Inhibition of ovulation in women by intranasal treatment with a luteinizing hormone-releasing hormone agonist

CONTRACEPTION

INHIBITIONOF OVULATION IN WOMEN BY INTRANASALTREATMENT WITH A LUTEINIZINGHORMONE-RELEASINGHORMONE AGONIST

Christer Bergquist,Sven Johan Nillius and Leif Wide

Departmentsof Obstetricsand Gynecologyand Clinical Chemistry, UniversityHospital,Uppsala, Sweden

ABSTRACT The potent and long-actingstim#atyv luteinizinghormone-releasing hormone LRH analogue D-Ser(TBU)-EA -LBH was administeredintranasally once daily in doses of between 87-600 ug to 39 normally ovulatingwomen over 26-35 days. All the 8 women who were treated with daily intranasaldoses of 87 or 174 vg, had signs of folliculargrowth during the treatmentand 6 of them had raised premenstrualprogesterone levels in blood. The progesteronevalues were low during 4 of the 6 presumptivelyovulatory cycles, indicatingdefectivecorpus luteum function.Three out of 5 women treated with 348 ug daily had anovulatory cycles and the other two very low progesteronevalues, indicating insufficientluteal function.Anovulationoccurred in all but 2 of the 26 women who received 400 or 600 ug of the analogue intranasally. None of the women had dysfunctionaluterine bleeding.Ten of the 26 women treated with 400 or 600 pg daily, did not have any bleeding during the five-weekstudy period. The remaining16 women experienced a menstrual-likebleeding after 26 days on average (range 21-34 days). Thirty-twoof the39 women have discontinuedintranasaltreatmentwith the analogue and all of them ovulatedwithin 4 weeks after discontinuation. The results suggest that it might be possible to us& inf6anasal administrationof the stimulator-y LRH analogue D-Ser(TBU)-EA -LRH to control fertility.

Accepted

for publication

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April

10, 1979

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INTRODUCTION Stimulator-y analogues of LRHwith potent and long-acting activities have been developed during recent years (1). They were produced for the purpose of simplifying LRHtreatment of infertility. However, in experimental animals LRHand its agonistic derivatives were found to exert paradoxical anti-fertility effects (1, 2). Recently we reported that daily6subfbtaneous administration of the potent LRHagonist D-Ser (TBU) -EA -LRH inhibited ovulation in regularly menstruating women (3) . In the present study we investigated effects of intranasal administration of various doses of this stimulatory LRHanalogue on ovulation in healthy women. We found that it was possible to inhibit ovulation 6 bylene daily intranasal spray application of 400-600 vg of D-Ser(TBU) EA -LRH. SUBJECTS ANDMETFKJDS

Thirty-nine healthy women, aged 22-37 years, volunteered for this study. They all had regular menstrual cycles with lengths of between 26-34 days. All but three women had previously been pregnant. Three womenwere sterilized and three women had intrauterine copper devices. The remaining 33 women did not use any contraceptives during the treatment period. The control cycle(s) before the study were normal ovulatory, as judged by basal body temperature (BBT) recordings and progesterone determinations in serum. Treatment The stimulatory LRHanalogue D-Ser(TBU)6-EA10-LRH (Hoe 766, Hoechst AG, Frankfurt&in, FRG) was administered intranasally once daily. The first nasal spray bottle had a concentration of 87 ug per spray application. Eight women (Group I) were given 87 or 174 Fig of the analogue and 5 women (Group II) 348 pg. Twenty-six women (Group III) were treated with a second batch of spray bottles which delivered 100 pg per spray application. Twelve women received 400 ug and fourteen 600 ug. Treatment was instituted within the first three days of the menstrual cycle and continued for 26-35 days or more. The treatment was monitored by clinical examinations, BBT recordings and frequent venous blood samples for determinations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol and progesterone. When doses of 87348 ug were given, blood samples were obtained twice a week 6 h after The basal hormone concentrations in the daily nasal administration. blood were determined once a week. When doses of 400 or 600 pg were given, only baseline blood samples were taken twice a week. Hormone assay methods Eez?ive FSH and LH in serum were assaved by a radioimmunosorbent technique with indirectly coupled antibodies’ (4).‘The results were expressed in pg per 1 using higly purified FSH and LH preparations as reference standards (5, 6). Immunoreactive estradiol in serum was

