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CORRECTED 
VERSION* 



per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 



Counterpart of reference /W 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 4 : 
C07J 31/00, A61K 31/575 



Al 



(11) International Publication Number: WO 88/ 01274 

(43) International Publication Date: 25 February 1988 (25.02.88) 



(21) International Application Number: PCT/AU87/0028 1 

(22) International Filing Date: 21 August 1987 (21.08.87) 

(31) Priority Application Number: PH 7614 

(32) Priority Date: 21 August 1986 (21.08.86) 

(33) Priority Country: AU 



(71) Applicant (for all designated States except US): 

BROADBENT, James, Meredyth [AU/AU]; Suite 2, 
227 Burwood Road, Hawthorn, VIC 3122 (AU). 

(71X72) Applicant and Inventor: KOSUGE, Yoshiki 
[JP/JP]; 3-4-18, Kamiashiarai, Shizuoka (JP). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): KOSUGE, Takuo 
[JP/JP1; 3-4-18, Kainiasfaiarai, Shizuoka (JP). TSUJI, 
Kuniro [JP/JP]; 2-11-17, Kamiashiarai, Shizuoka (JP). 
ISHIDA, Hitoshi [JP/JP]; 200-16, Sena, Shizuoka (JP). 



(74) Agents: SLATTERY, John, Michael et al.; Davies & 
Collison, 1 Little Collins Street, Melbourne, VIC 
3000 (AU). 



(81) Designated States: AT (European patent), AU, BE (Eu- 
ropean patent), CH (European patent), DE (Euro- 
pean patent), DK, FR (European patent), GB (Euro- 
pean patent), IT (European patent), JP, KR, LU (Eu- 
ropean patent), NL (European patent), SE (European 
patent), US. 



Published 

With international search report 



(54) Title: ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES 




(57) Abstract 

A compound of general formula (I), in substantially pure form, wherein A is a cation. A method for preparation is 
also disclosed, together with compositions and methods of use thereof. 



♦(Referred to in PCT Gazette No.05/1990, Section II) 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria. 


ES 


Spain 


MG 


Madagascar 


AD 


Australia 


FT 


Finland 


ML 


Mali 


BB 


Barbados 


FR 


France 


MR 


Mauritania 


BE 


Belgium 


GA 


Gabon 


MW 


Malawi 


BF 


Burkina Fnsso 


GB 


United Kingdom 


NL 


Netherlands 


BG 


Bulgaria 


HU 


Hungary 


NO 


Norway 


Bl 


Beam 


rr 


Italy 


RO 


Romania 


BR 


Brazil 


jp 


Japan 


SD 


Sudan 


CA 


dnada 


KP 


Democratic People's Republic 


SE 


Sweden 


CF 


Central African Republic 




of Korea 


SN 


Senegal 


CG 


Congo 


KR 


Republic of Korea 


su 


Soviet Union 


CH 


Switzerland 


U 


Liechtenstein 


TD 


Chad 


CM 


Cameroon 


LK 


Sri Lanka 


TG 


Togo 


DE 


Germany, Erdcral Republic of 


Ui 


Luxembourg 


US 


United States of America 


DfC 


Denmark 


MC 


Monaco 







per 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Internationa! Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 4 : 
C07J 31/00, A61K 31/575 



Al 



(11) International Publication Number: WO 88/ 01274 

(43) International Publication Date: 25 February 1988 (25.02.88) 



(21) International Application Number: PCT/AU87/0028I 

(22) International Filing Date: 21 August 1987 (21.08.87) 

(31) Priority Application Number: PH 7614 

(32) Priority Date: 21 August 1986 (21.08.86) 

(33) Priority Country : AU 



(71) Applicant (for all designated States except US): 

BROADBENT, James, Meredyth [AU/AU]; Suite 2, 
227 Burwood Road, Hawthorn, VIC 3122 (AU). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) : KOSUGE, Yoshiki 
[JP/JP]; KOSUGE, Takuo [JP/JP]; 3-4-18, Kamiashia- 
rai, Shizuoka (JP). TSUJI, Kuniro [JP/JP]; 2-11-17, 
Kamiashiarai, Shizuoka (JP). ISHIDA, Hitoshi [JP/ 
JP]; 200-16, Sena, Shizuoka (JP). 



(74) Agents: SLATTERY, John, Michael et al.; Davies & 
Collison, 1 Little Collins Street, Melbourne, VIC 
3000 (AU). 



