Plasma Gonadotropin and Gonadotropin-Releasing Hormone Levels After Intranasal Administration of Gonadotropin Releasing Hormone M. KATZ, B. L. PIMSTONE, P. J. CARR, AND S. HENDRICKS Departments of Obstetrics and Gynaecology, and Chemical Pathology and Medicine, University of Cape Town, Cape Town, South Africa GnRH. Plasma GnRH levels after intranasal administration failed to achieve the high peaks found after the intravenous route but maintained elevated levels for at least an hour and often longer. Despite the much lower plasma GnRH levels, intranasal GnRH produced a sustained effect on LH and FSH secretion, greater than GnRH given by the intravenous route. (J Clin Endocrinol Metab 43: 215, 1976)
ABSTRACT. Intranasal administration of gonadotropin-releasing hormone (GnRH) in doses ranging from 2-4 nig produced a consistently prolonged LH response in patients with secondary amenorrhea. In 4 cases, a delayed secondary rise occurred. A similarly prolonged FSH response was observed in the majority of patients. Six hours after intranasal GnRH, FSH and LH values were well above basal levels and were higher than those observed at a similar interval after intravenous
G
ONADOTROPIN-releasing hormone (GnRH) has proved a useful adjunct to other preparations in the assessment of the hypothalamic-pituitary axis (1,2) and of the pituitary-gonadal axis (3). More recently this hormone has been used successfully in the treatment of anovulation (4-6) and oligospermia and azoospermia (7). The need to administer GnRH parenterally 2 to 3 times daily for from one week (during induction of ovulation) to several months or years (in the treatment of hypogonadism) requires considerable doctor and patient motivation, which has, therefore, restricted its use. The availability of GnRH in the form of intranasal drops has brightened the therapeutic future of this substance. We have administered GnRH intranasally (in GnRH) in a series of patients with secondary amenorrhea in order to determine the duration, magnitude, and nature of plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) response and to compare plasma GnRH, FSH, and LH levels following intranasal GnRH to those observed following intravenous GnRH (iv GnRH).
Materials and Methods Thirteen patients with secondary amenorrhea (not ovarian failure) of 6 months to 2 years duration were Received August 25, 1975. Reprint requests to: Dr. M. Katz, Department of Obstetrics and Gynaecology, Medical School, Observatory 7900, Cape Province, South Africa.
chosen for the study. Written informed consent was obtained in each case. Ten patients received 50 or 100 /u,g iv GnRH. Those patients who received 50 ^tg iv GnRH were given 2 or 3 mg in GnRH at intervals varying from 1 day to 6 weeks later, while those who were given 100 /xg iv GnRH received 2, 3, or 4 mg in GnRH subsequently. The intranasal GnRH was dissolved in normal saline to give a solution containing 500 /u,g per drop of diluent. Both iv and in GnRH were supplied by Ayerst International. After basal (but non-fasting) samples ( — 15 and 0 min) were taken (some time between 8.00 and 10.00 AM), GnRH was administered intravenously as a single bolus, or intranasally with half the dose being instilled into each nostril. During intranasal administration, the head was tilted backwards and rotated to opposite sides at short regular intervals to ensure that at least a portion of the instilled GnRH entered the nasal sinuses. Blood was then taken into heparinized tubes at 15, 30, 60, 120, 180, 240, and 360 minutes and centrifuged immediately, and the plasma was stored at — 20 C until the time of assay. In patients receiving 50 /u,g iv and 4 mg in GnRH, the test was only continued for 120 minutes. Plasma FSH and LH were assayed by the doubleantibody radioimmunoassay technique (8,9), using the Second International Reference Preparation of Human Menopausal Gonadotropin as the reference standard for both FSH and LH. Human FSH and LH for labelling and anti-human FSH and LH sera were provided by Serono Immunochemicals. The sensitivities of both assays were 1 mlU/ml. Plasma GnRH was measured by radioimmunoassay, using a specific antiserum which detects the whole peptide and not fragments, as described elsewhere (10). The double-antibody method was used to separate free and antibody-bound labelled GnRH (11), with an assay sensitivity of 25 pg/ml. All specimens were run
215
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
il6
JCE & M • 1976 Vol 43 • No 1
COMMENTS
TABLE 1. Plasma luteinizing hormone (LH) before and at intervals after 50 or 100 pig GnRH intravenously
and 1-5 mg GnRH intranasally in patients with secondary amenorrhea % above baseline at (min)
Plasma LH (mlU/ml) at intervals (min)
240
360
182.1 306.6 115.8 57.5
17.3 117.9 39.6 11.2
23.5 27.4 66.3 82.0 — —
251.7 1,056.4 637.5 545.0
109.8 251.2 314.3 254.9
route
-15
0
15
30
60
90
120
180
240
360
1 2 3 4
50 fig iv
13.1 13.3 8.5 10.2
11.6 14.3 7.7 8.9
32.9 30.3 37.5 23.4
40.1 38.6 48.4 28.2
32.7 32.9 30.0 24.0
26.9 27.4 29.7 20.9
24.0 25.3 23.5 18.0
— — —
— — —
— — —
5 6 7 8 9
100 fig in
20.2 10.7 10.3 28.5 8.5
15.5 10.5 9.8 35.4 7.7
67.8 86.0 49.8 138.6 27.8
136.7 191.0 80.6 189.3 28.4
126.4 174.6 69.0 150.1 16.9
94.6 127.0 53.4 114.0 15.1
91.7 103.8 42.3 93.0 14.4
67.0 60.2 31.4 70.7 —
50.5 45.8 21.8 50.4 —
21.0 23.1 14.1 28.4 —
10 11
1 mg in
9.2 16.1
9.2 17.8
11.9 26.5
14.3 32.9
15.8 30.1
15.3 27.6
13.3 29.0
—
—
—
12
2 mg in
30.2 56.2 141.0 218.0 28.9 20.1
34.1 58.8 142.3 220.0 29.9 26.3
33.9 46.1 134.6 210.0 27.3 27.4
31.5 89.3 166.4 208.0 26.6 29.4
34.9 135.0 145.0 208.0 — —
39.4 90.2 118.0 149.0 — —
no.
