCLAF, Paris, France. The cross reactions with metabolites and natural steroids were less than 1% (11). Methodological blanks and accuracy studies have also been reported (11). Crystalline progesterone and estradiol were obtained from Sigma Chemical Co. 1,2,6,7-3H-progesterone (S.A., 96 Ci/mmole) and 1,2,6,7-3H-estradiol were bought from New England Nuclear. Antisera to progesterone and estradiol were a gift from Dr. E.D.B. Johansson of Uppsala, Sweden. The cross reaction, blanks and recoveries for the progesterone RIA are as previously described (11). Estradiol antiserum was raised in rabbits against 17@estradiol-6-(O-carboxymethyl)-oxime coupled to BSA. A dilution of 1:150,000 in phosphate buffered saline-0.1% gelatin bound about 50% of the tritiated estradiol. Two hundredN1 of serum were used for analysis of estradiol. The amount of labelled estradiol displaced from antiserum by 200~1 of serum from ovariectomized monkeys resulted in values of 3.9 f 0.5 pg in 7 assays. Recoveries of 50 pg unlabelled estradiol-17p from 200~11 ovariectomized monkey serum in 7 assays averaged 98.6 + 0.9% (mean + S.E.). The cross reaction of the antiserum with estrone was 23% so the RIA provided an estimate of the total estradiol-3H displacing activity extracted from serum. Cross reaction with other naturally occurring steroids and R2323 was less than 1%. The results are stated as pg of the standard, 17p estradiol. The depletion of R-2323 from each implant was estimated photometric analysis as described elsewhere (11).
by W
spectro-
RESULTS The Silastic B implants containing R-2323 were removed at 378 days from monkey 381, 371 days from monkeys 71 and 94 and 364 days from monkeys 242 and 480. Monkey 195 was released from the experiment at 210 days due to illness. The amount of steroid remaining after 12 months in the 9 capsules recovered intact ranged from 11.5 to 20.8 mg. The diffusion
MARCH 1977 VOL. 15 NO. 3
337
CONTRACEPTION
rate of R-2323 from the capsules averaged 14.0 + 1.2 pg/cm2/day (mean + S.E.) for the year with values ranging from 9.5 to 21.3 pg/cm2/day. The estimated diffusion rate from individual capsules as well as the total daily dose and dose per kg body weight received by each monkey are given in Table I. These represent the mean value for the entire period in situ and may not reflect the amount lost or dose received at specific times in the experiment. Materials recovery data are not available for monkeys 195 and 242 because the implants were not recovered intact.
Table I. Estimates of Diffusion Rate of R-2323 from Silasti@ Mean Dosage and Serum Concentrations Attained During 12 Months Monkey (a capsules) 381 94 71 480 242 195
(3) (2) (2) (2) (1) (1)
Diffusion Rate (&cm 2/day) 13.4, 15.7, 21.3 11.0, 15.0 14.9, 15.3 9.5, 9.8 * *
Dosage (ug/day) (nglkglday) 118 61 71 45
11.0 9.2 6.7 6.6
Capsules,
Mean Serum R-2323 ng/ml 0.68 0.53 0.47 0.30 0.36 0.57**
*capsules not recovered **average R-2323 serum levels for 210 days
The mean serum concentrations of R-2323 achieved by administration of of the steroid via subdermal capsules ranged from 0.30 to 0.68 ng/ml among the six monkeys (Table I). The variation between animals bearing two implants seems to parallel the apparent diffusion rates of their implants. R-2323 was found in all blood samples collected one hour after insertion of the capsules. Serum concentrations gradually increased and reached highest values by 3 days after implantation for all monkeys except 195 and 242 for which the highest value was attained on days 7 and 14 postimplantation,respectively (Figures la 6 lb).Serum R-2323, measured at weekly intervals, revealed that the drug did not remain at this high level but decreased throughout the observation period. However, the amount of decrease did not appear constant but could be roughly divided into three segments; days 3-14, days 14-90 and day 90 to the end of treatment. Regression lines for serum R-2323 concentrations during these three segments of the experiment were calculated by the method of least squares. The slopes of the regression lines and the average serum R-2323 level for each period are listed in Table II. During the first two weeks, the rate During days of fall was between 10 and 50 pg/ml/day, with one exception. 14-90 postimplantation, the rate of decline slowed to 0.8 to 10.5 pg/ml/ day and during the remainder of the experiment, R-2323 serum concentraIt can be tions were relatively stable, falling 2 pg/ml/day or less. seen that there is considerable variation in the slopes and the amount of change in the slopes for individual animals.
