Upsala J Med Sci 82: 21-26, 1977 Acute Effects of a New Stimulatory Luteinizing Hormone- re1 easi n g Hormone An a I og u e D-S er (TBU ) 6- EA lo- LR H on the Gonadotrophin and Gonadal Steroid Secretion in Women with Amenorrhoea SVEN JOHAN NILLIUS and L E I F WIDE From the Departments of Obstetrics and Gynaecology and Clinical Chemistry, University Hospital, Uppsala, Sweden ABSTRACT A new stimulatory luteinizing hormone-releasing hormone (LRH) analogue o-Ser(TBU)6-EA10-LRH was administered subcutaneously in a dose of 10 pg into 10 women with amenorrhoea. The injection resulted in a 20-fold increase of the LH level in blood and a 5-fold increase of the FSH level with maxima at 4 and 6 h after the administration, respec- tively. There was an evident biphasic pattern of LH release similar to that described after extended pituitary stimula- tion by constant infusions of LRH with early and late peaks of LH combined with a gradual, progressive FSH release. The duration of the effect on LH and FSH was at least 12 and 24 h, respectively. All the women responded with evi- dent oestradiol increases in blood during the last 18 h of the 24 h study period. A comparison between the effects of a single subcutaneous injection of 10 pg of the LRH analogue and 500 pg of LRH showed that the initial FSH and LH release was similar. However, the FSH and LH release dur- ing the remainder of the study period was significantly greater after administration of the analogue and so was the oestradiol increase. Thus, this study in women confirms previous studies in men that o-Ser(TBU)6-EA10-LRH is a potent stimulatory LRH analogue with prolonged biological activity. INTRODUCTION Luteinizing hormone-releasing hormone (LRH) was isolated from porcine hypothalami, structurally determined and synthesized by Schally and co- workers in 1971 (14). During recent years numerous analogues of LRH have been synthesized in at- tempts to ( a ) develop inhibitors of LRH useful for birth control and ( b ) to find superactive stimulatory analogues with prolonged biological activity (15, review). Such stirnulatory analogues should be more useful for therapeutic use than LRH itself, which has effects on the gonadotrophin secretion of short duration. The plasma clearance of exogenous LRH is very rapid with initial half-life of 4 min (2, 10). Present therapeutic regimens for treatment of hypogonadism in men and women therefore require 8-hourly injections of large doses of LRH over pro- longed periods of time to be effective (6, 8, 9). There is need for a superactive LRH analogue with prolonged activity for simplifying treatment with LRH . In the present study, acute effects of a new stimulatory LRH analogue D-Ser(TBU)6-EA10-LRH (Hoechst 766) on the gonadotrophin and gonadal steroid secretion were investigated in ten women with amenorrhoea. In five of these women the ef- fects of the LRH analogue were compared with those obtained after a large dose of synthetic LRH. PATIENTS AND METHODS Patients. Ten 20-29-year-old women with amenorrhoea volunteered for this study. Nine of them had secondary amenorrhoea of at least 2 years' duration, while one woman had primary amenorrhoea due to Kallman's syndrome (hypogonadotrophic hypogonadism with anos- mia). In seven of the women the amenorrhoea had oc- curred in a setting of self-imposed weight loss. Four of them had regained weight while three women were still underweight at the time of study. All the women had normal X-rays of the skull and pituitary fossa and their thyroid, adrenal, renal and hepatic function was normal. The patient with Kallman's syndrome was found to have a moderate hyperprolactinaemia (26 p g per I) while all the other women had normal prolactin levels in serum. The gonadotrophin levels in serum were low or normal and the endogenous oestrogen production was low. LRH treatment. The LRH analogue o-Ser(TBU)6-EA10- LRH (Hoechst 766) was administered subcutaneously in a dose of 10 p g to all the women. Venous blood sam- ples were obtained before and during the first 24 h after L ' ~ \ < I / < I J M c d S 1 182 22 S . J . Nillius and L . Wide . L - 3 - 2 - 1 -0 Arnenorrhoelc w m n n:10 I& D-SerlTBU)6-EAa-LRH SC I M I S E M -3,N3/1 - 2 . 