CONTRACEPTION
ANTI-FERTILITY EFFECTS HORMONE RELEASING Ronald
R. Humphrey,
Barbara
OF AN ANALOG OF LUTEINIZING HORMONE (LHRH) IN RATS L. Windsor,
Ferris
and Richard A. Edgren Department of Pharmacology, Parke, Davis Ann Arbor, Michigan 48106
G. Bousley, and
Co.
Abstract Like LHRH, its des-GlylO- ethylamide analog will terminate pregnancy in rats. Daily doses of 30 ug per day and above given morning and afternoon are completely effective in halting gestation when given over days l-7 and days 7-12. No clearly demonstrable effect was seen when peptide was administered on days l-3. Des-GlylO-LHRH-ethylamide is about 10 times more potent than LHRH in terminating pregnancy and some 3-5 times more potent in releasing LH. Introduction Synthetic luteinizing hormone releasing hormone (LHRH) interferes with pregnancy in rats and rabbits when given in large doses during early and mid gestation (1,2). In the rat, complete inhibition of pregnancy was demonstrated at daily doses of 1 mg LHRH administered on days l-7 or following implantation (days 7-12). Recently, Fujino et al. reported that replacement of the C-terminal glycine amide (GlyqHT') of LHRH by ethylamide resulted in an analog (des-GlylO-LHRH-ethylamide) with 3-5 times the potency of the parent peptide and with significantly more prolonged action in vivo (3-6). In view of the high doses of LHRH required to terminate pregnancy, the greater potency of this synthetic peptide prompted us to study its influence on gestation. Materials
and
Methods
Des-Gly lo-LHRH-ethylamide was synthesized in the Parke, Davis Chemistry Department by fragment condensation procedures. The product was purified by Dowex resin (acetate form) and Sephadex G-25 column chromatography. The nonapeptide [(pyre) Glu-His-TrpSer-Tyr-Gly-Leu-Arg-Pro-NH-Et] thus prepared was homogeneous in three thin-layer chromatographic systems. Microanalysis indicated that the product was a diacetate pentahydrate salt of molecular weight 1353.5 and had a [elDBof -59.6' (0.5% solution in 5% acetic acid).
Accepted
for publication
September
DECEMBER 1976 VOL. 14 NO. 6
22, 1976
625
CONTRACEPTION
Female Charles River CD rats (200-320 grams) were maintained on a 14:lO light-dark schedule (lighted, 0500 to 1900 hours) and allowed free access to Purina Lab Chow and tap water. On the evening they were cohabited with fertile males and insemination of proestrus, was confirmed the next morning by the presence of spermatozoa in the The pept ide was suspended in vaginal smear (day 1 of pregnancy). 0.1 ml corn oil and injected subcutaneously, twice daily over days Al 1 animals were ki Iled with carbon l-3, l-7 or 7-12 of gestation. dioxide on day 15; at autopsy the uterus was weighed and the number of embryonic sites was recorded. Results
and Discussion
Des-GlylO-LHRH-ethylamide was without statistically significant effect when administered over days l-3 of pregnancy (Table), although the results at the highest dose appear to be at the borderThus, the peptide appears not to infiuiine of meaningful effect. ence normal tubal transport or the zygotes during the preimplantation In contrast, administration from period at the doses employed. day 1 through 7 of pregnancy terminated gestation in all rats receivAutopsy on day 15 revealed empty uterine ing 30 ug or more per day. horns lacking any sign of implantation. The approximate EDso was When pregnancy continued at incompletely effective IO ug per day. doses, the implantation sites appeared abnormally colored and suggesting a vascular insult and early resorption. hemorrhagic, However, the number of conceptuses per pregnant rat was normal and the mothers appeared in good health while on medication. These findings suggest several interpretations. When adminisdes-Glylo-LHRH-ethylamide appears tered on days l-3 of gestation, Since this is the period of greatest estrogen sento be inactive. sitivity, the data do not suggest an action through increased estroActivity demonstrable when the administration period gen secretion. was extended to days 1-7 may result from a direct “toxic” effect on the embryo or the production of hormonal dysbalances that preclude nidation. The post nidatory effect, clearly demonstrated when compound was administered over days 7-12 of pregnancy (Table), appears quantitatively similar to the days l-7 period. In these ci rcumdai ly doses of 30 1-19and above were 100% effective stances, again, in terminating pregnancy and the EDso appears to be about 10 pg per day. In those animals that retained products of conception after No signifithere was evidence of incomplete resorption. treatment, cant decrease in the number or weight of conceptuses could be Thus, the compound disturbs gestation after implantademonstrated. tion but the mechanism of this effect remains obscure.