338
MARCH 1977 VOL. 15 NO. 3
pg’m’
“g/m’
Figure
-50 ._
I E2
Prog
la:
IO
30
40
50
60
70
80
90
EZ
t
+
2
4
6t
t
8
2
4
6
8
0
_
Days
IO 20
-
40 50 ._
60
mm
70
- Progesterone
0 Estradlol
post-~mplantot~on
30
X480c68Kgi TWO,mplanfr
m/ml IO 400
IO
Prog ng/ml
Serum concentrations of R-2323, progesterone and estrogens in six monkeys the first 90 days of treatment with subdermal implants containing R-2323. tal bars under the abscissa indicate episodes of uterine bleeding.
Days post-implantation
20
x 381wdiKpi
R-2323 ng/ml
90
during Horizon-
80
_
00
04
08
12
R-2323 na/ml
A
320
lb:
20
* ,BI
I6
E2 pg /ml
Figure
8I
Prog ng/ml
28
_
32
36 I.
40 .
48 I
52
08
,
hog ng/ml
16
E, pg /ml
20
I
28 I
-
.
32 I.
.
36
I,.
Estradlol
Progesterone
Weeks post-rmplantatton
24
.
1
I
*
48
52
00
04
1 i ?
#I *
1 108
R-2323 ng/ml
progesterone and estrogens in six monkeys bearing R-2323. Horizontal bars under the abscissa represent Arrows indicate removal of subdermal implants.
04
i
1
R-2323 ng/ml
Serum concentrations of R-2323, subdermal implants containing episodes of uterine bleeding.
44 .
Estradlol
0
Weeks post-Implantation
24 -
Progesterone
I
CONTRACEPTION
Table II. Rate of Decrease of Serum R-2323 Concentrations Bearing R-2323 Subdermal Implants Animal
381 94 71 480 242 195
R-2323, Mean (&ml) 1.41 1.33 1.06 0.85 0.50 0.79
Days l-14 Slope (pg/ml/day) -17.0 -48.3 -10.5 -50.3 + 2.4 -14.6
R-2323, Days 14-90 Mean Slope (ng/ml) (pg/ml/day) 1.01 0.74 0.70 0.41 0.41 0.62
-10.5 -6.8 -8.4 -1.0 -2.2 -0.8
in Monkeys
R-2323, Day 90 to End Mean Slope (&ml) (pg/ml/day) 0.54 0.42 0.36 0.23 0.33 0.49
-1.4 -0.4 +o.a -0.6 -0.6 -2.3
Serum estradiol and progesterone, shown in Figures la and lb, were measured by RIA. Ovulation and corpus luteum formation, as judged by progesterone concentrations greater than 2 ng/ml in weekly samples, appeared to be inhibited by the administration of R-2323 by subdermal implant for varying periods of time in monkeys 381, 71, 94 and 242. The subdermal implants prevented the preovulatory estradiol surge and subsequent progesterone secretion during the first month of treatment in all animals except 195 and 94. In the case of 94, the implants were inserted on day 9 of the cycle, after the preovulatory estradiol surge had occurred. It is interesting that the initially high levels of R-2323 did not prevent ovulation in animal 195 which continued to ovulate irregularly during the 13 weeks of observation. Animal 480 began having ovulatory cycles during the second month of treatment and these continued regularly for the rest of the year. Recovery of ovulation occurred near the 25th treatment week of monkey 242. Ovulation was prevented until the fortieth week, the eleventh month and the twelfth month postimplantation in animals 71, 94 and 381, respectively. During the anovulatory months, serum estradiol was less than 40 pg/ml and progesterone was less than 0.5 ng/ml. Uterine bleeding was absent or irregular during the period of anovulation. Monkeys 381 and 94 were completely amenorrheic until ovulation resumed. With the exception of 195, serum R-2323 levels were 0.30 to 0.35 ng/ml during the first month in which ovulation could be observed. R-2323 concentrations had declined from 0.4 ng/mlor more in the month preceding ovulation in monkeys 381, 94, 480 and 242. Once ovulation was reinstated, it appeared to occur regularly and cycles were of normal duration. DISCUSSION This study was conducted to ascertain the diffusion stability of progestogens delivered from subdermal Silasti& reservoirs. In addition we sought information regarding the minimum serum levels of R-2323 required to inhibit ovulation in rhesus monkeys and the reliability of this method of steroid administration in maintaining anovulation. The data demonstrate that the steroid diffuses rapidly from the implant into the peripheral circulation and achieves near maximum values within 24 hours. During the next 2-3 months, the steroid levels declined,
MARCH 1977 VOL. 15 NO. 3
341
CONTRACEPTION
rapidly at first and then more slowly, until a plateau was reached. The R-2323 levels in serum were quite stable during the latter 9 months of observation. Benagiano et aLhave previously reported that the urinary excretion of 14C of women bearing implants containing megestrol acetate14C substantially decreased during the first 3 months and then stabilized for the remainder of the study (12). Our data also confirm the direct observations by Weiner and Johansson of a high initial serum level of progestogen during the first 60 days in subjects implanted with d-norgestrel capsules (13). The initial rapid increase in serum R-2323 levels during the first few days after implantation is a phenomenon also noted by Benagiano and coworkers (12) and Weiner and Johansso (13). This can be explained in part by the saturation of the Silastl.