11.4
11.0
13
7.7
7.8
14 15 16 17
14.1 25.1 12.4 8.7
17.8 21.1 12.8 6.6
17.9 24.0 81.5 202.0 24.0 13.0
18 19
2.5 mg in
19.1 24.3
19.3 20.5
20.5 41.0
46.8 113.2
60.2 135.4
45.1 109.5
59.4 108.0
32.4 90.3
36.2 62.0
30.0 41.2
88.5 176.7
56.2 83.9
20 21 22 23
3 mg in
8.0 8.8 18.0 9.2 7.5 5.1
8.5 13.0 17.3 8.6 7.0 4.9
17.3 21.8 29.2 16.4 15.9 39.4
26.8 32.9 36.1 34.7 20.9 76.4
33.5 36.2 32.1 42.9 21.8 64.1
36.1 33.9 30.1 41.8 23.9 58.8
46.7 34.3 31.5 45.8 37.5 75.1
42.6 — 75.1 66.7 —
28.2
55.0
158.7
404.5
67.8 69.5 —
32.7 33.1 —
661.7 852.0
267.4 353.4
548.8
188.3
24
25 26 27
4 mg in
10.1 23.2
7.0 20.7
43.4 40.4
48.4 62.7
46.8 66.8
52.3 81.7
72.5 110.1
62.6 —
55.8 —
24.8 —
28
5 mg in
7.2
6.5
24.1
39.4
42.9
51.2
63.1
—
—
—
in the same assay. Intra-assay variation was less than 15%.
Results No clinical side effects were noted after intranasal GnRH. Table 1 demonstrates the individual, and Fig. 1 the mean, LH response following the administration of 50 and 100 fig iv GnRH and 1-5 mg in GnRH. The patterns obtained after intravenous administration were in keeping with those observed under similar conditions by other investigators (12,13). Plasma LH rose rapidly from baseline, reached a peak at 30 minutes and then dropped progressively. After in GnRH, a prolonged LH response pattern was observed in all patients at all dose levels. This response was characterized by a prompt rise of LH during the first 30-60 minutes followed by a
plateau. However patients 12, 18, 19, and 20 showed a secondary rise, 120-150 minutes after in GnRH. Whereas the 4 and 6 hour values after 100 fxg iv GnRH were 165% and 46.5% above mean basal levels, those after 2-4 mg in GnRH were 497.7% and 228.4% above baseline, respectively, reflecting a considerably more sustained response. The pattern of plasma FSH responses following in GnRH was similar to that observed following iv GnRH (Table 2, Fig. 2), except that FSH levels tended to remain elevated longer after in GnRH. Peak FSH levels were observed between 180 and 240 minutes in all except 2 cases in whom peak values occurred at 90 (case 5) and 120 (case 6) minutes. At 4 and 6 hours after intranasal administration, mean FSH values were 132.6% and 98.2% above baseline, respectively, after 2-4 mg in GnRH. Both were greater than
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS
217
TABLE 2. Plasma follicle-stimulating hormone (FSH) before and at intervals after 50 or 100 fig GnRH intravenously and 1-5 mg GnRH intranasally in patients with secondary amenorrhea % above baseline at( min)
Plasma FSH (mlU/ml) at intervals (min) no.
I3osc/ route
1
50 /xg iv
CftSG
2 3 4
5 6 7
100 /ig iv
8 9 10
1 mg in
11 12 13 14 15
2 mg in
16
17
-15
0
15
30
60
90
120
180
240
360
14.2 15.0 8.2
11.8 13.9 8.2
18.1 17.7 13.8 6.2
17.7 18.4
—
—
—
4.1
20.5 17.2 14.2 5.2
20.1 17.9 15.0
4.3
17.7 17.1 11.6 5.4
6.1
6.3
—
—
—
11.4 12.5 14.9 18.0
10.7 12.5 14.2 17.0
15.8 15.3 20.8 22.8
23.9 24.4 27.5 32.0
23.7 31.8 28.7 33.5
27.5 29.9 29.2 31.7
31.1 28.4 29.9 29.5
30.3 26.7 26.6
29.7 21.4 25.1 28.7
25.1 19.8 21.1 27.0
13.4 13.2
11.1 13.7
10.7 14.7
13.8 15.7
13.1 15.0
14.3 16.3
15.0 17.7
_ —
—
—
5.5 8.6 16.6 17.0 14.2 14.2
4.9 8.2 13.4 17.5 15.0 12.9
3.9 10.1 20.9 26.6 17.6 16.6
5.4 16.2 30.0 33.8 17.4 18.2
5.3 17.6 30.8 36.1 17.9 22.5
5.9 19.8 36.9
5.9 21.2 37.2
6.4
6.3
6.1
31.6 40.8 52.5
30.5 43.6 48.8
24.7 34.8 42.2
42.6 18.1
14.1
31.1
360
167.6 71.2 71.9 64.0
126.1 58.4 44.5 54.3
263.1 190.6 182.1
194.0 132.0 143.9
23.4
18.4 23.9
—
—
—
26.6 19.2
13.9 18.4
13.3 16.5
-38.5* 87.8
-41.2* 68.4
5.7 — 30.5 25.3
5.4 — 34.7 27.7
25.6
27.9
175.4 128.9
114.3 110.7
—
—
5.5 — 27.0 25.5 —
139.8
92.2
18 19
2.5 mg in
21.6 10.0
23.5 9.5
24.3 10.3
27.5 13.2
29.5 18.3
30.5 20.3
27.3 20.4
20 21 22 23 24 25
3 mg in
15.7
17.2
20.3
23.7
4.1
4.6
15.8 13.1 13.6 9.3
16.7 17.5 13.5 13.4
17.2 20.5 13.6 16.4
24.9 4.8 17.7 21.7 14.7 18.4
30.2
4.4
14.6 12.8 12.4 5.5
14.6 4.1 14.0 12.3 11.8 5.6
26 27
4 mg in
11.0 13.2
9.6
14.6
18.0 13.9
20.6
23.9
24.7
19.8
11.9
16.0 12.5
17.7
11.8
15.1
16.1
—
—
—
28
5 mg in
8.9
7.8
10.8
13.0
14.3
17.3
16.6
—
—
—
4.5
240
5.2
17.6 23.5 17.8 20.8
* For purposes of calculation of the mean per cent above baseline, the two negative results were assigned the value of zero.