(81) Designated States: AT (European patent), AU, BE (Eu- 
ropean patent), CH (European patent), DE (Euro- 
pean patent), DK, FR (European patent), GB (Euro- 
pean patent), IT (European patent), JP, KR, LU (Eu- 
ropean patent), NL (European patent), SE (European 
patent), US. 



Published 

With international search report. 



(54) Title: ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES 




(57) Abstract 

A compound of general formula (I), in substantially pure form, wherein A is a cation. A method for preparation is 
also disclosed, together with compositions and methods of use thereof. 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCT onthe frontpages of pamphlets publishing international appli- 



cations under the PCT. 










AT Austria 


FR 


France 


ML 


Mali 


ACT Australia 


GA 


Gabon 


MR 


Mauritania 


BB Barbados 


GB 


United Kingdom 


MW 


Malawi 


BE Belgium 


HU 


Hungary 


NL 


Netherlands 


B6 Bulgaria 


rr 


Italy 


NO 


Norway 


BJ Benin 


jp 


Japan 


RO 


Romania 


BR Brazil 


KP 


Democratic People's Republic 


SD 


Sudan 


CF Central African Republic 




of Korea 


SE 


Sweden 


CG Congo 


KR 


Republic of Korea 


. SN 


Senegal 


CH Switzerland 


LI 


Liechtenstein 


su 


Soviet Union 


CM Cameroon 


LK 


Sri Lanka 


TD 


Chad 


DE Germany, Federal Republic of 


LU 


Luxembourg 


TG 


Togo 


DK Denmark 


MC 


Monaco 


US 


United States of America 


H Finland 


MG 


Madagascar 







WO 88/01274 



1 



PCT/AU87/00281 



"ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES" 

This invention relates to the 
identification, isolation and preparation of an active 
principle by extraction from natural tissues, and in 
particular it relates to the identification, isolation 

5 and preparation of such an active principle by 

extraction from particular tissues of sharks. 

In Japan, a preparation known as "deep-sea 
shark liver oil" has been used as a folk remedy for a 
long time. It is an oil prepared from shark's liver 

10 and is. normally capsulated in soft capsules. The 

liver oil is said to be effective in treatment of many 
kinds of diseases, especially those which are related 
to the liver, such as hepatitis, nephritis, diabetes, 
etc. As well, when* used externally, it is widely 

15 recognised that the liver oil is effective in 

treatment of scalds, burns or other types of skin 
trouble, and also is ideal as an ingredient for 
cosmetics. 

The present inventors have been studying 
20 this material for many years, and recently have 
discovered the unexpected fact that an active 
substance exists in the aqueous component of shark ' s 
liver rather than the oil soluble component. This 
fact was recognised from a comparison of the practical 
25 use of the liver oil and a powder produced from the 



WO 88/01274 



2 



PCT/AU87/00281 



10 



15 



aqueous component of the liver by evaporation of the 
water* In comparative tests of a dosage of 9-00 mg of 
the liver oil per day and 60mg of the powder per day, 
the latter gave a better clinical result than the 
former.. Furthermore/ where the liver oil was 
thoroughly washed with water , the resulting oil showed 
almost no effect. These facts indicate that the 
active substance of deep-sea shark liver is not 
oil-soluble as previously believed, but is 
water-solub le - 

According to the present invention, there is 
provided an active principle which is isolated from an 
aqueous extract of the liver and/or gallbladder of a 
shark. 

In a first aspect of the invention/ there is 
provided a compound of the general formula I, in 
substantially pure form, 



20 



25 



OH 



i 2 oso 3 




X 



30 



35 



wherein A is a cation, such as a sodium, potassium, 
calcium or ammonium ion, or an organic amine. 

In other aspects, this invention provides a 
method for the preparation of a compound of general 
formula I in substantially pure form, together with 



compositions for pharmaceutical, dietary or cosmetic 
purposes which comprise such a compound. 

By using activity assays which are described 
in detail below, it has been shown that the active 
principle is water-soluble and does not exist in the 
oil-soluble component of shark 1 s liver. These assays 
have been used in a series of tests to ascertain 
whether the active principle exists only in the liver. 
All parts of the shark's body, such as the bones, 
meat, gallbladder, ovary, alimentary canal, etc., have 
been investigated, and it has been found that the 
gallbladder showed the same activities as liver in the 
assays. This result indicates that the active 
principle exists only in liver and gallbladder. 