& tubing with the steroid during manufacture and storage and in part by the high degree of vascularity of the surrounding tissue in reaction to injury and introduction of a foreign body, as well as by slower metabolism of the drug by the liver early in the postimplantation period. The rapid decline in serum R-2323 levels during the first two weeks after reaching the maximum value is probably due to decreasing concentration of the steroid in the capsular wall, decreasing vascularity of the tissue surrounding the capsule that occurs with wound healing as well as the induction of synthesis of enzymes required for the metabolism of the drug. The serum concentrations of R-2323 attained by administration of the steroid from subdermal Silastl.c@ reservoirs were suprisingly nonuniform in these monkeys. The source of variation appears to be the differences in diffusion rate of the steroid from the implants. Weiner and Johansson also found this variation of blood levels between subjects (13). Since in vitro relea e rates under controlled conditions are quite uniform for these Silasti A capsules (unpublished observations), it would seem that conditions at the implantation site may be important in determining the actual dose of steroid delivered to the circulation. One such factor is the degree of local reaction leading to fibrosis around the capsule; the more the fibrosis, the less is the absorption rate of the steroid (14). A thin layer of fibrous tissue was noticed around all capsules but no attempt was made to quantitate it. Another factor important in determining the diffusion rate of steroids from Silastia capsules is the state of the circulation around the area of implantation; this is in turn determined by the location of the implant and the general activity of the animal. Of the four monkeys with periods of anovulation, the inhibitory effect of R-2323 on menstruation and ovulation seems most prolonged in monkeys with high mean serum R-2323 levels. Ovulation resumed or was not suppressed when serum R-2323 was 0.3 to 0.4 ng/ml, apparently the threshold level of drug required to maintain inhibition. However, the R-2323 values in the months preceding the recovery of ovulation were not measurably greater. It is somewhat unexpected that ovulation would suddenly resume during a period of relatively stable drug levels. One possibility suggested by Croxatto to explain the failure of contraception following several months of protection even though the reservoir was not exhausted (6), is that the organism adapts to the continuous low levels of the steroid and can escape the inhibitory effect. More specifically, it can be theorized that greater
342
MARCH 1977 VOL. 15 NO. 3
CONTRACEPTION
binding by serum proteins may occur over prolonged treatment. Another possibility is the increasing appearance of metabolites that are biologically inactive but contribute to the apparent stable levels of radioassayThis is not probable in that the amounts of metabolites able R-2323. necessary to achieve substantial cross reaction is enormous since their cross reaction in the RIA is less than 1%. In conclusion, the direct measurement of blood levels of R-2323 have shown that contraceptive steroids can be delivered from subdermal implants at a constant rate over periods of at least 9 to 10 months after an initial 2 to 3 months in which an equilibrium between capsule diffusion capacity, Three other local tissue absorption and liver metabolism is established. characteristics of subdermal implant contraception have also been elucidated here which confirm studies using other progestogens (13): (a) Considerable variation exists in the ultimate diffusion rate and thus the drug level in the peripheral circulation once local tissue reaction is complete; (b) A wide margin in the absolute amount of drug is required by the individual to affect a biological parameter, e.g. ovulation; (c) The organism may achieve reversal of the biological endpoint even though seemingly constant amounts of drug are available.
MARCH 1977 VOL. 15 NO. 3
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CONTRACEPTION
REFERENCES
1.
Se&, S.J. and Croxatto, H. Paper presented at the 23rd Annual Meeting of The American Fertility Society, April 14-16, 1967, Washington, D.C.
2.
Kincl, F.A., Benagiano, G. and Angee, I. Sustained release hormonal preparations 1. Diffusion of various steroids through polymer membranes. Steroids 11:673-680, 1968.
3.
Tatum, H.J., Coutinho, E.M., Filho, A.J. and Sant'Anna, A.R.S. Acceptability of long-term contraceptive steroid administration by subcutaneous Silastic capsules. Amer. J. Obstet. Gynecol. 105:11391143, 1969.
4.
Croxatto, H., Diaz, S., Vera, R., Etchart, M. and Atria, F. Fertility control in women with a progestogen released in microquantities from subcutaneous capsules. Amer. J. Obstet. Gynecol. 105:1135-1138, 1969.
5.