the respective 93.7% and 70.8% observed after 100 /xg iv GnRH. No dose-related response of either FSH or LH could be demonstrated with doses of in GnRH which varied from 1-5 mg. Plasma GnRH (Table 3) was undetectable in all basal samples except in a few cases who had received iv GnRH on the previous day. The occasional persistence of detectable levels of GnRH for 24 hours after intravenous administration has been previously noted by us (14) and by others (15). After intravenous administration, plasma GnRH rose acutely to achieve very high levels (over 3 ng/ml) at 15 and 30 minutes, but declined sharply to reach low, but detectable levels at 60-120 minutes. In contrast, plasma GnRH after intranasal adminis-
tration rose to 150 pg/ml or less in all but one case (in which a peak of 1.0 ng/ml was found), and remained undetectable in a number of instances. In those cases in which GnRH was easily measurable, the levels remained sustained for 60-120 minutes, and occasionally longer.
Discussion This study has demonstrated that GnRH administered by the intranasal route in females with secondary amenorrhea produces prolonged LH and FSH response despite the relatively low concentration of plasma GnRH attained by this route of administration. A delayed secondary rise in plasma LH occurred in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1976 Vol 43 •. No 1
COMiMENTS
218 TABLE 3.
Plasma gonadotropin-releasing hormone (GnRH) before and at intervals after 50 or 100 /xg GnRH intravenously and 1-5 nig GnRH intranasally iin patients» with secondaryamenorrhea Plasma GnRH (pg/ml) at intervals (min) |-v
Case no. 1
/
LJOSdl
route 50 fig iv
30
0 0 0 0
>3,200 >5,000 >5,000 >5,000
2,200 >5,000 >5,000 1,600
0
0 0
>5,000 —
>5,000 >5,000
0
0
>3,200
250
200
0
2
0
3 4
0 0
5
100 /ig iv
6 7I
8 9 10
1 mg in
11
90
15
-15
0
0
60 0
40 80
120
180
240
360
0
— — — —
— — — —
— — — —
85 1,100
115
160
65
75 0 25
440
100
60
410
90
0 50
0 0
0
>5,000
50
50
50
55
90
0 50
_ —
_ —
— 40
0
0
25
25
25
0
0
0
90
60
55
50
0
0
50
55
50
50
140
55
0
0
13 14
65 0
80 0
0
160
85
140
160
0
0 65
15 16 17
0
0
0
0
0
85 50 0 0
0 240 0 0
0 — 0 0
0 0
0
0 90 25
0 85
0
0 60 55 0
—
—
0 90 — —
30 60
50 0
0 50
135 105
60 100
35 70
40 40
60
215 105
50 105
0
0
80 25
35
0
0 70
150 25 1,000
90
0 55 —
55 110 —
60 190
50 25
50
50
100
90
75
110
25
0
80
— — —
— — —
0 — — — —
12
2 mg in
18 19
2.5 mg in
20 21
3 mg in
22 23
140
—
24 25
26 27
4 mg in
0 0
75 0
100 25
80 40
50 30
50 0
25 0
0 —
0 —
0 —
28
5 mg in
0
0
0
120
25
25
0
—
—
—
some patients. This response was not previously shown after in GnRH in healthy males (16-18) or in amenorrheic women (19), and its significance in the small group of our patients is not clear. However, Bremner and Paulsen produced an almost identical pattern during continuous GnRH infusion (0.2 /xg/min) in 5 normal healthy males (20) and suggested the existence of two functional pools of LH in the human pituitary. The prolonged LH elevation after in GnRH may relate to the pattern of absorption from the nasal mucosa. The diluent, in which lyophilized GnRH was reconstituted, was normal saline and should not have delayed absorption. It is possible that the head of the patient was positioned and manipulated in such a way during GnRH administration that the decapeptide was successfully maneuvered into the
nasal sinuses, where it was pooled, and resulted in prolonged absorption. It is unlikely that the secondary rise resulted from the gut absorption of swallowed GnRH, as London (18) and Jeppson et al. (19) showed no LH response following oral and sublingual GnRH administration respectively. In an attempt to further prolong absorption, one patient was given 2.5 mg in GnRH twice, reconstituted on the second occasion in equal volumes (2 ml) of normal saline and methyl cellulose. However, similar gonadotropin responses and plasma GnRH levels were observed on both occasions in this single patient, suggesting that methyl cellulose as diluent had no additional value. Plasma levels of GnRH remained undetectable or achieved relatively low (but sustained) levels after intranasal compared with
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
219
COMMENTS W-GnRH
intravenous administration. The good biologic response to low but sustained levels of this hormone suggests that high plasma concentrations are not necessary for gonadotropin responses. However, they could have resulted from the persistence of biologically active, but immunologically inactive or poorly crossreacting, fragments of GnRH in the circulation, as our antiserum detects largely intact hormone (10). In patients 2, 4, 5, 6, and 8, plasma GnRH in the low picogram range was detectable 120 minutes or more after iv GnRH. In spite of a plasma half-disappearance time of 5 minutes or less (14,21,22), we and others (15,21,22) have observed a second slowly disappearing component of immunoreacting GnRH after iv infusion. The nature of this material is presently under investigation, as our technique does not give falsely elevated plasma values (10), which disappear after extraction. The therapeutic implications of the prolonged gonadotropin responses elicited by in GnRH may be important. In the doses of 2-4 mg in GnRH, plasma LH levels were still 497.7% above baseline values at 4 hours and 228.4% above baseline levels at 6 hours. The ease of administration and the prolonged action will considerably facilitate therapy with this hormone which, administered intranasally thrice daily, should maintain persistently elevated LH levels. However, the gonadal responses to endogenously released gonadotropin following in GnRH requires careful characterization before the therapeutic benefits can be fully assessed. Acknowledgments We are grateful to Dr. E. S. Polakow, Ayerst Laboratories, for the generous supply of GnRH for both intravenous and intranasal administration. Financial support for this study was obtained from the South African Medical Research Council and the South African Atomic Energy Board.
