Stimulation of LH release and ovulation in the rat by cutaneous application of luteinizing hormone-releasing hormone (LH-RH) in dimethyl sulfoxide (DMSO).

ENDOCRINE RESEARCH COMMUNICATIONS, 2(4&5), 327-333 (1975)

STIMULATION OF LH RELEASE AND OVULATION I N THE RAT BY CUTANEOUS APPLICATION OF LUTElNlZlNG HORMONE-RELEASING HORMONE (LH-RH) I N DIMETHYL SULFOXIDE (DMSO)

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J. R. Reel, R. R. Humphrey, J. W. Vaitkus and W. C. Dermody Department o f Pharmacology Research and Development D i v l s i o n Parke, Davis and Company Ann Arbor, Michigan 48106

Abstract The e f f i c a c y o f cutaneously a p p l i e d l u t e i n i z l n g hormone r e l e a s l n g hormone (LH-RH) i n s t i m u l a t i n g LH release i n the chronical l y ovariectomized, estrogen/progesterone-blocked r a t and o v u l a t i o n i n the chlorpromazine-blocked, proestrous r a t was investigated. Following the cutaneous a p p l i c a t i o n of 25 o r 100 pg o f LH-RH i n 100% dlmethyl s u l f o x i d e (DMSO), serum LH rose t o a peak a t 1 h r , then declined toward basal l e v e l s i n the ensuing 3 t o 4 hr. LH-RH applied cutaneously i n e i t h e r 100% DMSO o r 0.9% NaCl was a l s o capable o f inducing o v u l a t i o n i n the chforpromazlne-blocked, proestrous r a t ; however, LH-RH I n 0.9% NaCl d i d n o t y i e l d a l i n e a r l o g dose-response r e l a t i o n ship, t h e r e f o r e a v a l i d ED potency r a t i o f o r DMSO/O.g% NaCl could n o t be estlmated. Nhetheless, 0.9% NaCl was a consideraCutaneous b l y less e f f e c t i v e cutaneous v e h i c l e than DMSO. a p p l i c a t i o n o f LH-RH i n DMSO i n doses o f 100, 50, 25, 10, 5, 2.5 and 1 pg induced o v u l a t i o n i n loo%, loo%, go%, 73%, 30%, 10% and 0% o f the r a t s , r e s p e c t i v e l y . When the percent o v u l a t i o n response was transformed t o p r o b i t s and p l o t t e d against the l o g a r i t h m o f the LH-RH dose an approximately l i n e a r l o g dose-response was obtained; the same r e l a t i o n s h i p a l s o h e l d t r u e f o r o v u l a t i o n i n d u c t i o n f o l l o w i n g sc LH-RH adm i n i s t r a t i o n . I n turn, the E D o ' s o f these two p a r a l l e l doseresponse curves y i e l d e d a p o t e k y r a t i o estimate (sc/cutaneous) o f 0.025, suggesting t h a t 2.5% o f the cutaneously a p p l i e d LH-RH dose was absorbed through the s k i n i n a b i o l o g i c a l l y a c t i v e form. These data i n d i c a t e t h a t DMSO provides a convenient cutaneous v e h i c l e f o r the a d m i n i s t r a t i o n of LH-RH.

327 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.

REEL ET Az Previous s t u d i e s have e s t a b l i s h e d t h a t s y n t h e t i c LH-RH administered

n s e v e r a l d i f f e r e n t v e h i c l e s and by v a r i o u s r o u t e s

i n i t i a t e s the r e l e a s e o f LH and o v u l a t i o n i n a number o f mammalian s p e c i e s (1-7).

We f u r t h e r show h e r e t h a t LH-RH

e f f e c t i v e l y s t i m u l a t e s LH r e l e a s e and o v u l a t i o n i n t h e r a t when


a p p l i e d cutaneously i n 100% DMSO.

M a t e r i a l s and Methods

The a b i 1 i t y o f cutaneously a p p l i e d s y n t h e t i c LH-RH (ParkeDavis) t o r e l e a s e hypophyseal LH was assessed i n c h r o n i c a l l y o v a r i e c t o m i z e d s t e r o i d - b l o c k e d r a t s as p r e v i o u s l y d e s c r i b e d (1). The methods employed t o study o v u l a t i o n i n d u c t i o n i n t h e c h l o r p romazi ne-b locked proes t r o u s r a t f o 1 l o w i ng LH-RH t r e a t m e n t have a l s o been r e p o r t e d (1). a volume o f 50

I n a l l cases, LH-RH o r v e h i c l e i n

P I was a p p l i e d cutaneously t o a 1 cm2 shaved

area i n t h e s c a p u l a r region.

The 100% DMSO was o b t a i n e d from

Matheson Coleman S B e l l Co.