Tejuja, S. Use of subcutaneous Silastic capsules for long-term steroid contraception. Amer. J. Obstet. Gynecol. 107:954-957, 1970.
6.
Croxatto, H.B., Diaz, S., Atria, P., Cheriakoff, S., Rosatti, S. and Oddo, H. Contraceptive action of megestrol acetate implants in women. Contraception 4:155-167, 1971.
7.
Coutinho, E.M., Mattos, C.E.R., Filho, J.A., Silvin, M.C. and Tatum, H.J. Further studies on long-term contraception by subcutaneous Silastic capsules containing megestrol acetate. Contraception 5:389393, 1972.
8.
Coutinho, E.M. and Da Silva, A.R. One year contraception with norgestrienone subdermal implants. Fertility and Sterility 25:170-176, 1974.
9.
Coutinho, E.M., Da Silva, A.R., Carreira, C.M., Chaves, M.C. and Filho, J.A. Contraceptive effectiveness of Silastic implants containing the progestin R-2323. Contraception 11:625-636, 1975.
10.
G. R-2323: An original contraceptive Sakiz, E. and Azadian-Boulanger, compound. Excerpta Medica International Congress Series No. 219: Hormonal Steroids 865-871, 1970.
11.
Bahgat, M.R., Atkinson, L.E., Brinson, A.O. and Segal, S.J. Treatment of postpartum rhesus monkeys with progestogen: Appearance in milk and effects on lactation. Contraception 12:665-678, 1975.
12.
Benagiano, G., Ermini, M., Carenza, L. and Rolfini, G. Studies on sustained contraceptive effects with subcutaneous polydimethylsiloxane implants. I. Diffusion of megestrol acetate in humans. Acta Endocrinol. (Kbh) 73:335-346, 1973.
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CONTRACEPTION
13.
Weiner, E. and Johansson, E.D.B. Plasma levels of d-norgestrel, estradiol and progesterone during treatment with Silastic implants containing d-norgestrel. Contraception 14:81-92, 1976.
14.
Ermini, M., Carpino, F., Russo, M. and Benagiano, G. Studies on sustained contraceptive effects with subcutaneous polydimethylsiloxane implants 3. Factors affecting steroid diffusion in vivo and in vitro. Acta Endocrinol (Kbh) 73:360-373, 1973.
MARCH 1977 VOL. 15 NO. 3
345
CONTRACEPTIVE
STEROID ADMINISTRATION
SERUM CONCENTRATIONS AND PROGESTERONE
BY SUBDERMAL
OF R-2323,
IMPLANTS:
ESTROGEN
IN RHESUS MONKEYS"
M. Rasheed Bahgat**
and Linda E. Atkinson
The Population Council, Biomedical Division The Rockefeller University, New York, N.Y. 10021
ABSTRACT Silasti.I@ capsules containing R-2323 (13-ethyl-17ðinyl-17_hydroxygona-4,9,11-trien-3-one) were implanted subdermally in six normal female rhesus monkeys (Macaca mulatta) for one year. Serum concentrations of R-2323, estrogenspprogesterone were estimated by radioimmunoassay and the R-2323 remaining in the capsule reservoir was measured. The -in situ diffusion rate of R-2323 from the capsules averaged 14.0 + 1.2,&/cm2/day (mean + S.E.). Serum R-2323 was found one hour after insertion of the capsules and reached maximum values of 0.50-1.41 ng/ml within three days. Thereafter, the R-2323 serum concentrations declined, stabilizing after three months within a range of 0.23-0.54 ng/ml. Serum R-2323 values could not be predicted by the number of capsules inserted but reflected the differences in release of steroid from each implant. Ovulation and subsequent corpus luteum formation, as appraised by estrogen and progesterone blood levels, were inhibited for variable periods which were dependent on the serum R-2323 concentration attained.
Accepted
for
publication
January
13,
1977
*This work was undertaken as part of the contraceptive development research program sponsored and coordinated by the International Committee for Contraception Research of the Population Council. **Postdoctoral Fellow of The Ford Foundation Present address: Department of Biochemistry, Cairo University, Cairo,Egypt.
