U.S. patent application number 10/872463 was filed with the patent office on 2004-11-25 for pharmaceutical compositions having appetite suppressant activity.
This patent application is currently assigned to CSIR. Invention is credited to Horak, Roelof Marthinus, Learmonth, Robin Alec, Maharaj, Vinesh, Van Heerden, Fanie Retief, Vleggaar, Robert, Whittal, Rory Desmond.
Application Number | 20040234634 10/872463 |
Document ID | / |
Family ID | 25586362 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040234634 |
Kind Code |
A1 |
Van Heerden, Fanie Retief ;
et al. |
November 25, 2004 |
Pharmaceutical compositions having appetite suppressant
activity
Abstract
A pharmaceutical composition contains an extract obtainable from
a plant of the genus Trichocaulon or Hoodia containing an appetite
suppressant agent having the formula (1). A process for obtaining
the extract and a process for synthesizing compound (1) and its
analogues and derivatives is also provided. The invention also
extends to the use of such extracts and compound (I) and its
analogues for the manufacture of medicaments having appetite
suppressant activity. The invention further provides novel
intermediates for the synthesis of compound (1). 1
Inventors: |
Van Heerden, Fanie Retief;
(Fairland, ZA) ; Vleggaar, Robert; (Pretoria,
ZA) ; Horak, Roelof Marthinus; (Pretoria, ZA)
; Learmonth, Robin Alec; (Pretoria, ZA) ; Maharaj,
Vinesh; (Pretoria, ZA) ; Whittal, Rory Desmond;
(Pretoria, ZA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
CSIR
|
Family ID: |
25586362 |
Appl. No.: |
10/872463 |
Filed: |
June 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10872463 |
Jun 22, 2004 |
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10170750 |
Jun 14, 2002 |
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10170750 |
Jun 14, 2002 |
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10073357 |
Feb 13, 2002 |
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10073357 |
Feb 13, 2002 |
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09402962 |
Oct 13, 1999 |
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6376657 |
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09402962 |
Oct 13, 1999 |
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PCT/GB98/01100 |
Apr 15, 1998 |
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Current U.S.
Class: |
424/769 |
Current CPC
Class: |
C07J 15/00 20130101;
A61P 3/04 20180101; A61P 1/00 20180101; A61K 31/57 20130101; C07H
13/08 20130101; C07J 41/00 20130101; A61K 36/27 20130101; C07J
7/002 20130101; A61P 3/00 20180101; C07H 3/06 20130101; C07H 5/10
20130101; C07J 7/007 20130101; C07H 3/04 20130101; C07J 17/00
20130101; A61K 31/58 20130101; C07J 17/005 20130101; C07J 7/0025
20130101; A61K 31/704 20130101; C07J 7/0035 20130101; A61K 31/78
20130101; Y02P 20/582 20151101; A23L 33/105 20160801; A61K 36/27
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/769 |
International
Class: |
A61K 035/78 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 1997 |
ZA |
97/3201 |
Claims
1-121. Canceled
122. A method of suppressing appetite in a human or animal, said
method comprising administering to said human or said animal a
suitable dosage of an extract obtained from a plant of the genus
Trichocaulon or the genus Hoodia, wherein said extract contains an
effective amount of an appetite suppressant steroidal glycoside
from said plant and is optionally admixed with an excipient,
diluent or carrier.
123. A method of claim 122, wherein said extract is a free-flowing
powder.
124. A method of claim 123, wherein said powder is a spray-dried
extract of said plant.
125. A method of claim 123, wherein said powder is a freeze-dried
extract of said plant.
126. A method of claim 123, wherein said powder is a vacuum-dried
extract of said plant.
127. The method of claim 122, wherein said plant is of the species
Hoodia currorii, Hoodia gordonii or Hoodia lugardii.
128. The method of claim 122, wherein said suitable dosage is a
unit dosage.
129. The method of claim 127, wherein said suitable dosage is a
unit dosage.
130. A method of treating or combating obesity in a human or
animal, said method comprising administering to said human or said
animal a suitable dosage of an extract obtained from a plant of the
genus Trichocaulon or the genus Hoodia, wherein said extract
contains an effective amount of an appetite suppressant steroidal
glycoside from said plant and is optionally admixed with an
excipient, diluent or carrier.
131. The method of claim 130, wherein extract is a free-flowing
powder.
132. The method of claim 131, wherein said powder is a spray-dried
extract of said plant.
133. The method of claim 131, wherein said powder is a freeze-dried
extract of said plant.
134. The method of claim 131, wherein said powder is a vacuum-dried
extract of said plant.
135. A method of any one of claims 130, wherein said plant is of
the species Hoodia currorii, Hoodia gordonii or Hoodia
lugardii.
136. A method of any one of claims 130, wherein said suitable
dosage is a unit dosage.
137. The method of claim 135, wherein said suitable dosage is a
unit dosage.
Description
[0001] THIS INVENTION relates to steroidal glycosides, to
compositions containing such steroidal glycosides and to a new use
for these steroidal glycosides and the compositions containing
them. The invention further relates to a method of extracting and
isolating these steroidal glycosides from plant material, to a
method of synthetically producing these steroidal glycosides, and
to the products of such an extraction and such a synthesis
process.
[0002] In a particular application, the invention relates to an
appetite suppressant agent, to a process for synthetically
producing the appetite suppressant agent, to a process for
extracting the appetite suppressant agent from plant material, to
an appetite suppressant composition containing the appetite
suppressant agent, and to a method of suppressing an appetite.
[0003] According to the invention, there is provided a process for
preparing an extract of a plant of the genus Trichocaulon or of the
genus Hoodia, the extract comprising an appetite suppressant agent,
the process including the steps of treating collected plant
material with a solvent to extract a fraction having appetite
suppressant activity, separating the extraction solution from the
rest of the plant material, removing the solvent from the
extraction solution and recovering the extract. The extract so
recovered may be further purified, eg by way of suitable solvent
extraction procedures.
[0004] The invention also provides a plant extract made of plants
of the group comprising the genus Trichocaulon and the genus Hoodia
and having appetite suppressant activity.
[0005] The extract may be prepared from plant material such as the
stems and roots of said plants of the genus Trichocaulon or of the
genus Hoodia. The genus Trichocaulon and the genus Hoodia include
succulent plants growing in arid regions such as are found in
Southern Africa. In one application of the invention, the active
appetite suppressant extract is obtained from the species
Trichocaulon piliferum. The species Trichocaulon officinale may
also be used to provide an active appetite suppressant extract. In
another application of the invention, the active appetite
suppressant extract may be obtained from the species Hoodia
currorii, Hoodia gordonii or Hoodia lugardii. Bioassays conducted
by the Applicant on rats have indicated that certain of the
extracts possess appetite suppressant activity.
[0006] The plant material may be homogenised in the presence of a
suitable solvent, for example, a methanol/methylene chloride
solvent, by means of a device such as a Waring blender. The
extraction solution may then be separated from the residual plant
material by an appropriate separation procedure such as, for
example, filtration or centrifugation. The solvent may be removed
by means of the rotary evaporator, preferably in a water bath at a
temperature of 60.degree. C. The separated crude extract may then
be further extracted with methylene chloride and water before being
separated into a methylene chloride extract and a water extract.
The methylene chloride extract may have the solvent removed
preferably by means of evaporation on a rotary evaporator and the
resultant extract may be further purified by way of a
methanol/hexane extraction. The methanol/hexane extraction product
may then be separated to yield a methanol extract and a hexane
extract. The methanol extract may be evaporated to remove the
solvent in order to yield a partially purified active extract.
[0007] The partially purified active extract may be dissolved in
methanol, and may be further fractionated by column chromatography,
employing silica gel as an adsorption medium and a chloroform/30%
methanol mixture as an eluent.
[0008] A plurality of different fractions may be obtained, and each
may be evaluated, by suitable bioassaying procedures, to determine
the appetite suppressant activity thereof.
[0009] A fraction having appetite suppressant activity may
preferably be further fractionated such as by column chromatography
using silica gel as an adsorption medium and a 9:1
chloroform:methanol solvent, and the resultant sub-fractions
bioassayed for their appetite suppressant activity. A sub-fraction
displaying appetite suppressant activity may, if desired, be
further fractionated and purified, conveniently using a column
chromatographic procedure with silica gel as the adsorption medium
and a 9:1 ethylacetate:hexane solvent. The resultant purified
fractions may again be evaluated by suitable bioassay procedures
for their appetite suppressant activity.
[0010] The Applicant has found that at least one such purified
fraction has good appetite suppressant activity, and the active
principle in the fraction was identified by conventional chemical
techniques including nuclear magnetic resonance, and was found to
be a compound of the structural formula 2
[0011] In accordance with S.I. nomenclature, the active principle
(1) is the compound
3-O-[-.beta.-D-thevetopyranosyl-(1.fwdarw.4)-.beta.-D-cymaro-
pyranosyl-(1.fwdarw.4)-.beta.-D-cymaropyranosyl]-12.beta.-O-tigloyloxy-14--
hydroxy-14.beta.-pregn-50-en-20-one
(C.sub.47H.sub.74O.sub.15M.sup.+878)
[0012] According to another aspect of the invention, there is
provided a process for preparing an extract of a plant of the genus
Trichocaulon or of the genus Hoodia, the extract comprising an
appetite suppressant agent, the process including the steps of
pressing collected plant material to separate sap from solid plant
material and recovering the sap free of the solid plant material to
form the extract.
[0013] The extract may be dried to remove moisture, e.g. by
spray-drying, freeze-drying or vacuum drying, to form a
free-flowing powder.
[0014] The invention extends to a composition having appetite
suppressant activity comprising an extract as described above.
[0015] The composition may be admixed with a pharmaceutical
excipient, diluent or carrier and optionally it is prepared in unit
dosage form.
[0016] The invention also extends to the use of an extract as
described above in the manufacture of a medicament having appetite
suppressant activity, to an extract as described above for use as a
medicament having appetite suppressant activity, and to a method of
suppressing an appetite by administering to a human or animal an
effective dosage of a composition as described above.
[0017] Compound (1) is a novel compound and the invention extends
to compound (1) and certain analogues or derivatives of this
steroidal trisaccharide having appetite suppressant properties. The
molecules chosen as the analogues or derivatives are intended to
affect the properties of the steroidal trisaccharide with the aim
of increasing the activity of the active ingredient. The following
effects were taken into consideration when the analogues were
chosen:
[0018] (i) Hydrophobic Interactions and Lipophilicity
[0019] Functional group modifications of the active molecule is
intended to change the hydrophobicity and lipophilicity of the
molecule. Increased lipophilicity has been shown to correlate with
increased biological activity, poorer aqueous solubility, increased
detergency/cell lysis, increased storage in tissues, more rapid
metabolism and elimination, increased plasma protein binding and
faster rate of onset of action.
[0020] (ii) Electronic Properties and Ionization Constants
[0021] Functional group modification of the molecule is also
intended to change the acidity and basicity which would have a
major role in controlling the transport of the compound to its site
of action and the binding at this target site.
[0022] (iii) Hydrogen bonding
[0023] Functional group modifications of carboxyl and carbonyl
groups in the active molecule are intended to change the
interactions between the proteins in biological systems and the
chemically modified functional groups.
[0024] (iv) Steric Parameters
[0025] The purpose of changing the steric features of the molecule
is to increase binding to its receptor and thus increase its
biological activity.
[0026] The following chemical modifications to the molecule are
intended to affect the hydrophobicity and lipophilicity electronic
properties, hydrogen bonding and steric parameters on the
molecule:
[0027] a) Chemical modification of the C-12 group and ester
functionality;
[0028] b) Chemical modification of the 5,6-double bond, e.g.
hydrogenation and migration;
[0029] c) Chemical modification of the C-20 carbonyl and C-17
acetyl group;
[0030] d) Chemical modification of the "D" ring of the steroid or
aglycone ring;
[0031] e) Modification of the carbohydrates of the trisaccharide
moiety.
[0032] Accordingly, the invention provides a compound having the
general structural formula 3
[0033] in which R alkyl;
[0034] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, or any other organic
ester group;
[0035] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof; and in which the broken lines indicate the
optional presence of a further bond between C4-C5 or C5--C6.
[0036] The invention also provides a compound as described above
wherein there is a further bond between C.sub.5-C.sub.6, R=methyl,
R.sub.1=tigloyl,
R.sub.2=3-O-[-.beta.-D-thevetopyranosyl-(1.fwdarw.4)-.be-
ta.-D-cymaropyranosyl-(1.fwdarw.4)-.beta.-D-cymaropyranosyl] and
having the structural formula. 4
[0037] Further active analogues or derivatives of the appetite
suppressant compound (1) in accordance with the invention are
compounds having the following structural formulae: 5
[0038] in which R alkyl; and
[0039] R.sub.1.dbd.H, or benzoyl, or tigloyl, or any other organic
ester group 6
[0040] in which R=alkyl; and
[0041] R.sub.1.dbd.H, or tigloyl, or benzoyl, or any other organic
ester group 7
[0042] in which R=alkyl; and
[0043] R.sub.1.dbd.H, or tigloyl, or benzoyl, or any other organic
ester group 8
[0044] in which R alkyl; and
[0045] R.sub.1.dbd.H, or tigloyl, or benzoyl, or any other organic
ester group 9
[0046] in which R alkyl;
[0047] R.sub.1.dbd.H, or tigloyl, or benzoyl, or any other organic
ester group. 10
[0048] in which R alkyl; and
[0049] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, or any other organic
ester group;
[0050] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof; and in which the broken lines indicate the
optional presence of a further bond between C4-C5 or C5-C6. 11
[0051] in which R=alkyl; and
[0052] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, or any other organic
ester group;
[0053] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof; and in which the broken lines indicate the
presence of a further bond between C4-C5 or C5-C6. 12
[0054] in which R alkyl; and
[0055] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, or any other organic
ester group;
[0056] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof; and in which the broken lines indicate the
optional presence of a further bond between C4-C5 or C5-C6. 13
[0057] in which R=alkyl; and
[0058] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, or any other organic
ester group;
[0059] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof; and in which the broken lines indicate the
optional presence of a further bond between C.sub.4-C.sub.5, C5-C6
or C14-C15. 14
[0060] in which R=alkyl; and
[0061] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, any other organic
ester group;
[0062] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof; and in which the broken lines indicate the
optional presence of a further bond between C4-C5, C5-C6 or
C14-C15. 15
[0063] in which R alkyl; and
[0064] R.sub.1.dbd.H, alkyl, tigloyl, benzoyl, any other organic
ester group;
[0065] R.sub.2.dbd.H, or one or more 6-deoxy carbohydrates, or one
or more 2,6-dideoxy carbohydrates, or glucose molecules, or
combinations thereof;
[0066] and in which the broken lines indicate the optional presence
of a further bond between C4-C5, C5-C6 or C14-C15; and
[0067] R.sub.3.dbd.H, alkyl, aryl, acyl, or glucoxy. 16
[0068] in which R.dbd.H, alkyl, aryl or any steroid possessing a
C14 beta hydroxy group, or a C12 beta hydroxy functionality, or a
C17 acyl group, or a C5-C6 olefin, or combinations thereof.
[0069] The invention still further extends to a process for
synthetically producing a compound having appetite suppressant
activity.
[0070] The process uses a steroid as a starting material (or
intermediate or precursor), the steroid having the chemical formula
17
[0071] The steroid (15) can be prepared from a compound having the
formula (22) by a process which includes the steps of
[0072] (i) treating progesterone having the formula 18
[0073] with the micro-organism Calonectria decora to produce a
compound 12.beta., 15.alpha.-dihydroxy progesterone of the formula
19
[0074] (ii) treating compound (17) with tosyl chloride and pyridine
to produce a compound 12.beta.-hydroxy-15.alpha.-(p-toluene
sulfonyl)-progesterone of the formula 20
[0075] (iii) treating the compound (18) with collidine at
150.degree. C. to produce a compound
129-hydroxy-.DELTA..sup.14-progesterone of the formula 21
[0076] (iv) treating the compound (19) with acetyl chloride and
acetic anhydride at 120.degree. C., to produce a compound 3,
12.beta.-diacetoxypregna-3,5,14-trien-20-one of the formula 22
[0077] (v) treating the compound (20) with ethylene glycol and a
catalytic amount of p-toluene sulphonic acid, to produce a compound
3, 12.beta.-diacetoxy-20,20-ethylenedioxypregna-3,5,14-triene of
the formula 23
[0078] (vi) treating the compound (21) with NaBH.sub.4 to produce a
compound 3.beta.,
12.beta.-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-
-12-acetate of the formula 24
[0079] In a first alternative procedure, a process for the
preparation of steroid (15) according to the invention includes the
steps of
[0080] (a) treating compound (22) with a reducing agent, e.g.
LiAlH.sub.4, to produce a compound 3.beta.,
12.beta.-dihydroxy-20,20-ethylenedioxypreg- na-5,14-diene of the
formula 25
[0081] (b) treating compound (23) with N-bromoacetamide (NBA) and a
base, e.g. pyridine, to produce a compound 3.beta.,
12.beta.-dihydroxy-14,15-ep- oxy-20,20-ethylenedioxypregn-5-ene of
the formula 26
[0082] (c) treating compound (24) with a reducing agent, e.g.
LiAlH.sub.4/e.g. with refluxing, to produce a compound 3.beta.,
12.beta., 14.beta.-trihydroxy-20,20-ethylenedioxypregn-5-ene of the
formula 27
[0083] and (d) treating compound (25) with an acid, e.g. acetic
acid, and water to produce the steroid intermediate compound
3.beta., 12.beta., 14.beta.-trihydroxy-pregn-5-ene (15).
[0084] Reaction Scheme A depicts the procedure for the preparation
of steroid intermediate (15) from compound (22) according to "the
first alternative procedure" of the invention (and includes the
preparation of compound (22) from compound (16) for illustrative
purposes). 2829
[0085] In a second alternative procedure, a process for the
preparation of steroid (15) according to the invention includes the
steps of
[0086] (a) treating compound (22) (3.beta.,
12.beta.-dihydroxy-20,20-ethyl-
enedioxypregna-5,14-diene-12-acetate) with p-toluenesulfonyl
chloride and a base, e.g. pyridine, to produce a compound 3.beta.,
12.beta.-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-3-tosyl-12-acetat-
e of the formula 30
[0087] (b) treating compound (26) with potassium acetate in a
solvent, e.g. acetone, to produce a compound 6.beta.,
12.beta.-dihydroxy-20,20-eth-
ylenedioxy-3,5.alpha.-cyclopregnan-14-ene-12-acetate of the formula
31
[0088] (c) treating the compound (27) with a reducing agent, e.g.
LiAlH.sub.4, and e.g. tetrahydrofuran, to produce a compound
6.beta.,
12.beta.-dihydroxy-20,20-ethylenedioxy-3,5.alpha.-cyclopregnan-14-ene
of the formula 32
[0089] (d) treating the compound (28) with N-bromoacetamide,
optionally acetic acid, and a base, e.g. pyridine, to produce a
compound 6.beta.,
12.beta.-dihydroxy-20,20-ethylenedioxy-14,15-epoxy-3,5.alpha.-cyclopregna-
ne of the formula 33
[0090] (e) treating the compound (29) with a reducing agent, e.g.