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measured by a radioirrmrunological technique using an antiserum to an estradiol-6-oxime-BSA conjugate (7). Progesterone was assayed by a similar radioimmunological technique. RESULTS Hormonal response Intranasal administration of D-Ser(TBU)6-EA10-LRHproved to evoke a pronounced release of FSH and LH‘on the first da? of the treatment. This is illustrated by Fig. 1 which shows that the peak values of FSH and LH were obtained 4 h after the intranasal administration of the analogue. The maximumestrogen increase occurred after 6-12 h. The gonadotropin concentrations in serum returned to baseline 24 h after administration while the serum estrogen concentration remained increased.

D-Ser(TBU#EA’-OLRH

F9/1

X8~q

fig/l

n-

-13 -11

11-

Day of cycle :3 -9

9FSH o- -0 LH M

7.

-7

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1-

-1

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, .O-

600

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,

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24h

1. Serum levels of FSH, LH, estradiol and progesterone during the first 2% h @er intranasal administration of 348 ug of D-Ser- (TBU) -EA -LRH into a healthy 34-year-old womanon cycle day 3.

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J

M/l

17L,ug intranasally

50 days

4

31.0

11. 9-

-11

FSHo---o LH M

? I I I

rug/l -9

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-7

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.5

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.l

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0 \

P31 years

P

old

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: 600. ,b E

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10 Menstrual

20

30

40

50d

bleeding

Fig. 2. Serum levels of FSH, LH, estradioland progesterone6 h6aftyS daily intranasaladministrationof 174 pg of D-Ser(TBLJ) -EA LRH over 50 days in a healthy 31-year-oldwoman. Due to technicalproblemswith the spray application,this woman unintentionallyreceived a very large dose on treatmentday 1 (see text). One woman, who had initial technicalproblemswith her spray bottle, received a very large dose of the LRH analogue on the first treatment day (Fig. 2). She respondedwith a great gonadotropinsurge. Six hours after the administrationher LH level was 31.0 ug/l and her FSH level 10.4 vg/l. The average midcycle peak levels of LH and FSH in our

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CONTRACEPTION laboratory are 9.3 and 2.2 ug/l, respectively. The great gonadotropin release presumably induced rapid follicular maturation and ovulation, as evidenced by an estrogen-progesterone pattern similar to that seen during a normal ovulatory menstrual cycle (Fig. 2). A menstrual-like bleeding occurred already after 13 days of treatment. During @e following 37 days of the treatment with 174 pig D-Ser(TBU) -EA -LRH daily, there were no signs of ovulation.

FSH,ug/l

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D-SerITBdEA’~LRH

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LHE.lg/l 5 n=7 + =MtSEM

Js

i 0 Before

6h treatment

0 After

6h lwcek

of treatment

/+ 0

6h

After 2 weeks of treatment

Fig. 3. The geometric mean gonadotropin levels before and 6 $ aff&r intranasal administration of 87-348 pg of D-Ser(TBU) -EA -LRH into 7 healthy female volunteers before and after one and two weeks of treatment. Gonadotrophin responses to the LRHanalogue during the study were determined in 7 of the 13 women treated with daily doses of between 87-348 pg daily. Baseline FSH and LH levels and gonadotropin levels 6 h after administration of the analogue were determined before and after one and two weeks of treatment. All the 7 women had normal basal levels of FSH and LH. No obvious dose-related gonadotropin increase was observed in this small group of women. The results were therefore combined and mean gonadotropin values before and after administration of the analogue are shown in Fig. 3. Before treatment the mean LH response to the LRHanalogue was 3.6 pg/l and the mean FSH response 1.60 ug/l. After one and two weeks of treatment the FSH responses (0.20 and 0.19 ug/l, respectively) and LA responses (0.68 and 0.85

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pg/l) had decreasedsignificantly(p
n