MARCH 1977 VOL. 15 NO. 3
Faculty of Medicine,
335
CONTRACEPTION
INTRODUCTION The utility of subdermal Silasti.@ devices as reservoirs of contraceptive steroids has been envisioned and tested by investigators for several years (l-5). Potentially, these devices offer a constant controlled release of steroid into circulation that may provide fertility regulation by ovulation suppression or modification of other reproductive functions in women for a year or longer. Clinical testing of subdermal capsules containing megestrol acetate (174-acetoxy-6-methyl-pregna-4,6-diene-3,20dione), norgestrienone (13-methyl-1~-ethinyl-l7-hydroxy-gona-4,9,ll-trien3-one) or R-2323 have shown that at initially effective doses, pregnancy protection decreases with time -in situ (6-9). It has not been established if this effect is the result of depletion of the steroid reservoir, or a progressively declining diffusion rate of steroid from the device or variations in biological response to the available steroid. R-2323 is a totally synthesized contraceptive steroid. In animal studies it exhibits very low estrogenic, androgenic and progestomimetic activities, some anti-estrogenic and pituitary inhibitory activities and a striking antideciduogenic, antiprogestomimetic, antiprogestational and anti-implantation activities showing that it is a strong progesterone antagonist (10). The availability of a specific radioimmunoassay (RIA) for R-2323 (13ethyl-17LY-ethinyl-l7-hydroxy-gona-4,9,1l-trien-3-one) enabled us to directly measure the blood level and fluctuations of steroid achieved by diffusion from subdermal Silasti 8 capsules implanted in monkeys during a 12-month period. In addition, the concomitant biological effects of these concentrations of R-2323 on serum estrogens, progesterone and menstruation in rhesus monkeys were observed.
MATERIALS
AND METHODS
Animals. Six rhesus monkeys (Macaca mulatta) having 24- to 31-day -___ The last menstrual cycle was judged length menstrual cycles were selected. to be ovulatory by finding serum progesterone levels above two ng/ml durMonkey body weights ranged from 6 to ing the second half of the cycle. 10.7 kg. The monkeys were caged individually in a light and temperature regulated environment, provided with water ad libidum and fed a commercial monkey diet supplemented with fresh fruit. Their vaginas were swabbed daily with a cotton-tipped stick to detect uterine bleeding. contained 30.3 f0.2 mg (mean f S.E.) Implants. The Silasti'&capsules of R-2323 in a filled length of 31 mm. The tubing had an outer diameter The capsules were manufactured of 2.4 mm and an inner diameter of 1.6mm. by Laboratorios Gutfol, S.A., Mexico City, for the International Committee The capsules were sterilized by radiafor Contraception Research (ICCR). tion (Cobalt 60). Crystalline ~-2323 was obtained from Roussel, UCLAF, Paris, France. SilastiWis a registered land, Michigan, U.S.A.
336
trademark
of the Dow-Corning
Corporation,
Mid-
MARCH 1977 VOL. 15 NO. 3
CONTRACEPTION
Experimental Procedure. The Silastl.c@ capsules were implanted on days 5-9 of the menstrual cycle. Monkeys 195 and 242 received one R-2323 Monkeys 71,94 and 480 were given two capsules and monkey 381 implant. The capsules were inserted in the had three R-2323 capsules implanted. subcutaneous fat about 3 cm on either side of the umbilicus through separate incisions. Blood samples were collected at 1, 2, 4 and 24 hours postimplantation and then on days 7-15 of the menstrual cycle and every other day thereIn addition, after for 90 days and then every week for about one year. blood samples were obtained from monkeys 94 and 381 daily during the last two weeks of the experiment. Blood was drawn from the saphenous vein using a vacutainer syringe and the serum was separated by centrifugation and stored at -2O'C until analysis. Steroid Radioimmunoassays. Serum samples were analyzed for estrogen, progesterone and R-2323 by RIA using the charcoal-dextran method as previously described (11). Crystalline R-2323, 6,7-3H-R-2323 (S.A.) 45 Ci/mmole) as well as the antiserum to R-2323 were a gift from Roussel LJCLAF, Paris, France. The cross reactions with metabolites and natural steroids were less than 1% (11). Methodological blanks and accuracy studies have also been reported (11). Crystalline progesterone and estradiol were obtained from Sigma Chemical Co. 1,2,6,7-3H-progesterone (S.A., 96 Ci/mmole) and 1,2,6,7-3H-estradiol were bought from New England Nuclear. Antisera to progesterone and estradiol were a gift from Dr. E.D.B. Johansson of Uppsala, Sweden. The cross reaction, blanks and recoveries for the progesterone RIA are as previously described (11). Estradiol antiserum was raised in rabbits against 17@estradiol-6-(O-carboxymethyl)-oxime coupled to BSA. A dilution of 1:150,000 in phosphate buffered saline-0.1% gelatin bound about 50% of the tritiated estradiol. Two hundredN1 of serum were used for analysis of estradiol. The amount of labelled estradiol displaced from antiserum by 200~1 of serum from ovariectomized monkeys resulted in values of 3.9 f 0.5 pg in 7 assays. Recoveries of 50 pg unlabelled estradiol-17p from 200~11 ovariectomized monkey serum in 7 assays averaged 98.6 + 0.9% (mean + S.E.). The cross reaction of the antiserum with estrone was 23% so the RIA provided an estimate of the total estradiol-3H displacing activity extracted from serum. Cross reaction with other naturally occurring steroids and R2323 was less than 1%. The results are stated as pg of the standard, 17p estradiol. The depletion of R-2323 from each implant was estimated photometric analysis as described elsewhere (11).