1 .o o J T . . . . . . . . . . . . . , . , . , . . . . . , 0 2 L 6 8 M fz U 16 B 20 22 2 L h Fig. I . Mean serum levels of LH and FSH before and after subcutaneous administration of 10 p g of D - S e r ( n u ) ' - EA'O-LRH into 10 women with amenorrhoea. the administration and assayed for FSH, L H , oestradiol and progesterone. Five of the women were also studied by hormone assays before and after subcutaneous administration of 500 p g of LRH (Hoechst) which was given either one month before or after the administration of the analogue. Twenty-four hours before the administration of LRH or the analogue, the pituitary reserve capacity for gonadotrophin secretion was tested with 100 p g of LRH (7). Hormone assay methods. Immunoreactive FSH and LH in serum were assayed by a radioimmunosorbent technique with indirectly coupled antibodies (17). LH in serum was measured by utilizing human pituitary LH (12) labelled with lz5I and rabbit anti-human pituitary LH. The LH preparation had a biological activity of 9400 I U (2nd IRP-HMG) per mg. FSH in serum was measured by utiliz- ing human pituitary FSH ( 1 I ) labelled with 1251 and guinea pig anti-human pituitary FSH. The FSH preparation had a biological activity of 12000 IU (2nd IRP-HMG) per mg. The results are expressed in pg per 1. Immunoreactive oestradiol in serum was measured by a radioimmunological technique using an antiserum to an oestradiol-6-oxime-BSA conjugate ( 5 ) . Progesterone was assayed by a similar radioimmunological technique. Statistical methods. The hormone values were trans- formed into logarithms in the statistical calculations. The mean values given in the text are geometric means. For calculation of differences between mean values, formulas based on the Student's t-distribution were used. RESULTS Mean LH and FSH levels in serum before and after subcutaneous administration of 10 p g of D-Ser( T13U)6-EA10-LRH intor the ten women with amenorrhoea are shown in Fig. 1. The LH and FSH results from the preceding intravenous LRH test are shown in Fig. 2. Upsulu I Med Sci 82 The mean pretreatment LH level (0.18 p g per I ) was below the normal range for women of ,fertile age. All the women responded to the LRH analogue with a rapid increase of the LH level in blood with a first peak (2.7 pg per 1) at 45-60 min after the administration. The LH level remained unchanged for the next hour and then increafed again to a second peak with the highest mean value (3.7 pg per 1) at 4 h after the administration. After that the LH level slowly declined. The mean LH level was still significantly elevated over the pretreatment level at 12 ( p < O . O O l ) but not at 24 h (p>0.05) after the administration of the analogue. The mean pretreatment FSH level (0.60 pg per I ) was within the lower normal range for women of fertile age. FSH progressively increased after the injection of the LRH analogue and reached a peak (3.1 p g per I) at 6 h after t h e administration. Then the FSH level slowly declined. The mean FSH val- ues at 12 (1.9 p g per 1) and 24 ( I . 1 p g per I ) were significantly higher (y0.05) * A comparison between effects on the gonado- trophin secretion of subcutaneous administra- tion of 10 p g of D-Ser(T13U)6-EA10-LRH and 500 p g of LRH is i l h t r a t e d in Fig. 4 , which shows mean L H and FSH results from the five women who were given both LRH and the analogue on separate occasions. It can be seen from the figure that the results from the preceeding intravenous LRH tests did not differ on the two occasions. After administration of 500 p g of LRH the LH level rapidly increased to a peak 45 to 60 min later. The LH level remained essentially unchanged for about four hours and then slowly declined. L H was still significantly higher than the pretreatment level at 8 h (p<0.02) but not at 12 h after the administra- tion (p>0.05). The injection of the analogue re- sulted in a similar initial L H increase in blood, later followed by a second increase to a peak at 4 h after the administration. The LH level then slowly de- clined. Increased L H levels were observed at 12 (p0.05). The FSH level progressively increased in similar fashion after injection of LRH and the analogue. Peak levels were reached after 4 and 6 h, respective- ly. The FSH levels at 6, 8, 12 and 24 h after the administration of the analogue were significantly higher @<0.001) than those obtained after 500 p g of LRH. There were no significant differences (p>0.05) between the mean FSH levels during the first 4 h after the administration. The mean oestradiol level in blood was signifi- o J , , ~ , , . , . . . , , , , , . . . , . . . . . . t o x x x P. ,' ' \ -600 .yM F i g . 