In general terms, the two bioassays referred 
to herein and used to identify sources of the active 
principle and to assess the degree of purity of an 
extract, are designed to identify characteristic 
pharmacological activities of the substance. In 
particular, the bioassays, designated as (A) and (B) 
are based on the following activities: 

(A) The active principle prevents liver trouble in 
mice caused by carbon tetrachloride. 

(B) The active principle increases the respiration 
rate in mice when a toxic substances such as 
nicotine is administered. 

The present invention also provides a method 
for preparing an active principle as described above, 
which comprises the steps of preparing an aqueous 
extract of the liver and/or gallbladder of a shark, 
and isolating the active principle from the aqueous 
extract. 

The following description sets out general 
procedures for isolation of the active principle from 



WO 88/01274 



4 



PCT/AU87/00281 



the aqueous extract of the liver and/or gallbladder of 
a shark, involving the steps of extraction with polar 
organic solvents/ adsorption on suitable adsorbents 
and/or chromatography techniques. 

5 In order to determine whether the active 

principle is soluble in polar organic solvents, such 
as methanol r ethanol, acetone, etc., the powder 
obtained by freeze-drying of shark's bile was 
. extracted with polar organic solvent, then the {A) and 

10 (B) assays were applied to both the soluble part and 

the insoluble part* Activity was seen only in the 
assays on the soluble portion, thus establishing that 
active principle is soluble in polar organic solvents. 

In testing to determine whether the active 

15 principle can be isolated utilising adsorbents, many 

adsorbents were examined and it was found that the 
active principle can be adsorbed by ion exchange 
resins of basic anion exchange type, or by synthetic 
adsorbents such as XAD, HP-20, Sep-pak cl8, etc., or 

20 charcoal. This absorption test was performed by 

extracting shark r s liver and/ or gallbladder with 
water. Each adsorbent under test was added to the 
extract and left to stand overnight. The mixture was 
then filtered and each filtrate tested for activity by 

25 the (A) and (B) assays. The results indicate that the 

active substance is adsorbed by those adsorbents 
mentioned above. The active principle may be 
recovered from the adsorbent resins by extraction with 
acid, alkali or salts , and from the synthetic 

30 adsorbents and charcoal by extraction with polar 

organic solvents. 

Further purification of the active principle 
is achieved by chromatography, for example in a silica 
column, Sephadex LH-20 column, or by preparative TLC 



35 



(thin layer chromatography) or HPLC (high performance 
liquid chromatography) , etc. Each method gave 
satisfactory results, but HPLC gave the best 
purification. The active principle as isolated by 
HPLC was quite pure because it gave very sharp single 
peak and also gave a single spot of approximate 
representative Rf value of 0.36 on TLC. The active 
principle in its purified form is a white powder of 
melting point of 140 °C. 

Testing of the purified active principle by 
vanillin sulfuric acid gave a purple colour, 
indicating that it contains bile acid or bile alcohol 
in its structure. It has already been found that the 
bile of sharks contains a bile alcohol named scymnol. 
After partial acetylation of the active principle with 
acetic anhydride, followed by treatment of the crude 
product with dry dioxan-trichloroacetic acid for 
several days, scymnol was identified from the reaction 
mixture. The result indicated that the active 
principle is a scymnol derivative. It was the first 
isolation of the pure scymnol derivative contained in 
bile of shark, as the active principle. 

A preferred procedure for isolation of the 
active principle from the lyophilized bile of 
Rhizoprinodon acutus (obtained by homogenization and 
f reeze-drying of gall-bladders) , is set out in the 
following chart: 



WO 88/01274 



6 



PCT/AU87/Q0281 



Lyophilized bile of Khizoprionodon acutus 
extracted with 1. n-Hexane (100mlx3) 
2-. MeOH (100mlx3) 

Fraction I (MeOH-extract) 

1. dissolved in H 2 0 

2. Amberlite XAD-2 c.c, eluting with 

i. H 2 0 (400ml) 

ii. MeOH (400ml) 

Fraction II (MeOH-eluate) 

1 . dissolved in CHC1 3 -MeOH (1:1) 

2. Sephadex LH-20 c.c. eluted with 
■i. CKC1 3 -MeOH (1:1) (300ml) 

ii. MeOH * * (500ml) 

Fraction III 

HPLC; YMC-Pack A-324 (ODS) 

Colorless powder (compound I) 

As set out above , in this procedure the 
lyoophilized material is deflated with n-hexane, and 
then extracted with methanol. The concentrate thus 
obtained is applied to an Amberlite XAD-2 column in 

5 batches, using H 2 0, and ethanol as eluents. As the 

ethanol eluate contains the active principle (as 
determined by color reagent) , this fraction is 
successively subjected to gel filtration on Sephadex 
LH-20 with chloroform-methanol and methanol. The 

10 active principle is so effectively contained in the 

methanol eluate that its final purification is 
achieved by successive application of HPLC with a 
reverse phase column. 