50UG
N=5
15
FIG. 1. Plasma LH responses to different doses of GnRH given iv or intranasally (in).
3.
References 1. Mortimer, C. H., G. M. Besser, A. S. McNeilly, J. C. Marshall, P. Harsoulis, W. M. G. Tunbridge, A. Gomez-Pan, and R. Hall, Luteinizing hormone and follicle-stimulating hormone-releasing hormone test in patients with hypothalamicpituitary-gonadal dysfunction, Br Med J 4: 73, 1973. 2. Nakano, R., F. Kotsiyi, T. Mizuno, N. Hashiba, M. Washio, and S. Tojo, Response to luteinizing hormone releasing factor (LRF) in normal sub-
0 15 MINUTES
4.
5.
6.
jects and anovulatory patients, Ada Obstet Gynecol Scand 52: 171, 1973. Katz, M., and P. J. Carr, Plasma oestradiol response to synthetic follicle stimulating hormone/ luteinizing honnone releasing hormone in patients with secondary amenorrhoea, J Obstet Gynaecol Br Commonw 81: 791, 1974. Zarate, A., E. S. Canales, A. V. Serially, L. AyalaValdes, and A. J. Kastin, Successful induction of ovulation with synthetic luteinizing hormonereleasing hormone in anovulatory infertility, Fertil Steril 23: 672, 1972. Keller, P. J., Treatment of anovulation with synthetic luteinizing hormone-releasing honnone, Am J Obstet Gynecol 116: 698, 1973. Zanartu, J., A. Dabancens, A. J. Kastin, and A. V. Schally, Effect of synthetic hypo-hormone (FSH/
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1976 Vol 43 • No 1
220
lV-GnRH 20.
50UG
FIG. 2. Plasma FSH responses to different doses ofGnRH given iv or intranasally (in).
N=3 -15 0 15 30 MINUTES
7.
8.
9.
10.
11.
HOURS
LH-RH) in anovulatory sterility, Fertil Steril 25: 160, 1974. Zarate, A., F. Valdes-Vallina, A. Gonzalez, C. Perez-Ubierna, E. S. Canales, and A. V. Schally, Therapeutic effect of synthetic luteinizing hormone-releasing hormone (LH-RH) in male infertility due to idiopathic azoospermia and oligospermia, Fertil Steril 24: 485, 1973. Midgley, A. R., Radioimmunoassay: A method for human chorionic gonadotropin and human luteinizing hormone, Endocrinology 79: 10, 1966. Odell, W. D., A. F. Parlow, C. M. Cargille, and G. T. Ross, Radioimmunoassay for human folliclestimulating hormone: Physiological studies, J Clin Invest 47: 2551, 1968. Hendricks, S., R. Millar, and B. Pimstone, A specific radio-immunoassay for gonadotrophin-releasing hormone, S Afr MedJ 49: 1559, 1975. Morgan, C. R., and A. Lazarow, Immunoassay of insulin: Two antibody system. Plasma insulin
levels of normal, subdiabetic and diabetic rats, Diabetes 12: 115, 1963. 12. Kastin, A. J., C. Gual, and A. V. Schally, Clinical experience with hypothalamic releasing hormones. Part 2. Luteinizing hormone-releasing hormone and other hypophysiotropic releasing hormones, Rec Prog Honn Res 28: 201, 1972. 13. Besser, G. M., A. S. McNeilly, D. C. Anderson, J. C. Marshall, P. Harsoulis, R. Hall, B. J. Ormston, L. Alexander, and W. P. Collins, Hormonal responses to synthetic luteinizing hormone and follicle stimulating hormone-releasing hormone in man, Br MedJ 3: 267, 1972. 14. Pimstone, B., R. Millar, and S. Hendricks, A radioimmunoassay of gonadotrophin releasing hormone: The measurement of the hormone in rat hypothalamic extracts and in human plasma and urine, In Proceedings of the International Symposium on Hypothalamic Hormones, Milan, 1974, (Abstract).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS 15. Jeflcoate, S. L., Personal Communication, 1974. 16. Solbach, H. B., and VV. Wielgelmann, Intranasal application of luteinizing hormone releasinghormone, Lancet 1: 1259, 1973. 17. Bourguignon, J. P., II. G. Burger, and P. Franchimont, Radioimmunoassay of serum luteinizing hormone releasing hormone (LH-RH) after intranasal administration and evaluation of the pituitary gonadotrophic response, Clin Endocrinol 3: 437, 1974. 18. London, D. R., Effects of luteinizing hormone releasing hormone given intranasally,y Endocrinol 59: xiii, 1973. 19. Jeppson, S., S. Kullander, G. Rannevik, and J. Thorell, Intranasal administration of synthetic
221
gonadotrophin-releasing hormone, Br Med J 4: 231, 1973. 20. Bremner, W. J., and C. A. Paulsen, Two pools of luteinizing hormone in the human pituitary: Evidence from constant administration of luteinizing hormone-releasing hormone, J Clin Endocrinol Metab 39: 811, 1974. 21. Nert, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund, and VV. F. White, A radioimmunoassay for gonadotropin-releasing hormone (GnRH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 22. Ben-Jonathan, N., R. S. Mican, and J. C. Porter, Transport of LRF from CSF to hypophysial portal and systemic blood and the release of LH, Endocrinology 95: 18, 1974.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
ABSTRACT. Intranasal administration of gonadotropin-releasing hormone (GnRH) in doses ranging from 2-4 nig produced a consistently prolonged LH response in patients with secondary amenorrhea. In 4 cases, a delayed secondary rise occurred. A similarly prolonged FSH response was observed in the majority of patients. Six hours after intranasal GnRH, FSH and LH values were well above basal levels and were higher than those observed at a similar interval after intravenous
G
ONADOTROPIN-releasing hormone (GnRH) has proved a useful adjunct to other preparations in the assessment of the hypothalamic-pituitary axis (1,2) and of the pituitary-gonadal axis (3). More recently this hormone has been used successfully in the treatment of anovulation (4-6) and oligospermia and azoospermia (7). The need to administer GnRH parenterally 2 to 3 times daily for from one week (during induction of ovulation) to several months or years (in the treatment of hypogonadism) requires considerable doctor and patient motivation, which has, therefore, restricted its use. The availability of GnRH in the form of intranasal drops has brightened the therapeutic future of this substance. We have administered GnRH intranasally (in GnRH) in a series of patients with secondary amenorrhea in order to determine the duration, magnitude, and nature of plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) response and to compare plasma GnRH, FSH, and LH levels following intranasal GnRH to those observed following intravenous GnRH (iv GnRH).