LiAlH.sub.4, and e.g. tetrahydrofuran, to produce a compound
6.beta., 12.beta.,
14.beta.-trihydroxy-20,20-ethylenedioxy-3,5.alpha.-cyclopregnan- e
of the formula 34
[0091] and (f) treating compound (30) with an acid, e.g.
hydrochloric acid, and a solvent e.g. acetone, to produce compound
(15).
[0092] Reaction Scheme B shows the procedure for the preparation of
steroid intermediate (15) from compound (22) according to "the
second alternative procedure" of the invention. 35
[0093] Compound (1) may be synthesized from a first carbohydrate
intermediate in the form of an activated monosaccharide cymarose
moiety, which can be prepared from a compound having the formula
(36). Compound (36) can be prepared by a process which includes the
steps
[0094] (i) treating methyl-.alpha.-D-glucose having the formula
36
[0095] with benzaldehyde and zinc chloride to produce a compound
methyl-4,6-O-benzylidene-.alpha.-D-glucopyranoside of the formula
37
[0096] (ii) treating the compound (32) with tosyl chloride and
pyridine at 0.degree. C., to produce a compound
methyl-4,6-O-benzylidene-2-O-tosyl-.a- lpha.-D-glucopyranoside of
the formula 38
[0097] (iii) treating the compound (33) with NaOMe at 100.degree.
C. to produce a compound methyl
4,6-O-benzylidene-3-O-methyl-.alpha.-D-altropyr- anoside of the
formula 39
[0098] (iv) treating the compound (34) with N-bromosuccinamide
(NBS) to produce a compound methyl
6-bromo-4-O-benzoyl-3-O-methyl-6-deoxy-.alpha.-- D-altropyranoside
of the formula 40
[0099] and (v) treating the compound (35) with NaBH.sub.4 and
NiCl.sub.2, to produce a compound methyl
4-O-benzoyl-3-O-methyl-6-deoxy-.alpha.-D-alt- ropyranoside of the
formula 41
[0100] The invention extends to a process for the preparation of a
carbohydrate intermediate in the form of an activated
monosaccharide cymarose moiety which includes the steps of
[0101] (i) treating the compound (36) with PhSSiMe.sub.3, ZnI.sub.2
and Bu4.sup.+I.sup.- to produce a compound
4-O-benzoyl-3-O-methyl-6-deoxy-.al-
pha..beta.-D-phenylthioaltroside of the formula 42
[0102] (ii) optionally treating the compound (37) with
diethylaminosulphur trifluoride (DAST), e.g. at 0.degree. C., to
produce a compound
4-O-benzoyl-3-O-methyl-2-phenylthio-2,6-dideoxy-.alpha.-D-fluorocymaropyr-
anoside having the formula 43
[0103] or (iii) optionally, treating the compound (37) with
t-butyldimethylsilylchloride and imidazole in a solvent, e.g.
pyridine, to produce
4-O-benzoyl-3-0-methyl-2-O-t-butyldimethylsilyl-.alpha..beta.--
D-phenylthioaltroside having the formula 44
[0104] in which Z=TBDMS=t-butyldimethylsilyl
[0105] and (iv) treating the compound (39) with a base, e.g. sodium
methoxide, to produce
3-O-methyl-2-O-t-butyldimethylsilyl-.alpha..beta.-D-
-phenylthioaltroside having the formula 45
[0106] in which Z=TBDMS=t-butyldimethylsilyl.
[0107] Reaction Scheme C shows the procedure for the synthesis of
the activated monosaccharide cymarose moiety (40) from compound
(36) according to the invention (and includes the is preparation of
compound (36) from compound (31) for illustrative purposes). 46
[0108] The synthesis of compound (1) may also involve a second
carbohydrate intermediate in the form of an activated
monosaccharide thevetose moiety, which can be prepared from a
compound having the formula (47). Compound (47) can be prepared by
a process which includes the steps of
[0109] (i) treating .alpha.-D-glucose having the formula 47
[0110] with acetone and sulphuric acid to produce a compound
1,2:5,6-di-O-isopropylidene-.alpha.-D-glucofuranose of the formula
48
[0111] (ii) treating the compound (42) with NaH and MeI to produce
a compound
1,2:5,6-Di-O-isopropylidene-3-O-methyl-.alpha.-D-glucofuranose of
the formula 49
[0112] (iii) treating the compound (43) with acetic acid to produce
a compound 3-O-methyl-.alpha..beta.-D-glucopyranose of the formula
50
[0113] (iv) treating the compound (44) with methanol and
hydrochloric acid to produce a compound methyl
3-O-methyl-.alpha..beta.-D-glucopyranoside having the formula
51
[0114] (v) treating the compound (45) with benzaldehyde and zinc
chloride to produce a compound methyl
4,6-O-benzylidene-3-O-methyl-.alpha.-glucopy- ranoside having the
formula 52
[0115] (vi) treating the compound (46) with N-bromosuccinamide,
nickel chloride and sodium borohydride to produce a compound methyl
4-O-benzoyl-3-O-methyl-6-deoxy-.alpha..beta.-glucopyranoside having
the formula 53
[0116] The invention extends to a process for the preparation of an
activated monosaccharide thevetose moiety which includes the steps
of
[0117] (i) treating the compound (47) with
phenylthiotrimethylsilane and
trimethylsilyltrifluoromethanesulphonate to produce is a compound
4-O-benzoyl-3-O-methyl-1-phenylthio-6-deoxy-.alpha..beta.-glucopyranoside
having the formula 54
[0118] (ii) treating the compound (48) with pivaloyl chloride and a
solvent, e.g. pyridine, to produce a compound
4-O-benzoyl-3-O-methyl-2-O--
pivaloyl-1-phenylthio-6-deoxy-aglucopyranoside having the formula
55
[0119] and (iii) treating the compound (49) with a brominating
agent, e.g. N-bromosuccinimide, and diethylaminosulphur trifluoride
to produce a compound
4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-.beta.-gluc-
opyranoside occurring as stereo-isomers having the formula 56
[0120] Reaction Scheme D shows the procedure for the synthesis of
the activated monosaccharide thevetose moiety (50 (A) and 50(B))
from compound (48) according to the invention (and includes the
preparation of compound (47) from compound (41) for illustrative
purposes). 57
[0121] According to a still further aspect of the invention there
is provided a process of synthetically producing a compound of the
formula (1) and analogues and derivatives thereof which includes
the steps of synthesising a suitable steroid intermediate or
precursor and coupling the required number of suitable
monosaccharides with the steroid intermediate.
[0122] The invention also provides a process of coupling a
monosaccharide cymarose with the steroid intermediate, which
includes the steps of
[0123] (i) reacting a cymarose moiety (38) with a steroid
intermediate (15), e.g. at -15.degree. C., and in the presence of
tin chloride, in a solvent, e.g. ether, to produce a compound
3-O-[4-O-benzoyl-2-phenylthio--
g-D-cymaropyranosyl]-12,14-.beta.-dihydroxy-pregn-5-ene-20-one of
the formula 58
[0124] and (ii) treating the compound (51) with tiglic acid
chloride in pyridine and thereafter with a base, e.g. NaOMe, to
produce a compound
3-O-[-2-phenylthio-.beta.-D-cymaropyranosyl]-12.beta.-tigloyloxy-14-hydro-
xy-14.beta.-pregn-5-ene-20-one of the formula 59
[0125] The invention extends to a process which includes coupling a
monosaccharide cymarose moiety to a monosaccharide thevetose moiety
and coupling the resultant disaccharide with the combined steroid
product (52) to form compound (1).
[0126] The process of coupling the monosaccharide cymarose moiety
to the monosaccharide thevetose moiety and coupling the resultant
disaccharide to the combined steroid product (52) may include the
steps of
[0127] (i) coupling a selectively protected cymarose moiety (40)
and a selectively protected thevetose moiety (50 A) using tin
chloride (SnCl.sub.2) and silver trifluoromethanesulphonate, e.g.
at -15.degree. C., to produce a compound of the formula 60
[0128] in which Z=TBDMS=t-butyldimethylsilyl
[0129] (ii) treating compound (53) with tetrabutylammoniumfluoride
to produce a compound of the formula 61
[0130] (iii) treating compound (54) with diethylaminosulphur
trifluoride, e.g. at 0.degree. C., to produce a compound of the
formula 62
[0131] (iv) reacting compound (55) with compound (52) to produce a
compound of the formula 63
[0132] and (v) treating compound (56) in a Raney-Nickel reaction
and thereafter with a base, e.g. NaOMe, to produce compound (1) as
described above.
[0133] Reaction Scheme E shows the procedure for the synthesis of
intermediates (52) and (55) and coupling them to form compound
(56). 6465
[0134] According to the invention, an alternative process is
provided which includes coupling cymarose and thevetose moieties to
form a trisaccharide and coupling the trisaccharide onto a steroid
derivative to form a compound of the formula (1).
[0135] The process of forming the trisaccharide and coupling the
resultant trisaccharide to a steroid derivative may include the
steps of (i) coupling a selectively protected cymarose moiety (40)
and compound (45) using tin (II) chloride, AgOTf,
Cp.sub.2ZrCl.sub.2 to produce a compound of the formula 66
[0136] in which Z=TBDMS=t-butyldimethylsilyl
[0137] (ii) treating compound (57) with tetrabutylammoniumfluoride
and diethylaminosulphur trifluoride to produce a trisaccharide
compound having the formula 67
[0138] and (iii) coupling the trisaccharide (58) with a steroid
intermediate of the formula 68
[0139] using tin (II) chloride, AgOTf, Cp.sub.2ZrCl.sub.2 to
produce compound (1).
[0140] The steroid intermediate (59) may be produced by treating
steroid (15) with tiglic acid chloride.
[0141] Reaction Scheme F shows the procedure for the synthesis of
the trisaccharide (58) and the synthesis of compound (1) by
coupling the trisaccharide (58) with the steroid intermediate (59).
69
[0142] The intermediates (23), (24), (25), (27), (28), (29), (30),
(37), (38), (39), (40), (48), (49), (50), (51), (53), (54), (55),
(56), (57) and (58) described above are novel compounds and the
invention extends to these compounds as such.
[0143] Compound (1),
3-O-[-.beta.-D-thevetopyranosyl-(1.fwdarw.4)-.beta.-D-
-cymaropyranosyl-(1.fwdarw.4)-.beta.-D-cymaropyranosyl]-12.beta.-O-tigloyl-
oxy-14-hydroxy-14.beta.-pregn-5-en-20-one, and various analogues
and derivatives thereof have been found to have appetite
suppressing activity.
[0144] The invention extends also to a composition or formulation
having appetite suppressant activity, in which the active
ingredient is an extract obtained from a plant of the genus
Trichocaulon or the genus Hoodia.
[0145] The active ingredient may be a compound of the formula (1),
extracted from a plant of: the genus Trichocaulon or Hoodia or a
derivative thereof. The plant may be of the species Trichocaulon
officinale or Trichocaulon piliferum, or the species Hoodia
currorii, Hoodia gordonii or Hoodia lugardii.
[0146] The invention extends also to a composition or formulation
having appetite suppressant activity, in which the active
ingredient is a synthetically produced compound of the formula (1)
or a derivative or analogue thereof, as hereinbefore set out with
reference to compounds (2) to (14).
[0147] According to another aspect of the invention there is
provided a method of suppressing an appetite by administering to a
human or animal a suitable dosage of an appetite suppressant agent
comprising an extract of a plant of the genus Trichocaulon or
Hoodia. The extract may be incorporated in a composition or
formulation including also pharmaceutically acceptable other
ingredients.
[0148] The appetite suppressant agent may be an isolated natural
chemical or a synthetic chemical compound of the formula: 70
[0149] or derivatives or analogues thereof, as set out before.
[0150] The appetite suppressant composition or formulation may
consist of the appetite suppressant agent admixed with a
pharmaceutical excipient, diluent or carrier. Other suitable
additives, including a stabilizer and such other ingredients as may
be desired may be added.
[0151] The invention extends to the use of compound (1) or its
derivatives or analogues in the manufacture of a medicament having
appetite suppressant activity.
[0152] The invention further extends to compound (1), or its
derivatives or analogues as set out before, for use as a medicament
having appetite suppressant activity.
[0153] A method of suppressing an appetite by administering to a
human or animal an effective dosage of a composition as described
above is also provided.
[0154] A method has been described herein for extracting a
steroidal glycoside having appetite suppressant activity from plant
material obtained from a plant of the Trichocaulon or Hoodia genus.
The invention thus extends to an extract obtained from plant
material of the Trichocaulon or Hoodia genus and containing a
substantially pure steroidal glycoside of formula (1).
[0155] The invention extends also to a foodstuff or a beverage
containing an effective quantity of the steroidal glycoside of the
formula (1), or its derivatives or analogues as set out before, to
have an appetite suppressant effect when ingested.
[0156] Molecular genetic studies have led to a considerable
increase in the understanding of the regulation of appetite,
satiety and bodyweight. These studies have revealed numerous
central regulatory pathways, mediated by a number of neuropeptides.
The maintenance of a normal body weight is achieved by an intricate
balance between energy intake, food consumption, and energy
expenditure. Energy homeostasis is subject to a wide range of
influences, ultimately controlled by the brain. The different
signals include such things as sense of smell and taste and
gastro-intestinal signals such as distension of the
gastro-intestinal tract, chemical signals to the gastric mucosa and
blood-borne metabolites such as fatty acids and glucose.
[0157] Centrally, neuropeptide "Y" (NPY) which is negatively
regulated by leptin, has been established as one of the positive
regulators of feeding behaviour. Expression of the endogenous
antagonist for melanocortin receptors has also been shown to be the
basis for obesity in a particular model (the ob/ob mouse). Indeed
deficiency at the MC4 melanocortin receptor completely replicates
the obesity syndrome. Other mediators which have been shown to have
roles in the energy balance include bombesin, galonin and
glucagon-like peptide-1.
[0158] Without being bound by theory, the Applicant believes that
compound (1) and its analogues as described above act as an agonist
of the melanocortin 4 receptor. The effect of this is to regulate
NPY but also to increase cholecystokinin. The effect of
cholecystokinin amongst other things is to inhibit gastric
emptying.
[0159] Accordingly, the invention extends to a composition having
appetite suppressant activity comprising a melanocortin 4 receptor
agonist.
[0160] The agonist may be an extract or compound as previously
described, in particular the compound of formula (1). The
composition may be admixed with a pharmaceutical excipient, diluent
or carrier and is optionally prepared in unit dosage form.
[0161] The invention still further extends to the use of a
melanocortin 4 receptor agonist in the manufacture of a medicament
having appetite suppressant activity, to a melanocortin 4 receptor
agonist for use as a medicament having appetite suppressant
activity, to a method of suppressing an appetite by administering
to a human or animal an effective dosage of a composition
comprising a melanocortin 4 agonist as described above, and to the
use of a melanocortin 4 receptor agonist to suppress the appetite
of and/or to combat obesity in a human or animal.
[0162] The invention and its efficacy will now be further
described, without limitation of the scope of the invention, with
reference to the following examples and drawings.
[0163] In the drawings,
[0164] FIG. 1 shows a flow diagram of the general method of
extracting a first crude appetite suppressant extract and a
purified appetite suppressant extract from plant material of the
genus Trichocaulon or Hoodia;
[0165] FIG. 2 shows a graphical representation of a bioassay
carried out on rats using a partially purified methanol extract of
Trichocaulon piliferum;
[0166] FIGS. 3 and 4 together show a schematic representation of a
preferred embodiment of the process of the invention for producing
an extract of plant material of the genus Trichocaulon or Hoodia;
and
[0167] FIGS. 5 and 6 show a graphical representation of the
percentage change of body mass of rats for different groups for
days -7 to 7 and days 0 to 7 respectively in a repeat dose study
using a sap extract and a spray-dried sap extract of plant material
of the species Hoodia gordonii.
EXAMPLE 1
[0168] The general method of extracting a first crude appetite
suppressant extract and a purified appetite suppressant extract
from plant material of the genus Trichocaulon or of the genus
Hoodia is illustrated byway of the flow diagram of FIG. 1.
EXAMPLE 2
[0169] Bioassays carried out on rats using a partially purified
methanol extract obtained in the manner illustrated in Example 1,
indicated that the extract does in fact exhibit appetite
suppressant activity. The appetite suppressant activity of the
active extract can be illustrated by way of a typical example of
the effect of the methanol extract of Trichocaulon piliferum on
rats, by way of the graphic representation in FIG. 2.
[0170] It will be evident from FIG. 2 that the test group of rats
dosed with the extract on day 5 displayed a substantially
diminished food intake over the next two days, while a control
group did not disclose a comparable reduced food intake. The food
intake of the test group returned to normal, and in fact increased,
from day 8 onwards.
EXAMPLE 3
[0171] A preferred embodiment of a process in accordance with the
invention for producing an extract having appetite suppressant
activity is illustrated schematically by way of example in FIGS. 3
and 4, which two Figures together illustrate the comprehensive
process. However, various other procedures may be used, as will be
understood by persons skilled in the art.
[0172] Referring to FIG. 3, plant material of the genus
Trichocaulon or the genus Hoodia is fed into a blender 3, eg a
Waring blender, by way of feedline 1, with a solvent in the form of
a methylene chloride/methanol solution introduced via feedline 2.
The homogenised product is fed via line 4 into a separation stage
5, eg in the form of a filter or centrifuge, and the residual plant
material is removed via line 27.
[0173] The solvent/extract mixture is fed via line 6 into an
evaporation stage 7, where the solvent is removed, for example by
means of a rotor evaporator. The dried crude extract is fed via
line 8 into a further extraction stage 9 with the addition of a
methylene chloride/water solution introduced via feedline 29 for
further extraction, and then to a separation stage 13 by way of
line 11, where the water fraction is removed via line 31. The
dissolved extract fraction is fed via line 15 into a drier stage 17
where the solvent is evaporated, for example by a rotor
evaporator.
[0174] Referring to FIG. 4, the dried extract is fed via line 10
into an extraction stage 12. A methanol/hexane solution is also fed
via line 14 into the extraction stage 12 for further purification
and extraction of the dried extract. The extract/methanol/hexane
mixture is fed via line 16 into a separation stage 18, the hexane
fraction is removed via line 20, and the methanol/extract mixture
is then fed via line 22 into a drying stage 24. In the drying stage
24, the solvent is removed, eg by evaporation on a rotor
evaporator.
[0175] The dried, partially purified active extract is fed via line
26 and with the addition of methanol via line 28 into a solution
stage 30, and the dissolved fraction is fed via line 36 to a
chromatography column 38.
[0176] In the column 38 the methanol soluble fraction is further
fractionated, using silica gel and a chloroform/30% methanol
solvent, into different fractions schematically indicated as
fractions I to V. According to an actual fractionation procedure
carried out by the Applicant, the fractionation procedure yielded
the following fraction weights: I(3.9 g); II(2.6 g); III(2.1 g);
IV(1.1 g) and V(2.0 g). These fractions are individually evaluated
by a suitable bioassaying procedure (in a step not shown) and those
fractions identified as fractions I and II, displaying marked
appetite suppressant activity, are fed by feedlines 40 and 42 into
columns 44 and 46 respectively where they are further fractionated
and purified by column chromatography, again by using silica gel
and a 9:1 chloroform:methanol system.