WO 88/01274 PCT/AU87/00281 

7 

It has been suggested that scymnol might be 
in the form of a sulphate ester f but no positive 
information has been published about the position of 
attachment of the sulphate ester, because scymnol has 

5 six hydroxy 1 groups where the sulphate ester group 

might be attached. The present scymnol derivative has 
never been isolated as a pure substance. The active 
powder as purified by HPLC was subjected to elementary 
analyses. Results were anal: calcd for C2 7 H^^OgNS f 

10 C;57.34, H;9.02, N;2.47, S;5.66. Found C;57.23, 

H;8.92, N;2.45, S;5.30. These results suggested that 
the active compound has ammonium sulphate ester in the 
structure. Nuclear magnetic resonance spectroscopy of 
the active powder showed the following properties. 

15 1 H-NMR(in d 4 -MeOH) 6 (ppm) : 

4.22(dd, 1H, J=4.5 and lO.OKz), 

4.11(dd, 1H, J=10.0 and 16.7Hz) f 

4.00(bs, 1H) , 3.80(d, 1H, J=1.2Hz), 

3.60-3.80 (ia, 4H) , 3.30-3.45(m, 1H) , 0.72(s, 3H) . 

20 

13 C-NMR(in d 4 -MeOH) $(ppm) : 74.1(d), 72.9(d), 
71.3(d), 69.1(d), 66.7(t), 61.2(t), 48.4(d), 
47.8(d), 47.5(s), 43.1(d), 43.0(d), 41.0(d), 
40.4(t), 37.0(d), 36.5(t), 35.9(s), 35.8(t), 
25 33.3(t), 32.1(t), 31.2(t), 29.6(t), 28.8{t), 

27.9(d), 24.3(t), 23.2(q), 18.1(q), 13.1(q). 

13 

C-NMR spectrum shows that the active 



30 



compound has 27 carbon atoms made up of three methyl, 
11 methylene, 11 methine and two tertiary carbons. 
The signals at low field (0.72-2.35) in ^H-NMR 
spectrum suggest that it seems to be a coprostane 
derivative. At the higher field in 13 ONMR spectrum, 
signals at 74.1(d), 72.9(d), 71.3(d) and 69.1(d) are 



35 



8 

assignable to the methine carbon with hydroxy 1 group . 
And the two signals at 66.7 (t) and 61.2 (t) are 
ascribable to the O-substituted methylene carbon* 
2D0COSY0NMR spectra and C-H-shif t-COSY relationship 
indicate that these two carbons attach to a methine 
carbon and one of them with low chemical shift (66.7) 
has two unequivalent protons at 4.22 (dd) and 
4.14(dd)ppra in the ^H-NMR spectrum, which indicates 
that the active compound has the partial moiety of 
HOCE 2 -CH-CH 2 OR in the molecule. From the results of 
elementary analyses, R is -SO^NH^. 

From these NMR spectra and elementary 
analyses, the powder is characterised as 3a, 7a, 12a, 
24 5, 26-pentahydroxycoprostane-27-ammonium sulphate 
ester. The ammonium ion in the structure possibly 
came from the phosphate ammonium buffer used as mobile 
phase in HPLC, by replacement of a sodium ion. To 
verify this point, an active powder purified by XAD-2 
and then by column chromatography on Sepadex LH-20 was 
subjected to atomic absorption spectrophotometry for 
sodium and to elementary analysis for nitrogen. The 
results were, calcd. for C 27 E 47 0 9 SNa, Na?4.03, N;0.00, 
found Na;3.57, N;0.02. The stereochemistry of the 
C-24 position in the structure was determined as 24R 
by X-ray crystallographic analysis of scymnol and the 
specific rotation of sodium scymnol sulphate is 
positive. Accordingly, it is concluded that the 
active principle isolated from shark is 24R-(+)«3a, 
7a,12a,24,26 -pen t ahydr oxy copr ostane -2 7 - sodium sulphate 
ester. 