Materials and Methods Thirteen patients with secondary amenorrhea (not ovarian failure) of 6 months to 2 years duration were Received August 25, 1975. Reprint requests to: Dr. M. Katz, Department of Obstetrics and Gynaecology, Medical School, Observatory 7900, Cape Province, South Africa.
chosen for the study. Written informed consent was obtained in each case. Ten patients received 50 or 100 /u,g iv GnRH. Those patients who received 50 ^tg iv GnRH were given 2 or 3 mg in GnRH at intervals varying from 1 day to 6 weeks later, while those who were given 100 /xg iv GnRH received 2, 3, or 4 mg in GnRH subsequently. The intranasal GnRH was dissolved in normal saline to give a solution containing 500 /u,g per drop of diluent. Both iv and in GnRH were supplied by Ayerst International. After basal (but non-fasting) samples ( — 15 and 0 min) were taken (some time between 8.00 and 10.00 AM), GnRH was administered intravenously as a single bolus, or intranasally with half the dose being instilled into each nostril. During intranasal administration, the head was tilted backwards and rotated to opposite sides at short regular intervals to ensure that at least a portion of the instilled GnRH entered the nasal sinuses. Blood was then taken into heparinized tubes at 15, 30, 60, 120, 180, 240, and 360 minutes and centrifuged immediately, and the plasma was stored at — 20 C until the time of assay. In patients receiving 50 /u,g iv and 4 mg in GnRH, the test was only continued for 120 minutes. Plasma FSH and LH were assayed by the doubleantibody radioimmunoassay technique (8,9), using the Second International Reference Preparation of Human Menopausal Gonadotropin as the reference standard for both FSH and LH. Human FSH and LH for labelling and anti-human FSH and LH sera were provided by Serono Immunochemicals. The sensitivities of both assays were 1 mlU/ml. Plasma GnRH was measured by radioimmunoassay, using a specific antiserum which detects the whole peptide and not fragments, as described elsewhere (10). The double-antibody method was used to separate free and antibody-bound labelled GnRH (11), with an assay sensitivity of 25 pg/ml. All specimens were run
215
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
il6
JCE & M • 1976 Vol 43 • No 1
COMMENTS
TABLE 1. Plasma luteinizing hormone (LH) before and at intervals after 50 or 100 pig GnRH intravenously
and 1-5 mg GnRH intranasally in patients with secondary amenorrhea % above baseline at (min)
Plasma LH (mlU/ml) at intervals (min)
240
360
182.1 306.6 115.8 57.5
17.3 117.9 39.6 11.2
23.5 27.4 66.3 82.0 — —
251.7 1,056.4 637.5 545.0
109.8 251.2 314.3 254.9
route
-15
0
15
30
60
90
120
180
240
360
1 2 3 4
50 fig iv
13.1 13.3 8.5 10.2
11.6 14.3 7.7 8.9
32.9 30.3 37.5 23.4
40.1 38.6 48.4 28.2
32.7 32.9 30.0 24.0
26.9 27.4 29.7 20.9
24.0 25.3 23.5 18.0
— — —
— — —
— — —
5 6 7 8 9
100 fig in
20.2 10.7 10.3 28.5 8.5
15.5 10.5 9.8 35.4 7.7
67.8 86.0 49.8 138.6 27.8
136.7 191.0 80.6 189.3 28.4
126.4 174.6 69.0 150.1 16.9
94.6 127.0 53.4 114.0 15.1
91.7 103.8 42.3 93.0 14.4
67.0 60.2 31.4 70.7 —
50.5 45.8 21.8 50.4 —
21.0 23.1 14.1 28.4 —
10 11
1 mg in
9.2 16.1
9.2 17.8
11.9 26.5
14.3 32.9
15.8 30.1
15.3 27.6
13.3 29.0
—
—
—
12
2 mg in
30.2 56.2 141.0 218.0 28.9 20.1
34.1 58.8 142.3 220.0 29.9 26.3
33.9 46.1 134.6 210.0 27.3 27.4
31.5 89.3 166.4 208.0 26.6 29.4
34.9 135.0 145.0 208.0 — —
39.4 90.2 118.0 149.0 — —
no.
