LH AND FSH RELEASING POTENCY OF THE SYNTHETIC DECAPEPTIDE

p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

IN HUMAN BEINGS

K. THOMAS, M.D. J. DONNEZ, M.D.

J. FERIN, M.D.

Physiology of Human Reproduction Research Unit (Professor J. FERIN)

Department of Obstetrics and Gynecology (Professor J.A. SCHOCKAERT)

University Hospital B - 3000 LOUVAIN BELGIUM

ABSTRACT

The synthetic decapeptide p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NI¢ 2 was given to 15 healthy adult volunteers by rapid intravenous injection at dose levels of 10 to 100 /~g. Plasma LH and FSH concentrations were measured by radioimmunoassay during the hours fol lowing the injection.

In all subjects an increase of LH was observed. In five male volunteers a certain dose-response relationship for LH release was demonstrated. In six women it seems that differences may exist in hypophyseal responsiveness during the normal menstrual cycle. In four women using an oral contraceptive of combined typetthe pituitary responsiveness to the stimulation remained present.

In all volunteers the FSH release, if any, was quite small ; the subjects with the highest LH release also demonstrated in general the highest FSH release.

Research supported by the Ford Foundation, grant no. 660-0425.

Accepted for publ icat ion June 12, 1972

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INTRODUCTION

The chemical structure of hypothalamic porcine (1) and ovine (2) luteinizing hormone-releasing hormones has been demonstrated to be a decapeptide of the fol lowing amino acid sequence : p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2.

This decapeptide has since been synthetized in several laboratories and its biological activity on gonadotropin release investigated in animals. Recent reports (3, 4, 5) have revealed that the synthetic and natural materials have identical stimulating properties on pituitary gonadotropin release in animals and that the same decapeptide stimulates the secretion of both LH and FSH.

In human beings LH and FSH release has been observed with highly purified natural porcine LH-RH (6, 7) but observations with the synthetic decapeptide are still rare (8).

The purpose of the present study is to investigate the LH and FSH releasing potency of the synthetic decapeptide in adult human beings.

MATERIAL AND METHODS

Fifteen healthy volunteers, aged 23-40 years, were selected for this study. These subjects were divided into three groups : a) a group of five males.Details of clinical and experimental data are given in Table I. b) a group of six females demonstrating regular menstrual cycles.

In one subject the experiment was carried out on the day of onset of menses, in two during the follicular phase and in the three remaining subjects during the luteal phase of the cycle. Details of clinical and biological profiles are summarized in Table I I.

c) a group of four female volunteers using oral contraceptive means in the form of 0.250 mg d-norgestrel + 0.050 mg ethinyl-estradiol taken daily for 21-day periods separated by a 7-day treatment-free interval. The cycle and day of contraceptive treatment on which the experiment was performed as well as the dose of decapeptide

injected, are scheduled in Table III. All blood samples were collected in tubes containing powdered heparin. In most of the

subjects three control blood samples were taken by venipuncture at short intervals immediately before the experiment. After the injection of the decapeptide at zero time, blood was withdrawn from the antecubital vein at 5, 8, 12, 18, 25, 30, 45, 60, 75, 90, 105, 120, 180, 240, 360 minutes. All blood samples were immediately centrifuged and the plasma was frozen and stored at - 20 ° C prior to assay.

Plasma LH levels were measured using the dioxane radioimmunoassay method (9). Specificity and precision of the method have previously been reported (10, 11). The standard for the LH assay used throughout this study was the 2nd I RP-HMG.

For plasma FSH determinations the same dioxane radioimmunoassay technique as for LH was utilized (10). In the FSH assay procedure,the FSH preparation LER-1366 was used for iodination and LER-907 for standard, both donated by the NIH, Bethesda, U.S.A. The specificity of this assay system was demonstrated by its non-cross-reactivity

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T A B L E I : CLINICAL AND BIOLOGICAL DATA IN FIVE MALE VOLUNTEERS

Volunteer

S.J.