4. A comparison between effects of 10 Fg of D-Ser(TBU)6-EA'u-LRH and 500 pg of LRH on L H , FSH and oestradiol (E,) secretion in 5 women with amenorrhoea. Results from the preceding intravenous LRH tests are shown to the left on the figure. x xp<0.01, x x xp<0.001. .3a) xx) , , . I 0 2 L 6 8 10 12 K 16 1 8 2 0 2 2 T h Upsulu J Med Sci 82 24 S . J . Nillius and L . Wide L H LRH -W LRH S.C. LH m iv 0 - 0 1 0 p g O-*(TBU)-EA'-LRH s c M" L 4 P I - 3 - 2 - 1 - 0 - m Prol/l -m .50 .uxl -33 ..m € 2 F i g . 5. LH, FSH and oestradiol (E2) levels before and after administration of 10 p g of D-Ser(TBU)6-EA10-LRH and 500 k g of LRH in a 28-year-old woman with amen- oJ 1 0 orrhoea. cantly increased ( p < O . O O I ) over the pretreatment level at 6 h after the administration of both LRH and the analogue. Peak levels were reached after 6 and 12 h respectively. The oestradiol levels at 12 and 24 h after administration of the analogue were significantly higher ( p < O . O O l ) than those obtained after 500 p g of LRH. The mean oestradiol level at 24 h after administration of 500 p g of LRH was not significantly different (p>0.05) from the pretreat- ment level. There was no increase of the low pre- treatment progesterone level at 12 and 24 h after the administration of either the analogue or LRH. Blood levels of L H , FSH and oestradiol after administration of the analogue and LRH to an indi- vidual patient with amenorrhoea are shown in Fig. 5 . The gonadotrophin responses to intravenous LRH were similar on the two occasions. The sub- cutaneous injection of 500 p g of LRH resulted in a rapid LH increase to a peak at 45 min after ad- ministration followed by a plateau-like level for 2-3 hours and then a decline down to the pretreatment level which was reached at 12 h after the adminis- tration. The initial LH peak reached at 30-60 min after subcutaneous injection of the analogue was lower than obtained after 500 p g of LRH. However, U p ~ a l o J Med Sci 82 the LH level after the analogue injection increased further after 2 h and reached a second peak at 4-6 h after the administration followed by a slower fall down to basal levels. The FSH increase after subcutaneous administra- tion of 10 p g of the analogue was more pronounced than after 500 p g of LRH with a broad peak at 6-8 h and higher blood levels throughout the rest of the 24-hour-period. The oestradiol increase observed after injection of the analogue was also greater than that obtained after LRH (Fig. 5). N o side-effects were observed after the injection of LRH or the analogue. One of the women fainted in connection with venipuncture 3 h after adminis- tration of the analogue. DISCUSSION This study shows that the new LRH analogue D-Ser(TBU)6-EA'o-LRH was capable of increasing the gonadotrophin secretion in amenorrhoeic wom- en who had very low basal L H and low normal FSH levels. A single subcutaneous injection of 10 p g of the analogue evoked a 20-fold increase of the L H level in blood and a 5-fold increase of the FSH Acute effects ofa LRH analogue in amenorrhoea 25 level with maxima at 4 and 6 h after the administra- tion respectively. This analogue has previously proved to be high effective in raising gonadotro- phin secretion in experimental animals and normal male volunteers (3, 13, 18). Wiegelmann et al. (18) found significant L H increases after injection of 1 and 2.5 p g and dose-dependent LH increases after 5 and 10 p g of the analogue with peaks at 30 min after intravenous administration into normal men. FSH increases were only observed after the 5 and 10 pg doses with peaks after 120 min (18). Kuhl et al. (3) found that a subcutaneous injection of 5 p g of the analogue was as effective as 100 p g of LRH. How- ever, the analogue had a depot effect and the sub- cutaneous dose of 5 p g was effective for 6-8 h in normal men (3). In the amenorrhoeic subjects of the present study the duration of the effect on FSH was at least 24 h and the