11.4
11.0
13
7.7
7.8
14 15 16 17
14.1 25.1 12.4 8.7
17.8 21.1 12.8 6.6
17.9 24.0 81.5 202.0 24.0 13.0
18 19
2.5 mg in
19.1 24.3
19.3 20.5
20.5 41.0
46.8 113.2
60.2 135.4
45.1 109.5
59.4 108.0
32.4 90.3
36.2 62.0
30.0 41.2
88.5 176.7
56.2 83.9
20 21 22 23
3 mg in
8.0 8.8 18.0 9.2 7.5 5.1
8.5 13.0 17.3 8.6 7.0 4.9
17.3 21.8 29.2 16.4 15.9 39.4
26.8 32.9 36.1 34.7 20.9 76.4
33.5 36.2 32.1 42.9 21.8 64.1
36.1 33.9 30.1 41.8 23.9 58.8
46.7 34.3 31.5 45.8 37.5 75.1
42.6 — 75.1 66.7 —
28.2
55.0
158.7
404.5
67.8 69.5 —
32.7 33.1 —
661.7 852.0
267.4 353.4
548.8
188.3
24
25 26 27
4 mg in
10.1 23.2
7.0 20.7
43.4 40.4
48.4 62.7
46.8 66.8
52.3 81.7
72.5 110.1
62.6 —
55.8 —
24.8 —
28
5 mg in
7.2
6.5
24.1
39.4
42.9
51.2
63.1
—
—
—
in the same assay. Intra-assay variation was less than 15%.
Results No clinical side effects were noted after intranasal GnRH. Table 1 demonstrates the individual, and Fig. 1 the mean, LH response following the administration of 50 and 100 fig iv GnRH and 1-5 mg in GnRH. The patterns obtained after intravenous administration were in keeping with those observed under similar conditions by other investigators (12,13). Plasma LH rose rapidly from baseline, reached a peak at 30 minutes and then dropped progressively. After in GnRH, a prolonged LH response pattern was observed in all patients at all dose levels. This response was characterized by a prompt rise of LH during the first 30-60 minutes followed by a
plateau. However patients 12, 18, 19, and 20 showed a secondary rise, 120-150 minutes after in GnRH. Whereas the 4 and 6 hour values after 100 fxg iv GnRH were 165% and 46.5% above mean basal levels, those after 2-4 mg in GnRH were 497.7% and 228.4% above baseline, respectively, reflecting a considerably more sustained response. The pattern of plasma FSH responses following in GnRH was similar to that observed following iv GnRH (Table 2, Fig. 2), except that FSH levels tended to remain elevated longer after in GnRH. Peak FSH levels were observed between 180 and 240 minutes in all except 2 cases in whom peak values occurred at 90 (case 5) and 120 (case 6) minutes. At 4 and 6 hours after intranasal administration, mean FSH values were 132.6% and 98.2% above baseline, respectively, after 2-4 mg in GnRH. Both were greater than
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS
217
TABLE 2. Plasma follicle-stimulating hormone (FSH) before and at intervals after 50 or 100 fig GnRH intravenously and 1-5 mg GnRH intranasally in patients with secondary amenorrhea % above baseline at( min)
Plasma FSH (mlU/ml) at intervals (min) no.
I3osc/ route
1
50 /xg iv
CftSG
2 3 4
5 6 7
100 /ig iv
8 9 10
1 mg in
11 12 13 14 15
2 mg in
16
17
-15
0
15
30
60
90
120
180
240
360
14.2 15.0 8.2
11.8 13.9 8.2
18.1 17.7 13.8 6.2
17.7 18.4
—
—
—
4.1
20.5 17.2 14.2 5.2
20.1 17.9 15.0
4.3
17.7 17.1 11.6 5.4
6.1
6.3
—
—
—
11.4 12.5 14.9 18.0
10.7 12.5 14.2 17.0
15.8 15.3 20.8 22.8
23.9 24.4 27.5 32.0
23.7 31.8 28.7 33.5
27.5 29.9 29.2 31.7
31.1 28.4 29.9 29.5
30.3 26.7 26.6
29.7 21.4 25.1 28.7
25.1 19.8 21.1 27.0
13.4 13.2
11.1 13.7
10.7 14.7
13.8 15.7
13.1 15.0
14.3 16.3
15.0 17.7
_ —
—
—
5.5 8.6 16.6 17.0 14.2 14.2
4.9 8.2 13.4 17.5 15.0 12.9
3.9 10.1 20.9 26.6 17.6 16.6
5.4 16.2 30.0 33.8 17.4 18.2
5.3 17.6 30.8 36.1 17.9 22.5
5.9 19.8 36.9
5.9 21.2 37.2
6.4
6.3
6.1
31.6 40.8 52.5
30.5 43.6 48.8
24.7 34.8 42.2
42.6 18.1
14.1
31.1
360
167.6 71.2 71.9 64.0
126.1 58.4 44.5 54.3
263.1 190.6 182.1
194.0 132.0 143.9
23.4
18.4 23.9
—
—
—
26.6 19.2
13.9 18.4
13.3 16.5
-38.5* 87.8
-41.2* 68.4
5.7 — 30.5 25.3
5.4 — 34.7 27.7
25.6
27.9
175.4 128.9
114.3 110.7
—
—
5.5 — 27.0 25.5 —
139.8
92.2
18 19
2.5 mg in
21.6 10.0
23.5 9.5
24.3 10.3
27.5 13.2
29.5 18.3
30.5 20.3
27.3 20.4
20 21 22 23 24 25
3 mg in
15.7
17.2
20.3
23.7
4.1
4.6
15.8 13.1 13.6 9.3
16.7 17.5 13.5 13.4
17.2 20.5 13.6 16.4
24.9 4.8 17.7 21.7 14.7 18.4
30.2
4.4
14.6 12.8 12.4 5.5
14.6 4.1 14.0 12.3 11.8 5.6
26 27
4 mg in
11.0 13.2
9.6
14.6
18.0 13.9
20.6
23.9
24.7
19.8
11.9
16.0 12.5
17.7
11.8
15.1
16.1
—
—
—
28
5 mg in
8.9
7.8
10.8
13.0
14.3
17.3
16.6
—
—
—
4.5
240
5.2
17.6 23.5 17.8 20.8
* For purposes of calculation of the mean per cent above baseline, the two negative results were assigned the value of zero.