[0177] The sub-fractions II(A)-(C) obtained from column 44 do not,
when assayed, display a noteworthy appetite suppressant activity,
and may be recycled for further chromatography.
[0178] The sub-fractions I(A)-(L) obtained from column 46 are also
evaluated (by an assaying step not shown), and the sub-fraction
I(C) is found to have marked appetite suppressant activity.
[0179] The sub-fraction I(C) is fed-via line 48 into column 50 for
a further fractionation and purification, using silica gel and a
9:1 ethyl acetate:hexane eluent. Of the resultant purified
fractions, fraction I(C) (ii) is found, after assaying, to possess
marked appetite suppressant activity.
[0180] The purified product is identified by nuclear magnetic
resonance spectroscopy (as indicated in Tables 1 and 2 below), to
be compound (1).
1TABLE 1 .sup.1H (300.13 MHz) n.m.r. data for compound (1)
CDCl.sub.3 Compound (1) Hydrogen Atom J(HH)/Hz .delta..sub.H/p.p.m.
Aglycone-3 -- 3.522 m 6 -- 5.381 m 12 11.5, 4.1 4.607 dd 17 9.3,
9.3 3.157 dd 18 -- 1.029 s 19 -- 0.951 s 21 -- 2.164 s 3* 7.1, 1.5
6.888 qq 4* 7.1, 1.2 1.806 dq 5* 1.6, 1.2 1.853 dq Cym-1' 9.4, 2.1
4.816 dd 2'.sub.aq 13.8, 3.7, 2.1 2.055 ddd 2'.sub.ax 13.8, 9.4,
2.6 1.552 ddd 3' 3.7, 2.9, 2.6 3.776 ddd 4' 9.4, 2.9 3.179 dd 5'
6.3, 9.4 3.821 dd 6' 6.3 1.279 d.sup.a 3'-OMe -- 3.408 s.sup.d 1"
9.4, 2.1 4.730 dd 2" 13.8, 3.7, 2.1 2.108 ddd 2".sup.aq 13.8, 9.4,
2.6 1.601 ddd 3".sup.ax 3.7, 2.9, 2.6 3.755 ddd 4" 9.4, 2.9 3.239
dd 5" 6.3, 9.4 3.898 dd 6" 6.3 1.243 d.sup.b 3"-OMe -- 3.392
s.sup.e Thev-1"' 7.7 4.273 d 2"' 7.7, 8.0 3.469 dd 3"' 8.0, 2.9
3.099 dd 4"' 9.3, 2.9 3.179 dd 5"' 6.3, 9.3 3.351 dd 6"' 6.3 1.183
d'.sup.c 3"'-OMe -- 3.622 s .sup.a,b,cin each column may be
interchangeable. .sup.d,ein each column may be interchangeable,
*Refers to the tigloate group atoms
[0181]
2TABLE 2 Relevant .sup.13C (75.25 MHz) n.m.r. data for Compound (1)
in CDCl.sub.3 Aglycone moiety Sugar moiety Carbon
.delta..sub.c/p.p.m. Carbon .delta..sub.c/p.p.m. 1 37.04 T cym- 1'
95.84 D 2 29.44 T 2' 35.57 T 3 77.24 D 3' 77.05 D 4 38.62 T 4'
82.57 D 5 138.95 S 5' 68.48 D 6 131.90 D 6' 18.14 Q 7 27.30 T
3'-OMe 57.93 Q 8 35.30 D 1" 99.54 D 9 43.04 D 2" 35.17 T 10 37.22 S
3" 76.99 D 11 26.04 T 4" 82.52 D 12 75.88 D 5" 68.30 D 13 53.71 S
6" 18.36 Q 14 85.69 S 3"-OMe 57.09 Q 15 34.36 T Thev- 1"' 104.28 D
16 24.31 T 2"' 74.62 D 17 57.18 D 3"' 85.30 D 18 9.85 Q 4"' 74.62 D
19 19.27 Q 5"' 71.62 D 20 216.85 S 6"' 17.75 Q 21 33.01 Q 3"'-OMe
60.60 Q 1* 167.60 S 2* 128.69 D 3* 137.66 D 4* 14.41 Q 5* 12.08 Q
*Refers to the tigloate group atoms
[0182] Compound (1)
[0183] IR data: 3440 cm.sup.-1 (OH), 2910 cm.sup.-1 (CH), 1700
cm.sup.-1 (C.dbd.O) [.alpha..sub.D].sup.20.sub.589=12,67.degree.
(C=3, CHCl.sub.3) m.p. 147.degree. C.-152.degree. C.
[0184] Examples 4 to 13 illustrate the synthetic procedures whereby
the intermediate compounds and steroid (15) may be prepared
according to "the first alternative procedure".
EXAMPLE 4
[0185] 12.beta., 15.alpha.-Dihydroxy Progesterone (17)
[0186] Cultures of Calonectria decora (ATCC 14767) are prepared by
the inoculation of a culture medium comprised of sucrose (900 g),
K.sub.2HPO.sub.4 (30 g), Czapek concentrate (300 ml), corn steep
liquor (300 ml) and distilled water (30 i) (150.times.500 ml
flasks). After 5 days of shaking at 26.degree. C., progesterone
(16) (150 g) in a suspension of Tween 80 (0,1% soln., 1,5 l) is
added to the flasks. The cultures are incubated for a further 5
days and then worked-up by centrifugation, decantation, extraction
of the medium with chloroform, and then evaporation to yield the
dihydroxy progesterone (17) (75 g, 45%)
[0187] .sup.1H NMR (CDCl.sub.3): 5,71 (1H, s, H-4); 4,12-4,22 (1H,
m, H-15)
[0188] 4,43 (1H, br, s, OH); 3,46-3,53 (1H, dd, J=4, 6 Hz, H-12);
2,16 Hz (3H, s, H-21); 1,18 (3H, s, H-19); 0,74 (3H, s, H-18)
EXAMPLE 5
[0189] 12.beta.-Hydroxy-15.alpha.-(D-toluene Sulfonyl)-progesterone
(18)
[0190] The dihydroxy progesterone (17) (75 g, 0.22 mol) is
dissolved in dry pyridine (300 ml) and cooled to 0.degree. C.
p-Toluene sulfonyl chloride (46 g, 0,24 mol) in dry pyridine (200
ml) is added dropwise to the reaction mixture at 0.degree. C. The
reaction is stirred overnight at 0.degree. C., and quenched by the
addition of H.sub.2O (500 ml). The water layer is extracted with
ethyl acetate (1 f), and the organic extract washed with
hydrochloric acid. (6M, 3.times.1 f), aqueous saturated sodium
bicarbonate (500 ml), aqueous saturated sodium chloride (500 ml),
and water (500 ml). The organic layer is dried (MgSO.sub.4),
filtered and evaporated to yield p-toluene sulfonated progesterone
(18) (98 g, 92%) as a viscous dark yellow oil.
[0191] .sup.1H NMR (CDCl.sub.3): 7,7 (2H, d, J=14 Hz, H-2,6); 7,34
(2H, d, J=8, 4 Hz, H-3,5); 5,67 (1H, s, H-4); 4,86-4,93 (1H, m,
H-35); 3,45-3,50 (1H, dd, J=4, 6 Hz, H-12); 2,44 (3H, s, H-4Me);
2,15 (3H, s, H-21) 1,13 (3H, S, H-19); 0,74 (3H, s, H-18).
EXAMPLE 6
[0192] 12.beta.-Hydroxy-.DELTA..sup.14-progesterone (19)
[0193] A solution of the tosylated progesterone (18) (98 g, 0,19
mol) in 2,4,6-trimethyl collidine (500 ml) is refluxed at
150.degree. C. for 3 h. The reaction mixture is cooled and poured
into water (0.500 ml). The water layer is extracted with ethyl
acetate (1 l), after which the organic layer is washed with
hydrochloric acid (6M, 3.times.1 l), aqueous saturated sodium
bicarbonate (500 ml), aqueous saturated sodium chloride (500 ml),
and water (500 ml). After drying (MgSO.sub.4) and filtering, the
ethyl acetate is evaporated and the crude mixture is purified by
silica gel chromatography, eluting with acetone: chloroform (1:10)
to afford .DELTA..sup.14-progesterone (19) (50 g, 78 k) as a dark
red oil.
[0194] .sup.1H NMR (CDCl.sub.3): 5,73 (1H, s, H-4), 5,28 (1H, dd,
J=2, 2 Hz, H-15), 4,41 (1H, br, s, OH), 3,49-3,52 (1H, dd, J=4, 3
Hz, H-12), 2,80-2,84 (1H, dd, J=9, 2 Hz, H-17), 2,14 (3H, s, H-21),
1,19 (3H, s, H-19), 0.89 (3H, s, H-18).
EXAMPLE 7
[0195] 3,12.beta.-Diacetoxypregna-3,5,14-trien-20-one (20)
[0196] A solution of .DELTA..sup.14-progesterone (19) (50 g, 0,15
mol) in acetyl chloride (1,5 l) and acetic anhydride (750 ml) is
refluxed for 2 hours. The reaction mixture is poured into cold
ethyl acetate (1 l) and aqueous saturated sodium bicarbonate is
added with stirring until the effervescence ceases. The ethyl
acetate layer is separated from the sodium bicarbonate layer and
washed with further portions of aqueous sodium bicarbonate
(3.times.700 ml), thereafter with aqueous saturated sodium chloride
(700 ml) and finally with water (700 ml). The organic layer is
dried (MgSO.sub.4), filtered and evaporated to afford the
3,12.beta.-diacetoxypregna-3,5,14-trien-20-one (20) (60 g, 93 k) as
an orange oil.
[0197] .sup.1H NMR(CDCl.sub.3): 5,68 (1H, s, H-4), 5,44 (1H, m,
H-6), 5,31. (1H, dd, J=2, 2 Hz, H-0.1), 4,82-4,86 (1H, dd, J=4, 5
Hz, H-12), 3,10-3,18 (1H, t, J=9, 5 Hz, H-17), 2,18 (3H, s, 3-Ac),
2,11 (3H, s, 12-Ac), 2,08 (3H, s, H-21), 1,02 (3H, s, H-19), 1,01
(3H, s, H-18)
EXAMPLE 8
[0198] 3, 12.beta.-Diacetoxy-20,
20-ethylenedioxyprecna-3,5,14-triene (21)
[0199] The diacetoxy compound (20) (60 g, 0,14 mol) is dissolved in
benzene (1 l) and ethylene glycol (60 ml) and p-toluene sulfonic
acid (1 g) are added. (The benzene is previously refluxed with a
Dean-Stark trap). The mixture is refluxed with stirring and
azeotropic removal of water for 16 hours. Aqueous saturated sodium
bicarbonate solution (500 ml) is added to the cooled solution. This
is then washed with brine (500 ml), and with water (500 ml), and
dried (MgSO.sub.4). The solvent is evaporated and the crude mixture
purified by silica gel column chromatography, eluting with ethyl
acetate:hexane (2:8) to yield the ethylenedioxypregna-3,5,14-triene
(21) (35 g, 53%)
[0200] .sup.1H NMR (CDCl.sub.3): 5,68 (1H, s, H-4), 5,45 (1H, m,
H-6), 5,31 (1H, dd, J=2,2 Hz, H-15), 4,73-4,85 (1H, dd, J=4,4 Hz,
H-12), 3,78-3,98 (4H, m, ethylenedioxy), 2,16 (3H, s, 3-Ac), 2,04
(3H, s, 12-Ac), 1,29 (3H, s, H-21), 1,12 (3H, s, H-19), 1,02 (3H,
s, H-18).
EXAMPLE 9
[0201]
3.beta.-12.beta.-Dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12--
acetate (22)
[0202] The dienolacetate (21) (35 g, 0,077 mol) is suspended in
ethanol (500 ml) and sodium borohydride (2,8 g, 0.074 mol) is added
at 0.degree. C. The mixture is allowed to warm to room temperature
and stirred overnight. Most of the solvent is removed in vacuo and
the mixture is diluted with water (500 ml) and extracted with ethyl
acetate (500 ml). Work-up followed by chromatography on silica gel
with acetone/chloroform (1:10) yields the 3.beta.-alcohol (22) (25
g, 80%).
[0203] .sup.1H NMR (CDCl.sub.3): 5,41 (1H, m, H-6), 5,28 (1H, dd,
J=2,2 Hz, H-15), 4,72-4,81 (1H, dd, J=4,4 Hz, H-12), 3,82-4,02 (4H,
m, ethylene dioxy), 3,45-3,59 (1H, m, H-3), 2,03 (3H, s, 12-Ac),
1,28 (3H, s, H-21), 1,10 (3H, s, H-19), 1,01 (3H, s, H-18).
EXAMPLE 10
[0204] 3.beta.,
12.beta.-Dihydroxy-20,20-ethylenedioxypregn-5,14-diene (23)
[0205] The 3.beta.-alcohol (22) (25 g, 60.2 mmol) in dry
tetrahydrofuran (300 ml) is added dropwise to a suspension of
lithium aluminium hydride (2,7 g, 72,2 mmol) in dry tetrahydrofuran
(500 ml). The reaction mixture is stirred at room temperature for
24 hours after which water (2,7 ml) is carefully added and stirred
for a further 10 min. Sodium hydroxide (15% soln, 2,7 ml) is then
added and the suspension stirred. After 10 min, water (8,1 ml) is
added and the suspension stirred for 10 minutes, filtered, dried
(MgSO.sub.4), and the solvent evaporated to afford the 39, 129
dihydroxypregna-diene (23) (20 g, 90%).
[0206] .sup.1H NMR (CDCl.sub.3): 5,36 (1H, m, H-6), 5,23 (1H, dd,
J=2,2 Hz, H-15), 3,94-4,06 (4H, m, ethylene dioxy), 3,41-3,52 (1H,
m, H-3), 3,32-3,36 (1H, dd, J=4,3 Hz, H-12), 1,31 (3H, s, H) 1,01
(3H, s, H-19), 0,96 (3H, S, H-18).
[0207] .sup.13C NMR (CDCl.sub.3): 152,4 (c-14), 140,2 (c-5), 121,1
(c-15) 119,7 (c-6), 111,1 (C-20), 79,8 (C-12), 71,6 (C-3), 63,7 and
63,6 (ethylene dioxy), 58,8 (C-17), 19,0 (C-19), 11,9 (C-18).
[0208] 3.beta., 12.beta.-Dihydroxy-14 15-epoxy-20,
20-ethylenedioxypregn-5- -ene:
[0209]
3.beta.,12.beta.-Dihydroxy-56-epoxy-20,20-ethylenedioxypregn-14-ene
[0210] N-Bromoacetamide (211 mg, 1,5 mmol) is added to a stirred
solution of the 5,14-diene (23) (500 mg, 1,34 mmol) in acetone (100
ml), acetic acid (2,5 ml), and water (5 ml) at 0.degree. C. After
15 min sodium sulphite (5% soln, 50 ml) is added to the reaction
mixture. The acetone is evaporated, and the aqueous layer extracted
with dichloromethane (3.times.50 ml). The organic layer is dried
(MgSO.sub.4), filtered and evaporated. Pyridine (1 ml) is added to
the product, and stirred for 0,5 h. Dichloromethane (100 ml) is
then added to the reaction mixture, and the dichloromethane is
washed with citric acid (5% soln, 3.times.100 ml), saturated sodium
bicarbonate (50 ml), and water (50 ml). The organic layer is dried
(MgSO.sub.4), filtered and evaporated to give the mixture of 14,15-
and 5,6-epoxides (360 mg, 69%) as a white foam. The mixture of
epoxides could not be separated by silica gel column
chromatography.
EXAMPLE 11
[0211] 3.beta.,
12.beta.-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5 ene
(24)
[0212] The mixture of 14,15- and 5,6-epoxides (14,4 g, 37,0 mmol)
in dry tetrahydrofuran (200 ml) is added to a suspension of lithium
aluminium hydride (1,69 g, 44,4 mmol) in dry tetrahydrofuran (300
ml). The reaction mixture is stirred at room temperature for 24
hours, after which it is worked up as described earlier by the
addition of water (1,69 ml); and sodium hydroxide (15% soln, 1,69
ml). After filtration and evaporation of the solvent, the crude
product is purified by silica gel column chromatography using
methanol/chloroform (1:9) as solvent to give the unreacted 14,15
epoxy-20,20-ethylenedioxypregn-5-ene (24) (300 mg, 2,1%).
[0213] .sup.1H NMR (CDCl.sub.3): 5,31 (1H, m, H-6), 3,82-3,98 (4H,
m, ethylene dioxy), 3,43-3,52 (1H, m, H-3), 3,41 (1H, s, H-15),
3,31-3,35 (1H, dd, J=4, 3 Hz, H-12), 1,29 (3H, s, H-21), 1,17 (3H,
s, H-19), 1,02 (3H, s, H-18).
[0214] .sup.13C NMR (CDCl.sub.3): 139,8 (C-5), 120,8 (C-6), 112,1
(C-20), 77,2. (C-12), 75,4 (C-14), 61,0 (C-15), 22,3 (C-21), 19,2
(C-19), 9,5 (C-18).
EXAMPLE 12
[0215] 3.beta., 12.beta.,
14.beta.-Trihydroxy-20,20-ethylenedioxypregn-5-e- ne (25)
[0216] The 14,15-epoxide (24) (300 mg, 0,77 mmol) in dry
tetrahydrofuran (10 ml) is added to a suspension of lithium
aluminium hydride (300 mg, 7,89 mmol) in tetrahydrofuran and the
reaction refluxed for 48 h. After the addition of water (0,3 ml),
sodium hydroxide (15% soln, 0,3 ml) and filtration as described
earlier, the mixture is purified by silica gel column
chromatography using methanol:chloroform (1:9) as solvent to give
the trihydroxy pregnene (25) (250 mg, 83%)
[0217] .sup.1H NMR (CDCl.sub.3):5,38 (1H, m, H-6), 3,98 (4H, m,
ethylene dioxy), 3,43-3,53 (1H, m, H-3), 3,25-3,32 (1H, dd, J=4, 1
Hz, H-12), 1,32 (3H, s, H-21), 1,01 (3H, s, H-19), 0,98 (3H, s,
H-18)
[0218] .sup.13C NMR CDCl.sub.3): 139,1 (C-5), 122,1 (C-6), 112,2
(C-20), 85,1 (C-14), 75,1 (C-12), 71,6 (C-3), 23,4 (C-21), 19,4
(C-19), 8,9 (C-18)
EXAMPLE 13
[0219] 3.beta., 12.beta., 14.beta.-Trihydroxy-pregn-5-ene (15)
[0220] The ethylenedioxypregnene (25) (250 mg, 0,64 mmol) is
dissolved in acetic acid (13,4 ml) and water which after freeze
drying affords the trihydroxy steroid (15) (200 mg, 89%), m.p.:
228.degree.-235.degree. C. (lit 225.degree.-235.degree. C.), M+
348, [.alpha..sub.D).sup.20+35.degre- e. (lit
[.alpha..sub.D].sup.20+29.degree.).
