Oxytocin

OXYTOCIN Friedrich Nachtmann, Kurt Krummen, Friedrich M a d , and Erich Riemer 1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Conformation 1.6 Appearance, Colour, Odour 1.7 Biological Activity 2. Physical Properties 2.1 Infrared Spectrum 2.2 Ultraviolet Absorption 2.3 Circular Dichroism 2.4 Raman Spectra 2.5 Proton NMR 2.6 W-NMR 2.7 Solubility 2.8 Optical Rotation 2.9 Isoelectric Point 3. Production 3.1 Extraction from Gland Material 3.2 Chemical Synthesis 4. Stability 5. Metabolism 6. Analysis 6.1 Identity Tests 6.2 Quantitative Physicochemical Methods 6.3 Biological methods 6.4 Determination in Biological Material 6.5 Determination in Dosage Forms 7. References

564 564 564 565 565 565 567 567 567 567 568 568 572 573 573 573 576 576 576 576 577 578 58 1 582 582 584 590 592 595 596

FRIEDRICH NACHTMANN et al.

564

1.

Description

Oxytocin i s t h e c y c l i c octapeptide') hormone released by t h e p o s t e r i o r p i t u i t a r y and having uterotonic and galactagenic activity i n mmmls and h y p t e n s i v e a c t i v i t y i n birds.

Its 20-memberd ring i s composed of f i v e amino acids cystine, tyrosine, isoleucine, glutamine and asparagine -, and t h e s i d e chain contains a f u r t h e r 3 amino acids - proline, leucine and glycinamide. A l l the o p t i c a l l y active amino acids belong t o t h e L-series. The s t r u c t u r e of oxytocin w a s elucidated by du Vigneaud e t al., and indepndently by Tuppy i n 1953 ( 1 , 2 ) . The structure was confirmed by du Vigneaud e t al . by synthesis shortly afterwards ( 3 ) .

1.1 Nomenclature 1.11 Chemical names

L-Cysteinyl-L-tyrosyl-L-isoleucyl-L-glutaminylL-asparqinyl-L-cysteinyl-L-prolyl-L-leucyl-glycinamide c y c l i c (16) disulphide L-H€tni-cystinyl-L-tyrosy~-L-isoleucy~-L-g~utaminylL-asparaginyl-L-hemi-cystinyl-L-prolyl-L-leucylg lyc inamide 1.12

Generic name oxytocin [50-56-61

1.13

Brand names

The following brand names are listed i n t h e Merck Index ( 4 ) : Alpha-hypphamine; Ocytocin; Endop i t u i t r i n a ; Pitocin; Syntocinon; Nobitocin S; Orasthin; Oxystin; Partocon; Synpitan; Piton+; U teracon

.

1.2

Formula 1.21

Amino acid sequence 1

2

3

4

5

6

7

8

9

Cys-~-Ile
J

also known as a nonapeptide i n the l i t e r a t u r e . Instead of 1 cystine 2 cysteines can be used f o r the characterization of the amino acid sequence (see 1.1).

+) Oxytocin is

OXYTOCIN

565

1.22

Structural formula OH I

I

CH-CH3

CH2

y 2

!

I

L

S-CH2-CH-NH-CO-YH-NH-CO-CH-NH I

1

co I

CH2 I

CH2 I

CO-NH2

CH2 I

CO-NH2

N

/ \

iH2

CH-CO-NH-CH-CO-NH-CH2-CO-NH2

I

CH2 CH2

1.23

I

(72

CH-CH3 I CH3 Molecular formula C43H66N12012S2

1.3

Molecular wight 1,007.23

1.4

Elemental canposition C 51.28%

1.5

H 6.61%

M 16.69%

0 19.06%

S 6.37%

Conformation

Urry and Walter =re the first to propose a conformation for oxytocin in solution (5). A revised version was pblished by Gross and Meienhofer (6) according to thich the ring has an antiparallel pleated sheet conformation with intramolecular hydrogen bonds,and the

566

FRIEDRICH NACNTMANN ~t ul.

s i d e chain i s bent back t o t h e ring. Figure 1.1 i s a diagram of the proposed configuration.

Figure 1.1

Proposed conformations of oxytocin i n solution as published by Gross and Meienhofer ( 6 ) . A: i n dimethylsulphoxide

B: a t receptor sites

567

OXYTOCIN

1.6

Appearance, colour, d o u r

The f r e e base has not been obtained i n c r y s t a l l i n e form. By freeze-drying solutions of oxytocin a c i d i f i e d w i t h acetic acid t h e acetate i s obtained, a white f l u f f y powder w i t h a f a i n t odour of a c e t i c acid. 1.7

Biological a c t i v i t y

Oxytocin i s d i f f i c u l t t o prepare i n a pure form owing t o the canplex synthesis. Different biological a c t i v i t i e s have been reported i n the l i t e r a t u r e f o r allegedly pure oxytocin, thus suggesting t h a t the preparations i n question differed i n purity. Chan and du Vigneaud reported an a c t i v i t y of 507 f 23 units/mg ( 7 ) . Maxfield and Scheraga worked with oxytocin with an a c t i v i t y of 495 f 25 units/mg ( 8 ) . A similar a c t i v i t y , i.e. approximately 500 units/mg, was reported by Glickson e t a1 (9) and by C e r l e t t i and B e r d e (10) , while Deslauriers e t a1 described a compound having an a c t i v i t y of 510 f 23 u n i t s / q (11).Boissonnas and Huguenin reported an a c t i v i t y of 450 30 units/mg (12), and Photaki an a c t i v i t y of approximately 400 units/mg (13). Bockaert e t a l . described a 3H-oxytocin w i t h an a c t i v i t y of 440 units/mg ( 1 4 ) . The National I n s t i t u t e of Standards and Control (London, UK) indicates an a c t i v i t y of 595 units/mg f o r the Fourth International oxytocin Standard, a synthetically prepared and specially purified product.

. .

*

Oxytocin acetate w a s p l r i f i e d by preparative column+j3xomatcgraphy on silica gel and the e l u a t e was freeze-dried .The product so obtained had a biological a c t i v i t y determined by the r a t uterus method (15) of 591 f 23 units/mg. I f allowance is made f o r nonpeptide impurities (4.3% water, 8.7% acetic acid and 0.5% sodium), t h e a c t i v i t y is 684 f 27 units/mg pept i d e . The compound w a s sham by thin-layer chromatography (stationary phase: silica gel; solvent systan: chloroformmethanol 7:3 + 5% 0.2 N acetic acid) t o be hanogeneous, while HPLC (see section 6.27) shmed it t o contain less than 1% of detectable and presumably peptide byproducts. 2.

Physical properties 2.1.

Infrared spectrum

und described i n section The Infrared spectrum of t h e 1.7 was recorded f r m 4000 t o 600 ?using a Beckman Acculab +)

W e are grateful t o Mr. H. Bossert, Sandoz Ltd., f o r preparing t h e purified a c t i v e ccmpound.

FRIEDRICH NACHTMANN ct al.

568

8 apparatus. The spectrum of a KBr p e l l e t prepared with 1.5 g a c t i v e compound and 300 mg KBr i s shown i n Figure 2.1. Some regions of the spectrum d i f f e r from the catalogues of spectra ( 1 6 ) . The differences are a t t r i b u t a b l e t o the varying degrees of purity of the canpounds employed. 2.2

Ultraviolet absorption

The UV spectrum (Figure 2.2) of the product described i n 1.7 was recorded f o r an aqueous solution with a concentration of 0.3 mg/rnl over the range 210 - 320 nm using a we-Unicam apparatus, Type SP 1700. The absorption maximum was a t 275 nm and there was a shoulder a t 281 m. After correcting f o r nonpeptide impurities which do not absorb UV l i g h t a t 275 nm, t h e value i s 14.9 and the m l a r extinction coefficient is E& : 1500. The specis i n good agreement with the l i t e r a t u r e (16), but the mlar extinction coefficient is higher than t h a t reported i n t h e l i t e r a t u r e , ming t o the greater purity of the compound (16). 2.3

Circular dichroism

CD spectra f o r oxytocin have been described i n t h e literature by various authors (15,181. Beychok and Breslow investigated the CD spectrum a t various pH values of an oxytccin preparation W i t h an a c t i v i t y of 500 units/mg (17). The spectra obtained are shown i n Figure 2.3. The c h a r a c t e r i s t i c range of wavelengths is 215-310 nm.