The sodium or ammonium ion in the sulphate 
ester is easily replaced by other metal ions such as 
potassium, calcium, etc., or by organic amine cations 



WO 88/01274 



PCT/AU87/00281 



such as amino acids , etc., by means of well known 
procedures* 

The following Tables illustrate the activity 
of the aqueous extracts of this invention: 

TABLE I 



Dosage 



Bioassay (A) Bioassay (B) 



(Units) 



(Seconds) 



Oil-soluble part of 

shares liver 500mg 

Water-soluble part 

of shark's liver 50mg 

Control 



13,800 

9,500 
13,000 



21 

15 
22 



TABLE II 



Aqueous Extract of 
Shark's Gallbladder, 
Purified by: 



nn „ fl , Bioassay (A) Bioassay (B) 
Dosage (Units) (Seconds) 



Charcoal adsorption 


5mg 






15 


XAD-2 adsorption 


lmg 






16 


Anion-exchange resin 
adsorption 


0 . 5mg 


8 


,200 


14 


Purified active principle 


0 . 15mg 


9 


,600 


15 


Control 




14 


,000 


22 



Standard bioassays referred to in the above 
description were performed as follows: 
Bioassay (A) 

Biological test for protective activity against 



carbon tetrachloride (CCl^) -induced liver lesions in 
mice. 

Male Std:ddy mice (weight 30~35g) were used in 
groups of 5 animals. Samples of test materials were 
administered orally 7 days at a suitable daily dose 
and 0,1ml of 5% CC1 4 in olive oil was. orally 
administered at 24hrs after "the last sample 
administration. Blood was obtained from the orbital 
sinus at 24hrs after the CC1 4 administration. Serum 
was obtained by centrifugation (3,000 rpnu , lOmin) and 
glutamic pyruvic transaminase (GPT) activity was 
measured, by Reitman-Frankel-Momose method. Activity 
was expressed as a comparison of GPT values between 
the sample-administered groups and controls. 

Bioassay (B) 

Effect on respiration in nicotine administration 
to mice. 

Kale Stdtddy mice (weight 20-22g) were used in 
groups. of 5 animals. Nicotine tartrate (3mg) was 
injected subcutaneously. Samples of test materials 
were orally administered 3hrs before nicotine 
administration. The time taken for 30 respirations 
was counted 5 minutes after nicotine administration. 
Activity was expressed as -a comparison of the counted 
time between the sample-administered groups and 
controls : 

The present invention also provides a 
pharmaceutical composition comprising an active 
substance as described above, together with a 
pharmaceutically acceptable carrier or diluent 
therefor. By way of example, the active substance can 
be formulated as stable tablets after being mixed as a 



WO 88/01274 



PCT/AU87/00281 



11 

powder with a known carrier or bulking agent. 
Alternatively, the active substance can be 
incorporated into a lotion or cream base for topical 
application. In yet another alternative, the active 

5 substance can for example be filled in soft gelatin 

capsules, if desired after being admixed with shark's 
liver oil. Such pharmaceutical compositions may be 
used, for example, for the protection of the liver or 
activation of liver function in the treatment of 

10 diseases or conditions affecting the liver such as 

hepatitis, nephritis, diabetes, etc.. Such 
compositions may also be used for the activation of 
regeneration of skin tissue, for example, in the 
treatment of dermatitis, trauma or acne. 

15 Clinical tests which have been performed 

using compositions containing the active substance 
have specifically demonstrated its activity in 
restoration of the liver function, and in the 
treatment of seborrhea. 

20 in a further aspect of this invention, there 

is provided a dietary or health food composition which 
comprises the active principle described herein, 
together with one or more appropriate base or carrier 
materials. Such a composition may, for example, be 

25 useful in the treatment of a hangover. 

In another aspect, the present invention 
provides a cosmetic composition comprising the active 
principle as described above, together with a cosmetic 
base material. 

30 The compositions of the present invention 

may also incorporate known pharmaceuticals or other 
active ingredients, for example, antibiotics or other 
antibacterial substances. 



35 



WO 88/01274 PCT/AU87/00281 

12 

Further details of this invention will be 
apparent from the following Examples which illustrate 
the invention without, limiting it in any way. 