T.K.

C.B.

D,J.

V.F.

Age Weight

(years) (kg)

27 66

36 71

23 63

24 73

33 72

T A B L E II

Doseof LH RELEASE FSH RELEASE

Decapeptid~ max. increase injected %increase max. increase %increase

(#g) at 25 rain. in % at (rain.) at 25 min. in % at (min.)

100 550

50 440

25 316

10 255

10 198

610 18 56 64 18

440 25 48 72 18

316 25 3 35 45

255 25 21 21 25

204 30 15 52. 45

: CLINICAL AND BIOLOGICAL DATA IN SIX FEMALE VOLUNTEERS.

Volunteer

M.E. 37

D.L. 24

H.G. 40

G.R. 23

D.A. 23

V.R. 36

Age

(Years)

Dose of Weight Decapeptide Day of

(kg) injected cycle (Pg)

53 100 25 / 30

75 100 8 / 36

59 50 25 / 30

50 50 7 / 26

62 25 27 / 30

52 25 1 / 32

LH

% increase at 25 min.

470

220

670

241

360

637

RELEASE

max. increase

in % at (rain.)

480 18

250 18

720 30

241 25

394 18

637 25

FSH

increase at 25 min.

25

8

42

43

42

8

RELEASE

max. increase

in % at (min.)

83 45

23 45

60 18 /90

43 25

42 30

18 18 /30

T A B L E III : CLINICAL AND BIOLOGICAL DATA IN FOUR FEMALE VOLUNTEERS ON ORAL CONTRACEPTION (0.250 mg d-norgestrel + 0.050 mg ethin¥1-estradiol/day during 21 days; interval 7 days)

Volunteer

B.C.

C.M.

M.M.

D.N.

Age

(years)

26

27

26

24

LH RELEASE FSH RELEASE Dose of Cycle of Day of

Weight ~)ecapeptid( Treat- Treat- %increase max. increase %increase max. increase

(kg) injected ment ment at28min, in% fat(rain.) at25min, in% at(min.) (~g) ., ,,,

i

62 100 !second 21 130 152 45 57 57 25

63 50 second 10 200 220 18 20 46 45

58 50 : first 4 494 497 30 75 97 30

54 25 sixth 4 185 203 18 8 12 60

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:t N O R . . e-----e TX SO~ * - ' - ' ~ cs zs~

o , - . . - . -o v~ i o~

,c_ 30(

T,~ I .~ '~":::"','"*.- " - - ' - -.'~. \

I'J k . . . ~ . - , L : ~ ; ......... ~. , , - , , - ~ ~ ........... ",,,

0 5812 18 2530 4S 60 7~ 90 10~ 120 180 2¢0 360 Time in minutes

Fig. 1. Plasma LH increase after intravenous administration of the synthetic decapeptide

in five male volunteers

7~

_~SO(

5

.c_

~oc

,,', • ~ \ NORMAL WOMEN

t \ . . . . . . . . . . . I ' : I 0 - - -o oR s0~g ~ l~ ; i % ~-.-.-e OA ~ S ~ Z7130 I o . . . . . . .~ . . . . . . . . . . . . . .

t ,m[ct,ot~ .i '~ . ~ ~ i , " .

f . , , , , , , I . i , i . h i " ' . a - -

0 S812 le 2~30 t.S 60 75 90 105 120 " 180 2~0 360 Time in minutes

Fig. 2. Plasma LH increase after intravenous administration of the synthetic decapeptide in six normal menstruating women.

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with HLH, HCG and HTSH. The precision of the assay was determined by comparing differences between duplicate independent observations according to the formula : SD

= V £-~2D~N- ~ , where D = difference between duplicate sample counts and N = number of

duplicates. Caiculation from 92 duplicates revealed that the 95 % confidence limits were +_ 4 to 10 % of the FSH value determined in the steep portion of the standard curve.