Dose ug

Ovulatory cycles n

Anovulatory cycles n

I

8

87 or 174

6x

2

II

5

348

2xx

3

III

-26

Total -

39

400 or 600

-210

-24 29

XFour with defectiveluteal phase. xxDefectiveluteal phase -1nsufficent dose due to technicalproblemswith the nasal spray. All the 8 women who were treated with daily intranasaldoses of 87 or 174 pg (Group I) had evidence of folliculargrowth with raised estrogen levels in serum during the study period. Six of them had raised premenstrualserum progesteronelevels too. The progesteronevalues were low during 4 of these 6 presumptivelyovulatory cycles, indicating defectivecorpus luteum function. Three of the five women given 348 pg (Group II) had anovulatorytreatment cycles. The other two had low premenstrualprogesteronevalues, indicatingluteal phase insufficiency.Hormone levels during an anovulatorySO-day treatmentcycle in which a daily intranasaldose of 348 ng was administered,are shown in Fig. 4. The blood levels of FSH and LH before the daily administrationof the analoguewere lownormal and rather constant throughoutthe treatment.After 25 days of treatment,theserum estradiol concentrationincreasedindicating folliculargrowth and maturation.During this period the progesterone concentrationwas slightly raised (5.6 and 8.1 nmol/l) in two blood samples, indicatingpossible luteinizationof the follicle(s). A menstrual-likeanovulatorybleeding occurred after 44 days of treatment. Anovulationoccurred in all but 2 of the 26 women who received400 or 600 Figof the analoguedaily (Group III). The two women who had evidence of presumptiveovulation during the treatmenthad technical problems with their spray bottles and probably received insufficient doses of the analogue.After change of the spray bottle,bothwomen continued treatmentwith 400 and 600 pg, respectively,for two additional months and had no evidence of ovulation during that time.

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D-Ser (TBU)6-EA’o-LRH 348Ag intranasally 50 days

M/l 3.

J

FSH o--Q LH u

21-

0, pm01 /I

P34 years

v loo0

old

0 2

600 -

e z

200 *

I ,0--O’ o.o#o, ,o 0 ‘0-w

P-0

nmol/l ‘\

A’

? “o__a

t 20

,------_l 1

10

20

30

A

40

1 $

f.F

50 d A

Menstrual

Fig.

40

bleeding

4. Serum levels of FSH, LH, estradiol and progesterone bgfory0 daily intranasal administration of 348 I_rgD-Ser-(TBU) -EA LRH over 50 days in healthy 34-year-old woman.

Treatment has so far been discontinued in 32 of the 39 women. Most of them ovulated within 2 weeks and all ovulated within 4 weeks after discontinuation. Bleeding patterns Six of the eight women treated with 87-174 g (Group I) experienced a menstrual-like bleeding after 23 days on average (range 13-29 days), while two women had no bleedings during the 35-day treatment period. Two of the five subjects treated with 348 ug (Group II) did not bleed during the treatment period, one woman had short-lasting spottings and the remaining two women had menstrual-like bleedings after 24 and 34 days, respectively. Ten of the 26 women who received 400 or 600 pg daily (Group III) did not have any bleeding during the five-week study period. The remaining 16 women had a menstrual-like bleeding after 26 days on average (range 21-34 days). No obvious side effects spray was well tolerated

were observed during by all the patients.

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the treatment.

The nasal

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DISCUSSION This study shows that ovulation can be inhibited in women by one daily intryasa$Ospray administration of the stimulator-y LkH analogue D-Ser (TBU) -EA -LRH. Only 2 of the 26 volunteers who were treated with intranasal doses of 400-600 ug of the LRHagonist? had signs of presumptive ovulation during the five-week study period. The lack of effect in these two women could possibly be explained by initial technical problems with the spray administration. Both women had no evidence of ovulation during two additional treatment months. At daily intranasal dosages of less than 400 ug, 8 of 13 women presumably ovulated. However, most of these women had subnormal serum progesterone levels, suggesting luteal insufficiency. In further evaluation of the contraceptive properties of this LPtl agonist in women, it seems essential to use daily intranasal doses of 400 pg or more to make sure that ovulation is inhibited. The prolonged intranasal treatment with the analogue did not give rise to any side effects. Nor did side effects due to the induced anovulation, like dysfunctional uterine bleeding, occur. After discontinuation of treatment,ovulation rapidly returned. Acute intranasal spray administration of the LRHagonist resulted in a marked gonadotropin release but the pituitary responsiveness decreased during the course of the chronic treatment. The reduced reserve capacity for gonadotropin secretion probably prevented the release of a sufficient LH surge to induce ovulation and corpus luteum formation. Diminishing gonadotropin responses after repeated injections of this analogue have previously been described both in normal and amenorrheic women (3, 8, 9 10). The patterf: oflhhe gonadotropin release after a single injection of D-Ser(TBU) -I& -LRH is similar to that described after extended stimulation by constant infusions of LRH (11). In intact rhesus monkeys, LRHseems to be released in a pulsative fashion into the pituitary stalk portal blood (12). The prolonged stimulation by the LRHagonist is unphysiologic and may lead to exhaustion of the pituitary capacity to synthesize and release the gonadotropins during longterm treatment. Belchetz -et 2al (13) recently showed that intermittent administration of LRHwas optimal in reestablishing gonadotropin secretion in rhesus monkey with hypothalamic lesions, while constant infusions of LRHfailed to do so. They speculated that constant LRH administration led to a desensitization or down-regulation of the processes responsible for gonadotropin release (13). Such mechanisms6 mayOalso explain the decreasing gonadotropin responses to D-Ser(TBLJ) EA -LRH during the chronic treatment. ACKNOWLEDGMENTS We are indebted to Dr. Mrs. M. von der Ohe, Farbwerke Hoechst AG, for generous supply of the LRHanalogue and Mrs. Birgitta Bohman, Mrs. Ann Sandberg and technicians and engineers at the hormone laboratory for skilful technical assistance.