626
DECEMBER 1976 VOL. 14 NO. 6
CONTRACEPTION
TABLE INHIBITION
OF
PREGNANCY
IN
RATS
WITH
No. No.
Daily --Dose+
of
Rats
TREATMENT
Pe rcen t Pregnant
DES-GLYlo-LHRH-ETHYLAMIDE
of
Normal
Total Uterine Weight (gm) (M ? SE)
Em_bryos (M
f
SE)
l-3 OF PREGNANCY
DAYS
Oil
5
80
11.3 f 1.8
10.71 + I.10
1 119
5
100
9.6 + 1.8
8.33 f 1.42
3 ug
5
100
9.0 + 2.6
7.84 f 2.18
Corn
l0 119
5
80
II.5 ?:2.1
9.37 ? 1.06
30 1Jg
5
60
10.3 f 3.7
7.10 + 2.25
100 119
5
80
8.8 f 2.4
5.80 f 2.14
10.14 f I.19
TREATMENT
l-7 OF PREGNANCY 75
11.4 c 1.4
1 119
5
80
II.0 r I.1
7.60 f 1.19
311s
5
100
12.8 + 1.7
9.75 f 1.18
40
8.8 f 2.7
Corn
Oil
I2
l0 !Jg
IO
30 1-g
l0
0
0.55 f 0.05*
ANTI-FERTILITY EFFECTS HORMONE RELEASING Ronald
R. Humphrey,
Barbara
OF AN ANALOG OF LUTEINIZING HORMONE (LHRH) IN RATS L. Windsor,
Ferris
and Richard A. Edgren Department of Pharmacology, Parke, Davis Ann Arbor, Michigan 48106
G. Bousley, and
Co.
Abstract Like LHRH, its des-GlylO- ethylamide analog will terminate pregnancy in rats. Daily doses of 30 ug per day and above given morning and afternoon are completely effective in halting gestation when given over days l-7 and days 7-12. No clearly demonstrable effect was seen when peptide was administered on days l-3. Des-GlylO-LHRH-ethylamide is about 10 times more potent than LHRH in terminating pregnancy and some 3-5 times more potent in releasing LH. Introduction Synthetic luteinizing hormone releasing hormone (LHRH) interferes with pregnancy in rats and rabbits when given in large doses during early and mid gestation (1,2). In the rat, complete inhibition of pregnancy was demonstrated at daily doses of 1 mg LHRH administered on days l-7 or following implantation (days 7-12). Recently, Fujino et al. reported that replacement of the C-terminal glycine amide (GlyqHT') of LHRH by ethylamide resulted in an analog (des-GlylO-LHRH-ethylamide) with 3-5 times the potency of the parent peptide and with significantly more prolonged action in vivo (3-6). In view of the high doses of LHRH required to terminate pregnancy, the greater potency of this synthetic peptide prompted us to study its influence on gestation. Materials
and
Methods
Des-Gly lo-LHRH-ethylamide was synthesized in the Parke, Davis Chemistry Department by fragment condensation procedures. The product was purified by Dowex resin (acetate form) and Sephadex G-25 column chromatography. The nonapeptide [(pyre) Glu-His-TrpSer-Tyr-Gly-Leu-Arg-Pro-NH-Et] thus prepared was homogeneous in three thin-layer chromatographic systems. Microanalysis indicated that the product was a diacetate pentahydrate salt of molecular weight 1353.5 and had a [elDBof -59.6' (0.5% solution in 5% acetic acid).