A t acid pH values oxytocin displays a negative band a t 280 nm, a positive shoulder a t 250 nm and a large positive band a t 225 nm. m e n the solution i s neutralised (pH = 7.5) t h e negative band declines i n i n t e n s i t y , the shoulder a t 250nm becanes a true ~ i m m and the band a t 225 nm undergoes a s h i f t t o somewhat longer wavelengths. A t pH 10.6 (ionisation of tyrosine) a s t r i k i n g change occurs: a positive plateau appears a t 280 - 290 nm and there is a large positive band a t 245 nm. The a p t i c a l a c t i v i t y a t 280 nm i s contributed by tyrosine and t h e disulphide b n d , wlnile t h e optical a c t i v i t y i n t h e region of 225 nm i s a t t r i b u t a b l e to t r a n s i t i o n of tyrosine t o the ionised state.

WAVELENGTH

loo 90

IN MICRONS

2.5

3

3.5

4

4.5

5

5.5

6

65

I

I

I

I

I

1

I

I

1

l-7

7 1

75

8

1

,

9

10

11

t2

11

16

-70 -60 - 5 0

-40

-

30

- M

4000

3000

2000

I

I

1800

lboo

I

1400

I

I

1200

1000

,

10

! o

800

600

WAVENUMBER CM-'

Figure 2.1

Infra-red spectrum of oxytocin, activity 591

KBr pellet; spectrometer: Beckman Acculab 8

f

23 units/mg.

T

I

I

I

I

I

I

Wavelength [nrn]

I

I

I

1

I

Fiaure 2.2 UV spectrum of oxytocin, a c t i v i t y 591 Concentration, 0.3 mg/ml H 0 Spectrometer: Pye Unicam S8 1700

570

_+

23 units/mg

I

a, -

ri

2000

2000

1500

1500

1000

1000

500

500 0

0

E -500 0 .- 1 d, % 2000 a,

U

U

m

U

cn

2

1500 1000

500 0

l

I

l

1

Figure 2.3

I

E

I

I

I

I

1

C

I

l

l

1

D -

I

I

:

-500 2000

1500 1000

....

-500 -1000

1

220

240

260

280

-10.000

300

I 220

I

240

260

I

280

I-1000

I

300

x (mp) x (mp) CD spectra of oxytocln (activity 500 unlts/mgl and oxytccln analogues at various pH values; published by Beychok and Breslow (17) A Oxytocin -pH 2, pH 7.5, ----- pH 11.5 B 2,Isoleucine-oxytocin pH 2, PH 7 C De~nOQxytOCin pH 2, pH 7.5, ----- pH 11.5 D Deamino-2-isoleucine-oxytocin, pH 2

.....

~

.....

.....

FRIEDRICH NACHTMANN ef al.

572

2.4

Raman spectra

Investigations of the Raman spectra of oxytocin have been described i n t h e literature (8,19). Raman spectra are shown i n Figures 2.4 and 2.5. The incident radiation was the 514.5 nm l i n e of an argon ion laser (Spectrophysics, Model SP-1641, and the spectra were recorded with a Spex Ramalog 5.

OXYTOCIN AMIDE I

CH,

Tyr

AMIDE 111

SOLID

8%

I I.. I I LJ U L 1600 I400 1200 1000 800 600

d

Figure 2.4 Laser-Raman spectrum of oxytocin i n the s o l i d state Incident l i g h t : 150 mW, r e d u t i o n : 5 integration t i m e 2 s Scan rate: 6 cm-'/min; published by Tu e t al. (13).

I

I

1600

1

1

I400

1

1

1200

1

,

1000

1

1

800

1

I

I

600

F i v e 2.5 Laser Raman s p c t r u m of oxytocin i n aqueous solution and i n DqO L Incident l i g h t : 500 mW, other conditions as i n Figure 2.4; published by Tu e t a1 (19)

.

.

OXYTOCIN

2.5

573

Proton NMR

The spectrum of t h e ccmpound described i n 1.7 w a s recorded a t 360 PlRz w i t h a Bruker WH-360 spectraneter (Figure 2.6)'). 5 mg oxytocin was dissolved i n 400 p l d DMSO and one drop of mixture of C E 1 3 and lBlS was added to I&solution. This accounts for the small signal for CHC13 a t 8.31 p p . 1F6, 6 = 0 p p , was used as t h e i n t e r n a l standard. The spectrum i s i n good agreement with t h e data i n the literature (9,20). The IH-NMR spectrum of oxytocin has k e n discussed i n detail by Glickson e t al. ( 9 ) . A l l the amino acids were unoquivocally assigned to the speeral features. 2.6

13C-NMR

The spectrum (Figure 2.7) was recorded a t 90.5 MHz w i t h a Bruker WH-360 spectraneter.') 100 mg of the ccrnpound described i n 1.7 w a s dissolved i n 2.5 ml D20 and the pD w a s adjusted t o 3.6 w i t h CH COOH. Diaxane, 8 = 67.8 p p , was used as the i n t e r n a l standard. The spectrum i s i n very good agreement with spectra published i n t h e l i t e r a t u r e . For t h e assignment of t h e s i g n a l s and i n t e r p r e t a t i o n of spectrum, t h e reader is referred t o papers by a number of authors (11,21-25). A l l t h e amino acids were unequivocally assigned t o the spectral features. 2.7

Solubility

The s o l u b i l i t y of the ccmpound described i n 1.7 (freezedried oxytocin as t h e acetate) w a s determined i n three solvents. The s a t u r a t e d s o l u t i o n s were assayed by HPUJ (see 6.27), and t h e r e s u l t s are shown i n T a b l e 2.1. T a b l e 2.1

Solubility of oxytocin (as the acetate)

')

Solvent

Unit s / m l

Methanol

86 400

W e are g r a t e f u l t o Mr. M. L o o s l i , Sandoz L t d . , recording the spectrum.

for

Figure 2.6

'H-NMR-Spectrum of oxytccin, activity 591 t 23 units/mg, in d6-DMS0 Apparatus: Bruker WH-360

L L

180

170

--r----'-------

70

Figure 2.7

60

160

.-

r-v-

l-'-'--

50

13C-NMR-spectm

140

150

40

--------

of oxytocin, a c t i v i t y 531

Apparatus: Bruker '41-360

r--'

30

f

* c

ijo

IL

-

--,-.-r--7--,-7-T

120

77-1

20

23 units/mg, D 0 ($ 2

-

b -T -

7 - 7 - -

10

= 3.6)

-

FRIEDRICH NACHTMANN et al.

576

2.8

Optical r o t a t i o n

Optical r o t a t i o n values for oxytccin f r a n t h e l i t e r a t u r e are given i n T a b l e 2.2. Table 2.2

@tical r o t a t i o n of oxytocin Value

Conditions

Reference

l,Oo

c = 0,53; water

26

+)

c = 0.53; water

26

-24.0'

c = 0.5; 1 N acetic acid

13

-23.1'

c = 0.51;l N acetic acid

27

-26.1 -26.2'

f

-23'

28

+) n a t u r a l oxytocin

2.9

Isoelectric p o i n t

Oxytocin i s an amphoteric canpound. Accordingly, the isoelectric p i n t reported i n the l i t e r a t u r e is a t p H 7.7 (26,28, 2 9 ) , c o n s i s t e n t w i t h the presence i n the mlecule of a free amino group and a free phenol group. 3.