5 EXAMPLE 1 - Preparation of Crude Active Principle 

280g of a mixture of liver and gallbladder 
isolated from 4kg of shark was homogenised in 300ml of 
water , and the mixture was centrifuged at 12 r 000 rpm 
for 30 minutes to obtain a clear aqueous layer. 50g 

10 of ion exchange resin of basic anion exchange type was 

added to the aqueous . layer and the mixture was left to 
stand overnight. The resin was removed by filtration 
and washed with water « The resin was then extracted 
with 200ml of 0.5% sodium chloride solution. lOOg of 

15 XAD2 was added to the extracted solution. XAD2 was 

removed by filtration and washed with water. XAD2 was 
extracted with 200ml of ethanol. From the extract, 
ethanol was removed by distillation to obtain 45mg of 
crude active powder. 



20 



25 



30 



EXAMPLE 2 — Silica gel column chromatography 

lOOg of crude active compound, obtained by 
adsorption on a XAD-2 column was subjected to 
chromatography on a. silica gel column f using MeOH- 
CHCl 3 -H 2 O(30:70:6) as solvent, to afford white powder 
(40g>. 

EXAMPLE 3 - Thin layer chromatography (TLC) 

Crude active compound was subjected to TLC on a 
precoated silica gel 60 thin layer plate (Merck) , 
using the system (parts by volume) : 
n-BuOH(85)-AcOH(10)-H 2 O(5) and MeOH(40) -CHC1 3 (60) - 
H 2 O{10)* The active principle showed as a single spot 



35 



WO 88/01274 PCT/AU87/00281 

13 

on TLC, and was visualized by spraying with vanillin 
sulfuric acid reagent. 

EXAMPLE 4 - High performance liquid chromatography 

5 (HPLC) 

Final purification of crude active powder was 
achieved by successive application of preparative HPLC 
with a reverse phase column. 31g of the active 
compound in the form of white powder , mp.l40°, was 

10 obtained from lOOg of XAD-2 purified sample. The 

approximate representative retention time of the 
active compound was 16 minute. The conditions for 
HPLC were as follows: column: YMC-Pack A-324 (ODS) ; 
flow rate: 20ml/min.; mobile phase: CH 3 CN-0.02N 

15 phosphate ammonium buff er (pH 7.45) (8:2); detector: 

refractive index. 



EXAMPLE 5 - Column chromatography on Sgphadex LH-20 
Crude active compound (lOOg) obtained by 

20 adsorption on a XAD-2 column was subjected to gel 

filtration on Sephadex LH-20 column, using 
MeOH-CHCl 3 (1:1) and then MeOH as eluents, to afford 
white powder (45g) from the MeOH fraction. 
Rechromatography on the same column afforded 30g of 

25 almost pure white powder. 



EXAMPLE 6 

Gall-bladders (65g) , obtained from 5 sharks of 
the species Rhizoprionodon acutus (ca 8Kg weight) , 
30 were homogenized and then f reeze-dried. This material 

(10.25g) was used as a source of the active principle, 
sodium scymnol sulphate. After defatting the material 
with refluxing n-hexane (100ml x3) , it was extracted 
with methanol (100ml x3) under reflux for Ih. The 



35 



WO 88/01274 



PCT/AU87/00281 



14 

concentrate (3.67gJ was dissolved in H 2; 0 (80ml), and 
applied to an Amberlite XAD-2 column (3.0 x 16.0cm). 
The column was eluted with HjO (400ml) and then with 
ethanol (400ml). Then, the ethanol eluate (1.95g) was 

5 applied to Sephadex LH-20 column (3.0 x 32.0cm), 

chloroform and methanol (1:1). After elution with 
chlproform and methanol (200ral) r the column was 
developed with methanol in batches of 50ml. 
Concentration of the methanol eluate containing the 

10 sodium salt gave a white gum (1.06g) . Purification of 

this material (120mg) by HPLC yielded 85.6mg of sodium 
scymnol sulphate as white powder. The conditions for 
HPLC were as follows: column, YMC-Pack A-324(0DS) 
10x300mm; flow rate, 2ml/min? mobile phase, 35% 

15 CH 3 CN-0.1N Sodium Phosphate Buffer (pH 6.43); 

detector/ Refractive Index, Sodium scymnol sulphate 

25 

has the following physical datat White powder; [a] D 
« 21. 75(0.50, in MeOH) ; Anal.: Calcd. for C 2? H 47 0 9 SNa 
: C;56.82 H;8.30 ' S;5.62 Na;4.03. Found: C;56.99 
20 H;8.79 S;5.62 Na;4.23. SIMS mass (m/e) : 