Accepted
for publication
September
DECEMBER 1976 VOL. 14 NO. 6
22, 1976
625
CONTRACEPTION
Female Charles River CD rats (200-320 grams) were maintained on a 14:lO light-dark schedule (lighted, 0500 to 1900 hours) and allowed free access to Purina Lab Chow and tap water. On the evening they were cohabited with fertile males and insemination of proestrus, was confirmed the next morning by the presence of spermatozoa in the The pept ide was suspended in vaginal smear (day 1 of pregnancy). 0.1 ml corn oil and injected subcutaneously, twice daily over days Al 1 animals were ki Iled with carbon l-3, l-7 or 7-12 of gestation. dioxide on day 15; at autopsy the uterus was weighed and the number of embryonic sites was recorded. Results
and Discussion
Des-GlylO-LHRH-ethylamide was without statistically significant effect when administered over days l-3 of pregnancy (Table), although the results at the highest dose appear to be at the borderThus, the peptide appears not to infiuiine of meaningful effect. ence normal tubal transport or the zygotes during the preimplantation In contrast, administration from period at the doses employed. day 1 through 7 of pregnancy terminated gestation in all rats receivAutopsy on day 15 revealed empty uterine ing 30 ug or more per day. horns lacking any sign of implantation. The approximate EDso was When pregnancy continued at incompletely effective IO ug per day. doses, the implantation sites appeared abnormally colored and suggesting a vascular insult and early resorption. hemorrhagic, However, the number of conceptuses per pregnant rat was normal and the mothers appeared in good health while on medication. These findings suggest several interpretations. When adminisdes-Glylo-LHRH-ethylamide appears tered on days l-3 of gestation, Since this is the period of greatest estrogen sento be inactive. sitivity, the data do not suggest an action through increased estroActivity demonstrable when the administration period gen secretion. was extended to days 1-7 may result from a direct “toxic” effect on the embryo or the production of hormonal dysbalances that preclude nidation. The post nidatory effect, clearly demonstrated when compound was administered over days 7-12 of pregnancy (Table), appears quantitatively similar to the days l-7 period. In these ci rcumdai ly doses of 30 1-19and above were 100% effective stances, again, in terminating pregnancy and the EDso appears to be about 10 pg per day. In those animals that retained products of conception after No signifithere was evidence of incomplete resorption. treatment, cant decrease in the number or weight of conceptuses could be Thus, the compound disturbs gestation after implantademonstrated. tion but the mechanism of this effect remains obscure.
626
DECEMBER 1976 VOL. 14 NO. 6
CONTRACEPTION
TABLE INHIBITION
OF
PREGNANCY
IN
RATS
WITH
No. No.
Daily --Dose+
of
Rats
TREATMENT
Pe rcen t Pregnant
DES-GLYlo-LHRH-ETHYLAMIDE
of
Normal
Total Uterine Weight (gm) (M ? SE)
Em_bryos (M
f
SE)
l-3 OF PREGNANCY
DAYS
Oil
5
80
11.3 f 1.8
10.71 + I.10
1 119
5
100
9.6 + 1.8
8.33 f 1.42
3 ug
5
100
9.0 + 2.6
7.84 f 2.18
Corn
l0 119
5
80
II.5 ?:2.1
9.37 ? 1.06
30 1Jg
5
60
10.3 f 3.7
7.10 + 2.25
100 119
5
80
8.8 f 2.4
5.80 f 2.14
10.14 f I.19
TREATMENT
l-7 OF PREGNANCY 75
11.4 c 1.4
1 119
5
80
II.0 r I.1
7.60 f 1.19
311s
5
100
12.8 + 1.7
9.75 f 1.18
40
8.8 f 2.7
Corn
Oil
I2
l0 !Jg
IO
30 1-g
l0
0
0.55 f 0.05*