Production 3.1

Extraction f r a n gland material

Today the e x t r a c t i o n of cocytocin fran p o s t e r i o r p i t u i t a r y gland mterial is of l i t t l e p r a c t i c a l importance and l a r g e l y of h i s t o r i c a l i n t e r e s t . The first experiments w i t h hypphyseal extracts were carried out by Oliver and Schafer and date back t o 1895 (30). A t that tine the extracts contained t h e pressor p r i n c i p l e vasopressin as w e l l as oxytocin. Preparations having both oxytocic and pressor activity were k n m as p i t u i t r i n and wre

OXYTOCIN

577

employed i n medicine.

Kamm (31) described a method of preparing hypphyseal extracts without pressor a c t i v i t y : the gland material is dried w i t h acetone and extracted with hot 0.25% acetic acid, and a crude product i s s a l t e d out f r a n t h e concentrated extracts w i t h m n i u m sulphate. This product i s extracted w i t h acetic acid, and t h e a c t i v e material consisting of equal parts of oxytocin and vasopressin is precipitated by addition of a mixture ether/petroleum ether. The two canponents are separated by exploiting their d i f f e r e n t solubilities i n organic solvents. Ether i s added t o an acetic acid solution of the active material t o p r e c i p i t a t e the vasopressin which i s f i l t e r e d o f f . Oxytocin i s abtained a s a s o l i d substance w i t h a rubbery consistency by addition of a l i t t l e water and petroleum ether. The pressor e f f e c t of the h o m n e so obtained i s only 3 - 4% of its oxytocic a c t i v i t y . 3.2

Chenical synthesis

The f i r s t synthesis by du Vigneaud e t a l . (3,32) was followed by f u r t h e r syntheses within the next years (33-36) d i f f e r i n g i n the protective groups used, the peptide linkage methods employed and t h e plan followed i n building up t h e mlecule. The protective groups which h m e been mainly employed i n the large-scale production of oxytocin are t o s y l , carbobenzoxy and t-butyloxycarbonyl residues f o r the amino group and t h e benzyl residue f o r the mercapto group. The methods which have proved of value f o r effecting peptide linkage are the mixed anhydrides method, the active esters method using p-nitrophenol, hydroxysuccinimide o r hydroxybenztriazole i n ccmbinat i o n w i t h dicyclohexylcarbdiimide, and the a i d e method. The o p n chain N- and S-protected nonapeptide which i s s t h e precursor of oxytocin may be constructed on the 6 + 3 or the (5 + 2) + 2 plan, these intermediates being synthesised s t e p by step. In the method employing tosyl and carbobenzoxy residues, t h e last stages consist i n cleavage of t h e protective groups w i t h sodium i n liquid ammonia followed by Oxidation with a i r t o close the ring, yielding oxytocin. As an example of a 3 + 6 plan, the synthesis according t o Boissonnas (33) i s outlined i n the following scheme.

FRIEDRICH NACHTMANN cf nl.

578

H-Gln-Asnqs (BZL) -Pro-Leu-Gl y-NH2

le-N3 Z-Cys(BZL) -v-I

Tripeptide

I

I

Hexapeptide

\i

Z - C y s (BZL) -TJr-Ile-Gln-Asn-Cys

(BZL) -Pro-Leu-Gly-NH2

Nonapeptide 1. Na/NH3 O2 ( a i r )

2. 'i

OXrrCCIN Stability Freeze-dried oxytocin acetate m y be kept i n a r e f r i g e r a t o r (2 6OC) without special precautions f o r several years w i t h no s i g n i f i c a n t loss of oxytocic a c t i v i t y (37). However, R e s s l e r and popence (28) mention that inactivation nay occur by disulphi.de interchange. The shelf l i f e of aqueous solutions is g r e a t l y dependent on the pH (Figure 4.1). 4.

-

X

20

-

5

f

L

C

15

-

P

%

8

E" 8

a

0

10

-

B

5

0

3 -----€I-

4

5

6

7

a

9

lot 1 -PH

_ _ _ - _ _ %-----lot 2

Figure 4 . 1 Loss of a c t i v i t y as a function of pH of oxytocin solutions containing 200 units/ml which had been boiled f o r 30 minutes.

OXYTOCIN

579

Figure 4 . 1 shows the residual content of oxytocin, assayed by

HPLC, i n solutions a t d i f f e r e n t pH values, which had been

boiled f o r 30 minutes (38).

The apthum p H range i s 3 - 5. I n strongly acid solutions t h e peptide linkages undergo hydrolysis. Under neutral and w a k l y a l k a l i n e conditions, dimeric and polymeric compounds are formed, especially i n concentrated solutions, by conversion of t h e intrarnolecular disulphide bridges of two o r mre oxytocin m n m r s t o intermolecular bridges (disulphide interchange) ( 2 8 ) . A sterile aqueous concentrate of oxytocin a t optimum pH, containing a preservative, w i l l keep f o r several years i n a refrigerator. Figure 4.2 shows the oxytocic a c t i v i t y (rat uterus) of axytocin concentrate which had been stored a t various tanperatures.

The concentrate contained 200 u n i t s oxytocin/ml i n a sterile solution a t pH 3.5 , containing trichlorobutanol (39) . Concentrates which had been kept i n t h e r e f r i g e r a t o r showed no loss of oxytocin a c t i v i t y . Concentrates kept a t 21OC showed a s l i g h t loss of a c t i v i t y (approx. 1.5% per year) , whereas concentrates kept a t 3OoC showed a marked loss of a c t i v i t y (approx. 10% per year). This indicates that t h e concentrate has a shelf l i f e of a t l e a s t 3 years even a t 2 l o C , but should not be exposed t o higher tenperatures, Similar r e s u l t s were abtained w i t h d i l u t e injections of oxytocin (37). In l i n e with these findings, sane pharmacopoeias') specify the following shelf lives f o r oxytccin injection:

Eur. P.:

73: Ccmpendium Medicamentom: B.P.

2 years a t 25OC a t least 3 years a t 2

-

10°C

5 years a t 15OC

+) Abbreviations of t h e p h a n n a c o p i a s

as i n Martindale,

The Extra Pharmacopoeia, 27th Edition

FRIEDRICH NACHTMANN et 01

580

IlY *.*

i

t

t

100

w

80

I+

Months

0

24

re

20-22%

e4 j

Months

0

\

110

-

24

48

\

'\

\

\

\

3OoC

Figure 4.2

Shelf life of axytocin concentrates

OXYTOCIN

5.

581

Metabolism

The natural concentration of oxytocin i n human plasma i s low. According t o Chard e t a l . the level i s less than 0.75 p u n i t s / m l i n healthy men and women ( 4 0 ) , while Leake and Weitzmann published values of 1.4 - 1.7 punits/ml ( 4 1 ) . During labour t h e r e i s a marked rise i n oxytocin level: Kumaresan e t a l . found a concentration of 82 punits/ml (42) and Leake and Weitzrrann published a value of 6 punits/ml (41). The h a l f - l i f e of oxytocin i n the blood i s only a few minutes (43,44), t h e reason being t h a t it i s rapidly degraded mainly by the liver and kidneys. Tbm main e n z p s y s t m s are respnsible f o r t h e inactivation of axytocin. The removal of glycinamide fran t h e C - t e r m i n a l end of the oxytocin mlecule has been demonstrated i n a l l animal species investigated and i n man, b u t t h e removal of Leu-Gly-NH2 i s confined t o a few species (44,45,46). F u r t h e m r e , numerous other organs display peptidase activity and are able t o s p l i t t h e oxytocin molecule. &tracts of r a t brain i n a c t i v a t e oxytocin by uns p e c i f i c peptidases (47) and similarly microsanal and soluble f r a c t i o n s of uterus and pancreas (48,49) and testis (50) inactivate oxytocin. Hcmogenised testis i n a c t i v a t e s oxytocin by reduction of the disulphide bridge and cleavage of the ensueing cysteine-tyrosine peptide sequence. Plasma-oxytocinase is formed i n t h e uterus of pregnant women and released i n t o t h e plasma. This aminopeptidase hydrol y s e s t h e hemicystinyl-tyrosine peptide bond t o y i e l d an acyclic ccmpound (51). The biological sites of inactivation of oxytocin according t o P l i s k a and Rudinger (52) are Shawn i n t h e following diagram: 6 5 4 b b b Cys-Tyr-11e-Gln-Asn-Cys-Pro-LeuGly-NH2 I I

,r

la

1 a: SS-SH transhydrogenase 1 b: aminopeptidase, s p l i t s t h e m l e c u l e between t h e h m i 2 3 4

cystinyl-tyrosine residues

C I

J

6

: serum oxytocinase : tyrosinase : carboxamidopeptidase

f : endopeptidases

582

6.