Determination for LH and FSH were carried out in duplicate and the mean value expressed as milli-lnternational Units (mlU) of equivalents of the 2nd IRP-HMG per ml of plasma (Figs. 4 and 5). In Tables I, II and III and in Figures 1,2 and 3, values of LH and FSH are expressed as % increase on the mean of the three basal levels measured prior to the experiment. This mean is indicated as 0 %. In order to eliminate interassay errors, all samples from each volunteer were measured in the same assay set. Furthermore, using the dioxane precipitation technique, which permits the processing of a great number of samples, it was possible to measure all samples of the 15 volunteers in the same assay set, avoiding in this way interassay errors between subjects.

The decapeptide (pyro)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 was prepared by classical method of peptide synthesis according to the proposed amino-acid sequence of porcine LH-RH (1). This pure synthetic material was dissolved in isotonic sodium chloride solution, rendered free of bacteria by passage through a 0.22 micron mhlipore fi l ter and then tested to demonstrate that it was sterile and nonpyrogenic. It was administered to the volunteers by a single and rapid injection into the antecubital vein at dose levels ranging from 10 to 100 microgrammes per subject (see Tables I, II and II I) .

R ESU LTS

In no subject were side-effects observed either during or after the experiment,

a) Normal men (Table I and Fig. 1)

A significant increase in LH levels was measured in the five male volunteers. Maximal increase in LH levels was generally obtained 18 to 30 minutes after injection of the synthetic decapeptide. Then plasma LH concentrations decreased slightly but remained approximately constant up to 60 minutes after the injection ; they had returned near to zero levels within 3 to 6 hours. I t appears from the illustration that there exists a certain dose-response relationship in men, and this for the maximal LH increase at 25 minutes (Table I) as well as for the total LH release (Fig. 1). The elevations in plasma FSH concentrations are not so clearly defined ; when they do occur, they are much lower than in the case of LH. The maximal increase of FSH occurred in several cases later than the maximal increase of LH (Table I).

b) Normal women (Table II and Fig. 2)

Maximal LH levels occurred 18, 25 or 30 minutes after injection. In general after this maximum, high plateau levels were maintained for 90 minutes. Only after 4 to 6 hours

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500

o

5 3o0

WOMEN USING ORAL CONTRACEPTIVE ¢=~ ( d - n o r g e s t r e l + e t h i n y l - e s t t e d i o l ) i/ll " " ' ~ o ;o_ -o-- 8CcM 10O~so~g

I o . . . . . . o ON 2S ~=lJ I " I ~" I "~

o- .o...~. #~ ~¢- .o~¢~..-~- ,~.... o

IVINJECTION i ~* / % " '~ 0F THE I o # %% ".

o,=.~.,,o,| o .><~ ..-=-..

tO0 • " o . ' < ~ " " o . . .

o s~l~ 1~ ~o /,~ d~ ~ 9'0 ~ds 1~o "-"~o 2;.0 Time in minutes

3~o

Fig. 3. Plasma LH increase after intravenous administration of the synthetic decapeptide in four women using an oral contraceptive of combined type.

8C

o

2 E d $C

i /.C

3C

2C

i i i i .~l i i t 5812 18 2t530 ~5 60 7~3 g=0 105 120 180 2/,0 360

Time in minutes

Fig. 4. Plasma LH levels after rapid intravenous injection of 100 #g of the synthetic decapeptide in four adult subjects.

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were values near zero levels observed again.

The maximal increase in LH (720 %) was found in subject H.G. with 50 #g decapeptide. No dose-response relationship was observed in this group. However, volunteers M.E. and .H.G., on whom the experiment was performed during the luteal phase, demonstrated a much greater LH increase than subjects D.L. and G.R., on whom the injection was carried out during the follicular phase ; the same doses of decapeptide was injected to M.E. and D.L. (100 #g) and to H.G. and G.R. (50 #g). On the day of onset of the menstruation volunteer V.R., given only 25 #g of decapeptide, demonstrated a strong LH release. In this group, as in the men, the FSH release was much lower and the maximum sometimes occurred later (Table I I). In general the subject showing the highest LH release, also showed the highest FSH release.

c) Women using oral contraception (Table III and Fig. 3)

In all four subjects a significant rise in LH levels occurred. In volunteer M.M. a very large release was observed and 6 hours after zero time high levels were still measured. As in groups a) and b) only slight increase in plasma FSH concentrations was noted, and this was greatest in subject M.M. who also showed the highest LH increase.