by W
spectro-
RESULTS The Silastic B implants containing R-2323 were removed at 378 days from monkey 381, 371 days from monkeys 71 and 94 and 364 days from monkeys 242 and 480. Monkey 195 was released from the experiment at 210 days due to illness. The amount of steroid remaining after 12 months in the 9 capsules recovered intact ranged from 11.5 to 20.8 mg. The diffusion
MARCH 1977 VOL. 15 NO. 3
337
CONTRACEPTION
rate of R-2323 from the capsules averaged 14.0 + 1.2 pg/cm2/day (mean + S.E.) for the year with values ranging from 9.5 to 21.3 pg/cm2/day. The estimated diffusion rate from individual capsules as well as the total daily dose and dose per kg body weight received by each monkey are given in Table I. These represent the mean value for the entire period in situ and may not reflect the amount lost or dose received at specific times in the experiment. Materials recovery data are not available for monkeys 195 and 242 because the implants were not recovered intact.
Table I. Estimates of Diffusion Rate of R-2323 from Silasti@ Mean Dosage and Serum Concentrations Attained During 12 Months Monkey (a capsules) 381 94 71 480 242 195
(3) (2) (2) (2) (1) (1)
Diffusion Rate (&cm 2/day) 13.4, 15.7, 21.3 11.0, 15.0 14.9, 15.3 9.5, 9.8 * *
Dosage (ug/day) (nglkglday) 118 61 71 45
11.0 9.2 6.7 6.6
Capsules,
Mean Serum R-2323 ng/ml 0.68 0.53 0.47 0.30 0.36 0.57**
*capsules not recovered **average R-2323 serum levels for 210 days
The mean serum concentrations of R-2323 achieved by administration of of the steroid via subdermal capsules ranged from 0.30 to 0.68 ng/ml among the six monkeys (Table I). The variation between animals bearing two implants seems to parallel the apparent diffusion rates of their implants. R-2323 was found in all blood samples collected one hour after insertion of the capsules. Serum concentrations gradually increased and reached highest values by 3 days after implantation for all monkeys except 195 and 242 for which the highest value was attained on days 7 and 14 postimplantation,respectively (Figures la 6 lb).Serum R-2323, measured at weekly intervals, revealed that the drug did not remain at this high level but decreased throughout the observation period. However, the amount of decrease did not appear constant but could be roughly divided into three segments; days 3-14, days 14-90 and day 90 to the end of treatment. Regression lines for serum R-2323 concentrations during these three segments of the experiment were calculated by the method of least squares. The slopes of the regression lines and the average serum R-2323 level for each period are listed in Table II. During the first two weeks, the rate During days of fall was between 10 and 50 pg/ml/day, with one exception. 14-90 postimplantation, the rate of decline slowed to 0.8 to 10.5 pg/ml/ day and during the remainder of the experiment, R-2323 serum concentraIt can be tions were relatively stable, falling 2 pg/ml/day or less. seen that there is considerable variation in the slopes and the amount of change in the slopes for individual animals.
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pg’m’
“g/m’
Figure
-50 ._
I E2
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IO
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40
50
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70
80
90
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_
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IO 20
-
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- Progesterone
0 Estradlol
post-~mplantot~on
30
X480c68Kgi TWO,mplanfr
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IO
Prog ng/ml
Serum concentrations of R-2323, progesterone and estrogens in six monkeys the first 90 days of treatment with subdermal implants containing R-2323. tal bars under the abscissa indicate episodes of uterine bleeding.
Days post-implantation
20
x 381wdiKpi
R-2323 ng/ml
90
during Horizon-
80
_
00
04
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R-2323 ng/ml
progesterone and estrogens in six monkeys bearing R-2323. Horizontal bars under the abscissa represent Arrows indicate removal of subdermal implants.
04
i
1
R-2323 ng/ml
Serum concentrations of R-2323, subdermal implants containing episodes of uterine bleeding.
44 .