[0221] .sup.1H NMR (CDCl.sub.3): 5,39 (1H, m, H-6), 3,56-3,62 (1H,
t, J=8, 1 Hz, H-17), 3,42-3,51 (1H, m, H-3), 3,28-3,39 (1H, dd,
J=4, 3 Hz, H-12), 2,23 (3H, s, H-21), 1,01 (3H, s, H-19), 0,90 (3H,
s, H-18)
[0222] .sup.13C NMR (CDCl.sub.3): 217,7 (C-20), 138,9 (C-5), 122,2
(C-6), 85,5 (C-14), 73,6 (C-12), 71,6 (C-3), 57,0 (C-17), 55,1
(C-13), 43,6 (C-9), 42,1 (C-4), 37,3 (C-1), 36,8 (C-10), 35,9
(C-0.8), 34,5 (C-15), 32,9 (C-21), 31,5 (C-16), 30,1 (C-2), 27,4
(C-7), 24,4 (C-11), 19,4 (C-19), 8,3 (C-18)
[0223] Examples 14 to 19 illustrate the synthetic procedures
whereby the intermediate compounds and steroid (15) may be prepared
according to "the second alternative procedure".
EXAMPLE 14
[0224]
20,20-Ethylenedioxy-3.beta.-toluene-p-sulphonyloxy-pregn-5,14-diene-
-12.beta.-ol acetate (26)--A solution of p-toluenesulphonyl
chloride (650 mg, 3.4 mmol) in pyridine (10 ml) was added dropwise
to a mixture of the
20,20-Ethylenedioxypregna-5,14-diene-3.beta.,12.beta.-diol
12-acetate (22) (1.3 g, 3.1 mmol) in pyridine (15 ml) at 0.degree.
C. The reaction mixture was left stirring at room temperature for
24 hours after which water was added to the reaction mixture. The
solution was extracted with ethyl acetate (2.times.50 ml), the
ethyl acetate layer was washed citric acid (5.times.50 ml),
saturated sodium bicarbonate solution (100 ml), saturated sodium
chloride solution (100 ml) and water (100 ml). The: ethyl acetate
was dried (MgSO.sub.4), filtered, and evaporated and purified by
flash column chromatography using hexane-ethyl acetate (8:2 v/v) as
the eluant to give the .beta.-O-tosyl steroid (26), (1.5 g, 84%),
as a yellow oil, (Found M 570.271, C.sub.32H.sub.42O.sub.7S
requires: M 570.273).
[0225] .delta..sub.H 1.021 (3H, s, 19-H), 1.131 (3H, s, 18-H),
1.282 (3H, s, 21-H), 2.021 (acetateOCH.sub.3), 2.431 (3H, s,
Ar--CH.sub.3), 3.883 (4H, m, OCH.sub.2CH.sub.2O), 4.750 (1H, dd, 3
J 10.8 Hz, 5.2 Hz, 12-H), 4.890 (1H, m, 30H), 5.281 (1H, dd, 3 J
4.2 Hz, 2.1 Hz, 15-H), 5.388 (1H, m, 6-H), 7.341 (2H, d, .sup.3 J
8.2 Hz, ArH), 7.746 (2H, d, 3 J 8.2 Hz, ArH).
[0226] .delta..sub.c 13.493Q (C-18), 19.002Q (C-19), 21.612Q
(Ar-methyl)*, 21.671Q (C-21)*, 24.175Q (acetate methyl), 63.401T
(ethylenedioxy), 63.498T (ethylenedioxy), 71.531S(C-13), 80.912D
(C-12), 82.531D (C-3), 111.363S (C-20), 120.881D (C-15), 121.461D
(C-6), 123.715-133.917 (Aromatic), 139,903S(C-14), 151,722S(C-5),
170.819S (ester carbonyl).
[0227] * may be interchanged
EXAMPLE 15
[0228] 20, 20-Ethylenedioxy-3.alpha.,
5-cyclo-5.alpha.--pregn-14-ene-6.bet- a., 12.beta.-diol-12-acetate
(27)--A solution of 3.beta.-toluene-p-sulphon-
yloxy-pregn-5,14-diene (26) (1.2 g, 2.1 mmol) and potassium acetate
(2.2 g, 22.4 mmol) in water (250 ml) and acetone (500 ml) was
refluxed at 60.degree. C. for 16 hours. The acetone was evaporated
and the water was extracted with ethyl acetate (200 ml). The ethyl
acetate was dried (MgSO.sub.4), filtered, and evaporated. Flash
chromatographic separation of the mixture using chloroform-acetone
(9:1 v/v) as the eluant gave the 3.alpha., 5-cyclo derivative (27),
(530 mg, 61%) as a yellow oil, (Found M 416.262,
C.sub.25H.sub.36O.sub.5 requires M 416.263).
[0229] .delta..sub.H 0.288 (1H, dd, .sup.3 J 8.1 Hz, 4.9 Hz,
4-H.sub.a), 0.477 (1H, dd, .sup.3 J 4.44 Hz, 4-H.sub.b), 1.025 (3H,
s, 19-H)., 1.121 (3H, s, 18-H), 1.256 (3H, s, 21-H), 1.989 (3H, s,
acetate-CH.sub.3), 3.302 (1H, dd, .sup.3 J 2.8 Hz 2.8 Hz, 6-H),
3.784-3.947 (4H, m, OCH.sub.2CH.sub.2O), 4.721 (1H, dd, 3 J 8.5 Hz,
5.6 Hz, 12-H), 5.232 (1H, dd, 3 J 3.9 Hz, 1.9 Hz, 15-H).
[0230] .delta..sub.c 11.678T(C-4), 12.298Q(C-18), 19.971Q (C-19),
23.623Q (C-21), 24.153Q (acetate methyl), 63.700T (ethylenedioxy),
63.788T (ethylenedioxy), 73.591D.(C-6), 80.551D (C-12),
111.126S(C-20), 118.778D (C-15), 152.959S(C-14), 170.991S (ester
carbonyl).
EXAMPLE 16
[0231] 20,20-Ethylenedioxy-3.alpha.,
5-cyclo-5.alpha.-pregn-24-ene-6.beta.- ,12.beta.-diol (28)--A
solution of the 3.alpha.,5-cyclo derivative (27), (500 mg, 1.2
mmol) in tetrahydrofuran (20 ml) was added dropwise to a suspension
of lithium aluminium hydride (50 mg, 1.3 mmol) in tetrahydrofuran
(10 ml). The reaction mixture was stirred for 4 hours and quenched
by the addition of water (50 .mu.l). After 30 minutes, sodium
hydroxide was added (15% solution, 50 .mu.l) and stirring continued
for a further 30 minutes. Water (150 .mu.l was added and the
reaction mixture was filtered. The tetrahydrofuran was dried
(MgSO.sub.4) filtered and evaporated and flash chromatographic
purification using chloroform-acetone (8:2 v/v) as the eluant to
give the diol (28), (370 mg, 83%) as an oil, (Found M 374.250,
C.sub.23H.sub.34O.sub.4 requires: M 374.252)
[0232] .delta..sub.H 0.298 (1H, dd, 3 J8.1 Hz, 4.9 Hz, 4-H.sub.2),
0.510 (1R, dd, 3 J 4.4 Hz, 4.4 Hz, 4-H.sub.b), 0.985 (3H, s, 19-H),
1.055 (3H, s, 18-H), 1.325 (3H, s, 21-H), 3.318 (1H, dd, 3 J 3.0
Hz, 3.0 Hz, 6-H),), 3.363 (1H, dd, 3 J 11.4 Hz, 4.2 Hz, 12-H),
4.019 (4H, m, OCH.sub.2Ch.sub.2O) 4.622 (1H, s, OH), 5.255 (1H, dd,
3 J 3.9 Hz, 1.9 Hz, 15-H)
[0233] .delta..sub.c 11.681T(C-4), 12.243Q(C-18), 19.844Q (C-19),
23.604Q(C-21), 63.620T (ethylenedioxy), 63.733T (ethylenedioxy),
73.569D (C-6), 77.478D (C-12), 11.125S(C-20), 118.702D (C-15),
152.912S(C-14).
EXAMPLE 17
[0234] 20, 20-Ethylenedioxy-14, 15.beta.-epoxy-3.alpha.,
5-cyclo-5.alpha., 14.beta.-pregnane-6.beta., 12.beta.-diol
(29)--N-bromoacetamide (150 mg, 1.1 mmol) was added to a solution
of the 20,20-ethylenedioxy-3.alpha.,5-c-
yclo-5.beta.-pregn-14-ene-6.beta., 12.beta.-diol (28) (340 mg, 0.91
mmol) in acetone (20 ml), water (0.25 ml) and acetic acid (0.25 ml)
at 0.degree. C. After 15 min., sodium sulphite (5% solution, 20 ml)
was added to the reaction mixture. The acetone was evaporated under
reduced pressure and the remaining solution was extracted with
dichloromethane (3.times.30 ml). The dichloromethane layer was
dried (MgSO.sub.4), filtered and evaporated to a concentrated
volume (50 ml). Pyridine (0.5 ml) was added to the mixture and
stirred for a further 1 hour after which the dichloromethane layer
was washed with a citric acid solution (5%, 3.times.30 ml),
saturated sodium bicarbonate solution (30 ml) and water (30 ml).
The dichloromethane layer was dried (MgSO).sub.4), filtered and
evaporated and purified by flash column chromatography using
chloroform-methanol (9.5:0.5 v/v) as the eluant to give the epoxide
(29) (180 mg, 51% as a foam, (Found M 390.245,
C.sub.23H.sub.34O.sub.2 requires: M 390.247).
[0235] .delta..sub.H 0.287 (1H, dd, .sup.3 J 8.1 Hz, 4.9 Hz,
4H.sub.a), 0.501 (1H, dd, 3 J 4.4 Hz, 4.4 Hz, 4-H.sub.b), 0.978
(3H, s, 19-H), 1.048 (3H, s, 18-H), 1.321 (3H, s, 21-H), 30.318
(1H, dd, 3 J 3.1 Hz, 3.1 Hz, 6-H),), 3.355 (1H, dd, 3 j 11.2 Hz,
4.1 Hz, 12-H), 3.491 (1H, S, 15-H), 4.001 (4H, m,
OCH.sub.2Ch.sub.2O), 4.901 (1H, s, OH). .delta..sub.c 11.668T(C-4),
11.973Q(C-18), 19.515Q (C-19), 23.519Q(C-21), 59.910D (C-15),
63.601T (ethylenedioxy), 63.713T (ethylenedioxy), 72.501S(C-14),
73.571D (C-6), 77.471D (C-12), 111.085S(C-20).
EXAMPLE 18
[0236] 20,20-Ethylenedioxy-6.beta.,12.beta., 14-triydroxy-3.alpha.,
5-cyclo-5.alpha.,14.beta.-pregnane (30)--A solution of the epoxide
(29) (170 mg, 0-0.44 mmol) in tetrahydrofuran (10 ml) was added to
a suspension of lithium aluminium hydride (20 mg, 0.53 mmol) in
tetrahydrofuran (5 ml). The reaction mixture was refluxed for 2
hours after which water (20 .mu.l) was added and stirring continued
for 05 hour. Sodium hydroxide solution (15%, 20 .mu.l) was added
and stirring continued for a further 0.5 hour. A further quantity
of water was added (60 Al) and the suspension was stirred for 1
hour. After filtration, the suspension was dried (MgSO.sub.4)
filtered, and the tetrahydrofuran was evaporated. Flash
chromatographic separation of the resulting mixture eluting with
chloroform-methanol (9:1 v/v) gave the required triol (30), (90 mg,
s3') as a clear oil, (Found M 392.261, C.sub.23H.sub.38O.sub.5
requires: M 392.263).
[0237] .delta..sub.H 0.287 (1H, dd, 3 J 8.1 Hz, 4.9 Hz, 4-H.sub.2),
0.510 (1H, dd, .sup.3 J 4.4 Hz, 4.4 Hz, 4-H.sub.b), 0.971 (3H, s,
19-H), 1.042 (3H, s, 18-H), 1.319 (3H, s, 21-H), 3.321 (1H, dd, 3 J
3.0 Hz, 3.0 Hz, 6-H), 3.321 (1H, dd, .sup.3 J 11.1 Hz, 3.9 Hz,
12-H), 3.561 (1H, s, OH), 4.084 (4h, m, OCH.sub.2Ch.sub.2O) 4.671
(1H, s, OH).
[0238] .delta..sub.c 11.668T (C-4), 11.971Q (C-18), 19.511Q (C-19),
23.520Q (C-21), 63.612T (ethylenedioxy), 63.711T (ethylenedioxy),
73.483D (C-6), 76.051D (C-12), 84.307S (C-14), 111.099S(C-20).
EXAMPLE 19
[0239] 3.beta., 12.beta.,14-Trihydroxy-14.beta.-pregn-5-en-20-one
(15)--A mixture of the triol (30) (80 mg, 0.20 mmol) in acetone (20
ml) and hydrochloric acid (1M, 10 ml) was refluxed at 60.degree. C.
for 2 hours. The reaction mixture was cooled and saturated sodium
bicarbonate solution (20 ml) was added. The acetone was evaporated
and the aqueous layer extracted with chloroform (3.times.20 ml),
the chloroform layer was dried (MgSO.sub.4), filtered and
evaporated to give the epimeric trihydroxy steroids (15a, 15b) (42
mg, 61%). Separation of the epimeric mixture (15a, 15b) (15 mg) was
achieved by flash chromatographic separation using chloroform
methanol (9:1 v/v) as the eluant to give the pure 17.beta.-epimer
(15a), (10 mg), m.p. 224-229.degree. C. (acetone), (lit.
226-223.degree.), (Found M 348.234, C; 72.32, H 9.21%
C.sub.21H.sub.32O.sub.4 requires: C, 72.38; H 9.26%, M 348.236),
and the 17.alpha.-epimer (15B) (3 mg), m.p. 183-191.degree. C.
(acetone), (lit 184-196.degree.).
[0240] 3.beta.,12.beta., 14-Trihydroxy-14.beta.-pregn-5-en-20-one
(15a): .delta..sub.H 0.963 (1H, s, 19-H), 1.192 (3H, s, 18-H),
2.236 (3H, s 21-H), 3.325 (1H, dd, 3 J 11.2 Hz, 3.9 Hz, 12-H),
3.464 (1H, s, OH), 3.5140 (1H, m, 3-H), 3.598 (1H, dd, 3 J 9.6 Hz,
9.6 Hz, 17-H), 4.255 (1H, s, OH), 5.383 (1H, m, 5-H).
[0241] .delta..sub.c 8.275Q (C-18), 19.414Q (C-19), 24.400T (C-11)
24.581T (C-16), 27.443T (C-7), 30.062T (C-2), 32.972Q (C-21),
34.543T (C-15), 35.864D (C-8), 36.975S(C-10), 37.337T (C-1),
42.144T (C-4), 43.565D (C-9), 55.101S (C-13), 57.038D (C-17),
71.597D (C-3), 73.558D (C-12), 85.566S(C-14), 122.223D (C-6),
138.932S(C-5), 217.011S(C-20)
[0242] 3.beta., 12.beta., 14-Trihydroxy-14.beta.-pregn-5-en-20-one
(15b): .delta..sub.H 0.996 (1H, s, 19-H), 1.144 (3H, s, 18-H),
2.221 (3H, s 21-H), 3.339 (1H, dd, 3 J 9.4 Hz, 9.4 Hz, 17-H), 3.492
(1H, m, 3-H), 3.629 (1H, dd, 3 J11.1 Hz, 3.9 Hz, 12-H), 3.712 (1H,
s, OH), 4.325 (1H, s, OH), 5.383 (1H, m, 5-H).
[0243] Examples 20 to 28 illustrate the procedures whereby the
intermediate compounds may be prepared to form the first
monosaccharide (40).
EXAMPLE 20
[0244] Methyl-4,6-O-benzylidene-.alpha.-D-glucopyranoside (32)
[0245] A mixture of methyl-.alpha.-D-glucopyranoside (30 g, 0,15
mol), benzaldehyde (70 ml) and zinc chloride (20 g) is stirred at
room temperature for 24 hours. The reaction product is poured into
ice water and stirring continued for 15 min. The white precipitate
is filtered and washed with diethyl ether. The solid material is
stirred with a solution of sodium metabisulphite (10% soln), for 15
min, filtered and washed with water. The solid material is
crystallized from chloroform and ether to yield the benzylidene
product (32) (31 g, 72)
EXAMPLE 21
[0246]
Methyl-4,6-O-benzylidene-2-O-tosyl-.alpha.-D-glucopyranoside(33)
[0247] p-Toluene sulfonyl chloride (25 g, 1,2 eq) in pyridine (100
ml) is added dropwise to a solution of the benzylidene glucose (32)
(31 g, 0.12 mol) in pyridine (100 ml) at 0.degree. C. The reaction
is stirred at room temperature for 48 hours. Ice is added to the
reaction mixture. The resulting white solid material is washed with
water and recrystallized from hot ethanol to yield the tosylated
glucose (33) (28 g, 60%).
EXAMPLE 22
[0248]
Methyl-4,6-O-benzylidene-3-O-methyl-.alpha.-D-altropyranoside
(34)
[0249] The tosylate (33) (28 g, 64 mmol) in a solution of sodium (7
g) in methanol (150 ml) is heated at 110.degree. C. for 48 hour in
an autoclave. The re-action vessel is cooled and solid carbon
dioxide is added to the reaction mixture. After filtration, the
methanol is evaporated and the solid material is then taken up in
water. The aqueous layer is extracted with chloroform (.times.3).
The chloroform is dried (MgSO.sub.4), filtered and evaporated. The
crude mixture is purified by silica gel column chromatography
eluting with chloroform:acetone (9:1) to yield the altroside (34)
(10 g, 52%)
EXAMPLE 23
[0250]
Methyl-6-bromo-4-O-benzoyl-3-O-methyl-6-deoxy-.alpha.-D-altropyrano-
side (35)
[0251] The benzylidene altroside (34) (10 g, 33 mmol) is added to a
solution of N-bromosuccinimide (7.6 g) and barium carbonate (20 g)
in carbon tetrachloride and the reaction mixture is refluxed at
75.degree. C. for 3 hours. The reaction mixture is filtered and the
carbon tetrachloride layer is washed with water. The organic layer
is dried (MgSO.sub.4), filtered and evaporated to yield
6-bromo-altroside (35), (9 g, 69%)
EXAMPLE 24
[0252]
Methyl-4-O-benzoyl-3-O-methyl-6-deoxy-.alpha.-D-altropyranoside
(36)
[0253] Sodium borohydride (18 g) in water (30 ml) is added dropwise
to a solution of the bromoaltroside (35) (9 g, 23 mmol) and nickel
chloride (18 g) in ethanol (300 ml) at 0.degree. C. The reaction
mixture is refluxed at 75.degree. C. for 1 hour and then it is
filtered. The ethanol is evaporated and the remaining aqueous layer
is extracted with chloroform (.times.3). The chloroform is dried
(MgSO.sub.4), filtered and evaporated, to yield the
6-deoxy-altroside (36) (5 g, 72 !).