DISCUSSION AND CONCLUSIONS

There is no doubt that the synthetic decapeptide used in this study possesses a marked LH-releasing potency in human beings, in men as well as in women. Also in women using oral contraceptio%a hypophyseal LH release can be brought about similar to that in untreated women. Doses of decapeptide of 10 #g are capable of producing measurable LH increase.

For FSH the releasing potency is not nearly as clearly demonstrated. The increment of plasma FSH concentrations, if any, is in most cases very slight and the maximal increase found in this study amounted to 97 % (subject M.M.). Using natural highly purified porcine LH-RH also, much lower FSH release than LH release has been reported previously (6, 7).

The synthetic decapeptide used in the present study, did not stimulate the pituitary release of TSH (THOMAS, BECKERS, and FERIN, unpublished).

In men a certain dose-response relationship for LH was observed, confirming earlier data with natural porcine LH-RH (7), but individual variability can not be excluded. In the fertile woman this relationship was not demonstrated; however, it must be mentioned that at the same doses of decapeptide, the two subjects in luteal phase showed a much stronger response of LH release if expressed as % increase, than the two women on days 7 and 8 of the cycle. By sheer chance, the two subjects investigated during the luteal phase were older. Nevertheless, this observation is surprising and raises the question as to whether some difference in the pituitary responsiveness to a standard dose of LH-RH might exist during the normal ovulatory cycle. Only a greater number of

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20,

0

cJ

"6 15

E

I "1o c

(M

'5 5 g - r m U_

SUaJECT OL ~ (.yc~, d°y B/36) ~ " " Y x , , , , ~

o.____..//.------ - - - ~

'3'o sb ?'s 9'o lds JJ Time in minutes

Fig. 5. Plasma FSH levels after rapid intravenous injection of 100 /~g of the synthetic decapeptide in four adult subjects. (Same volunteers as in Fig. 4).

observations and serial experiments during the same cycle in the same subjects may shed light on this point. In animals pituitary responsiveness can vary throughout the cycle (12) and can depend on basal estrogen impregnation (13).

In general, the maximal increase in LH concentrations was found 25 minutes after injection, and for clinical testing this moment may be suggested as the optimal time to find highest increase in LH. For FSH, highest increase may occur only at 45 or 60 minutes.

Considerable differences in the magnitude of hypophyseal response to the same dose of decapeptide, due probably in part to "basal hormonal state" were observed. This is clearly shown in Fig. 4 for LH and in Fig. 5 for FSH. The four subjects receiving the same dose of 100/~g are compared in these two figures, where absolute LH and FSH levels are presented. The two volunteers M.E. and B.J. demonstrating the highest LH release also show the highest FSH release. The maximal values found in these subjects are comparable to the values found during spontaneous midcycle LH and FSH discharges using our radioimmunoassay system. These results and similar data obtained with lower doses of

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decapeptide suggest that a dose of 50 to 100 #g may be necessary in order to obtain a pituitary stimulation of physiological magnitude. Further studies will be needed to show whether these doses are optimal and whether doses of more than 100 /~g may be necessary to obtain a higher release of pituitary FSH. It has been claimed that much lower doses of LH-RH produce measurable increase in plasma LH (8).

No disturbance in the physiological process of the menstrual cycle was observed following the administration of these single doses. Repeated injections during the normal cycle of 50 pg of decapeptide (every 5 days) with a dose given even the day preceding the spontaneous LH surge, did not seem to influence the spontaneous LH discharge nor the physiological events of the cycle (THOMAS and FERIN, unpublished).