FRIEDRICH NACHTMANN e t a / .

Analysis 6.1

Identity tests

6.11 General tests The mthds of detection described i n section 6.122 (thinlayer chrmatqraphy) which produce various colour reactions or fluorescence, m y be regarded as general, unspecific identif i c a t i o n tests f o r peptides. 6.12 Specific i d e n t i f i c a t i o n tests 6.121 Infrared spectrum See section 2.1 6.122 Thin-layer c h r m t c g r a p h y The Rf value and visualisation by various detection procedures i s a s p e c i f i c c r i t e r i o n of identity. A nunher of solvent systans, mainly based on butanolacetic acid-water mixtures, are enployed t o develop silica gel thin-layer plates.

The solvent s y s t m kutanol-acetic acid-water (4:l:l p a r t s by volume) i s r e c m n d e d i n t h e l i t e r a t u r e f o r use with silica gel p l a t e s (53-55). According t o Hase and Walter (54) who used Pauly reagent o r chlorine-o-toluidine reagent t o detect t h e spts oxytocin has a n Rf value of 0.45. Another butanol-acetic acid-water systan ( 4 :1:5) (upper phase) i s stated by a number of authors (56-60) t o be a s u i t a b l e mobile phase.

Khan and Sivanandaiah (58) , who used kutanol - 0 . 1 N acetic acid-pyridine (5:11:3, upper phase) and s i l i c a gel G p l a t e s , reported an Rf value of 0.68 f o r oxytocin. A solvent system canprising butanol-acetic acidpyridine-water (15:3:10:12) f o r use With silica gel G p l a t e s has also been described (57,59,60). The last-mntioned author employed t h e ninhydrin o r chlorine-o-toluidine reaction t o visualise the spots.

Flouret e t 61. (61) barked With Eastrnan Chrcmagram s i l i c a gel thin-layer sheets, developing the chromatograms with methanol-chlorofonn-acetic acid-water (38:62:2:2) and using the Pauly and/or the chlorine-o-toluidine colour reaction t o detect t h e spots (Rf = 0.4). A solvent s y s t m derived from t h e above and modified as follows may be used t o distinguish oxytocin f r m other nona-

OXYTOCIN

583

peptides such as [8-lysine]vasopressin, [ 8-ornithine]vasopressin, 2-phenylalanineI 8-lysinel vasopressin, [des- 1-amino] oxytocin ( 6 2 ) . Mobile phase: methanol-chlorofonn-acetic acid-water 30:70:1:6

Thin-layer plate: Canmrcial silica gel p l a t e s 60 F254, MERcK, D m s t a d t , Federal Republic of Germany, thickness 0.25 m Suitable methcds t o d e t e c t t h e spots are the FolinCiocalteau's reagent (MERCK), fluorescarnine (63) and t h e usual ninhydrin and chlorine-o-toluidine reactions. Samejima e t a l. (64) have developed a highly sensitive fluorescence spray reagent (phenylacetaldehyde-ninhydrin)

.

N a k a m u r a and Pisano (65) have described TIC systems f o r separating several peptides, including mytocin, derivatised with fluorescamine. The canpound i s dissolved i n h f f e r solution, s p t t e d on Merck c m r c i a l silica gel 60 p l a t e s and derivatised a t t h e start l i n e by d e v e l o p n t with o r immersion i n an acetone-hexane solution of fluorescamine. The Rf values were 0.46 with chlorofom-isopropanol-water (2: 8: 1) and 0.89 with acetone-ethyl acetate-mthanol-water (3:2:1:1) a s the m b i l e phase. 6.123

Electrophoresis

Electrophoresis was carried out as described by Miihlemann e t 61. (53) i n a mist chamber apparatus (CAM?G, Muttenz, Switzerland) using cel lulose-coated p l a t e s with a thickness of 0 . 1 m (MACHERY-NAGEL, Diiren, Federal R e p b l i c of Gemany) and applying a potential gradient of approximately 23 V/cm f o r 45-60 minutes. Fyridine-acetic acid-water 1:10:90, pH 3.6 w a s enployed as the electrolyte. The m b i l i t y of mytocin (relative t o arginine) w a s reported t o be m * 0.29. Arg. Flouret e t 61. (61) described the thin-layer electrophoresis of oxytocin on Eastman chranagram s i l i c a gel t h i n layer sheets i n a Brinkmann-Desaga apparatus (400 V, 2 hours; using 0.1 N pyridine-acetic acid h f f e r pH 5.6). 6.124

Amino acid analysis

Detection of the amino acids a f t e r hydrolysis of the peptide may be regarded as an i n d i r e c t methcd of identification. Oxytocin i s normally hydrolysed w i t h 6 N hydrochloric acid a t 115OC f o r 1 6 hours i n a sealed tube. The amino acids formed are separated by ion exchange chranatography, assayed a f t e r dericolorimetrical l y (ninhydrin) or f luorimetriv a t i s a t i o n ec a l l y (Fluram , o-phthalaldehyde) and i d e n t i f i e d by canparing t h e retention times with those of a given amount of a test

FRIEDRICH NACHTMANN ct al.

584

mixture. This method m y be used t o determine t h e amino acid ratio and the peptide content (see section 6.25). 6.125 R a t uterus roethd

- see section 6.31 Under t h e conditions described i n t h e sections on assay methods i n various pharmacopoeias, a solution of mytocin induces cont r a c t i o n s i n uterine muscle. 6.2

m a n t i t a t i v e physico-chemical mthcds 6.21

-

see section 2.2

6.22

-

Ultraviolet spectranetry F l u o r h e t r i c mthcds

see section 6.27

6.23

Colorimetric analysis

The peptide has keen assayed using t h e well-knawn colour reactions, e.g. t h e ninhydrin reaction (66) and t h e Folin-Lany reactions ( 6 7 ) . The rnethod of Ellmann (68) has been used t o determine t h e sulfhydryl content. 6.24

Determination of nitrogen (Kjehldahl)

The organic nitrogen of t h e peptide i s converted t o m n i u m sulphate by the Kjehldahl mthcd using concentrated sulphuric acid and a suitable catalyst. The solution is rendered a l k a l i n e and t h e m n i a i s steam d i s t i l l e d i n t o a receiver containing boric acid. The boric acid i s then titrated potentianetrically with hydrochloric acid and the nitrogen, o r t h e peptide content, calculated fran t h e result. 6.25

Amino acid analvsis

- see section

6.124

Amino acid analysis m y be used to determine t h e amino acid ratio as w e l l as t h e peptide content. Analysis of mytocin reveals t h e presence of aspartic acid, glutamic acid, proline, glycine, isoleucine and leucine i n equimolecular proportions. Cystine and tyrosine must a l s o be detectable, but since they undergo p a r t i a l deccmposition during hydrolysis, the quantity of these two amino acids i s only about

585

OXYTOCIN

70% of theory.