the respective 93.7% and 70.8% observed after 100 /xg iv GnRH. No dose-related response of either FSH or LH could be demonstrated with doses of in GnRH which varied from 1-5 mg. Plasma GnRH (Table 3) was undetectable in all basal samples except in a few cases who had received iv GnRH on the previous day. The occasional persistence of detectable levels of GnRH for 24 hours after intravenous administration has been previously noted by us (14) and by others (15). After intravenous administration, plasma GnRH rose acutely to achieve very high levels (over 3 ng/ml) at 15 and 30 minutes, but declined sharply to reach low, but detectable levels at 60-120 minutes. In contrast, plasma GnRH after intranasal adminis-
tration rose to 150 pg/ml or less in all but one case (in which a peak of 1.0 ng/ml was found), and remained undetectable in a number of instances. In those cases in which GnRH was easily measurable, the levels remained sustained for 60-120 minutes, and occasionally longer.
Discussion This study has demonstrated that GnRH administered by the intranasal route in females with secondary amenorrhea produces prolonged LH and FSH response despite the relatively low concentration of plasma GnRH attained by this route of administration. A delayed secondary rise in plasma LH occurred in
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1976 Vol 43 •. No 1
COMiMENTS
218 TABLE 3.
Plasma gonadotropin-releasing hormone (GnRH) before and at intervals after 50 or 100 /xg GnRH intravenously and 1-5 nig GnRH intranasally iin patients» with secondaryamenorrhea Plasma GnRH (pg/ml) at intervals (min) |-v
Case no. 1
/
LJOSdl
route 50 fig iv
30
0 0 0 0
>3,200 >5,000 >5,000 >5,000
2,200 >5,000 >5,000 1,600
0
0 0
>5,000 —
>5,000 >5,000
0
0
>3,200
250
200
0
2
0
3 4
0 0
5
100 /ig iv
6 7I
8 9 10
1 mg in
11
90
15
-15
0
0
60 0
40 80
120
180
240
360
0
— — — —
— — — —
— — — —
85 1,100
115
160
65
75 0 25
440
100
60
410
90
0 50
0 0
0
>5,000
50
50
50
55
90
0 50
_ —
_ —
— 40
0
0
25
25
25
0
0
0
90
60
55
50
0
0
50
55
50
50
140
55
0
0
13 14
65 0
80 0
0
160
85
140
160
0
0 65
15 16 17
0
0
0
0
0
85 50 0 0
0 240 0 0
0 — 0 0
0 0
0
0 90 25
0 85
0
0 60 55 0
—
—
0 90 — —
30 60
50 0
0 50
135 105
60 100
35 70
40 40
60
215 105
50 105
0
0
80 25
35
0
0 70
150 25 1,000
90
0 55 —
55 110 —
60 190
50 25
50
50
100
90
75
110
25
0
80
— — —
— — —
0 — — — —
12
2 mg in
18 19
2.5 mg in
20 21
3 mg in
22 23
140
—
24 25
26 27
4 mg in
0 0
75 0
100 25
80 40
50 30
50 0
25 0
0 —
0 —
0 —
28
5 mg in
0
0
0
120
25
25
0
—
—
—
some patients. This response was not previously shown after in GnRH in healthy males (16-18) or in amenorrheic women (19), and its significance in the small group of our patients is not clear. However, Bremner and Paulsen produced an almost identical pattern during continuous GnRH infusion (0.2 /xg/min) in 5 normal healthy males (20) and suggested the existence of two functional pools of LH in the human pituitary. The prolonged LH elevation after in GnRH may relate to the pattern of absorption from the nasal mucosa. The diluent, in which lyophilized GnRH was reconstituted, was normal saline and should not have delayed absorption. It is possible that the head of the patient was positioned and manipulated in such a way during GnRH administration that the decapeptide was successfully maneuvered into the
nasal sinuses, where it was pooled, and resulted in prolonged absorption. It is unlikely that the secondary rise resulted from the gut absorption of swallowed GnRH, as London (18) and Jeppson et al. (19) showed no LH response following oral and sublingual GnRH administration respectively. In an attempt to further prolong absorption, one patient was given 2.5 mg in GnRH twice, reconstituted on the second occasion in equal volumes (2 ml) of normal saline and methyl cellulose. However, similar gonadotropin responses and plasma GnRH levels were observed on both occasions in this single patient, suggesting that methyl cellulose as diluent had no additional value. Plasma levels of GnRH remained undetectable or achieved relatively low (but sustained) levels after intranasal compared with
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
219
COMMENTS W-GnRH
intravenous administration. The good biologic response to low but sustained levels of this hormone suggests that high plasma concentrations are not necessary for gonadotropin responses. However, they could have resulted from the persistence of biologically active, but immunologically inactive or poorly crossreacting, fragments of GnRH in the circulation, as our antiserum detects largely intact hormone (10). In patients 2, 4, 5, 6, and 8, plasma GnRH in the low picogram range was detectable 120 minutes or more after iv GnRH. In spite of a plasma half-disappearance time of 5 minutes or less (14,21,22), we and others (15,21,22) have observed a second slowly disappearing component of immunoreacting GnRH after iv infusion. The nature of this material is presently under investigation, as our technique does not give falsely elevated plasma values (10), which disappear after extraction. The therapeutic implications of the prolonged gonadotropin responses elicited by in GnRH may be important. In the doses of 2-4 mg in GnRH, plasma LH levels were still 497.7% above baseline values at 4 hours and 228.4% above baseline levels at 6 hours. The ease of administration and the prolonged action will considerably facilitate therapy with this hormone which, administered intranasally thrice daily, should maintain persistently elevated LH levels. However, the gonadal responses to endogenously released gonadotropin following in GnRH requires careful characterization before the therapeutic benefits can be fully assessed. Acknowledgments We are grateful to Dr. E. S. Polakow, Ayerst Laboratories, for the generous supply of GnRH for both intravenous and intranasal administration. Financial support for this study was obtained from the South African Medical Research Council and the South African Atomic Energy Board.