LH level remained elevated over the basal level for at least 12 h after a single subcutaneous injection of 10 p g of the analogue. There was an evident biphasic pattern of LH release after the single injection of the LRH ana- logue with early and late peaks of LH combined with a gradual, progressive FSH release. This pat- tern of gonadotrophin release is similar to that de- scribed by Bremner & Paulsen ( 1 ) after continous intravenous infusions of LRH into normal men. The results from their study suggested the existence of two functional pools of L H in the human pituitary, one that is acutely releasable and another that re- quires longer stimulation to be released (1). In wom- en, this biphasic pattern of LH response to ex- tended stimulation by constant infusion of LRH is most evident during the early follicular phase of the cycle (16). The amenorrhoeic subjects had oestrogen levels similar to those found in the early follicular phase of the cycle. When they were given a single injection of the large dose of 500 p g of LRH, there was a pi onounced first initial peak of acutely releaseable LH followed by continued L H release for the next 3 4 hours but no evident second peak of LH release was observed in contrast to what was found after administration of the analogue. A single injection of 10 p g of the LRH analogue resulted in a comparable initial LH peak but then it also evoked a great re- lease of L H from the second pool. This pool may be regarded as a pituitary reserve with a component of yet unmeasurable amounts of newly synthesized gonadotrophins (4). The great activation of the sec- ond pool by the single dose of the LRH analogue shows that it exerts a more extended pituitary stimulation than a single large dose of LRH. This prolonged action of the LRH analogue may be due to a better binding to the pituitary receptors, a slower inactivation or a combination of both factors ( 1 5 ) . The gonadotrophins released into the blood by the LRH analogue stimulated the ovaries to an in- creased steroid secretion and all the women had evident oestradiol elevations in blood during the last 18 hours of the 24 h study period with maximum at 12 h after the administration. The gonadal re- sponse was more pronounced after administration of the analogue than after the large dose of LRH, another illustration of the more prolonged and po- tent action of the LRH analogue. Thus, the present study in women confirms pre- vious studies in men and shows that D-Ser(mU)'- EA'O-LRH is a potent stimulatory LRH analogue with prolonged biological activity. The marked acute effects on the gonadotrophin and gonadal steroid secretion in amenorrhoeic women suggest that this LRH analogue will be useful therapeuti- cally. It might be possible to simplify the present therapeutic regimens by using this potent and long-acting LRH analogue for chronic LRH treat- ment. ACKNOWLEDGEMENTS This study was supported by the Swedish Medical Re- search Council (Grant No. 13X-3145). We are indebted to Dr Mrs M. von der Ohe, Farbwerke Hoechst AG, Frankfurt (Main) for generous supply of the LRH analogue and LRH, to Professor Paul Roos, at the Institute of Biochemistry for supplying the highly puri- fied FSH and LH preparations and to Mrs Anna-Lena Barmark, Mr Christer Bengtsson, Mrs Birgitta Bohman, Mrs Rose-Marie Lindquist and Mrs Ann Sandberg for skilful technical assistance. REFERENCES Bremner, W. J . & Paulsen, C. A,: Two pools of luteinizing hormone in the human pituitary: Evidence from constant administration of luteinizing hormone- releasing hormone. J Clin Endocrinol Metab 39: 81 1 , 1974. Keye, W. R., Kelch, R . P., Niswender, G. D. & Jaffe, R. 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J., Gemzell, C . & Roos, P.: Radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. Acta Endocrinol, Suppl. 174: 1, 1973. 18. Wiegelmann, W . , Solbach, H . G., Kley, H. K . , Nieschlag, E., Rudorff, K . H. & Kriiskemper, H. L.: Effect of new LH-RH analogue (D-Ser(TBU)'- EA"'-LH-RH) on gonadotrophin and gonadal I secretion i n men. Hormone Res 7: 1 , 1976. Received Februury 2, 1977 Address for reprints: Sven Johan Nillius, M.D. Department of Obstetrics and Gynaecology University Hospital S-750 14 Uppsala 14 Sweden ;teroid U m u l a J Mrd S c i 82