Estradlol
0
Weeks post-Implantation
24 -
Progesterone
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CONTRACEPTION
Table II. Rate of Decrease of Serum R-2323 Concentrations Bearing R-2323 Subdermal Implants Animal
381 94 71 480 242 195
R-2323, Mean (&ml) 1.41 1.33 1.06 0.85 0.50 0.79
Days l-14 Slope (pg/ml/day) -17.0 -48.3 -10.5 -50.3 + 2.4 -14.6
R-2323, Days 14-90 Mean Slope (ng/ml) (pg/ml/day) 1.01 0.74 0.70 0.41 0.41 0.62
-10.5 -6.8 -8.4 -1.0 -2.2 -0.8
in Monkeys
R-2323, Day 90 to End Mean Slope (&ml) (pg/ml/day) 0.54 0.42 0.36 0.23 0.33 0.49
-1.4 -0.4 +o.a -0.6 -0.6 -2.3
Serum estradiol and progesterone, shown in Figures la and lb, were measured by RIA. Ovulation and corpus luteum formation, as judged by progesterone concentrations greater than 2 ng/ml in weekly samples, appeared to be inhibited by the administration of R-2323 by subdermal implant for varying periods of time in monkeys 381, 71, 94 and 242. The subdermal implants prevented the preovulatory estradiol surge and subsequent progesterone secretion during the first month of treatment in all animals except 195 and 94. In the case of 94, the implants were inserted on day 9 of the cycle, after the preovulatory estradiol surge had occurred. It is interesting that the initially high levels of R-2323 did not prevent ovulation in animal 195 which continued to ovulate irregularly during the 13 weeks of observation. Animal 480 began having ovulatory cycles during the second month of treatment and these continued regularly for the rest of the year. Recovery of ovulation occurred near the 25th treatment week of monkey 242. Ovulation was prevented until the fortieth week, the eleventh month and the twelfth month postimplantation in animals 71, 94 and 381, respectively. During the anovulatory months, serum estradiol was less than 40 pg/ml and progesterone was less than 0.5 ng/ml. Uterine bleeding was absent or irregular during the period of anovulation. Monkeys 381 and 94 were completely amenorrheic until ovulation resumed. With the exception of 195, serum R-2323 levels were 0.30 to 0.35 ng/ml during the first month in which ovulation could be observed. R-2323 concentrations had declined from 0.4 ng/mlor more in the month preceding ovulation in monkeys 381, 94, 480 and 242. Once ovulation was reinstated, it appeared to occur regularly and cycles were of normal duration. DISCUSSION This study was conducted to ascertain the diffusion stability of progestogens delivered from subdermal Silasti& reservoirs. In addition we sought information regarding the minimum serum levels of R-2323 required to inhibit ovulation in rhesus monkeys and the reliability of this method of steroid administration in maintaining anovulation. The data demonstrate that the steroid diffuses rapidly from the implant into the peripheral circulation and achieves near maximum values within 24 hours. During the next 2-3 months, the steroid levels declined,
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rapidly at first and then more slowly, until a plateau was reached. The R-2323 levels in serum were quite stable during the latter 9 months of observation. Benagiano et aLhave previously reported that the urinary excretion of 14C of women bearing implants containing megestrol acetate14C substantially decreased during the first 3 months and then stabilized for the remainder of the study (12). Our data also confirm the direct observations by Weiner and Johansson of a high initial serum level of progestogen during the first 60 days in subjects implanted with d-norgestrel capsules (13). The initial rapid increase in serum R-2323 levels during the first few days after implantation is a phenomenon also noted by Benagiano and coworkers (12) and Weiner and Johansso (13). This can be explained in part by the saturation of the Silastl.& tubing with the steroid during manufacture and storage and in part by the high degree of vascularity of the surrounding tissue in reaction to injury and introduction of a foreign body, as well as by slower metabolism of the drug by the liver early in the postimplantation period. The rapid decline in serum R-2323 levels during the first two weeks after reaching the maximum value is probably due to decreasing concentration of the steroid in the capsular wall, decreasing vascularity of the tissue surrounding the capsule that occurs with wound healing as well as the induction of synthesis of enzymes required for the metabolism of the drug. The serum concentrations of R-2323 attained by administration of the steroid from subdermal Silastl.c@ reservoirs were suprisingly nonuniform in these monkeys. The source of variation appears to be the differences in diffusion rate of the steroid from the implants. Weiner and Johansson also found this variation of blood levels between subjects (13). Since in vitro relea e rates under controlled conditions are quite uniform for these Silasti A capsules (unpublished observations), it would seem that conditions at the implantation site may be important in determining the actual dose of steroid delivered to the circulation. One such factor is the degree of local reaction leading to fibrosis around the capsule; the more the fibrosis, the less is the absorption rate of the steroid (14). A thin layer of fibrous tissue was noticed around all capsules but no attempt was