EXAMPLE 25
[0254]
4-O-Benzoyl-3-O-methyl-6-deoxy-.alpha..beta.-D-phenylthioaltropyran-
oside (37)
[0255] Phenylthiotrimethylsilane (5 ml) and
trimethylsilyltrifluoromethane sulphonate (2 ml) are added at
0.degree. C. to a solution of the 6-deoxy-altroside (36) (5 g, 17
mmol) in dichloromethane (200 ml). The reaction mixture is stirred
at room temperature for 6 hours. Saturated sodium bicarbonate is
added to the reaction mixture. The dichloromethane layer is dried
(MgSO.sub.4), filtered and evaporated. The crude mixture is
purified by silica gel column chromatography eluting with
chloroform:acetone (9:1) to yield the
.alpha..beta.-phenylthioaltroside (37) (4 g, 63%).
EXAMPLE 26
[0256]
4-O-Benzoyl-3-O-methyl-2-phenylthio-2,6-dideoxy-.alpha..beta.-D-flu-
orocymaropyranoside (38)
[0257] Diethylaminosulphurtrifluoride (0,65 g) is added rapidly to
a solution of the .alpha..beta.-phenylthioaltroside (37) (0,5 g,
1,33 mmol) in dichloromethane at 0.degree. C. The reaction is
stirred for 0,5 h at 0.degree. C. and then saturated sodium
bicarbonate is added. The dichloromethane is separated from the
aqueous layer, dried (MgSO.sub.4), filtered and evaporated to yield
the .alpha..beta.-fluorocymarose (38) (450 mg, 90
EXAMPLE 27
[0258] 4-O-Benzoyl-3-O-methyl-2-O--
t-butyldimethylsilyl-.alpha..beta.-D-p- henylthio-altroside
(39)
[0259] The 6-deoxy altroside (37) (5 g) is silylated using
t-butyldimethylsilylchloride (3 g) and imidazole (3 g) in pyridine
(50 ml). The reaction is worked-up by extracting with ethyl
acetate, washing the ethyl acetate with hydrochloric acid (6 N),
then with sodium bicarbonate, and finally with water. The ethyl
acetate layer is dried (MgSO.sub.4), filtered and evaporated to
yield the silylated benzoyl phenylthioaltroside (39) (80%).
EXAMPLE 28
[0260]
3-O-methyl-2-O-t-butyldimethylsilyl-.alpha..beta.-D-phenylthioaltro-
side (40)
[0261] The silylated benzoyl phenylthioaltroside (39) (6 g) is
treated with sodium methoxide (100 ml) for 4 hours. The methanol is
evaporated and water is added to the reaction. The water layer is
acidified (pH 5, ACOH) and extracted with ethyl acetate. The ethyl
acetate is washed with water, dried (MgSO.sub.4) filtered and
evaporated to yield silylated methyl phenylthioaltroside (40)
(75%).
[0262] Examples 29 to 37 illustrate the procedures synthetic
whereby the intermediate compounds may be prepared to form the
second monosaccharide (50).
EXAMPLE 29
[0263] 1,2:5,6-Di-O-isopropylidene-.alpha.-D-glucofuranose (42)
[0264] Sulfuric acid (40 ml) is added dropwise to a solution of
.alpha.-D-glucose (41) (50 g, 0,28 mol) in acetone (1 l) at
0.degree. C. The reaction mixture is stirred for 24 h and then it
is neutralized using sodium hydroxide (6 M). The acetone is
evaporated and the aqueous layer is extracted with chloroform
(.times.2). The chloroform is dried (MgSO.sub.4) filtered and
evaporated. Crystallization from cyclohexane yielded the
di-isopropylidene glucose (42) (41 g, 57%).
EXAMPLE 30
[0265]
1.2:5.6-Di-O-isopropylidene-3-O-methyl-.alpha.-D-glucofuranose
(43)
[0266] The .alpha.-D-glucofuranose (42) (41 g, 0,16 mol) in
tetrahydrofuran (300 ml) is added dropwise to a suspension of
sodium hydride (5 g) in tetrahydrofuran (200 ml). After 0,5 h,
methyl iodide (25 g) in tetrahydrofuran (100 ml) is added dropwise
to the reaction mixture which is then stirred for 24 h. Water is
added to the reaction mixture which is then extracted with ether
(.times.3). The ether layer is dried (MgSO.sub.4), filtered and
evaporated to yield the methyl protected glucose (43) (38 g,
83%).
EXAMPLE 31
[0267] 3-O-Methyl-.alpha..beta.-D-glucopyranoside (44)
[0268] The methyl diisopropylidene compound (43) (38 g, 0,14 mol)
is dissolved in acetic acid (50%, 700 ml) and the solution refluxed
for 18 h. After cooling the acetic acid is evaporated. The crude
product is purified by column chromatography eluting with
chloroform:methanol:aceton- e:water (70:27:2:1) to yield
3-O-methyl-.alpha..beta.-glucopyranoside (44) (13 g, 50%).
EXAMPLE 32
[0269] Methyl 3-O-methyl-.alpha..beta.-D-glucopyranoside (45)
[0270] The 3-O-methyl-.alpha..beta.-glucopyranoside (44) (10 g) is
dissolved in methanol (50 ml) and HCl (conc.) (1 ml) and refluxed
overnight. Solid NaHCO.sub.3 is added and the reaction is filtered.
The methanol is evaporated to give
1,3-di-O-methyl-.alpha..beta.-D-glucopyran- oside (45), (95%).
EXAMPLE 33
[0271] Methyl
4,6-O-benzylidene-3-O-methyl-.alpha..beta.-glucopyranoside (46)
[0272] The glucopyranoside (45) (8 g) is stirred at room
temperature in a solution of benzalaldehyde (20 ml) and zinc
chloride (5 g). After 24 hours, ice is added and the aqueous layer
is extracted with chloroform. The chloroform layer is dried
(MgSO.sub.4), filtered and evaporated. The benzalaldehyde is
removed by vacuum distillation and the product is purified by
silica gel column chromatography eluting with acetone:chloroform
(0,5:9,5), to yield benzylidene-.alpha..beta.-glucopyr- anoside
(46) (60%).
EXAMPLE 34
[0273] Methyl
4-O-benzoyl-O-methyl-6-deoxy-.alpha..beta.-glucopyranoside (47)
[0274] The benzylidene compound (46) (5 g) is refluxed at
80.degree. C. in a mixture of N-bromosuccinimide (3,7 g) and barium
carbonate (4 g) in carbon tetrachloride. After 4 hours, the
reaction is filtered and the carbon tetrachloride is washed with
water, dried (MgSO.sub.4), filtered and evaporated to give the
bromo compound (70%).
[0275] The bromo compound (4,3 g) is dissolved in a solution of
ethanol (300 ml) and nickel chloride (8,6 g) at 0.degree. C. To
this solution, sodium borohydride (8,6 g) in water (50 ml) is added
dropwise over; a period of 15 minutes. The reaction mixture is
refluxed at 100.degree. C. for 45 minutes, cooled, filtered and
evaporated. Chloroform is added, and the chloroform layer is washed
with water, dried (MgSO.sub.4), filtered and evaporated to give the
6-deoxy sugar. (47) (70%)
EXAMPLE 35
[0276]
4-O-Benzoyl-3-O-methyl-1-phenylthio-6-deoxy-.alpha..beta.-glucopyra-
noside (48)
[0277] The 6-deoxy glucopyranoside (47) (3 g) is dissolved in
dichloromethane (50 ml). To this solution,
phenylthiotrimethylsilane (2 g) and
trimethylsilyltrifluoromethanesulphonate (0,2 ml) are added. The
solution is stirred at room temperature overnight, after which
saturated sodium bicarbonate is added. The dichloromethane layer is
dried (MgSO.sub.4), filtered and evaporated. The product is
purified by silica gel column chromatography eluting with ethyl
acetate:hexane (2:8), to give the compound (48) (60%).
EXAMPLE 36
[0278] 4-O-Benzoyl-3-O-methyl-2-O-pivaloyl-1-phenylthio-6-deoxy
.alpha..beta.-glucopyranoside (49)
[0279] To a solution of the glucopyranoside (48) (2 g) in pyridine
(20 ml), pivaloyl chloride (2 ml) is added. The solution is stirred
at room temperature overnight after which water is added. The
aqueous layer is extracted with ethyl acetate, and the organic
layer is washed with HCl (6 N). The organic layer is dried
(MgSO.sub.4), filtered and evaporated to give the pivaloyl ester
(49) (80%).
EXAMPLE 37
[0280]
4-O-Benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-8-glucopyranos-
ide (50)
[0281] N-Bromosuccinimide (1,2 g) and diethylaminosulphur
trifluoride (1,2 g) are added to a solution of the pivaloyl ester
(49) (2 g) in dichloromethane (100 ml) at 0.degree. C. After 1
hour, saturated sodium bicarbonate is added. The dichloromethane
layer is dried (MgSO.sub.4), filtered and evaporated. The
.beta.-fluoropyranoside (50) is purified by silica gel column
chromatography eluting with ethyl acetate:hexane (2:8), (yield
45%).
[0282] Example 38 illustrates the synthetic procedure whereby the
compound
3-O-(4-O-benzoyl-2-phenylthio-.beta.-D-cymaropyranosyl]-12,14.beta.-dihyd-
roxy-pregnan-5-ene-20-one(51) may be prepared.
EXAMPLE 38
[0283] 3-O-[4-O-benzoyl-2-phenylthio-.beta.-D-cymaropyranosyl]-12,
14.beta.-dihydroxy-pregn-5-en-20-one (51)
[0284] Tin chloride (190 mg, 1 mmol) is added to a solution of
3,12,14.beta.-trihydroxy pregnan-5-ene-20-one (15) (100 mg, 0,28
mmol) and the fluorocymaropyranoside (38) (210 mg, 0,56 mmol), in
dry diethyl ether and 4 .ANG. molecular sieves at -15.degree. C.
The reaction mixture is maintained at -15.degree. C. for 3 days.
Saturated sodium bicarbonate is added to the reaction mixture. The
ether layer is dried (MgSO.sub.4), filtered and evaporated. The
product is purified by silica gel column chromatography eluting
with chloroform methanol (9, 5:0,5) to yield the glycoside (51) (30
mg, 15
[0285] Examples 39 to 41 illustrate the synthetic procedures
whereby the cymarose and thevetose moieties may be coupled.
EXAMPLE 39
[0286] Thevetose-cymarose Dissaccharide (53)
[0287] A solution of thevetose (50 A) (1,5 g), cymarose (40) (1,3
g), and molecular sieves 4A in dichloromethane is stirred at room
temperature for 1 hour. The reaction mixture is cooled to
-15.degree. C., and tin (II) chloride (0,8 g) and silver
trifluoromethanesulphonate (1,1 g) are added. The mixture is
stirred at -15.degree. C. for 16 hours, after which triethylamine
(0,5 ml) is added. The reaction product is filtered and the
dichloromethane is evaporated. The dissaccharide (53) is purified
by silica gel column chromatography eluting with ethyl
acetate:hexane (2:8), yield 15%.
EXAMPLE 40
[0288] Thevetose-cymarose Dissaccharide (54)
[0289] To a solution of the dissaccharide (53) (200 mg) in
tetrahydrofuran (20 ml), tetrabutylammonium fluoride (0,4 ml) is
added. The mixture is stirred at room temperature for 1 hour, after
which saturated sodium bicarbonate is added. The reaction mixture
is extracted with ethyl acetate and the ethyl acetate layer is
dried (MgSO.sub.4), filtered and evaporated. The dissaccharide (54)
is purified by silica gel column chromatography
(acetone:chloroform, 0,5:9,5) yield 60%.
EXAMPLE 41
[0290] Thevetose-cymarose Dissaccharide (55)
[0291] To a solution of the dissaccharide (54) (80 mg) in
dichloromethane (10 ml), diethylamino sulphur trifluoride (80
.mu.l) is added at 0.degree. C. After stirring at 0.degree. C. for
0,5 hour, saturated sodium bicarbonate and more dichloromethane are
added. The dichloromethane is dried (MgSO.sub.4), filtered and
evaporated. Purification by silica gel column chromatography (ethyl
acetate:hexane 1:9), gives the dissaccharide (55) in a 65%
yield.
EXAMPLE 42
[0292] The results of the following three bioassays on the appetite
suppressant are set out below, viz.
[0293] a) Irwin Test;
[0294] b) Acute Toxicity Test; and
[0295] c) Oral Dose Anorectic Test.
[0296] a) Irwin Test
[0297] The purpose of this test was to evaluate the appetite
suppressant of the invention produced from a plant extract as
hereinbefore described, according to the reduced animal Irwin test
for tranquillising and sedative action.
[0298] Experimental Procedure
[0299] The appetite suppressant was extracted from plant material
by the Applicant by the method as hereinbefore described and
administered to two of four groups of three animals each: one group
receiving no treatment, one group receiving the solvent
dimethylsulfoxide (DMSO), one group receiving the test sample at 50
mg/kgt and one group receiving the test sample at 300 mg/kg.
Treatment took place by intraperitoneal injection, and observations
were made at specific intervals up to five hours post treatment.
Only symptoms other than those observed in the DMSO-treated animals
were used in the interpretation of the results.
[0300] Results
[0301] It was clear that the solvent, DMSO, had a marked effect on
the animals, especially on the heat regulating mechanism. Body
temperatures of all the animals treated with the solvent, alone or
together with the test sample, showed a marked drop.
[0302] Animals in the low dose group showed decreased dispersion in
the cage and decreased locomotor activity, as in all the other
groups, including the control group. Apathy was seen in the same
degree as in the DMSO-treated group. Decreased respiration was
observed 15-60 minutes after treatment. Ptosis (closing of the
eyelids) was also observed to a larger degree than in the DMSO
group. A pinna (ear) response was seen as well as a positive finger
response, indicating fearfulness. Body temperature dropped to
32,7.degree. C. after treatment.
[0303] Animals in the high dose group showed as in the other groups
an initial decreased dispersion in the cage and decreased locomotor
activity, but showed increased dispersion and locomotor activity
before death, which occurred approximately 1 hour after treatment.
Severe clonic symmetrical convulsions occurred 30 minutes after
treatment. Respiration decreased initially, but increased before
death. A pinna (ear) response was delayed and a positive finger
response was observed, indicating fearfulness, both as observed in
animals in the low dose group. Body temperature dropped to
30,7.degree. C. after treatment. Increased positional passivity was
observed as well as decreased body tone. Abnormal limb rotation was
observed, the grip strength decreased, no pain response was present
and loss of righting reflex occurred.
[0304] Discussion
[0305] When compared with the control and DMSO-treated animals,
animals receiving the low dose (50 mg/kg) only showed decreased
respiration and an increased degree of ptosis. Animals receiving
the high dose (300 mg/kg) of the test sample reacted very intensely
by showing convulsions and death. All other observations made in
these animals can be ascribed to the animals being in convulsions
and dying. Signs suggestive of tranquillising and sedative actions
such as marked decreased dispersion in the cages, decreased
locomotor activity and apathy in the test groups that could be
ascribed to the test sample were not seen.
[0306] It can therefore be concluded that the test sample is lethal
to mice at 300 mg/kg and has respiratory suppressive effects on
mice at 50 mg/kg, when given intraperitoneally with DMSO as
solvent.
[0307] b) Acute Toxicity Test
[0308] The purpose of this test was to gain information on the
toxicity of the test sample.
[0309] Experimental Procedure
[0310] A plant extract prepared in accordance with the invention as
hereinbefore described, and having appetite suppressive action was
purified and one test sample was tested at increasing doses by oral
treatment in mice. Two animals were used per dose group, except in
the highest dose group where only one animal was treated. Animals
were examined for good health and their body masses determined on
the day of treatment.
[0311] Doses ranged from 100 mg/kg up to 3 028,5 mg/kg. The dose
was calculated and mixed into prepared potato starch, so that each
animal received a total dose of 0,2 ml. Animal 13 received 0,25 ml.
Potato starch was prepared by mixing 20 g starch into a small
volume of cold water, and adding it to boiling water, to make up a
volume of 1 litre. The suspension was allowed to cool to room
temperature before dosing.
[0312] Animals in groups 1 and 2 were treated on the same day. They
were observed for 24 hours and if no signs of toxicity developed,
the next group was treated. The same approach was followed until
all the animals were treated. This schedule was followed to ensure
that animals were not unnecessarily treated when an acute toxic
dose had been reached in the previous group.
[0313] Animals were observed for clinical signs of toxicity
immediately (1-2 hours) after treatment and daily thereafter. Body
mass was determined once a week and total food and water intakes of
each animal were measured.
[0314] Surviving animals were euthanased by intraperitoneal
injection of pentobarbitone sodium (commercially available under
the trade name Euthanaze, Centaur %) on day 14 of the experiment. A
post-mortem examination was performed on these animals, as well as
on the one animal which died during the experiment. Samples for
histopathology were collected.
[0315] Results
[0316] Group 1 (Control Group)
[0317] No clinical signs of toxicity were observed during the
14-day observation period. Food and water intakes were within the
normal parameters. Changes in body mass were also within normal
parameters. No histopathological changes were recorded in the liver
samples.
[0318] Group 2 (100 mg/kg)
[0319] No clinical signs of toxicity were observed during the
observation period. Food and water intakes were normal and changes
in body mass over the observation period were also normal. No
macroscopical pathology was observed and no histopathological or
morphological changes were recorded in the liver samples.
[0320] Group 3 (200 mg/kg)
[0321] Animals in this group showed no clinical symptoms of
toxicity during the experiment. Food and water intakes were normal,
as was the change in body mass. No macroscopic pathology was
observed, but the livers showed histopathological changes on
examination. Cloudy swelling of the hepatocytes was mild in animal
6, but moderate in animal 5. Moderate hydropic degeneration also
occurred in the hepatocytes of animal 5.
[0322] Group 4 (400 mg/kg)
[0323] No clinical signs of toxicity were observed during the
observation period, and no macroscopic pathology was observed
during the post-mortem examination. Moderate cloudy swelling and
mild hydropic changes of the hepatocytes were observed on
histology.
[0324] Water and food intakes and the increase in body mass in
animal 7 were normal. Animal 8 consumed almost double the total
food intake of animal 7 (144,6 g and 73,9 g respectively), but the
increase in body mass was only 0,81 g compared to 2,7 g.
[0325] Group 5 (800 mg/kg)
[0326] One animal (animal 10) died three hours after dosing without
showing any specific signs. The other animal (animal 9) survived
the entire observation period without any signs of toxicity. Water
intake in the surviving animal was normal (42,42 ml), while food
intake was high (134,2 g). The body mass increased by 2,85 g which
was the highest of all animals in the experiment.
[0327] At the post-mortem examination of animal 10, which died
shortly after oral dosing, the lungs were congested. No foreign
body reaction which would have indicated inhalation of test
material was present. No macroscopic pathology was observed in
animal 9. Mild cytoplasmic vacuolisation (hydropic degeneration)
was present in animal 10, but moderate in animal 9. The glandular
cytoplasmic appearance of the liver was classified as moderate in
both animals.
[0328] Group 6 (1 600 mg/kg)
[0329] None of the animals presented any clinical signs of toxicity
during the duration of the experiment. No macroscopic pathology was
observed at post-mortem examination, but moderate degenerative
changes in the liver of animal 11 were observed at
histopathological examination. Animal 12 showed moderate cloudy
swelling and mild hydropic changes of the hepatocytes. Food and
water intakes were normal, as was the increase in body mass over
the experimental period.