50UG
N=5
15
FIG. 1. Plasma LH responses to different doses of GnRH given iv or intranasally (in).
3.
References 1. Mortimer, C. H., G. M. Besser, A. S. McNeilly, J. C. Marshall, P. Harsoulis, W. M. G. Tunbridge, A. Gomez-Pan, and R. Hall, Luteinizing hormone and follicle-stimulating hormone-releasing hormone test in patients with hypothalamicpituitary-gonadal dysfunction, Br Med J 4: 73, 1973. 2. Nakano, R., F. Kotsiyi, T. Mizuno, N. Hashiba, M. Washio, and S. Tojo, Response to luteinizing hormone releasing factor (LRF) in normal sub-
0 15 MINUTES
4.
5.
6.
jects and anovulatory patients, Ada Obstet Gynecol Scand 52: 171, 1973. Katz, M., and P. J. Carr, Plasma oestradiol response to synthetic follicle stimulating hormone/ luteinizing honnone releasing hormone in patients with secondary amenorrhoea, J Obstet Gynaecol Br Commonw 81: 791, 1974. Zarate, A., E. S. Canales, A. V. Serially, L. AyalaValdes, and A. J. Kastin, Successful induction of ovulation with synthetic luteinizing hormonereleasing hormone in anovulatory infertility, Fertil Steril 23: 672, 1972. Keller, P. J., Treatment of anovulation with synthetic luteinizing hormone-releasing honnone, Am J Obstet Gynecol 116: 698, 1973. Zanartu, J., A. Dabancens, A. J. Kastin, and A. V. Schally, Effect of synthetic hypo-hormone (FSH/
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1976 Vol 43 • No 1
220
lV-GnRH 20.
50UG
FIG. 2. Plasma FSH responses to different doses ofGnRH given iv or intranasally (in).
N=3 -15 0 15 30 MINUTES
7.
8.
9.
10.
11.
HOURS
LH-RH) in anovulatory sterility, Fertil Steril 25: 160, 1974. Zarate, A., F. Valdes-Vallina, A. Gonzalez, C. Perez-Ubierna, E. S. Canales, and A. V. Schally, Therapeutic effect of synthetic luteinizing hormone-releasing hormone (LH-RH) in male infertility due to idiopathic azoospermia and oligospermia, Fertil Steril 24: 485, 1973. Midgley, A. R., Radioimmunoassay: A method for human chorionic gonadotropin and human luteinizing hormone, Endocrinology 79: 10, 1966. Odell, W. D., A. F. Parlow, C. M. Cargille, and G. T. Ross, Radioimmunoassay for human folliclestimulating hormone: Physiological studies, J Clin Invest 47: 2551, 1968. Hendricks, S., R. Millar, and B. Pimstone, A specific radio-immunoassay for gonadotrophin-releasing hormone, S Afr MedJ 49: 1559, 1975. Morgan, C. R., and A. Lazarow, Immunoassay of insulin: Two antibody system. Plasma insulin
levels of normal, subdiabetic and diabetic rats, Diabetes 12: 115, 1963. 12. Kastin, A. J., C. Gual, and A. V. Schally, Clinical experience with hypothalamic releasing hormones. Part 2. Luteinizing hormone-releasing hormone and other hypophysiotropic releasing hormones, Rec Prog Honn Res 28: 201, 1972. 13. Besser, G. M., A. S. McNeilly, D. C. Anderson, J. C. Marshall, P. Harsoulis, R. Hall, B. J. Ormston, L. Alexander, and W. P. Collins, Hormonal responses to synthetic luteinizing hormone and follicle stimulating hormone-releasing hormone in man, Br MedJ 3: 267, 1972. 14. Pimstone, B., R. Millar, and S. Hendricks, A radioimmunoassay of gonadotrophin releasing hormone: The measurement of the hormone in rat hypothalamic extracts and in human plasma and urine, In Proceedings of the International Symposium on Hypothalamic Hormones, Milan, 1974, (Abstract).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS 15. Jeflcoate, S. L., Personal Communication, 1974. 16. Solbach, H. B., and VV. Wielgelmann, Intranasal application of luteinizing hormone releasinghormone, Lancet 1: 1259, 1973. 17. Bourguignon, J. P., II. G. Burger, and P. Franchimont, Radioimmunoassay of serum luteinizing hormone releasing hormone (LH-RH) after intranasal administration and evaluation of the pituitary gonadotrophic response, Clin Endocrinol 3: 437, 1974. 18. London, D. R., Effects of luteinizing hormone releasing hormone given intranasally,y Endocrinol 59: xiii, 1973. 19. Jeppson, S., S. Kullander, G. Rannevik, and J. Thorell, Intranasal administration of synthetic
221
gonadotrophin-releasing hormone, Br Med J 4: 231, 1973. 20. Bremner, W. J., and C. A. Paulsen, Two pools of luteinizing hormone in the human pituitary: Evidence from constant administration of luteinizing hormone-releasing hormone, J Clin Endocrinol Metab 39: 811, 1974. 21. Nert, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund, and VV. F. White, A radioimmunoassay for gonadotropin-releasing hormone (GnRH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 22. Ben-Jonathan, N., R. S. Mican, and J. C. Porter, Transport of LRF from CSF to hypophysial portal and systemic blood and the release of LH, Endocrinology 95: 18, 1974.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:34 For personal use only. No other uses without permission. . All rights reserved.