Further amino a c i d s are n o t d e t e c t a b l e i n s y n t h e t i c oxytocin. Hcwever, the presence of f o r e i g n amino acids, such as arginine, l y s i n e , and phenylalanine, m y serve as a crit e r i o n of p u r i t y for oxytocin of natural o r i g i n (see s e c t i o n 6.34). The peptide content i s c a l c u l a t e d from t h e y i e l d of i n t a c t amino acids present. 6.26

Gel f i l t r a t i o n

Various authors have described gel f i l t r a t i o n nrethds using Sephadex 6 1 5 and G-25 (54,56,57,61). This method which separates o f f dimers and p l p r s is mainly employed as a means of p u r i f i c a t i o n . 6.27

High performance l i q u i d chromatography (HPLC)

Since t h e introduction of chanically mdified silica g e l s as the s t a t i o n a r y phase, H E W o n reversed phase C8 or C18 columns has cane t o the fore as the m t h d of choice for t h e assay of oxytocin. Krummn and F r e i estimated the oxytccin content of i n j e c t i o n s , tablets and oxytocin concentrate (69). Isocratic e l u t i o n and short wavelength UV d e t e c t i o n a t 200-220 nm were adequate for the purpose. Figure 6 . 1 shows a t y p i c a l chromatogram (70) Since t h e mbile phase (acetonitrile/phosp h a t e Ixlffer) r e a d i l y transmits UV l i g h t a t 200-220 nm, the lower l i m i t of d e t e c t i o n is approximately 30 ng (aplprox. 30 pmol) per i n j e c t i o n (69). The HPLC results correlate very w e l l w i t h those chtained by bioassay. Figure 6.2 illustrates the gocd agreement between HPIC v a l u e s and t h e results obt a i n e d i n t h e rat u t e r u s test for 38 d i f f e r e n t batches of oxytocin (70). The c o r r e l a t i o n c o e f f i c i e n t s and critical values are s h a m i n T a b l e 6.1.

.

T a b l e 6.1 Correlation c o e f f i c i e n t s and critical values for t h e

r e s u l t s i n Figure 6.2, p b l i s h e d by K r m n e t al. (70). Samples Liquid Solid

Liquid + Solid

-~

+)

I EEZ~E~I

Correlation Coefficient

25

0.9962 0.9995 0.9969

Critic?) Value 0.618 0.801 0.513

For n-2 degrees of freedan a t t h e 0.1% level.

HPLC i s so highly selective t h a t it W i l l also separate canpounds closely releated i n s t r u c t u r e . Figure 6.3 shows by way of example the separation of oxytocin and 3 Stere0i-s having 1 or 2 amino acids w i t h the D-configuration (70).

i’

TR ICHLOROBUTANOL

OWOCIN

BY-PRODUCT

SOLVENT

Figure 6 . 1

olranatogran of 50 p l of oxytocin concentrate (200 units/rnl) Conditions were as follows: Column RP 18, 1 0 pn, 250 x 4 . 6 mm I D , isocratic e l u t i o n w i t h 18%a c e t o n i t r i l e i n phosphate buffer s o l u t i o n (1/15 ml) pH 7, roan tenperatwe, f l a w rate 2.0 rnl/min, pressure a t column i n l e t 150 bars, W monitor a t 210 m.

OXYTOCIN

587

RAT UTERUS I.U./ML OR I.U./MG 300

200

100

I

I

I

100

200

300

HPLC 1. U. /ML

OR

I.U./MG

Figure 6.2

Correlation between the results of r a t uterus and HPLC assays of oxytocin Correlation coefficients and critical values for the results i n Table 2.1

FRIEDRICH NACHTMANN ct al.

588

1

L

2 1

I

f

I

I

20

16

12

8

I

4

TIME (MINI Figure 6.3

Qlrcmatqram of 50 p l of a mixture of oxytccin and diastereoisomers (= 100 pg/rnl each). Conditions as f o r Figure 6.1 Larsen e t al. (71,59) separated oxytocin f r m 7 of its stereoisomers (cf. T a b l e 6.2) on a p Bondapak C18 column. Mixtures of 10% tetrahydrofuran or 18%a c e t o n i t r i l e or 16% dioxane i n acetate buffer =re used as the mbile phase. The nature of t h e organic solvent and t h e pH a f f e c t t h e separation. H E W was found t o be much superior to the classical separation on Sephadex 6 2 5 .

OXYTOCIN

589

Table 6.2 The e f f e c t of t h e solvent on the separation of oxytocin and its diastereaners by reversed-phase HPK; published by Larsen e t al. (59)

.

1

16%Dioxane LO% THF 1 8 W a stereoisaners 30%0.05M NH40Ac 82%0. O l M NH40Ac 84%0.05M NH40Ac IH 4.0 pH 4.0 pH 4.0 L.6 ml/min 2.0 rnl/min 1.5 ml/min k' c1 k' a k' a 7.73 1.00 7.31 1.00 7.70 1.00 9.12 1.18 8.33 1.14 10.4 1.42 9.12 1.18 9.66 1.25 9.43 1.29 11.1 1.44 9.28 1.27 14.6 2.00 13.1 1.69 13.3 1.72 13.7 1.77 12.0 1.64 17.7 2.29 12.7 1.67 10.7 1.46 Oxytocin m y also be separated fran other peptide hormones, e. g r8-lysinel or (8-arginine] vasopressin by reversed-phase HPIC. The separation m y be effected i s o c r a t i c a l l y (72) or by gradient e l u t i o n (73) with mixtures of aqueous buffers and a c e t o n i t r i l e , methanol, dioxane or tetrahydrofuran. The mobile phase must contain a minimum concentration of salt, since otherwise t h e separation efficiency i s low. The chranatographic separation is influenced by the nature of the organic solvent and also by the pH and salt concentration, but these e f f e c t s are smaller f o r oxytocin than f o r other similar peptides (69).

.

Nachtrnann employed an isocratic mthcd and short wavelenght W detection (74) to test t h e p r i t y of intermediates used i n t h e synthesis of corytocin. The lower l i m i t of det e c t i o n was i n the ng range b t h f o r f r e e and f o r protected peptides. The p r h r y m i n e group of t h e N-tenninal,hemicystine of oxytocin may be derivatised with FLURAMR and assayed fluorimetrically. Gruber e t al. (75) carried out the derivatisation i n phosphate buffer a t pH 7 before chranatographic separation on P a r t i s i l ODs with a l i n e a r gradient of 15 t o 50% acetone i n 0.03% m n i u m formate and 0.01% thiodiglycol. 15 pmol of the oxytocin derivative gave an e a s i l y detectable peak with a signal t o noise ratio of 15:l. This methcd w a s used by Live e t a l . t o test t h e p u r i t y of synthetic oxytocin (76,601. I t was possible i n t h i s way t o separate oxytocin frcm 16 similar peptides. Radhakrishnan e t al. separated oxytocin fran other polypeptides, such as [8-arginine]vasopressin, on P a r t i s i l SCX cation exchangers using volatile pyridine acetate buffers (77). An autanated fluorescamine column m n i t o r i n g system was used f o r detection.

FRIEDRICH NACHTMANN c’t al.

590

Postcolumn d e r i v a t i s a t i o n with FLURAMR was employed by F r e i e t al. (78,79) and K r m n e t 61. (70). The lower l i m i t of detection was 5 - 1 0 ng (5 - 1 0 p l ) per injection. The ccmpound was chranatographed on reversed-phase C 8 o r C 18. Since the sample solutions are concentrated i n t h e column, very d i l u t e solutions can s t i l l be determined with great

precision. 6.3

Biological mthods

The a c ~ v i t yof a smple of axytocin is determined by canparing it with t h e International Standard Preparation o r with a preparation which has been standardised against t h e International Standard. A t t h e present t i m e the standard preparation f o r determination of axytocin a c t i v i t y i s t h e Fourth International Standard f o r Oxytocin f o r Bioassay (80). The standard preparat i o n f o r t h e determination of vasopressor a c t i v i t y i s the F i r s t International Standard f o r Lysine Vasopressin ( 8 0 ) . Both standards (highly p r i f i e d peptides of synthetic origin) supersede t h e Third International Standard for oxytocin and Vasopressin, Bovine, f o r Bioassay, which was an acetone-dried extract of posterior lobes.

Numerous biological assays have been h o w n f o r mny years and have been t h e subject of detailed reviews (81-87). For t h e expximental procedures t h e reader i s referred to these papers and t o t h e mthcds described i n t h e various phannacopoeias. Only methods included i n t h e mst important pharmacopoeias will l x discussed here. Oxytocin as an active principle i s not described i n any of t h e pharmacopoeias. The stipulated contents as a percentage of t h e declared canposition relate t o oxytocin injections. 6.31 R a t uterus m t h d

The OOntraCtiOnS induced i n isolated rat uterus by t h e oxytocin sample are canpared with those induced by a standard preparation and evaluated as described i n t h e pharmacopoeias. Pharmacopoeia +) B.P. Eur.P.