[0330] Group 7 (3 028.5 mg/kg)
[0331] Only one animal was treated at this dose. This animal showed
no signs of toxicity during the observation period, and no
macroscopic pathology was observed. At histopathological
examination, moderate cloudy swelling and hydropic degeneration of
the hepatocytes was observed. The animal showed a loss of body mass
over the observation period (-0,82 g), but food and water intakes
were normal.
[0332] Discussion
[0333] Since a very small number of animals were used in each dose
group, it is difficult to make any conclusions. The fact that only
one animal died at a low dose rate, without showing any symptoms,
might indicate that death was not related to the test sample, but
due to stress during and/or after treatment. No animals in higher
dose groups died or showed any signs of toxicity, which further
supports this assumption.
[0334] The increased food intake observed in animal 8 could
possibly be ascribed to excessive spillage of food as was reflected
in the small increase in body mass. It should be kept in mind that
all the animals in this experiment were only treated once, and that
it is unlikely that an appetite suppressor will have a marked
influence on either the food or water intakes, or body mass over a
14 day period, as was the case in this experiment.
[0335] From the histopathological examination of the liver samples,
it was clear that the pathological changes were dose related, with
animals receiving higher doses showing the extensive changes. The
pathology observed was not metabolic of nature, but possibly test
sample-induced. The changes were only degenerative and therefore
reversible. No signs of irreversible hepatocellular changes were
observed.
[0336] It can, therefore, be concluded that only one animal died at
a lower dose (800 mg/kg), but that the death was possibly not test
sample related. None of the other animals in any of the dose groups
showed any signs of toxicity during the 14 day observation period
after treatment, or died as result of the treatment. A single oral
dose of the test sample induced reversible dose-related
hepatocellular changes.
[0337] c) Oral Dose Anorectic Test
[0338] The purpose of this test was to determine the activity of a
plant extract prepared in accordance with the invention, and the
minimum effective dose, and at the same time investigate any
possible side-effects such as respiratory suppression, as
experienced in the Irwin Test (referred to above).
[0339] Experimental Procedure
[0340] Animals were allocated to treatment groups using
randomisation tables. Each treatment group consisted of three
animals, with 6 animals in the control group. The test sample was
dosed to young female rats with body weight 100-150 g at
acclimatisation, for three consecutive days. Animals were
identified by means of metallic ear tags and KMnO.sub.4 skin
markings for easy identification. Animals were housed individually
in standard rodent polycarbonate cages, and water and powdered
commercial rodent pellets were available ad libitum. Water and food
intakes were measured and calculated for each day. In order to find
the minimum effective dose of the test sample, five doses were
tested. Treatment was by oral gavage, with the test sample
suspended in potato starch.
[0341] The test substance was compound (1), a white granular powder
prepared from an extract from plant material in accordance with the
invention, and the measured quantity of the test sample was mixed
with prepared potato starch and dosed. Mixing with potato starch
took place immediately before dosing on each day. Before withdrawal
of the dosing volume for each animal, the suspensions were mixed
thoroughly using a Vortex.
[0342] A range of five doses was tested, with a control group
receiving only the carrier substance. Doses were chosen on the
basis of the effects observed in the aforedescribed Irwin Test and
were:
[0343] Group 1: 0,00 mg/kg (Control Group)
[0344] Group 2: 6,25 mg/kg
[0345] Group 3: 12,50 mg/kg
[0346] Group 4: 25,00 mg/kg
[0347] Group 5: 37,50 mg/kg
[0348] Group 6: 50,00 mg/kg
[0349] Results
[0350] Treatment did not affect the health of the animals during
the study period. Animals treated with the test sample in all dose
groups, showed a significantly reduced mean body mass gain over the
total study period, and animals in three of the five treatment
groups actually lost body mass.
[0351] Mean food intakes for all the treatment groups were reduced
over the study period. Animals in the higher dose groups showed an
increased water consumption.
[0352] Respiratory rate in none of the animals in any dose group
was significantly effected.
[0353] Animals in all dose groups presented with friable livers at
post-mortem examination, but no macroscopic pathology was
observed.
[0354] Discussion
[0355] Data collected during the acclimatisation period confirmed
that all animals included in the experiment were healthy and body
mass gain was comparable between the animals.
[0356] The reduction, and in some animals even a loss, in body mass
gain, in combination with the reduced food intake is strongly
indicative of suppression of the appetite centre.
[0357] Reduced food intake and reduced body mass gain was
experienced even with the lowest dose group (6,25 mg/kg). Actual
loss in body mass was experienced in the 12,50 mg/kg group.
[0358] It is important to note that the treatment groups all had an
increased water consumption when feed consumption decreased (FIG.
2). This could be due to a diuretic effect of the test sample, or
to stimulation of the thirst centre in the brain.
[0359] The fact that no respiratory suppression occurred as had
been observed in the acute toxicity test referred to above, with
the intraperitoneal route, is seen as a positive aspect. This could
be due to reduced absorption from the gastrointestinal tract, with
consequent reduced bioavailability. The bioavailability at the oral
doses tested was, however, sufficient for the test sample to be
effective. The slight reduction in respiratory rate 1 hour post
treatment in most groups could be ascribed to filling of the
stomach with the dose volume and consequent passivity of the
animals.
[0360] The friable livers observed in the treatment groups could be
due to a change in the energy metabolism secondary to the reduced
food intake, causing increased fat metabolism and overload on the
liver. If this was indeed the case, these changes could possibly be
regarded these changes as transitory which might recover with time
after a steady state had been reached, or after withdrawal of the
test sample. The possible effect on the liver also needs further
investigation.
[0361] Since this study was intended primarily as a screening test,
small groups of test animals were used. This makes statistical
interpretation of the data difficult, especially where individual
animals react totally differently. However, the data indicates that
the test sample has appetite suppressive action, even at the lowest
dose tested (6,25 mg/kg). No clinical signs of respiratory
suppression occurred at the doses tested.
EXAMPLE 43
[0362] Harvested Hoodia plants received either from the natural
environment or through a cultivation programme are first stored at
4.degree. C. for a maximum of 48 hours. The plants are washed in
tap water and thereafter sliced into .+-.1 cm slices. The sliced
pieces are all combined and then pressed through a hydraulic press
at 300 bar pressure for a minimum of 0.5 hour per pressing. During
the pressing the sap of the plant is collected separately. The sap
is stored at -18.degree. C. until further processing is
required.
[0363] The sap is spray-dried under suitable conditions to obtain a
free flowing powder. The moisture content in the powder is
preferably less than 5 after spray drying and, if necessary, it is
further dried in a vacuum oven or using a fluid bed drier.
[0364] Both the sap and the spray-dried material have been shown
effective as an appetite suppressant in biological assays in
rats.
[0365] Experimental
[0366] 50 kg of Hoodia gordonii plants were washed with tap water
and thereafter sliced into 1 cm slices. The sliced plants were then
pressed through a hydraulic press at 300 bar for a minimum of 0.5
hour per batch. The sap was collected and the mass was found to be
10 kg when Hoodia gordonii plants from the environment were used,
and 20 kg when Hoodia gordonii plants from the cultivation
programme was used. The sap (500 g) was spray-dried using the
following conditions:
3 Flow rate 2.85 ml/min Inlet temperature 110.degree. C. Outlet
temperature 70.degree. C. Chamber temperature 78.degree. C.
[0367] The spray-dried powder obtained was a free flowing powder
(22 g) with a moisture content of 6.9%.
[0368] The spray dried powder was analysed for active ingredient
concentration using HPLC techniques. The concentration of the
active was determined to be 13 g/kg of spray dried powder.
[0369] HPLC Analysis Method
4 Eluant Acetonitrile: water (7:3), isocratic Column Reverse phase
C-18 UV absorbance 225 nm Flow rate 1 ml/min Injection volume 10
.mu.l
[0370] Method
[0371] Spray-dried powder (10 mg) was dissolved in water (0.5 ml)
and acetonitrile (0.5 ml) 10 .mu.l of this solution was injected
into the HPLC and the concentration of the active compound (1) was
determined using a standard curve which was prepared from the pure
compound (1)
EXAMPLE 44
[0372] The results of a study designed to assess the possible
anorectic effects of compound (1) in the rat are presented below.
In the following, the samples tested are pure sap (Sample 1),
spray-dried sap (Sample 2) and active moiety (Sample 3). Samples 1
and 2 are the sap and the spray-dried sap respectively, as
described in Example 43 above. Sample 3 is solvent-extracted
compound (1) of .gtoreq.95% purity.
[0373] Sample 1 to 3 were each administered as a single oral dose
to male Wistar rats Two additional control groups received vehicle
(distilled water or DMSO). Orally administered fenfluramine (7.5
mg/kg) was included as a reference standard. Sample 1 (pure sap)
administered orally, produced dose-dependent reductions in food
consumption which were statistically significant at doses of 1600
mg/kg and above when compared with vehicle-treated controls.
Concomitant reductions in bodyweight (or growth rate) were also
recorded. On the day of dosing, statistically significant increases
in water consumption were recorded at 3 hours post-dose (6400 and
10000 mg/kg) and 6 hours post-dose (10000 mg/kg). Between 24 and 48
hours post-dose, statistically significant reductions in water
consumption were recorded at doses of 3200 mg/kg and above.
[0374] Sample 2 (spray-dried sap) administered orally at 76 mg/kg
also produced statistically significant reductions in food
consumption and bodyweight when compared with vehicle-treated
animals. No statistically significant effectson water consumption
were recorded.
[0375] Sample 3 (active moiety) produced statistically significant
reductions in food consumption at an oral dose of 5.0 mg/kg. No
statistically significant effects on bodyweights were produced by
the active moiety although examination of the data revealed a
slight delay in growth when compared with vehicle-treated control
animals. No statistically significant effects on water consumption
were recorded.
[0376] The reference standard, fenfluramine (7.5 mg/kg), produced
statistically significant reductions in food consumption at 6 and
24 hours post-dose when compared with the relevant vehicle-treated
control group. No statistically significant effects on water
consumption or bodyweight were recorded.
[0377] No treatment-related effects on the livers were
recorded.
5 TEST SUBSTANCE Identity Sample 1 (pure sap) Sample 2 (spray-dried
sap) Sample 3 (active moiety) Appearance Brown liquid Powder White
powder Storage conditions -20.degree. C. in the dark Room
temperature in the 4.degree. C. in the dark dark Purity Pure sap
Pure spray-dried sap .gtoreq.95% Vehicle Distilled water Distilled
water Dimethylsulphoxide (DMSO)
[0378] Experimental Procedure
[0379] Fifty-five male Wistar rats were used for the study.
[0380] Bodyweights, food consumption (food hopper weight) and water
consumption (bottle weight) were recorded daily at the same time
each day from the day of arrival until the termination of the
study.
[0381] On Day 1, the rats received a single oral (gavage) dose
according to the following table:
6 Dose Group n Oral treatment (mg/kg) 1 5 Vehicle (distilled water)
-- 2 4 Sample 1 (pure sap) 800 3 5 Sample 1 (pure sap) 1600 4 5
Sample 1 (pure sap) 3200 5 5 Sample 1 (pure sap) 6400 6 5 Sample 1
(pure sap) 10000 7 5 Sample 2 spray-dried sap 38 8 5 Sample 2
spray-dried sap 76 9 5 Sample 3 (active moiety) 2.5 10 5 Sample 3
(active moiety) 5.0 11 3 Fenfluramine 7.5 12 3 Vehicle (DMSO)
--
[0382] Groups 1-8 were dosed using a constant dose volume of 10
ml/kg and groups 9-12 were dosed using a dose volume of 1
ml/kg.
[0383] Food and water consumption were also measured at 1,3 and 6
hours after dosing on Day 1.
[0384] Following the measurements of Day 8, the animals were killed
by carbon dioxide asphyxiation, and the livers excised and placed
in 10% buffered formalin, prior to histology.
[0385] Paraffin wax sections of each liver were taken at 4-5 .mu.m
and stained with haematoxylin and eosin. Additional sections were
cut on a cryostat at 12 .mu.m and stained for fat with Oil Red O
(ORO).
[0386] Data Analysis
[0387] The post-dose food and water consumption measurements and
bodyweights at each time-point for the P57-treated animals were
compared with those for the relevant, similarly-treated vehicle
control group using analysis of variance followed by Williams' test
for comparisons with controls.
[0388] The data for the fenfluramine-treated animals was compared
with that for the vehicle-treated control group using Student's t
test.
[0389] Results
[0390] The results are summarised in the tables.
[0391] Sample 1 (pure sap) administered orally produced marked,
dose-related reductions in daily food consumption. The duration and
amplitude of these reductions in food consumption were
dose-dependent. At 24 hours post-dose, Sample 1 (pure sap) produced
statistically significant reductions in food consumption at doses
of 1600 mg/kg and above when compared with vehicle-treated
controls. The highest dose of Sample 1 (sap) (10000 mg/kg) produced
statistically significant reductions in food consumption on a daily
basis up to 5 days post-dose.
[0392] Sample 2 (spray-dried sap) and Sample 3 (active moiety)
produced marked and statistically significant reductions in food
consumption at oral doses of 76 and 5.0 mg/kg respectively. In both
cases the effects lasted 48 hours post-dose. The reference
standard, fenfluramine (7.5 mg/kg, p.o.) produced statistically
significant reductions in food consumption at 6 and 24 hours
post-dose when compared with the relevant vehicle-treated control
group (Group 12).
[0393] Sample 2 (spray-dried sap) and Sample 3 (active moiety)
produced no marked, dose-related effects on water consumption. On
the day of dosing, the pure sap produced statistically significant
increases in water consumption at 3 hours post-dose (6400 and 10000
mg/kg) and 6 hours post-dose (10000 mg/kg). Two days after dosing
however, statistically significant decreases in water consumption
were recorded in animals receiving Sample 1 (sap) at 3200, 6400 and
10000 mg/kg. These reductions however, were not clearly
dose-related and only occurred between 1 and 2 days post-dose. The
biological significance of these effects therefore remains
unclear.
[0394] Sample 1 (pure sap) produced dose-related, statistically
significant effects on bodyweights when compared with the
vehicle-treated control group (Group 1). When administered orally
at doses of 3200 mg/kg and above, Sample 1 (pure sap) produced
statistically significant reductions in bodyweight or decreased
growth rates when compared with vehicle-treated animals. These
effects were statistically significant from 48 hours post-dose
until the end of the study.
[0395] Sample 2 (spray-dried sap) administered orally at 76 mg/kg
also produced statistically significant reductions in growth of the
animals when compared with the vehicle-treated control group (Group
1). These effects were statistically significant between Days 3 (48
hours post-dose) and 5 inclusive.
[0396] Although Sample 3 (active moiety) appeared to delay the
growth of the animals at the highest dose (5.0 mg/kg) when compared
with the relevant vehicle-treated control group (Group 12), this
effect was not statistically significant.
[0397] Fenfluramine, (7.5 mg/kg) produced no marked or
statistically significant effects on water consumption or
bodyweights when compared with the vehicle-treated control group
(Group 12).
[0398] No treatment-related effects on the livers were
recorded.
7TABLE 1a Effects of oral administration on food consumption in the
rat (daily pre-dose data) Group mean food consumption (g .+-. sd)
between Days: Group Oral treatment Dose (mg/kg) -6--5 -5--4 -4--3
-3--2 -2--1 1 Vehicle (water) -- 27.8 .+-. 1.54 24.2 .+-. 1.83 27.6
.+-. 3.67 28.3 .+-. 3.50 29.4 .+-. 2.66 2 Sample 1 sap 800 28.3
.+-. 1.43 24.9 .+-. 0.82 27.7 .+-. 0.76 28.4 .+-. 1.51 30.1 .+-.
0.27 3 Sample 1 sap 1600 29.0 .+-. 1.39 25.0 .+-. 2.16 27.4 .+-.
1.96 28.8 .+-. 0.61 29.5 .+-. 1.55 4 Sample 1 sap 3200 27.2 .+-.
2.33 25.1 .+-. 2.46 26.0 .+-. 2.52 28.5 .+-. 2.29 27.6 .+-. 1.15 5
Sample 1 sap 6400 28.7 .+-. 1.64 25.3 .+-. 1.73 27.3 .+-. 1.45 29.2
.+-. 1.09 30.3 .+-. 0.90 6 Sample 1 sap 10000 28.5 .+-. 2.38 23.7
.+-. 2.73 26.0 .+-. 2.31 27.0 .+-. 3.50 28.7 .+-. 2.26 7 Sample 2
spray-dried 38 28.1 .+-. 1.24 23.9 .+-. 1.79 24.5 .+-. 2.30 27.6
.+-. 1.61 28.5 .+-. 1.87 8 Sample 2 spray-dried 76 28.7 .+-. 0.91
26.5 .+-. 1.55 27.1 .+-. 1.01 28.7 .+-. 1.99 28.9 .+-. 1.37 9
Sample 3 active moiety 2.5 28.8 .+-. 1.49 26.4 .+-. 3.12 29.0 .+-.
1.99 29.4 .+-. 1.76 29.5 .+-. 2.81 10 Sample 3 active moiety 5.0
28.3 .+-. 2.1 25.8 .+-. 1.86 28.1 .+-. 2.65 28.0 .+-. 2.65 28.5
.+-. 3.03 11 Fenfluramine 7.5 29.1 .+-. 0.66 25.3 .+-. 4.03 27.0
.+-. 1.53 30.8 .+-. 0.54 29.7 .+-. 2.84 12 Vehicle (DMSO) -- 27.9
.+-. 1.8 26.7 .+-. 2.11 28.7 .+-. 1.99 28.1 .+-. 4.06 30.5 .+-.
2.54 sd Standard deviation
[0399]
8TABLE 1b Effects of oral administration on food consumption in the
rat (daily post-dose data) Group mean food consumption (g .+-. sd)
between Days: Group Oral treatment Dose (mg/kg) 1-2 2-3 3-4 4-5 1
Vehicle (water) -- 29.5 .+-. 3.15 29.6 .+-. 2.84 30.6 .+-. 3.49
31.8 .+-. 3.21 2 Sample 1 sap 800 26.1 .+-. 0.98 29.3 .+-. 1.49
30.7 .+-. 1.15 30.9 .+-. 0.60 3 Sample 1 sap 1600 22.6 .+-. 3.17**
26.9 .+-. 2.06 30.9 .+-. 2.54 30.9 .+-. 1.22 4 Sample 1 sap 3200
20.1 .+-. 1.39** 19.0 .+-. 1.88** 22.8 .+-. 1.77** 28.0 .+-. 3.14 5
Sample 1 sap 6400 18.2 .+-. 4.18** 14.8 .+-. 1.75** 18.4 .+-.
0.97** 22.4 .+-. 3.01** 6 Sample 1 sap 10000 15.1 .+-. 2.98** 12.4
.+-. 2.61** 16.0 .+-. 3.15** 19.7 .+-. 4.31** 7 Sample 2
spray-dried 38 25.6 .+-. 2.85 27.3 .+-. 0.95 30.3 .+-. 2.06 31.0
.+-. 2.13 8 Sample 2 spray-dried 76 24.2 .+-. 3.25* 25.2 .+-. 3.24*
29.9 .+-. 1.85 30.2 .+-. 2.28 9 Sample 3 active moiety 2.5 26.8
.+-. 3.33 29.1 .+-. 3.43 31.7 .+-. 3.08 34.0 .+-. 2.95 10 Sample 3
active moiety 5.0 .sup. 22.1 .+-. 2.19.sup..dagger..dagger. .sup.