R e s u l t s and Discussion F o l l o w i n g cutaneous a p p l i c a t i o n o f 25 o r 100 ug o f LH-RH i n DMSO t o chron i ca 1 l y o v a r i e c t o m i zed es trogen/proges terone-blocked r a t s t h e r e i s a r a p i d i n c r e a s e i n serum LH (Fig.

1A).

Peak

l e v e l s o f serum LH a r e achieved by one h r p o s t - a p p l i c a t i o n , f o l l o w e d by a d e c l i n e toward basal l e v e l s o v e r t h e n e x t t h r e e t o


STIMULATION OF LH RELEASE BY CUTANEOUS LH-RH

TIME (HR.)

329

TIME (HR.)

FIGURE 1 .

Time course of LH r e l e a s e i n c h r o n i c a l l y o v a r i e c t o m i r e d ,

estrogen/progesterone-blocked r a t s f o l l o w i n g cutaneous (Panel A) o r S C (Panel 6 ) a d m i n i s t r a t i o n o f s y n t h e t i c LH-RH. A, 0 0, 100 pg LH-RH i n 100% DMSO; 0 0, 25 pg LH-RH i n 100% DMSO; and A A, 50 p1 o f 100% DMSO. B, A A, 100 ug LH-RH i n 0.9% NaCl and A A , 50 p1 o f 0.9% NaCI. Each p o i n t r e p r e s e n t s t h e mean f o r 5 r a t s and the v e r t i c a l b a r s r e p r e s e n t the s t a n d a r d e r r o r o f t h e mean.

-

f o u r hrs.

-

-

-

-

The LH r e l e a s e response (area under t h e serum

LH curve) behaves i n a d o s e - r e l a t e d manner i n t h a t the i n c r e a s e i n serum LH a f t e r t h e 100 pg LH-RH a p p l i c a t i o n i s a p p r o x i m a t e l y f o u r times g r e a t e r than t h a t observed w i t h t h e 25 pg dose.

That

a s m a l l p r o p o r t i o n o f cutaneously a p p l i e d LH-RH r a p i d l y p e n e t r a t e s the s k i n t o promote p i t u i t a r y LH r e l e a s e was f u r t h e r suggested by the f i n d i n g t h a t a 100 pg sc i n j e c t i o n of LH-RH i n

0.9% NaCl i n i t i a t e d a t i m e course o f LH r e l e a s e q u a l i t a t i v e l y s i m i l a r , b u t q u a n t i t a t i v e l y 8 - f o l d g r e a t e r (area under t h e serum LH curve) than t h a t f o r cutaneous a p p l i c a t i o n o f 100 pg o f LH-Rli ( F i g .

16).

REEL ET AL.

330 TABLE I

l n d u c t l o n o f Ovulatlon I n Chlorpromazine-blocked, Proestrous Rats Followlng Cutaneous A p p l i c a t i o n o f S y n t h e t i c LH-RH Treatment


1.

1

100% DMSO Control, 50v1 LH-RH, lOOpg LH-RH, 50pg LH-RH, 25vg LH-RH, lOpg LH-RH, 5vg LH-RH, 2.5~9 LH-RH, 1.0119

No. o f Rats Ovulated Per Total No.

% Rats Ovulated

Ova Per Ovulated Rat

0/18

10/10 10/10 9/10 11/15 3/10 1/10 0/10

100 100 30 10

1 3.020.9 1 3,220.7 12.020.4 10.6+1.0 10. E2.6 6.0

41

11.721.3

40 10

1 3,020.4 12.0

90

73

I I , 0.9% NaCl Control, Sop1 LH-RH, lOOpg LH-RH, 501.19 LH-RH, 25pg LH-RH, 1Ovg

0/18 7/15 4/10 1/10 0/10

'On the day o f proestrus a l l r a t s received sc 1 mg o f chlorpromazine a t 1100 h r and LH-RH o r v e h i c l e alone was a p p l i e d cutaneously t o a 1 cm2 shaved area I n the scapular region a t 1500 hr. Ova counts were performed a t 0800 h r on the following mornlng.

Me ants E M The r e s u l t s summarlzed i n Table I i n d i c a t e t h a t LH-RH a p p l i e d cutaneously I n e i t h e r DMSO o r 0.9% NaCl i s a l s o capable o f lnduclng o v u l a t i o n i n the chlorpromazlne-blocked,

proestrous

r a t ; however, a t comparable doses, LH-RH applied cutaneously I n 0.9% NaCl Induced o v u l a t i o n I n a smaller percentage o f the r a t s than d l d LH-RH I n DMSO. dose-response

Due t o the l a c k o f a l i n e a r l o g

r e l a t l o n s h l p f o r the s a l i n e data, a v a l i d E D

potency estlmate could n o t be obtalned; therefore, an E D potency r a t l o f o r DMS0/0.9% NaCl could n o t be estlmated.