This report represents a preliminary contribution to the extensive physiological investigatiofi of gonadotropin-releasing decapeptide which wil l have to be carried out before this compound is used for therapy and contraception.

REFERENCES

1. MATSUO, H., BABA, Y., NAIR, R.M.G., ARIMURA, A. and SCHALLY, A.V. Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Comm 43: 1334- 1339 (1971)

2. BURGUS, R., BUTCHER, M., LING, N., MONAHAN, M., RIVIER, J., FELLOWS, R., AMOSS, M., BLACKWELL, R., VALE, W. et GUILLEMIN, R. Structure mol& culaire du facteur hypothalamique (LR F) d'origine ovine contrblant la s~cr~tion de I'hormone gonadotrope hypophysaire de lut~inisation (LH). C R Acad Sc S~rie D273 : 1611 - 1613(1971)

3. SCHALLY, A.V., KASTIN, A.J. and ARIMURA, A. Hypothalamic follicle- stimulating hormone (FSH) and luteinizing hormone (LH)- regulating hormone: structure, physiology, and clinical studies. Fertil Steril 22 : 703 - 721 (1971)

4. GAY, V.L. The hypothalamus : physiology and clinical use of releasing factors. Fertil Steril 23 : 50 - 63 (1972)

B. ARIMURA, A., MATSUO, H., BABA, Y., DEBELJUK, L., SANDOW, J. and SCHALLY, A.V. Stimulation of release of LH by synthetic LH-RH in vivo : I. A comparative study of natural and synthetic hormones. Endocrinology 90 : 163 - 168 (1972)

6. KASTIN, A.J., SCHALLY, A.V., GUAL, C., MIDGLEY, Jr., A.R., BOWERS, C.Y. and GOMEZ-PEREZ, F. Administration of LH-releasing hormone to selected subjects. Amer J Obst Gynec 103 : 177 " 182 (1970)

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7.

8.

9.

10

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13.

KASTIN, A.J., SCHALLY, A.V., GUAL, C., MIDGLEY, Jr., A.R., MILLER, M.C. and CABEZA, A. Dose-response relationship of luteinizing hormone to luteinizing hormone-releasing hormone in man. J Clin Invest 50 : 1551 - 1553 (1971)

SCHALLY, A.V., ARIMURA, A., KASTIN, A.J., MATSUO, H., BABA, Y., REDDING, T.W., NAIR, R.M.G., DEBELJUK, L. and WHITE, W.F. Gonadotropin- releasing hormone : one polypeptide regulates secretion of luteinizing and follicle- stimulating hormones. Sciences 173 : 1036 - 1038 (1971)

THOMAS, K. and FERIN, J. A new rapid radioimmunoassay for HCG (LH, ICSH) in plasma using dioxan. J Clin Endocr 28 : 1667 - 1670 (1968)

THOMAS, K., NASH, D. and FERIN, J. Chemical precipitation techniques for the radioimmunologic quantitation of gonadotrophins with special reference to the organic solvent dioxane. Acta endocr (Kbh) 63, suppl 142 : 279 - 299 (1969)

THOMAS, K., WALCKIERS, R. and FERIN, J. Biphasic pattern of LH midcycle discharge. J Clin Endocr 30 : 269 - 272 (1970)

REEVES, J.J., ARIMURA, A. and SCHALLY, A.V. Pituitary responsiveness to purified luteinizing hormone-releasing hormone (LH-RH) at various stages of the estrous cycle in sheep. J Anita Sci 32 : 89 - 91 (1971)

REEVES, J.J., ARIMURA, A. and SCHALLY, A.V. Changes in pituitary respon- siveness to luteinizing hormone- releasing hormone (LH-RH) in anestrous ewes pretreated with estradiol benzoate. Biology of Reproduction 4 : 88 -92 (1971)

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