made to quantitate it. Another factor important in determining the diffusion rate of steroids from Silastia capsules is the state of the circulation around the area of implantation; this is in turn determined by the location of the implant and the general activity of the animal. Of the four monkeys with periods of anovulation, the inhibitory effect of R-2323 on menstruation and ovulation seems most prolonged in monkeys with high mean serum R-2323 levels. Ovulation resumed or was not suppressed when serum R-2323 was 0.3 to 0.4 ng/ml, apparently the threshold level of drug required to maintain inhibition. However, the R-2323 values in the months preceding the recovery of ovulation were not measurably greater. It is somewhat unexpected that ovulation would suddenly resume during a period of relatively stable drug levels. One possibility suggested by Croxatto to explain the failure of contraception following several months of protection even though the reservoir was not exhausted (6), is that the organism adapts to the continuous low levels of the steroid and can escape the inhibitory effect. More specifically, it can be theorized that greater
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binding by serum proteins may occur over prolonged treatment. Another possibility is the increasing appearance of metabolites that are biologically inactive but contribute to the apparent stable levels of radioassayThis is not probable in that the amounts of metabolites able R-2323. necessary to achieve substantial cross reaction is enormous since their cross reaction in the RIA is less than 1%. In conclusion, the direct measurement of blood levels of R-2323 have shown that contraceptive steroids can be delivered from subdermal implants at a constant rate over periods of at least 9 to 10 months after an initial 2 to 3 months in which an equilibrium between capsule diffusion capacity, Three other local tissue absorption and liver metabolism is established. characteristics of subdermal implant contraception have also been elucidated here which confirm studies using other progestogens (13): (a) Considerable variation exists in the ultimate diffusion rate and thus the drug level in the peripheral circulation once local tissue reaction is complete; (b) A wide margin in the absolute amount of drug is required by the individual to affect a biological parameter, e.g. ovulation; (c) The organism may achieve reversal of the biological endpoint even though seemingly constant amounts of drug are available.
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REFERENCES
1.
Se&, S.J. and Croxatto, H. Paper presented at the 23rd Annual Meeting of The American Fertility Society, April 14-16, 1967, Washington, D.C.
2.
Kincl, F.A., Benagiano, G. and Angee, I. Sustained release hormonal preparations 1. Diffusion of various steroids through polymer membranes. Steroids 11:673-680, 1968.
3.
Tatum, H.J., Coutinho, E.M., Filho, A.J. and Sant'Anna, A.R.S. Acceptability of long-term contraceptive steroid administration by subcutaneous Silastic capsules. Amer. J. Obstet. Gynecol. 105:11391143, 1969.
4.
Croxatto, H., Diaz, S., Vera, R., Etchart, M. and Atria, F. Fertility control in women with a progestogen released in microquantities from subcutaneous capsules. Amer. J. Obstet. Gynecol. 105:1135-1138, 1969.
5.
Tejuja, S. Use of subcutaneous Silastic capsules for long-term steroid contraception. Amer. J. Obstet. Gynecol. 107:954-957, 1970.
6.
Croxatto, H.B., Diaz, S., Atria, P., Cheriakoff, S., Rosatti, S. and Oddo, H. Contraceptive action of megestrol acetate implants in women. Contraception 4:155-167, 1971.
7.
Coutinho, E.M., Mattos, C.E.R., Filho, J.A., Silvin, M.C. and Tatum, H.J. Further studies on long-term contraception by subcutaneous Silastic capsules containing megestrol acetate. Contraception 5:389393, 1972.
8.
Coutinho, E.M. and Da Silva, A.R. One year contraception with norgestrienone subdermal implants. Fertility and Sterility 25:170-176, 1974.
9.
Coutinho, E.M., Da Silva, A.R., Carreira, C.M., Chaves, M.C. and Filho, J.A. Contraceptive effectiveness of Silastic implants containing the progestin R-2323. Contraception 11:625-636, 1975.
10.
G. R-2323: An original contraceptive Sakiz, E. and Azadian-Boulanger, compound. Excerpta Medica International Congress Series No. 219: Hormonal Steroids 865-871, 1970.
11.
Bahgat, M.R., Atkinson, L.E., Brinson, A.O. and Segal, S.J. Treatment of postpartum rhesus monkeys with progestogen: Appearance in milk and effects on lactation. Contraception 12:665-678, 1975.
12.
Benagiano, G., Ermini, M., Carenza, L. and Rolfini, G. Studies on sustained contraceptive effects with subcutaneous polydimethylsiloxane implants. I. Diffusion of megestrol acetate in humans. Acta Endocrinol. (Kbh) 73:335-346, 1973.
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13.
Weiner, E. and Johansson, E.D.B. Plasma levels of d-norgestrel, estradiol and progesterone during treatment with Silastic implants containing d-norgestrel. Contraception 14:81-92, 1976.
14.
Ermini, M., Carpino, F., Russo, M. and Benagiano, G. Studies on sustained contraceptive effects with subcutaneous polydimethylsiloxane implants 3. Factors affecting steroid diffusion in vivo and in vitro. Acta Endocrinol (Kbh) 73:360-373, 1973.
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