50

50 It


331

I

6-

5 z5-


0

4-

341

I

i s obvious, however, t h a t 0.9% NaCl is a considerably less e f f e c t i v e cutaneous v e h i c l e f o r LH-RH than i s DMSO.

I n the case

o f LH-RH i n DMSO, the percent o v u l a t i o n response (quanta1 response) can be transformed t o p r o b i t s and p l o t t e d against the logarithm o f the LH-RH dose t o y i e l d an approximately l i n e a r l o g dose-response curve (Fig.

2).

This same r e l a t i o n s h i p a l s o

holds t r u e f o r our p r e v i o u s l y pub1 ished o v u l a t o r y response data f o l l o w i n g sc LH-RH a d m i n i s t r a t i o n (1) and i s included i n Fig. 2.

As i l l u s t r a t e d i n Fig. 2 p r o v i s i o n a l l i n e a r regression l i n e s (8) were f i t t e d by eye t o the e m p i r i c a l p r o b i t s f o r the cutaneous and sc LH-RH treatment groups (excluding the 0% and 100% o v u l a t i o n response groups),

thus p r o v i d i n g a simple graphical method o f

REEL ET AL.

332 e s t i m a t l n g a potency r a t i o (sc/cutaneous).

The potency r a t i o was

determined from estimated ED50 values (cutaneous,

0.185 pg).

The potency r a t i o (0.185 pg/7.5

7.5

pg and

SC,

pg) o f 0.025 implies

t h a t 2.5% o f the cutaneously applied LH-RH dose was absorbed through the s k i n i n a b i o l o g i c a l l y a c t i v e form.


As demonstrated herein,

DMSO appears t o enhance the movement

o f LH-RH through the skin, as has been shown f o r o t h e r substances

(9-11).

Cutaneous a p p l i c a t i o n o f LH-RH i n DMSO does e f f e c t i v e l y Therefore, DMSO,

s t i m u l a t e LH release and o v u l a t i o n i n r a t s .

by v i r t u e of i t s unique s o l v e n t and p e n e t r a t i n g p r o p e r t i e s , provides a convenient cutaneous v e h i c l e f o r the a d m i n i s t r a t i o n o f LH-RH and p o s s i b l y o t h e r peptides.

ACKNOWLEDGEMENTS The authors thank D r . Richard A. Edgren f o r h e l p f u l e d i t o r i a l suggestions. We a l s o wish t o thank Mrs. Suzanne M c C a l l i s t e r f o r her time and e f f o r t i n t y p i n g t h i s manuscript.

Re f e re nces

1.

Humphrey, R. R., Dermody, W. C., Brink, H. o., Bousley, F. G., Schottin, N. H., Sakowski, R., Vaitkus, J. W., Veloso, H. T. and Reel, J. R., Endocrlnology,z: 1515,

1973. 2.

Dermody, W. C., Humphrey, R. R. and Reel, J. R., I n Gual, C., and E. Rosenberg (eds.), Hypothalamic Hypophysiot r o p i c Hormones, C l i n i c a l and P h y s i o l o g l c a l Studies, Excerpta Medica I n t . Congr. Series No. 263, 1973, p. 56.

3.

Beck, C. C., Reel, J. R., J. Ani. Sci.,x: 300,

4.

Weiner, R. I . , Zerkel, J., Blake, C. A., Schally, A. V. and Sawyer, C. H., Neuroendocrinology,l: 261, 1972.

Dermody, W.

C.

and Coppock, R. W.,

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5.

K e l l e r , P. J. and Gerber, C.,

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64,


1973. 6.

Gonzalez-Barcena, D., Kastin, A. J., Schlach, D. S., Bermudez, J. A., Lee, D., Arimura, A., Ruelas, J., Zepada, 1 . and Schally, A. V., J. Clin. Endocrinol 481, 1973. Metab.,=:

7.

London, D. R., B u t t , W. R., Lynch, S. S., Marshall, J. Dwusu, S., Robinson, W. R. and Stephenson, J. M., J. C 1 In. Endocrinol. Metab.,=: 829, 1973.

8.

Goldstein, A., B i o s t a t i s t i c s : An I n t r o d u c t o r y Text, The MacMillan Co., New York, 1964, p. 173.

9.

Weyer, E. M . (ed.), Ann. N. Y. Acad. Sci., Vol. 141, The New York Academy o f Sciences, New York, 1967.

10.

C.,

Jacob, S. W. and Herschler,R (eds.), Ann. N. Y. Sci., Vol. 243, The New York Academy o f Sciences, New York,

1975. 11.

Kligman, A.

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