21.0 .+-. 3.07.sup..dagger..dagger. 27.6 .+-. 5.26 30.5 .+-. 3.33
11 Fenfluramine 7.5 .sup. 22.4 .+-. 3.19.sup..dagger. 31.9 .+-.
0.84 32.7 .+-. 2.50 33.0 .+-. 2.55 12 Vehicle (DMSO) -- 29.9 .+-.
3.36 30.6 .+-. 4.43 30.1 .+-. 4.17 32.4 .+-. 5.26 Group mean food
consumption (g .+-. sd) between Days: Group Oral treatment Dose
(mg/kg) 5-6 6-7 7-8 1 Vehicle (water) -- 30.7 .+-. 2.24 31.7 .+-.
3.03 32.9 .+-. 3.18 2 Sample 1 sap 800 33.3 .+-. 1.69 32.7 .+-.
0.80 40.1 .+-. 13.40 3 Sample 1 sap 1600 34.1 .+-. 1.36 33.7 .+-.
1.69 33.8 .+-. 1.61 4 Sample 1 sap 3200 31.4 .+-. 2.82 32.3 .+-.
2.91 33.0 .+-. 3.01 5 Sample 1 sap 6400 26.9 .+-. 2.81 31.0 .+-.
2.31 32.0 .+-. 2.34 6 Sample 1 sap 10000 22.6 .+-. 5.70* 30.1 .+-.
4.79 32.6 .+-. 5.90 7 Sample 2 spray-dried 38 31.8 .+-. 1.63 31.1
.+-. 1.94 31.8 .+-. 2.45 8 Sample 2 spray-dried 76 31.2 .+-. 2.26
32.3 .+-. 1.44 33.1 .+-. 0.61 9 Sample 3 active moiety 2.5 34.4
.+-. 4.32 33.1 .+-. 4.11 34.8 .+-. 3.71 10 Sample 3 active moiety
5.0 33.0 .+-. 3.16 32.4 .+-. 3.25 33.0 .+-. 3.84 11 Fenfluramine
7.5 30.4 .+-. 0.23 32.7 .+-. 1.90 32.4 .+-. 1.60 12 Vehicle (DMSO)
-- 31.8 .+-. 3.08 32.8 .+-. 3.98 33.3 .+-. 3.76 sd Standard
deviation Groups 2-8 were compared with vehicle Group 1: *p <
0.05, **p < 0.01 Groups 9-11 were compared with vehicle Group
12: .sup..dagger.p < 0.05, .sup..dagger..dagger.p < 0.01
[0400]
9TABLE 2a Effects of oral administration on water consumption in
the rat (daily pre-dose data) Group mean water consumption (g .+-.
sd) between Days: Group Oral treatment Dose (mg/kg) -6--5 -5--4
-4--3 -3--2 -2--1 1 Vehicle (water) -- 40.9 .+-. 4.61 34.8 .+-.
4.15 37.6 .+-. 5.63 33.5 .+-. 7.42 32.2 .+-. 6.32 2 Sample 1 sap
800 38.6 .+-. 1.96 37.1 .+-. 9.74 36.4 .+-. 4.81 28.1 .+-. 1.83
30.4 .+-. 4.75 3 Sample 1 sap 1600 43.4 .+-. 10.53 35.9 .+-. 3.84
38.4 .+-. 4.56 31.1 .+-. 4.47 36.5 .+-. 5.39 4 Sample 1 sap 3200
40.1 .+-. 5.58 33.3 .+-. 3.01 37.3 .+-. 4.46 31.3 .+-. 3.48 31.7
.+-. 3.18 5 Sample 1 sap 6400 43.8 .+-. 8.57 36.3 .+-. 9.02 35.4
.+-. 8.18 34.0 .+-. 6.62 35.1 .+-. 5.72 6 Sample 1 sap 10000 37.4
.+-. 5.34 32.7 .+-. 3.35 33.2 .+-. 4.86 29.0 .+-. 5.11 32.2 .+-.
3.27 7 Sample 2 spray-dried 38 40.0 .+-. 4.36 35.8 .+-. 4.92 34.7
.+-. 3.20 30.2 .+-. 1.88 31.4 .+-. 2.98 8 Sample 2 spray-dried 76
38.6 .+-. 1.98 37.0 .+-. 1.96 48.8 .+-. 21.5 31.6 .+-. 4.56 39.0
.+-. 17.27 9 Sample 3 active moiety 2.5 42.0 .+-. 6.70 37.0 .+-.
5.05 34.1 .+-. 3.16 28.0 .+-. 2.58 31.6 .+-. 3.12 10 Sample 3
active moiety 5.0 40.9 .+-. 4.48 34.2 .+-. 3.00 32.7 .+-. 1.26 28.2
.+-. 1.65 33.1 .+-. 4.82 11 Fenfluramlne 7.5 47.0 .+-. 5.3 35.5
.+-. 7.49 34.7 .+-. 3.73 30.9 .+-. 2.12 31.6 .+-. 2.80 12 Vehicle
(DMSO) -- 43.3 .+-. 5.67 34.5 .+-. 4.97 35.2 .+-. 4.34 28.3 .+-.
4.64 31.4 .+-. 6.44 sd Standard deviation
[0401]
10TABLE 2b Effects of oral administration on water consumption in
the rat (daily post-dose data) Group mean water consumption (g .+-.
sd) between Days: Group Oral treatment Dose (mg/kg) 1-2 2-3 3-4 4-5
1 Vehicle (water) -- 34.9 .+-. 5.45 36.9 .+-. 6.06 38.0 .+-. 7.59
37.2 .+-. 6.18 2 Sample 1 sap 800 30.9 .+-. 3.77 34.4 .+-. 8.12
38.2 .+-. 13.71 35.9 .+-. 13.51 3 Sample 1 sap 1600 29.2 .+-. 1.66
31.7 .+-. 5.35 41.3 .+-. 11.21 34.6 .+-. 4.10 4 Sample 1 sap 3200
35.9 .+-. 5.88 26.2 .+-. 2.66* 30.5 .+-. 2.44 34.1 .+-. 4.80 5
Sample 1 sap 6400 33.4 .+-. 12.04 27.4 .+-. 8.13* 32.6 .+-. 10.67
35.4 .+-. 10.78 6 Sample 1 sap 10000 31.7 .+-. 12.74 28.5 .+-.
8.85* 32.4 .+-. 8.87 36.6 .+-. 6.50 7 Sample 2 spray-dried 38 36.0
.+-. 6.02 34.5 .+-. 1.79 38.2 .+-. 7.16 39.6 .+-. 7.09 8 Sample 2
spray-dried 76 45.0 .+-. 19.03 39.1 .+-. 16.59 46.9 .+-. 18.34 35.9
.+-. 3.40 9 Sample 3 active moiety 2.5 32.2 .+-. 4.01 36.1 .+-.
12.42 38.3 .+-. 11.71 41.5 .+-. 16.60 10 Sample 3 active moiety 5.0
33.9 .+-. 2.40 31.5 .+-. 8.12 35.1 .+-. 3.82 37.7 .+-. 5.99 11
Fenfluramine 7.5 34.1 .+-. 3.60 37.2 .+-. 1.48 36.7 .+-. 3.92 33.8
.+-. 2.89 12 Vehicle (DMSO) -- 40.7 .+-. 9.10 33.8 .+-. 9.37 32.9
.+-. 7.07 35.2 .+-. 11.49 Group mean water consumption (g .+-. sd)
between Days: Group Oral treatment Dose (mg/kg) 5-6 6-7 7-8 1
Vehicle (water) -- 37.7 .+-. 5.54 35.3 .+-. 2.86 36.5 .+-. 5.85 2
Sample 1 sap 800 39.5 .+-. 11.20 28.8 .+-. 1.22 31.8 .+-. 5.58 3
Sample 1 sap 1600 48.1 .+-. 12.27 37.8 .+-. 7.28 36.9 .+-. 9.28 4
Sample 1 sap 3200 45.8 .+-. 18.54 51.0 .+-. 35.21 42.6 .+-. 13.88 5
Sample 1 sap 6400 45.2 .+-. 8.72 36.2 .+-. 6.72 35.9 .+-. 9.58 6
Sample 1 sap 10000 40.7 .+-. 11.51 38.0 .+-. 6.66 37.5 .+-. 6.21 7
Sample 2 spray-dried 38 42.7 .+-. 9.74 45.6 .+-. 17.15 46.1 .+-.
9.49 8 Sample 2 spray-dried 76 41.9 .+-. 12.37 36.9 .+-. 8.47 38.1
.+-. 8.93 9 Sample 3 active moiety 2.5 34.7 .+-. 7.57 33.0 .+-.
4.20 35.3 .+-. 8.70 10 Sample 3 active moiety 5.0 39.5 .+-. 7.78
37.4 .+-. 11.07 37.8 .+-. 6.42 11 Fenfluramine 7.5 33.7 .+-. 5.43
32.1 .+-. 1.93 33.6 .+-. 2.50 12 Vehicle (DMSO) -- 33.8 .+-. 9.82
32.3 .+-. 7.44 32.0 .+-. 7.22 sd Standard deviation Groups 2-8 were
compared with vehicle Group 1: *p < 0.05 Groups 9-11 were
compared with vehicle Group 12 (no significances)
[0402]
11TABLE 3a Effects of oral administration on bodyweight in the rat
(daily pre-dose data) Group mean bodyweight (g .+-. sd on Day:
Group Oral treatment Dose (mg/kg) -5 -4 -3 -2 -1 1 Vehicle (water)
-- 130.9 .+-. 5.56 150.7 .+-. 5.37 157.3 .+-. 5.29 168.1 .+-. 6.20
177.5 .+-. 6.70 2 Sample 1 sap 800 131.6 .+-. 4.34 150.1 .+-. 4.84
158.5 .+-. 4.35 169.6 .+-. 4.99 177.7 .+-. 4.10 3 Sample 1 sap 1600
130.1 .+-. 4.3 148.6 .+-. 6.59 156.7 .+-. 6.38 167.5 .+-. 6.04
176.6 .+-. 6.37 4 Sample 1 sap 3200 130.8 .+-. 6.19 147.7 .+-. 7.56
154.4 .+-. 8.06 165.2 .+-. 8.43 175.8 .+-. 9.10 5 Sample 1 sap 6400
132.6 .+-. 7.01 151.3 .+-. 7.23 158.4 .+-. 8.50 169.0 .+-. 8.79
178.1 .+-. 7.75 6 Sample 1 sap 10000 132.3 .+-. 6.75 151.8 .+-.
9.08 157.3 .+-. 9.37 167.1 .+-. 10.41 175.4 .+-. 10.90 7 Sample 2
spray-dried 38 131.7 .+-. 8.28 149.0 .+-. 5.85 156.2 .+-. 5.81
166.7 .+-. 5.54 175.6 .+-. 8.42 8 Sample 2 spray-dried 76 130.0
.+-. 6.99 146.1 .+-. 6.00 155.9 .+-. 6.59 166.0 .+-. 6.87 175.1
.+-. 6.55 9 Sample 3 active moiety 2.5 132.6 .+-. 7.63 148.9 .+-.
8.51 157.3 .+-. 8.91 169.8 .+-. 8.96 179.4 .+-. 8.71 10 Sample 3
active moiety 5.0 133.5 .+-. 6.45 150.5 .+-. 9.55 158.8 .+-. 8.48
171.0 .+-. 7.72 179.0 .+-. 9.20 11 Fenfluramine 7.5 133.2 .+-. 9.21
152.7 .+-. 9.09 160.0 .+-. 9.82 170.0 .+-. 9.15 182.8 .+-. 10.21 12
Vehicle (DMSO) -- 129.1 .+-. 3.17 147.3 .+-. 4.37 155.0 .+-. 6.29
166.0 .+-. 5.91 174.8 .+-. 8.26 sd Standard deviation
[0403]
12TABLE 3b Effects of oral administration on bodyweight in the rat
(daily post-dose data) Group mean bodyweight (g .+-. sd) on Day:
Group Oral treatment Dose (mg/kg) Pre-dose (1) 2 3 4 5 1 Vehicle
(water) -- 185.4 .+-. 7.77 192.6 .+-. 7.16 202.0 .+-. 10.17 211.2
.+-. 7.98 220.2 .+-. 10.35 2 Sample 1 sap 800 186.0 .+-. 4.90 187.0
.+-. 4.55 198.5 .+-. 4.20 206.8 .+-. 5.91 214.8 .+-. 4.65 3 Sample
1 sap 1600 185.0 .+-. 6.67 186.0 .+-. 8.28 193.2 .+-. 6.42 204.0
.+-. 6.40 212.4 .+-. 5.81 4 Sample 1 sap 3200 181.8 .+-. 9.18 184.6
.+-. 8.88 186.2 .+-. 8.67* 189.8 .+-. 9.99** 199.2 .+-. 9.34** 5
Sample 1 sap 6400 186.6 .+-. 7.96 185.6 .+-. 6.39 183.8 .+-. 6.87**
185.2 .+-. 9.18** 191.2 .+-. 7.89** 6 Sample 1 sap 10000 182.8 .+-.
12.22 181.4 .+-. 14.06 179.8 .+-. 15.85** 180.6 .+-. 13.85** 185.6
.+-. 11.28** 7 Sample 2 spray-dried 38 183.4 .+-. 8.11 185.8 .+-.
9.23 195.8 .+-. 7.79 205.6 .+-. 9.79 214.4 .+-. 9.61 0 Sample 2
spray-dried 76 180.6 .+-. 6.47 183.4 .+-. 7.57 188.6 .+-. 6.73*
198.2 .+-. 8.50* 206.0 .+-. 9.43* 9 Sample 3 active moiety 2.5
188.2 .+-. 9.42 191.2 .+-. 11.15 200.0 .+-. 11.25 209.6 .+-. 12.28
219.6 .+-. 12.95 10 Sample 3 active moiety 5.0 186.4 .+-. 10.02
192.0 .+-. 9.93 192.4 .+-. 9.84 201.0 .+-. 11.27 209.4 .+-. 12.70
11 Fenfluramine 7.5 190.3 .+-. 9.71 190.3 .+-. 10.97 197.7 .+-.
7.37 207.7 .+-. 7.23 217.7 .+-. 10.69 12 Vehicle (DMSO) -- 183.3
.+-. 8.33 190.3 .+-. 10.26 199.0 .+-. 10.82 207.7 .+-. 12.66 215.7
.+-. 14.05 Group mean bodyweight (g .+-. sd) on Day: Group Oral
treatment Dose (mg/kg) 6 7 8 1 Vehicle (water) -- 227.2 .+-. 10.26
235.8 .+-. 11.82 242.8 .+-. 11.97 2 Sample 1 sap 800 222.8 .+-.
4.99 231.5 .+-. 3.70 240.0 .+-. 3.65 3 Sample 1 sap 1600 223.0 .+-.
6.33 232.6 .+-. 7.70 240.4 .+-. 6.66 4 Sample 1 sap 3200 210.6 .+-.
10.21** 219.0 .+-. 11.29* 228.4 .+-. 12.18* 5 Sample 1 sap 6400
201.0 .+-. 6.89 213.0 .+-. 6.96** 222.0 .+-. 7.94** 6 Sample 1 sap
10000 192.2 .+-. 10.99** 203.4 .+-. 11.68 212.4 .+-. 11.35** 7
Sample 2 spray-dried 38 222.6 .+-. 9.34 231.4 .+-. 10.62 239.6 .+-.
11.46 0 Sample 2 spray-dried 76 214.0 .+-. 9.51 222.0 .+-. 9.49
232.2 .+-. 9.68 9 Sample 3 active moiety 2.5 229.4 .+-. 13.69 238.4
.+-. 14.50 247.0 .+-. 14.35 10 Sample 3 active moiety 5.0 219.8
.+-. 11.86 228.2 .+-. 12.28 236.0 .+-. 13.95 11 Fenfluramine 7.5
224.3 .+-. 10.12 234.3 .+-. 12.70 243.3 .+-. 9.24 12 Vehicle (DMSO)
-- 222.3 .+-. 14.84 230.7 .+-. 15.95 239.0 .+-. 17.35 sd Standard
deviation Groups 2-8 were compared with vehicle Group 1: *p <
0.05, **p < 0.01 Groups 9-11 wore compared with vehicle Group 12
(no significances)
[0404] Histopathology Report
[0405] Histological examination was restricted to the liver. No
treatment-related changes were detected for Sample 1 (liquid),
Sample 2 (spray-dried sap), Sample 3 (active moiety), fenfluramine
or the DMSO control group.
[0406] The findings recorded were of a similar incidence in control
and treated groups.
13TABLE Microscopic pathology incidence summary Group 1 Group 2
Group 3 Group 4 Group 5 Group 6 0 800 1600 3200 6400 10000 Sex:
Males mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Males on study 5 4 5 5 5
5 Animals completed 5 4 5 5 5 5 Liver 5 Examined 5 4 5 5 5 0 No
abnormalities detected 0 0 1 2 3 3 Parenchymal inflammatory cell
foci (Total) 0 1 0 0 0 3 Minimal 0 1 0 0 0 1 Hepatocyte hypertrophy
- centrilobular (Total) 0 0 0 0 0 1 Minimal 0 0 0 0 0 0
Extramedullary haemopoiesis (Total 2 0 0 0 0 0 Minimal 2 0 0 0 0 0
Hepatocyte necrosis - focal (Total) 1 0 0 0 0 0 Minimal 1 0 0 0 0 0
Portal lymphoid infiltration (Total) 3 4 4 3 2 2 Minimal 3 4 4 3 2
2 Eosinophilic hepatocytes - focal (Total) 1 0 0 0 0 0 Minimal 1 0
0 0 0 0 Portal fibrosis (Total) 0 0 1 0 0 0 Minimal 0 0 1 0 0 0
Liver (ORO stain) Examined 5 4 5 5 5 5 No abnormalities detected 2
3 2 4 3 3 Hepatocyte fat - centrilobular (Total) 3 1 2 1 2 2
Minimal 3 1 2 1 2 2 Hepatocyte fat - periportal (Total) 0 0 1 0 0 0
Minimal 0 0 1 0 0 0 Group 7 Group 8 Group 9 Group 10 Group 11 Group
12 38 76 2.5 5 7.5 0 Sex: Males mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Males on study 5 5 5 5 3 3 Animals completed 5 5 5 5 3 3 Liver
Examined 5 5 5 5 3 3 No abnormalities detected 2 2 0 1 0 2
Parenchymal inflammatory cell foci (Total) 0 0 0 0 0 1 Minimal 0 0
0 0 0 1 Hepatocyte necrosis - focal (Total) 0 0 1 0 0 0 Minimal 0 0
1 0 0 0 Portal lymphoid infiltration (Total) 3 3 5 4 3 1 Minimal 3
3 5 4 3 1 Portal leucocytes (Total) 0 0 1 0 0 0 Minimal 0 0 1 0 0 0
Liver (ORO stain) Examined 5 5 5 5 3 3 No abnormalities detected 5
3 3 3 2 2 Hepatocyte fat - centrilobular (Total) 0 2 2 2 1 1
Minimal 0 2 2 2 1 0
EXAMPLE 45
[0407] A further bioassay, which employed the same test samples as
described in Example 44, is described below. Animals in this study
received a restricted diet i.e. animals only received food between
12:00 and 3:00 pm daily. This is different from all other
biological assays conducted thus far, whereby food was available to
the rats at lib. Animals were acclimatised over a seven day period
(days -7 to -1), dosing took place from day 0 to day 6 at 9:00am by
oral gavage. The recovery period was from days 7 to day 13. Dosage
groups are described in Table 1 below. It should be noted that the
actual control group is labelled Group 09. Group 5 is a controlled
group which received a diet equivalent to that of Group 4. The
purpose of this group was to evaluate the effect a restricted diet
has on the lives of the animals.