Swiss Nord

.

+)

S t i p l a t e d potency of injections (Confidence limits, P = 0.95)

90

- 111%

(80

- 125%)

Abbreviations of the pharmacopoeias as i n Martindale, The Ektra Pharmacopoeia, 27th Edition

OXYTOCIN

59 1

6.32 Chicken b l o d pressure method The depressor e f f e c t s of t h e sample and standard on the chicken blood pressure are masured and evaluated i n accordance with d e t a i l e d instructions given i n t h e following pharmacopoeias. Pharmacopoeia +)

S t i p l a t e d potency of injections (Confidence limits, P = 0.95/L = confidence i n t e r v a l )

B.P. W.P.

90

- 111%

(80

. U.S. Jap .

85 85

-

(L K0.20) (L G0.15)

Swiss Nord

120% 120%

- 125%)

6.33 Milk e j e c t i o n assay This method i s based on measurement of the milk e j e c t i o n pressure i n a l a c t a t i n g rat and i s proposed i n t h e 1978 addendum t o the 1973 B r i t i s h Pharmacopoeia as an a l t e r n a t i v e t o the other two methods (rat uterus and chicken blood pressure).

6.34 R a t blood pressure assay This mthcd masures the pressor e f f e c t of oxytocin samples. It sets a l i m i t t o t h e content of vasopressin which may occur as a impurity i n a y t o c i n of natural origin. This test is not required when the cocytocin i s prepared by synt h e s i s and t h e absence of foreign amino acids i s demonstrated by amino acid analysis (88,891. Hcwever, synthetic cocytocin i t s e l f has a mall i n t r i n s i c pressor e f f e c t i n nmunals (88,90,91). The synthetic highly p u r i f i e d oxytocin described i n section 1.7, which was shown of peptide impurities, had a by HPLC t o contain less than 1% vasopressor a c t i v i t y amounting t o 0.9% and 1.09% of its oxyt o c i n a c t i v i t y , according t o the r e s u l t s obtained i n 4x0 independent laboratories (92). Belcw are shcwn requirements l i s t e d i n the mst important pharmacopoeias. The USP limit i s inconsistent with t h e abovementioned i n t r i n s i c vasopressor a c t i v i t y of pure oxytocin. +) Abbreviations of t h e pharmacopeias

as in Martindale,

The Extra Pharmacopoeia, 27th Edition

FRIEDRICH NACHTMANN vt al.

592

Pharmacopoeia +)

6 5% 6 2.5%

B.P.

Eur.P. Swiss Nord U.S. Jap

s 5% 6 5% s 1% < 5%

. .

6.4

S t i p l a t d ximu mum vasopressor a c t i v i t y as a percentage of oxytccic a c t i v i t y

Determination i n biological mterial

6.41 Bioassays The mst c m n l y enployed bioassays f o r oxytccin i n biol q i c a l f l u i d s are based on the c o n t r a c t i l e e f f e c t on t h e myonetrim or mnmry myoepithelium i n vitro o r i n vivo. Measurement of t h e depressor e f f e c t i n t h e chicken is less sensitive. The various m e t h d s which d i f f e r i n t h e i r s e l e c t i v i t y , sensitivity and precision have been evaluated by Munsick as shown i n T a b l e 6.3 (87). The d e t a i l s m y be found i n t h e numerous reviews (83, 87,93,94). The rat milk ejection test is t h e mst s e n s i t i v e methcd, the lower l i m i t of detection being 2.5 - 5 Vunits/rnl (87,94). Despite t h e r e l a t i v e l y high s p e c i f i c i t y of t h e bioassay m e t h a , accanpanying substances frequently i n t e r f e r e so t h a t p r i o r extraction procedures are necessary and these are usually time consuming and not p a r t i c u l a r l y reproducible. Affinity chranatography on agarose-bound neurophysin m y be a s u i t a b l e alternative (see 6.42).

of t h e pharmacopoeias as i n Martindale, The Extra Pharmacopoeia, 2 7 t h Edition

+) Abbreviations

T a b l e 6.3

A q u a l i t a t i v e evaluation of several mthcds 3 + mst) ; p b l i s h d by Munsick (87)

SpeciSpeciYcity t o ' i c i t y t o k y t o c i n txytocin

.

vs Other Jeuro-

Isolated r a t

-

uterus

0 0.5 mM Mg2+ Rabbit, guinea

pig o r rat milk-ejection, i i R a t or guinea pig milk-eject i o n , i a. Avian vasodepressor

.

'ypo)hysial 'eptides

vs. other

extraneous sub-

stances

bnsitivity to )xytocin

used t o assay oxytocin (1 + least;

Rapid creening Assay for gtocin

Expense Of

Equip m n t for 'recision ExperiExpof mntal j rience Required setup Assay I

S e t up

Time per

Assay

Prepa-

ration

. -

3+ 2+ 2+ 2+ 2+

1+ 1+ 2-3+ 2+ 2-3+

1-2+ 1-2+

3+ 3+

2+

2+

3+

1+

2+ 2+

1-2+ 1-2+

1

1-2+ 1-2+

1-2+ 1-2+

~

I

2-3+

3+

i I

2+

2+

1+

2+

3+

i Ii

3+

3+

2+

2-3+

2+

1

1+

2+

FRIEDRICH NACHTMANN et al.

594

6.42 Radioimmunoassays (RIA)

Owing to its low m l e c u l a r w i g h t a y t o c i n i s not a good antigen and i n i t i a l l y t h e production of antibodies w i t h a high t i t r e presented d i f f i c u l t i e s . Gilliland and Prout shaved t h a t antibodies could be produced by administering natural unconjugated oxytocin with Freund's adjuvant t o rabbits (95). Hmver, t h e antibody t i t r e was low. Despite t h i s handicap, a radioimmunoassay was developed using t h i s technique, b u t it was no mre sensitive than t h e best bioassay methods (96,97).

Better r e s u l t s e r e obtained w i t h oxy-tocin adsorbed on carbon microparticles (40,98) and w i t h oxytocin conjugated with bovine serum albumin (99). The latter methcd has been taken up by several authors (100,101). Rabbits are used t o produce the antibodies. Nevertheless, only i n a few laboratories has it proved possible t o prepare usable antibodies (1021, and t h i s has so f a r prevented t h e application of RIA for oxytocin on a wider scale (103). Ncw, however, oxytocin a n t i s e m i s available c m r e r c i a l l y (104). The h o m n labelling is usually accanplished by iodination w i t h f251 using t h e chloramine-T method (103) Dawood e t a1 labelled oxytocin by the lactoperoxidase method (105).

.

.

For d e t a i l s of RIA methodology, t h e reader i s referred t o t h e original papers cited and t o t h e various reviews (88, 93,103,106). Problems are also encountered i n extracting oxytocin f r a n blood serum. Qlard e t 61. have p b l i s h d a method i n which oxytocin is adsorbed fran plasma on F u l l e r ' s e a r t h and eluted with aqueous acetone ( 4 0 ) . However, recovery i s not particul a r l y good (50 - 60%) and t h e reproducibility leaves sanething t o be desired (101). Sane authors therefore m i t t h e extraction s t e p and use blood serum d i r e c t l y f o r t h e RIA (100,104). A n extract based on a f f i n i t y chranatography on agarosebound neurophysin has recently been described (107). Oxytocin can be extracted frm plasma, urine and cerebrospinal f l u i d w i t h high recovery and high s p e c i f i c i t y by this methcd.