654 ^27 H 47? 0 9^ {C 2 H 6°>2 1 ' 574 tC 27 H 47°6 ^(C^O) % . 
IRv ml^ cnl : 3420, 2950, 1470, 1380, 1230, 1070, 980, 
9 10 ,- 810. H-NMR (in CD 3 OD) ; 6 (ppm) : 
. 4.22(lE,dd,J=4.5, 10.0Hz), 4.11 (lH,dd ,J=6.6, 10.0Hz), 

25 4.00 (IE, broad) , 3.-80 (1H, m) , 3 .80-3 .62 (3H, m) , 

3.45-3.30 (1H, m), 2 .35-2 . 15 (2H, m.) , 2.05-1.02 (23H, 
m) , 1.02(3H, d, J=6.2Hz) , 0.92(3H, s) , 0.72(3H, 
s) . 13 C-NMR (inCD 3 OD); «(ppm): 74.1(d), 72.9(d), 
71.4(d) , 69.1(d) , 66.7(t), 61.2(t), 48 .3 (d) , 47 .8 (d) , 

30 47 . 5(a), 43.1(d), 43.0(d), 41.0(d), 40.3(t) r 37.0(d), 

36.5(t), 35.9(b), 35.8(t), 33.3(t), 32.1(t), 31.2(t), 
29.5 (t), 28.8 (fc), 27.9(d), 24.3 (t), 23. 2 (q), 18.1(q), 
13.1(q). 



WO 88/01274 



15 



PCT/AU87/00281 



EXAMPLE 7 

Trials have been conducted using the active 
principle of this invention in an antiseborrheous 
lotion applied topically by 40 male and female 

5 patients affected by long established (72) years 

facial hyperseborrhea. The trials were conducted as 
double blind trials with 20 patients. applying a 
placebo and 20 patients applying the lotion containng 
the active principle. 

10 in these trials, the treatment was applied 

three times daily (morning, midday and evening) over a 
period of 20 days, and an evaluation of seborrhea 
(Seborrhea Index) made at days 0, (prior to 
treatment), 10 and 21, (at end of treatment). 

15 The results showed a significantly greater 

improvement in the seborrhea for patients using the 
lotion containing the active principle than for 
patients using the placebo. It was also observed that 
this improvement was shown in both male and female 

20 patients. 



25 



30 



35 



WO 88/01274 



PCT/AU87/00281 



16 



EXAMPLE 8 - Compositions 
1 . Cold cream 

Spenaacetti 6.0g 

Beeswax 6.0g 

Carbopol 934 10. Og 

Sodium Carbonate 4.75g 

Rose water 5* 0ml 

Rose oil . 0.02ml 

Expressed almond oil 56.0g 

Active principle 0.05g 

Distilled water 20,0g 



2 . 



Tonic 
Ethanol 

Active principle 
Flavour 

Distilled water - sufficient quantity to 

make 100ml 



30ml 
20mg 
q*s, 



WO 88/01274 



PCT/AU87/00281 



17 

CLAIMS ; 

1 . A compound of the general formula I , in 

substantially pure form, 




Wherein A is a cation ♦ 

2 . A compound according to claim 1 , wherein the 
cation is a sodium, potassium, calcium or ammonium 
ion, or an organic amine, 

3 . A method for the preparation of a compound 
of the general formula I as defined in claim 1, in 
substantially pure form, which comprises the steps of 
preparing an aqueous extract of the liver and/or 
gall-bladder of a shark, and isolating the said 
compound from said aqueous extract. 

4 . A method according to claim 3 , wherein said 
step of isolation from the aqueous extract comprises 
at least one step selected from solvent extraction, 
adsorption and chromatography. 



WO 88/01274 



PCT/AU87/00281 



18 

5. A pharmaceutical composition, comprising a 
compound of the general formula I as defined in claim 
1, together with a pharmaceutical^ acceptable carrier 
or diluent therefor/ 

6. A pharmaceutical composition according to 
claim 5, in the form of a tablet, capsule, lotion or 
cream* 

7 * Use of a compound of the general formula I 

as defined in claim 1, for the protection of the liver 
or activation of liver function in the treatment of 
diseases or conditions affecting the liver. 

8» A composition for the treatment of the skin 

comprising a compound of the general formula I as 
defined in claim 1 , together with a topically 
acceptable carrier or diluent therefor. 

9 - A composition according to claim 8, further 

comprising an antibiotic or other antibacterial 
substance. 

10 • A cosmetic composition comprising a compound 

of the general formula I as defined in claim 1, 
together with a cosmetic base material. 