[0408] Results
[0409] The results generated during the study showed that the
acclimatization period was too short. Rats feed mainly during the
night and the sudden change to a restricted access to feed for 3
hours during day-time, resulted in low daily intakes. The daily
intake of feed was still increasing in most groups at the end of
the acclimatization period when dosing with the test items started.
As a result of this, the effect of the test materials did not
significantly affect the food intake of the rats during the period
of dosing.
[0410] The mean body masses for the different groups for day -7 to
-1 and days 0 to 6 are shown in the Table D1 and Table D2. The
effect of the different dosages of the sap and spray-dried sap is
shown in the accompanying graphs as % change in body mass day 0 to
7 (FIG. 5), and % change in body mass day -7 to 7 (FIG. 6). The
loss in body mass is clearly dose-related especially with the
higher dosages.
[0411] The histopathological examination of the livers did not show
any significant pathology in the groups receiving the test
items.
[0412] Food
[0413] Food consumption was measured daily, during acclimatization
and during the study. Food was available for a 3 hour feeding
period daily, starting at 12:00 and ending at 15:00. The animals
were fasted for the remainder of the time. Animals in Group 5
received a measured quantity food on Day 1, equivalent to the
average food consumption of Group 4 on Day 0. This controlled
feeding pattern for Group 5, as determined from the average food
consumption of Group 4 from the previous day, was followed for Days
1-7.
[0414] Water
[0415] Water was provided in standard containers. Water (Magalies
Water Board Tap Water, suitable for human consumption) was
available ad libitum. Water consumption was measured once daily, at
the same time each day, after food consumption determination.
[0416] Acclimatization
[0417] The animals were acclimatized for seven days before the
start of the study, during which time food and water consumption
were determined as described above. The body masses were determined
on a daily basis during this time.
[0418] Study Design and Procedures
14TABLE 1 STUDY DESIGN GROUP TEST NUMBERS DOSE TEST ITEM 01 6.male.
001-006 100 mg/kg Frozen sap 02 6.male. 007-012 400 mg/kg Frozen
sap 03 6.male. 013-018 1600 mg/kg Frozen sap 04 6.male. 019-024
3200 mg/kg Frozen sap 05 6.male. 025-030 CONTROL Elga Option 4
Purified Water 06 6.male. 031-036 .sup. 2.2 mg/kg Spray-dried sap
07 6.male. 037-042 .sup. 8.8 mg/kg Spray-dried sap 08 6.male.
043-048 35 mg/kg Spray-dried sap 09 6.male. 049-054 CONTROL Elga
Option 4 Purified Water
[0419] Route of Administration
[0420] The test items were administered on a daily basis for seven
days, using an intra-gastric needle. Animals were fasted for 18
hours prior to the item administration (starting at 09:00).
[0421] Duration of Treatment
[0422] Animals were treated for seven consecutive days (from Day
0-Day 6). Three animals of each group were sacrificed 24 hours
after the last dosing (Day 7). The remaining three animals were
sacrificed 7 days after the last treatment (Day 13). This procedure
was followed for all the groups except for Group 5 where three
animals were sacrificed 24 hours after the last controlled feeding
(Day 8), the remaining three animals were sacrificed 7 days after
the last treatment (Day 13).
[0423] Body Masses
[0424] Body masses were determined daily, at approximately the same
time each day for the duration of the study, including during the
acclimatization period.
[0425] Euthanasia
[0426] Three animals of each group were sacrificed 24 hours after
the last dosing (Day 7).
[0427] The remaining three animals were sacrificed 7 days after the
last treatment. This procedure was followed for all the groups
except for Group 5 where three animals were sacrificed 24 hours
after the last controlled feeding (Day 8), the remaining three
animals were sacrificed 7 Days after the last treatment (Day 13).
The animals were euthanased at the end of the study period with
CO.sub.2 gas.
[0428] Ophthalmoscopic Examinations
[0429] Ophthalmoscopic examinations, using an ophthalmoscope, were
done prior to the first adminstration of the test item and at
termination, in all animals in all groups.
[0430] Macroscopic Pathology
[0431] A full post mortem examination was performed on every animal
which was euthanased at the end of the study period.
[0432] Histopathology
[0433] Histopathological examination was performed on the liver of
each of the animals.
15TABLE D.1 MEAN BODY MASSES/GROUP/WEEK Mean body masses (g) &
Standard deviation Group Oral treatment Dose (mg/kg) Day -7 Day -6
Day -5 Day -4 01 Sample 1 (Sap) 100 203.38 .+-. 95.39 197.13 .+-.
90.63 192.75 .+-. 89.49 188.62 .+-. 86.75 02 Sample 1 (Sap) 400
192.53 .+-. 65.60 183.92 .+-. 61.20 178.25 .+-. 59.37 173.17 .+-.
58.10 03 Sample 1 (Sap) 1600 149.25 .+-. 54.80 142.87 .+-. 51.89
136.85 .+-. 52.17 132.37 .+-. 49.64 04 Sample 1 (Sap) 3200 224.15
.+-. 80.70 214.45 .+-. 77.25 207.10 .+-. 76.38 201.82 .+-. 75.42 05
Elga Option 4 -- 214.55 .+-. 74.90 204.85 .+-. 72.41 198.57 .+-.
71.79 193.48 .+-. 68.49 purified water (control) 06 Sample 2
(Spray-dried sap) 2.2 208.65 .+-. 65.74 199.37 .+-. 62.49 193.18
.+-. 61.18 188.25 .+-. 60.89 07 Sample 2 (Spray-dried sap) 8.8
256.95 .+-. 77.55 246.02 .+-. 73.67 237.47 .+-. 73.53 232.62 .+-.
71.73 08 Sample 2 (Spray-dried sap) 35 194.37 .+-. 43.74 185.83
.+-. 42.70 177.53 .+-. 41.10 172.05 .+-. 40.13 09 Elga Option 4 --
171.52 .+-. 69.81 162.67 .+-. 62.68 154.95 .+-. 61.83 151.38 .+-.
59.48 purified water (control) Mean body masses (g) & Standard
deviation Group Oral treatment Dose (mg/kg) Day -3 Day -2 Day -1 01
Sample 1 (Sap) 100 184.95 .+-. 84.80 182.48 .+-. 83.47 182.25 .+-.
82.57 02 Sample 1 (Sap) 400 170.82 .+-. 57.42 168.25 .+-. 58.40
169.37 .+-. 59.25 03 Sample 1 (Sap) 1600 131.50 .+-. 49.50 129.67
.+-. 48.89 131.12 .+-. 48.22 04 Sample 1 (Sap) 3200 198.25 .+-.
74.82 194.83 .+-. 75.34 196.77 .+-. 74.56 05 Elga Option 4 --
192.40 .+-. 67.48 190.87 .+-. 67.39 190.15 .+-. 65.24 purified
water (control) 06 Sample 2 (Spray-dried sap) 2.2 186.22 .+-. 59.98
184.55 .+-. 58.86 185.97 .+-. 58.76 07 Sample 2 (Spray-dried sap)
8.8 229.78 .+-. 71.76 228.07 .+-. 69.88 228.45 .+-. 68.81 08 Sample
2 (Spray-dried sap) 35 170.10 .+-. 39.49 167.25 .+-. 37.61 168.00
.+-. 38.83 09 Elga Option 4 -- 149.63 .+-. 57.66 148.30 .+-. 57.12
149.07 .+-. 56.01 purified water (control)
[0434]
16TABLE D.2 MEAN BODY MASSES/GROUP/WEEK Mean body masses (g) &
Standard deviation Group Oral treatment Dose (mg/kg) Day 0 Day 1
Day 2 Day 3 01 Sample 1 (Sap) 100 183.87 .+-. 83.33 175.83 .+-.
81.82 175.72 .+-. 79.05 175.48 .+-. 77.54 02 Sample 1 (Sap) 400
173.45 .+-. 60.73 164.58 .+-. 58.52 164.75 .+-. 58.37 166.22 .+-.
57.69 03 Sample (Sap) 1600 134.38 .+-. 46.01 129.20 .+-. 44.74
127.53 .+-. 43.20 127.20 .+-. 41.36 04 Sample (Sap) 3200 199.60
.+-. 75.16 196.38 .+-. 73.96 192.20 .+-. 71.20 189.05 .+-. 69.11 05
Elga Option 4 -- 194.27 .+-. 67.46 187.93 .+-. 65.48 181.97 .+-.
65.01 177.53 .+-. 64.73 purified water (control) 06 Sample 2
(Spray-dried sap) 2.2 189.07 .+-. 60.15 181.52 .+-. 58.99 181.48
.+-. 57.79 184.42 .+-. 55.64 07 Sample 2 (Spray-dried sap) 8.8
230.28 .+-. 69.32 221.55 .+-. 68.02 220.17 .+-. 66.63 221.80 .+-.
63.88 08 Sample 2 (Spray-dried sap) 35 169.10 .+-. 38.40 164.42
.+-. 38.03 162.50 .+-. 36.81 162.75 .+-. 36.36 09 Elga Option 4 --
151.02 .+-. 55.45 146.55 .+-. 53.77 148.10 .+-. 52.67 149.70 .+-.
52.05 purified water (control) Mean body masses (g) & Standard
deviation Group Oral treatment Dose (mg/kg) Day 4 Day 5 Day 6 01
Sample 1 (Sap) 100 175.53 .+-. 76.20 177.95 .+-. 73.99 178.43 .+-.
72.68 02 Sample 1 (Sap) 400 166.55 .+-. 57.79 169.93 .+-. 57.47
171.77 .+-. 57.29 03 Sample (Sap) 1600 126.70 .+-. 39.19 128.00
.+-. 39.22 128.07 .+-. 38.66 04 Sample (Sap) 3200 186.57 .+-. 66.29
186.05 .+-. 67.45 185.68 .+-. 65.73 05 Elga Option 4 -- 174.73 .+-.
61.08 172.85 .+-. 58.63 171.45 .+-. 56.79 purified water (control)
06 Sample 2 (Spray-dried sap) 2.2 185.75 .+-. 55.29 189.35 .+-.
54.66 189.68 .+-. 53.70 07 Sample 2 (Spray-dried sap) 8.8 222.82
.+-. 63.56 224.82 .+-. 62.38 224.90 .+-. 62.05 08 Sample 2
(Spray-dried sap) 35 162.52 .+-. 36.93 164.30 .+-. 37.69 164.22
.+-. 37.18 09 Elga Option 4 -- 152.58 .+-. 50.37 155.82 .+-. 49.91
157.85 .+-. 49.70 purified water (control)
[0435]
17TABLE D.3 MEAN BODY MASSES/GROUP/WEEK (CONTINUED) Dose Mean body
masses (g) & Standard deviation Group Oral treatment (mg/kg)
Day 7 Day 8 Day 9 Day 10 01 Sample 1 (Sap) 100 185.38 .+-. 72.64
234.73 .+-. 62.44 236.73 .+-. 62.39 234.07 .+-. 62.09 (GHA I 35A)
02 Sample 1 (Sap) 400 178.83 .+-. 58.24 225.63 .+-. 13.05 277.13
.+-. 14.18 227.10 .+-. 14.03 (GHA I 35A) 03 Sample 1 (Sap) 1600
132.22 .+-. 37.08 133.80 .+-. 55.17 135.23 .+-. 455.74 134.53 .+-.
54.96 (GHA I 35A) 04 Sample 1 (Sap) 3200 188.57 .+-. 66.14 199.63
.+-. 61.07 198.90 .+-. 57.48 198.70 .+-. 54.55 (GHA 9 35A) 05 Elga
Option 4 -- 173.97 .+-. 54.29 172.98 .+-. 52.06 157.80 .+-. 58.62
158.87 .+-. 57.76 purified water (control) 06 Sample 2 (Spray-dried
sap) 2.2 196.00 .+-. 53.09 190.27 .+-. 27.78 190.27 .+-. 29.54
192.60 .+-. 29.09 (GHA I 59) 07 Sample 2 (Spray-dried sap) 8.8
231.30 .+-. 61.91 177.27 .+-. 24.48 178.17 .+-. 23.79 180.67 .+-.
25.04 (GHA I 59) 08 Spray-dried sap 35 167.48 .+-. 36.75 164.90
.+-. 22.54 166.63 .+-. 23.08 168.43 .+-. 22.66 (GHA I 59) 09 Elga
Option 4 -- 165.50 .+-. 49.27 193.73 .+-. 22.37 196.87 .+-. 21.86
198.07 .+-. 21.02 purified water (control) Dose Mean body masses
(g) & Standard deviation Group Oral treatment (mg/kg) Day 11
Day 12 Day 13 01 Sample 1 (Sap) 100 236.33 .+-. 62.31 239.07 .+-.
60.24 238.43 .+-. 59.85 (GHA I 35A) 02 Sample 1 (Sap) 400 229.43
.+-. 16.97 234.93 .+-. 18.35 236.20 .+-. 15.97 (GHA I 35A) 03
Sample 1 (Sap) 1600 138.30 .+-. 53.03 139.30 .+-. 51.10 142.80 .+-.
49.51 (GHA I 35A) 04 Sample 1 (Sap) 3200 194.73 .+-. 52.78 194.93
.+-. 50.78 197.93 .+-. 51.57 (GHA 9 35A) 05 Elga Option 4 -- 160.80
.+-. 57.67 163.40 .+-. 56.27 167.80 .+-. 58.49 purified water
(control) 06 Sample 2 (Spray-dried sap) 2.2 194.73 .+-. 29.68
196.97 .+-. 29.04 198.60 .+-. 30.18 (GHA I 59) 07 Sample 2
(Spray-dried sap) 8.8 182.03 .+-. 25.31 185.10 .+-. 24.60 189.73
.+-. 23.58 (GHA I 59) 08 Spray-dried sap 35 171.67 .+-. 24.42
174.90 .+-. 25.70 178.57 .+-. 23.58 (GHA I 59) 09 Elga Option 4 --
199.83 .+-. 20.21 204.93 .+-. 18.65 207.13 .+-. 18.22 purified
water (control)
[0436]
18TABLE 1 HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS
Sample 1 Animal no Hepatic lesions GROUP 1: 100 mg/kg Sample 1 Day
7 01 NPL 02 NPL 03 NPL C1 + Day 13 04 NPL MLC 05 FHS1 + 06 NPL
GROUP 2: 400 mg/kg Sample 1 07 FHS1 + 08 NPL C1 + 09 NPL Day 13 10
DHS1 + 11 NPL 12 DHS1 + GROUP 3: 1600 mg/kg Sample 1 Day 7 13 NPL
14 NPL 15 NPL Day 13 16 NPL 17 DHS1 + 18 NPL GROUP 4: 3200 mg/kg
Sample 1 19 NPL 20 NPL 21 NPL Day 13 22 DHS1 + 23 FHS1 + 24 NPL
GROUP 5: CONTROL: ELGA OPTION 4 PURIFIED WATER: RESTRICTED FOOD
INTAKE GROUP 5: Control: Elga option 4 purified water Day 7 25 NPL
MLC 26 NPL 27 NPL Day 13 28 DHS1 + 29 DHS1 + 30 NPL Legend: C =
Congestion DHS = Diffuse hydropic cell swelling FHS = Focal
hydropic cell swelling NPL = No parenchymal lesions MLC = Minimal
lymphocytic cuffing 1+ = mild 2+ = moderate 3+ = severe
[0437]
19TABLE 2 HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS
Sample 2 Animal no Hepatic lesions GROUP 6: 2.2 mg/kg Sample 2 Day
7 31 NPL 32 NPL MLC 33 FHS1 + Day 13 34 NPL 35 DHS1 + 36 NPL GROUP
7: 8.8 mg/kg Sample 2 37 NPL 38 NPL 39 NPL C1 + Day 13 40 DHS1 + 41
NPL 42 MLC FHS1 + GROUP 8: 35 mg/kg Sample 2 Day 7 43 NPL 44 NPL 45
NPL Day 13 46 NPL 47 NPL C1 + 48 MLC FHS1 + GROUP 9: CONTROL: ELGA
OPTION 4 PURIFIED WATER GROUP 9: Control: Elga option 4 purified
water Day 7 49 NPL 50 NPL 51 FHS1 + Day 13 52 DHS1 + 53 NPL 54 FHS1
+ Legend: C = Congestion DHS = Diffuse hydropic cell swelling FHS =
Focal hydropic cell swelling NPL = No parenchymal lesions MLC =
Minimal lymphocytic cuffing 1 + = mild 2 + = moderate 3 + =
severe
[0438] No specific lesions were recorded in the liver sections from
the experimental rats which received the frozen sap as well as the
spray-dried sap that could be attributed to the oral adminstration
of the abovementioned chemicals. The hydropic cell swelling
recorded in both control and experimental rats may indicate normal
metabolic cell swelling and anoxic changes. Minimal foci of
lymphocytic perivascular cuffing were found in some animals and is
most likely an incidental observation. In a few rats congestion of
mild degree is present in the hepatic sinusoids and should be
regarded as an incidental observation.
[0439] An important feature of the invention shown by the results
of this study is that no tolerance to any of the samples developed
over the test period. This may provide considerable benefit,
particularly in relation to the use of the compounds and
compositions of the invention in the treatment of obesity.
[0440] While the compounds and compositions of the invention have
primarily been described in relation to their properties as
appetite suppressants, it should be noted-that this
expression--"appetite suppressant"--is used herein to denote
activity which tends to limit appetite and/or increase the sense of
satiety, and thus tends to reduce total calorific intake; this in
turn tends to counteract obesity. Accordingly, this invention
extends to a method of treating, preventing or combating obesity in
a human or non-human animal which comprises administering to said
human or non-human animal an obesity treating, preventing or
combating amount of a compound of formula (2). A preferred
embodiment of this aspect of the invention utilises a composition
or extract containing a compound of formula (1).
[0441] The term "animal" as used herein extends to, but is not
restricted to, companion animals, e.g. household pets and
domesticated animals; non-limiting examples of such animals include
cattle, sheep, ferrets, swine, camels, horses, poultry, fish,
rabbits, goats, dogs and cats.
[0442] As an anorectic agent or in the treatment or prevention of
obesity in a human, a compound of formula (2), preferably of
formula (1), or the composition defined in any one of claims 9 and
25-31 hereafter, is advantageously administered to said human in a
dosage amount of from about 0.01 mg/kg/day to about 10 mg/kg/day. A
preferred dosage range is 0.05 mg/kg/day to 0.5 mg/kg/day. When
using the spray dried powder form of the extract of this invention,
a preferred dosage range is 0.1 mg/kg/day to 20 mg/kg/day;
especially preferred is 0.5 mg/kg/day to 5 mg/kg/day.
* * * * *