The lower limit of detection of RIA has been variously reported as 2.0 punits/ml (104) and 0.05 punits/ml (103). 6.43 High performance liquid chranatcgraphy (HPLC) G r u b e r e t 61. (75) determined t h e oxytccin content of r a t p o s t e r i o r p i t u i t a r i e s using the methcd described i n 6.27. These authors p i n t out t h a t the methcd can be improved by using a

OXYTOCIN

595

new fluorescence reagent. HPLC may be expected t o play an important part i n future i n t h e assay of oxytocin i n biological material.

6.5

Determination i n dosage forms

Before HPIC becme available, oxytocin i n pharmaceutical dosage fonns was usually assayed by t h e chicken blood pressure method or rat uterus mthod, which were adopted by most pharnacopeias (cf 6.3). HPLC has recently been developed and is now the mthod of choice. It i s simpler as regards t h e apparatus needed, cheaper and mre rapid than t h e bioassays and it i s appreciably more accurate (70,108). It is also s u f f i c i e n t l y s p e c i f i c t o separ a t e oxytocin frun byproducts of synthesis (e.g. stereoisaners of t h e active canpound) o r related peptides (e.g. 8-lysine vasopressin) (cf. 6.27). This mthod m y also be used f o r s t a b i l i t y tests on t h e various oxytocin dosage forms (69,70).

FRIEDRICH NACHTMANN ef a1

596

References

1. du V i g n e a u d V.

, Ressler

Ch.

, Tripett

S.

, J.

B i o l . Chm.

205, 949 (1953) 2. G p y H., B l o c h e n . B i a p h y s . A c t a 11, 449 (1953) 3. du V i g n e a u d V. , R e s s l e r Ch. , Swan?.M. , R o b e r t s C.W. , Katsoyannis P.G. and Gordon S., J. Am. Chm. SOC. 75, 4879 (1953) 4. The Merck Index, 9th ed., Merck u. Co. Inc., Rahway, W, 1976, p. 904 5. U r r y D.W., Walter R., Proc. N a t . Acad. Sci. USA, 68, 956 (1971) 6. G r o s s E., Meienhofer J., The Peptides, V o l 1, A c a d . Press. Inc. , 1979, p. 5 7. C3anW.Y. and du V i g n e a u d V., E n d o c r i n o l o g y 71, 977 (1962) 8. Maxfield F.R. and Scheraga H.A., Biochanisw-16, - 4443 (1977) 9. G l i c k s o n J.D., R m a n R., P i t n e r T.P., D a d o k J., bthnerBy A.A. and Walter R., B i o c h a n i s t r y 15, 1111 (1976) 10. C e r l e t t i A. and Berde B., C1. T e r a p . 15, 313 (1958) 11. Deslauriers R., Walter R. and S m i t h I 7 . P . , Proc. N a t . A c a d . Sci. USA 71, 265 (1974) 12. Boissonnas R . A . x d H u g u e n i n R.L. , H e l v . C h h . A c t a 43, 182 (1960) 13. Photaki I., J. Am. Chm. SOC. 88, 2292 (1966) 14. Bockaert J. , Imbert PI. , Jard SFand M o r e l F. , Mol P h m a col. 8, 230 (1972) 15. B r i t i z h Pharmacopeia, L o n d o n H e r Majesty's Stationery O f f i c e 1973, p. 339 16. D i h b e m W.H. , UV and I R Spectra of Sane Important D r u g s , Editio C a n t o r A u l e n d o r f 1978 17. Beychok S. and B r e s l m E. , J. B i o l . Chm. 243, 151 (1968) 6, m 3 (1968) 18. Goodman M. and T o n i o l o C., B i o p o l y m r s 19. Tu A.T., B j a r n a s o n J.B. and H r u b y V.J., B i o c h h n . e t Biophys. A c t a 533, 530 (1978) 20. Feeney J., Roberts G.C.K., Rockey J.H. and B u r g e n A.S.V. N a t u r e New B i o l o g y 232, 108 (1971) 21. Deslauriers R., Walter R. and S m i t h I . C . P . , Biochern. B i o phys. Res. C m . 48, 854 (1972) 22. B r e w s t e r A.I.R. , W-y V. J. , Spatola A.F. and Bovey F.A. , B i o c h e m i s t r y 1 2 , 1643 (1972) 23. Lyerla J . R . J r 7 a n d Freedman M.H. , J. B i o l . Chern. 247, 8183 (1972) 24. Walter R., P r a s d K.U.M., Deslauriers R. and S m i t h I.C.P., Proc. N a t . A c a d . Sci. USA 70, 2086 (1973) 25. Hruby V.J. , Deb K.K. , Spatza A.F. , Upson D.A. and Yamamot0 D., J. Am. Chm. Soc. 101, 202 (1979) 26. du V i g n e a u d V., H a r v e y Lett-s 50, 1 (1954-55)

.

OXYTOCIN

597

27. R e s s l e r Ch. and du V i g n e a u d V., J. Am. Chem. Soc. 79, 4511 (1957) 28. R e s s l e r Ch. and popnoe E.A., Methods Invest. Diagn. E n d o c r i n o l . 1973, 2A (Pept.Horm.) , 681 29. H a n c 0. and P a d r Z., Chenie und B i o l o g i e der Hormone, VEB Gustav Fischer V e r l a g , Jena 1979, p. 221 30. O l i v e r G. and Schafer E.A., J. Physiol. 18, 277 (1895) 50, 573 (1928r 31. Kamm O., J. Am. Chem. Soc. 32. du V i g n e a u d V., R e s s l e r Ch., Swan J.M., Roberts C.W. and Katsoyannis P.G., J. Am. Chem. Soc. 76, 3115 (1954) 33. Boissonnas R.A., Guttmann St., JaqueGud P.-A. and Waller J.-P., H e l v . C h b . A c t a 38, 1491 (1955) 34. V e l l u z L., B u l l . Soc. France 1 4 m (1956) 35. B e y e r m a n n H.C., Bontekoe J.S. and Koch A.C., Rec. Trav. Chim. 78, 935 (1959) 36. BodansG M. and du V i g n e a u d V., J. Am. Chem. Soc. 81, 183, 1324 (19g) 2504 (1959), 81, 5688 (1959), N a t u r e 37. Sandoz AG B a s x , unpublished results 38. Nachtmann F., unpublished results 39. Sanabo Ges.m.b.H., W i e n , unpublished r e s u l t s 40. Chard T. , Ebyd N.R.H. , Forsling M.L. , M c N e i l 1 A.S. and Landon J., J. E n d o c r . 48, 223 (1970) 6, 41. Leake R.D. and WeitzmG R.E., C l i n i c s i n Pharmacology 65 (1979) 42. Kumaresan P., Han G.S., Anandarangam P.B. and V a s i c k a A., O b s t . Gynecol. 46, 272 (1375) 19, 189 (1959) 43. Chaudhury R.R. and Walker J.M., J. E n d o c r i n . 44. Walter R., B i c p h y s . A c t a 422, 138 (1976) 45. Walter R. and Bawman R.H., Endocrinology 92, 189 (1973) 46. Walter R. and Shlank H., Endocrinology 897990 (1971) 47. Marks N., A b r a s h L. and Walter R., P r o c y S o c . Exp. B i o l . Med. 142, 455 (1973) 48. B r z e z i n s k a - S l e b d z i n s k a E., J. E n d o c r . 63, 361 (1974) 49. B r z e z i n s k a - Slebdzinska E., Endocr. J2xp710, - 259 (1976) 50. W o j c i k K. and B r z e z i n s k a - Slebdzinska E., B u l l . Acad. pol. Sci., Ser. Sci. B i o l . 23, 577 (1975) 51. Ferrier B .M., H e n d r i e J . M . and B r a n d a L. A., Can. J. B i o chen. 52, 60 11974) 52. P l i s k a T : a n d ’ R u d i & e r J., C l i n . E n d o c r . 5 Suppl., 73s (1976) 53. Mtihlemann M., T i t o v M.I., Schwyzer R. and R u d i n g e r J., H e l v . Chim. A c t a 55, 2854 (1972) 5, 54. H a s e S. and Walterk., Int. J. P e p t i d e Protein Res. 283 (1973) 55. P e t t i t G.P., Synthetic Peptides, V o l m e 4, E l s e v i e r , Amsterdam 1976 87, 56. Manning M. and du V i g n e a u d V., J. Am. Chem. Soc. 3978 (1965)

598

FRIEDRICH NACHTMANN cl a / .