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REFERENCES 1.

Schally,A.V.: Aspects of hypothalamicregulationof the pituitary gland. Science 202:18-28 (1978)

2.

Corbin, A., Beattie, C.W., Tracy, J., Jones, R., Foell, T.J., pharmacology Yardley, J. and Rees, R.W.A.: The anti-reproductive of LH-RH and agonisticanalogues.Int J Fertil 23:81-92 (1978)

3.

Nillius, S.J., Bergquist,C. and Wide, L.: Inhibitionof ovulation in women by chronic treatmentwith a stimulatoryLRH analogue - A new approach to birth control? Contraception17:537545 (1978)

4.

Wide, L., Nillius, S.J., Gemzell, C. and Roos, P.: Radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. Acta Endocrinol Suppl 174:1-58 (1973)

5.

Roos, P.: Human follicle-stimulating hormone. Acta Endocrinol Suppl 131:1-93 (1968)

6.

Roos, P., Nyberg, L.. Wide, L. and Gemzell, C.: Hhan pituitary luteinizinghormone. Isolationand characterizationof four glycoproteinswith luteinizingactivity.Biochim et Biophys Acta 405:363-379 (1975)

7.

Lindberg,B.S., Lindberg,P., Martinsson,K. and Johansson,E.D.B.: Radioinununological methods for the estimation of oestrone, oestradiol-176and oestriol in pregnancyplasma. Acta Obstet Gynecol Stand Suppl 32:5-19 (1974)

8.

Dericks-Tq, J.S.E.,lBammer,E. and Taubert, H.-D.: The effect of D-Ser(TBU)-LH-RH-EA upon gonadotropinrelease in normally cyclic women. J Clin EndocrinolMetab 45:597-600 (1977)

9.

Nillius, S.J. and Wide, L.: Acute and chronic effects of the stimu$atoB luteinizinghormone-releasinghormone analogue D-Ser (TBU) -EA -LRH on the gonadotrophinand gonadal steroid secretion in women with amenorrhoea.Acta Endocrinol (Kbh.) Suppl 212:138 (1977)

10.

Hanker, J.P., Bohnet, H.G., Miihlenstedt, D., Nowack, C. and Schneider,H.P.G.: Gongdotrophinrelease during chronic administration of D-Ser(TBU) LH-RH-EA in functionalamenorrhoea. Acta Endocrinol 89:625-631 (1978)

11.

Nillius, S.J. and Wide. L.: Acute effects of a new stimulaEory10 luteinizinghormone-releaseing hormone analogueD-Ser(TBU)-EA LRH on the gonadotrophinand gonadal steroid secretion in women with amenorrhoea.Ups J Med Sci 82:21-26 (1977)

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12.

Carmel, P.W., Araki, S. and Ferin, M.: Pituitary stalk portal blood collection in rhesus monkeys: Evidence for pulsatile release of gonadotropin-releasing hormone (C&H): Endocrinology 99: 243-248 (1976)

13.

Belchetz, P.E., Plant, T.M., Nakai, Y., Keogh, E.J. and Knobil, E.: Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631632 (1978)

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