11. Use of a compound of the general formula I 

as defined in claim 1, for the treatment of the skin. 



INTERNATIONAL SEARCH REPORT 

International Aoolrcation No PCT/AU 87/00281 





A<cof«in« to tnttrnational ^aunt Claiurtcatwi <I*C1 or to ootn Nation** Claiatfcation and I'C 




Int. CI. 


C07J 31/00, A61K 31/575 






M«A<mym Documentation JearcntO f 




ClaaetAcauon Sfmoo** 




IPC 

US CI. 


C07J 31/00 
260/397.2 




td tht Cit«m nut men Oo«u»»mi *n l»«M»d w »t wwn 



AU : IPC as above 



lit OOCUMtWT» COHSIOIMO TO It Mt«VANT» , 1 

A US,A, 4296109 (LAURENT et al ) 20 October 1981 (1) 
(20.10.81) 

A US.A, 3994878 (PARTRIDGE, Jr. et al) 30 November (1) 
1976 (30.11.76) 



• Special catagona* of ©ttd document! : 

-A- document dtftmntj tn* ctnt?a» ««• ol tno t« ***** 

cootidtf to 10 Ot ol 04«i«uUf itttetoet 
-r ttrtitr document Owl puOtitflt* oa o# tfitr t*o mutation** 

fifcna tatt 

■V document w*** mt? tfire» doubt* on e^oMv djimjil 
.men it eittd to oitOiito too ouohcouoy «tt of tnotno* 
esfttie* — tintf aeectaJ rtoaon tot totofiedj 

-O - document rtlttnno, to in oral diaclOOu'O. «to, tifefettion or 
otnar meant 

-f document ouMtantd »no*to tfia mttmatienol ^"0 doit Out 
tata# tnan tht enonty data cttimtd 


-T- lotot document B«ol.into iftoMM ^rSTifcSWS 

at ortoMf dttt tn« not m COnmct wit* tnt *ooi*ctt«o« 
cVttd £Vo"'*ia«« w»o o««eiolo or moory ««««rt-«9 <*• 
invention 

•x- document ol temcritr rtitvonoo: wo claimed 'fl****** 
X »o eonVdtrtd novo! o< Miwtot »o eont.«trt« to 

Mivolto to mvtnuvt «tao 
-v- eaeumoM ol sortttvlar ft»e*tnce; tne doimtd 
Y t^t.Yifao to *»Ot»o ti» mwntree ttto **tn tnt 
Si^M ft^ymSSto vl or more otne* eucn doc*,- 
tVfSTi-Vi corner. otto« 00*0.0 to . otrw* 
in int oit 

-4- tocumtnt mtmotf tt Iftt to»t oottnt 1%mib 


tV. CtWTWCATIQW - _ 

Out ot tne Actual Comoituon of t«a Inttrnttiontl Setftn 

17 November 1987 (17.11.87) 


Dttt tl Mtum« ol mit IMtwottoool Sotrcft «toon 


Inta'futtoftal Seafcnmg Autnontf 

Australian Patent Office 


m 




AwtAoruti omctr 

J - G - HANS0N 



form PCTHSAtW u.cont u«r>waor ' M5I 



ANNEX TO THE IMERNSTIQNMj SESRCH REPORT ON 
INlERNftTIQNftL APELICATICIN NO. PCT/AU 87/00281 



This Annex lists the loom "A" publication, level patent family 
nwrihers relating to the patent documents cited in the abcroe-nentioned 
International search report . The Australian Patent Office is in no way Ti*w«a 
far these particulars which are merely given foe the p ur po se of information. 



Patent Document 
Cited in Search 
Report 



Patent Fami ly Members 



US 4296109 



AU 51423/79 
EK 4048/79 
GB 2034715 
CE 2932166 



CA 1127630 
EP 10056 
JP 55051100 



DE 2843690 
ES 484706 
SU 818489 



US 3994878 



AT 


7513/76 


AT 


5464/79 


AT 


5463/79 


AT 


5465/79 


BE 


847131 


CH 


628907 


CH 


634337 


DE 


2645527 


FR 


2351998 


FR 


2407941 


GB 


1564806 


GB 


1564807 


GB 


1564808 


GB' 


1564809 


GB 


1564810 


IT 


1068692 


JP 


52046061 


NL 


7611155 


CH 


626096 


CH 


626097 


US 


4038272 



END OF ANNEX