57. S p a t o l a A., Cornelius D. and Hruby V., J. Org. Chm. 391 15, 2207 (1974) 58. Khan S.A. and Sivanandaiah K.M., I n t . J. P e p t i d e P r o t e i n Res. 12, 164 (1978) 59. Larsen'B. , Fox B.L., Burke M.F. and Hruby V. , I n t . J. P e p t i d e P r o t e i n Res. 13, 1 2 (1979) 60. Live D.H. , Agosta W.CTand C m h r n D., J. Org. Chm. 42, 22, 3556 (1977) 61. Flouret G., Terada S., Kato F., Gualtieri R. and L i p k o w s k i A., I n t . J. P e p t i d e P r o t e i n Res. 13, 137 (1979) 62. SANDOZ AG, Q u a l i t y C o n t r o l , Basle Switzerlas, unpublished 63. E'ujiki H. and Zurek G., J. Chranatogr. 140, 129 (1977) 64. Samejima K. , Dairman W., S t o n e J. and U m r i e n d S. , Anal. Biochem. 42, 237 (1971) 65. N a k a m u r a H. and-bano J.J., J. Chrmatogr. 121, 33 (1976) 66. Moore S. and S t e i n W.H., J. B i o l . Chan. 1 7 6 , 3 6 7 (1948) 67. Lowry O.H. , Rosebrough N.J. , F a r r A.L. a x a n d a l l R.J. J. B i o l . Chem. 193, 265 (1951) 68. Ellman G.L., A r z Biochen. Biophys. 82, 70 (1959) 69. K r m n K. and F r e i R.W. , J. C h m a t s . 132, 429 (1977) 70. K r m n K., Max1 F. and Nachtmann F., Pharmaceutical Technology, Sept. 1979, 77, (1979) 71. Larsen B. , Viswanatha V., Chang S.Y. and Hruby V. J., J. Chranatogr. S c i . 16, 207 (1979) 72. K r m n K. and F r e i KW., J. Chrcmatogr. 132, 27 (1977) 73. O ' H a r e M.J. and Nice E.C., J. Chrmatogr.171, 209 (1979) 74. Nachtmann F., J. Chranatogr. 176, 391 ( 1 9 7 r 75. Gruber K.A., S t e i n S. , Brink L .,Radhakrishnan A.N. a n d Udenfriend S. , Proc. N a t . A c a d . S c i (USA) 73, 1314 (1976) (Goodman M. 76. Live D.H., Pept., Proc. Am. Pept. Symp., 5=., and Meienhofer J., Ed.), Wiley N.J. 1977, p. 44 77. Radhakrishnan A.N., S t e i n S., Licht A., G r u b e r K.A. and Udenfriend S., J. C h r m a t o g r . 132, 552 (1977) 78. F r e i R.W. , Michel L. and S a n t i x , J. Chmatogr. 126, 665 (1976) 79. F r e i 'R.W., Michel L. and S a n t i W. , J. Chranatogr. 142, 261 (1977) 80. WHO ECBS 30th R e p r t 1 9 7 9 , Techn. Rep. Ser. N o 638, 24 81. Sawyer W.H. i n Harris G. W. and Donovan B .T. (ed. ) , The P i t u i t a r y Gland, V o l . 3, B u t t e m r t h s , London 1966, p. 288 82. Walker J.M. i n Gray C.H. and Bachxach A.L. (ed.) , Hormones i n B l o o d , V o l . 1, Acadanic P r e s s , New York 1967, p. 145 83. Stiirmer E. i n Berde B. (ed.), Handboak of Experimental Phamacology, V o l . 23, S p r i n g e r , New York 1968, p. 130 84. F i t z p a t r i c k R.J. and B e n t l e y P.J. i n Ekrde B. (ed.), H a n d b o c k of Experimental Pharmacology, V o l . 23, S p r i n g e r , New York 1968, p. 190

OXYTOCIN

599

85. Smith M.W. i n Heller H. and Pickering B.T. (ed.) , I n t e r n a t i o n a l Encyclopaedia of Pharmacology and Therapeutics, Sect. 41, V o l . 1, P e r g m n Press, Oxford 1970, p. 173 86. Chard T. and Forsling M.L. i n Antoniades H.N. (ed.) , Hormones i n Human Blood, Harvard Univ. Press, Cambridge 1976, p. 488 87. Munsick R.A. i n JosimoVich J.B. (ed.), Uterine Contraction Side E f f e c t s of S t e r o i d a l Contraceptives (Problems of H m n Reproduction, V o l . I ) , J. Wiley, New York 1973 88. Mhme H. and Hartke K., EuropZisches Arzneibuch, Band 3 , Kcmrentar, Wissenschaftliche Verlagsgesellschaft, S t u t t g a r t , Germany (1979) 89. Calm D.H., Proc. Analyt. Div. man. SOC. 5, 130 (1976) 90. Boissonnas R.A., Guttrnann S t . , Berde B. and Konzett H., E k p r i e n t i a V o l . M I , 9, p. 377 (1961) 91. Hruby V.J., Flouret G. and du Vigneaud V., J. B i o l . Chan. 244, 1 4 , 3890 (1969) 92. 2 AG, Quality Control, Basle, Switzerland, unpublished data 93. Berde B., i n Van Cawnberge H. and F r a n c h h n t P. ( e d . ) , Assay of P r o t e i n and Polypeptide H o m n e s V o l . 33., Pergamon P r e s s , Oxford 1970, p. 128 94. F i t z p a t r i c k R.J., Methods Invest. Diagn. Endocrinol. 1973 2A (Pept. Horn.), 694 95. G i l l i l a n d P. and Prout T., Metabolism 1 4 , 912 (1965) and 14, 918 (1965) 96. zeeler M. , Kogan A. and Glick S.M., Clin. Res. 14, 479 (1966) 97. Glick S., hheeler M., Kogan A. and Kumaresan P., S u m e r i e s , I n t e r n a t i o n a l Symposium on the Pharmacology of H o m n a l Polypeptides and Proteins, Milan 1967, p. 48 98. Chard T.M., Kitau J. and Landon J., J. Endocrinol. 46, 269 (1970) 99. Glick S.M. , i n J o s h v i c h J.B. (ed.), Uterine Contraction - Side E f f e c t s of S t e r o i d a l Contraceptives (Problems of Human Reproduction, V o l . I ) , J. Wiley, N.Y. 1973 100. Bossuyt-Piron C. and Bossuyt A., I n t . J. Nucl. Ekd. B i o l . 5, 1 4 4 (1978) 101. k g h a v a n K.S. , Jogender Singh and Chabra J . K . , Indian J. Med. Res. 66, 787 (1977) 102. Piron-Bossu-yt C., Bossuyt A , , Braunmann H. and Van Den Driessche R., Annales d'Endocrinologie (Paris) 27, 389 (1976) 103. Kagan A. and Glick S.M., i n Jaffee/Bennann (ed.), M e t h c d s of Hormone Radioimmunoassay, 2nd Ed., A c a d g S i c Press 1979

13,

600

FRIEDRICH NACHTMANN ct al.

104. Gorewit R.C., Proc. Soc. exp. B i o l . (N.Y.) 160, 80 (1979) 105. Dam& M.Y., Faghavan K.S. and Pociask C. , J. Endocrinol. 76, 2 6 1 (1978) 106. E n Cauwenberge H., Legros J.J. and F r a n c h b n t P. i n Van Cauwenberge and F r a n c h h n t P. (ed.) , Assay of P r o t e i n and Polypeptide Hormones, V o l . 33, P e r g m n Press, Oxford 1970, p. 136 107. Robinson I.C.A.F. and Walker J . M . , J. Endocrinol. 80, 1 9 1 (1979) 108. Max1 F. and Krunmn K., P h m . Acta Helv. 53, 207 (1978)