U.S. patent application number 10/752057 was filed with the patent office on 2004-07-15 for antiprotozoal saponins.
This patent application is currently assigned to Janssen Pharmaceutica N.V.. Invention is credited to De Kimpe, Norbert G. M., Germonprez, Nils Albert Gilbert, Maes, Louis Jules Roger Marie, Ngoc Ninh, Tran, Van Puyvelde, Luc Emiel Mathilde.
Application Number | 20040138151 10/752057 |
Document ID | / |
Family ID | 8234537 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040138151 |
Kind Code |
A1 |
Maes, Louis Jules Roger Marie ;
et al. |
July 15, 2004 |
Antiprotozoal saponins
Abstract
Saponins of formula 1 a stereoisomeric form thereof or a
pharmaceutically acceptable addition salt thereof, wherein R.sub.1
to R.sub.12 have the meaning given in the description, can be
isolated from plants of the family Myrsinaceae and used to decrease
the infectiousness of and reduce the mortality associated with
protozoan parasites of the genus Leishmania which are responsible
for a group of conditions known as leishmaniases.
Inventors: |
Maes, Louis Jules Roger Marie;
(Wechelderzande, BE) ; Germonprez, Nils Albert
Gilbert; (Blankenberge, BE) ; Van Puyvelde, Luc Emiel
Mathilde; (Waasmunster, BE) ; De Kimpe, Norbert G.
M.; (Destelbergen, BE) ; Ngoc Ninh, Tran;
(Hanoi, VN) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Janssen Pharmaceutica N.V.
National Center for Science and Technology
Universiteit Gent
|
Family ID: |
8234537 |
Appl. No.: |
10/752057 |
Filed: |
January 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10752057 |
Jan 6, 2004 |
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09868755 |
Sep 12, 2001 |
|
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09868755 |
Sep 12, 2001 |
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PCT/EP99/10177 |
Dec 15, 1999 |
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Current U.S.
Class: |
514/33 ;
536/18.1 |
Current CPC
Class: |
A61P 33/02 20180101;
C07H 15/256 20130101; C07H 17/08 20130101; Y02A 50/30 20180101;
A61K 36/185 20130101 |
Class at
Publication: |
514/033 ;
536/018.1 |
International
Class: |
C07H 015/24; A61K
031/704 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 1998 |
EP |
982044091 |
Claims
1. A process for the isolation of triterpene saponins from plants
belonging to the family Myrsinaceae, characterized in that said
process comprises the steps of (a) exracting the dried plant parts
with an alcohol and concentrating the extract, (b) removing the
apolar fraction from the extract by liquid-liquid extraction with
an apolar solvent, and (c) further purifying the saponins in the
alcohol extract by liquid-liquid extraction, filtration and
chromatography.
2. A process according to claim 1 wherein the alcohol is methanol,
ethanol, isopropanol, butanol, each optionally admixed with
water.
3. A process according to claim 1 wherein the saponins of the
alcohol extract are further purified by (c6) extracting the aqueous
fraction with butanol satured with water, (c7) evaporating the
organic layer to dryness, (c8) washing the residue in a ketone, and
(c9) filtering off the crude saponin mixture.
4. A process according to claim 1 wherein the saponins are isolated
from the plant species Maesa balansae, and the chromatography
comprises straight phase chromatography liquid chromatography on
silicagel or reversed-phase liquid chromatography with gradient
eluent system using A: 0.5% ammonium acetate in water B: methanol
C: acetonitrile wherein at t=0, (A:B:C)=(60:20:20) and t=end,
(A:B:C)=(0:50:50).
5. A triterpene saponin obtainable by a process according to anyone
of claims 1 to 4.
6. A triterpene saponin according to claim 5 wherein said saponin
is isolated from the plant species Maesa balansae, and the
chromatography comprises reversed-phase liquid chromatography with
gradient eluent system using A: 0.5% ammonium acetate in water B:
methanol C: acetonitrile wherein at t=0, (A:B:C)=(60:20:20) and
t=end, (A:B:C)=(0:50:50), and wherein said saponin has the
following characteristics: Compound 1: MW=1532,
.lambda..sub.max=228.6 nm, .lambda..sub.max2=273.3 nm; Compound 2:
MW=1510, .lambda..sub.max=223.9 nm, .lambda..sub.max2=274.5 nm;
Compound 3: MW=1532, .lambda..sub.max=279.2 nm,
.lambda..sub.max2=223.9 nm; Compound 4: MW=1510,
.lambda..sub.max=280.4 nm, .lambda..sub.max2=222.7 nm; Compound 5:
MW=1574, .lambda..sub.max=276.8 nm, .lambda..sub.max2=225.0 nm; or
Compound 6: MW=1552, .lambda..sub.max=279.2 nm,
.lambda..sub.max2=223.9 nm.
7. A triterpene saponin having the formula 5wherein R.sub.2 is
--(C.dbd.O)C.sub.6H.sub.5 or
--O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3, R.sub.3 is (E) or (Z)
--O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5, and R.sub.4 is hydrogen or
--C.dbd.O)CH.sub.3.
8. A compound according to claim 7 wherein in compound 1, R.sub.2
is --O(C.dbd.O)C.sub.6H.sub.5, R.sub.3 is (Z)
--O(C.dbd.O)CH.dbd.CH--C.sub.6- H.sub.5, R.sub.4 is hydrogen; in
compound 2, R.sub.2 is --O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3,
R.sub.3 is (Z) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5, R.sub.4 is
hydrogen; in compound 3, R.sub.2 is --O(C.dbd.O)C.sub.6H.sub.5,
R.sub.3 is (E) --O(C--O)CH.dbd.CH--C.sub.6H.sub.5, R.sub.4 is
hydrogen; in compound 4, R.sub.2 is
--O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3, R.sub.3 is (E)
--O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5, R.sub.4 is hydrogen; in
compound 5, R.sub.2 is --O(C.dbd.O)C.sub.6H.sub.5, R.sub.3 is (E)
--O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5, R.sub.4 is
--(C.dbd.O)CH.sub.3; in compound 6, R.sub.2 is
--O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3, R.sub.3 is (E)
--O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5, R.sub.4 is
--(C.dbd.O)CH.sub.3.
9. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and as an active ingredient a triterpene
saponin as defined in claim 5, 6, 7 or 8.
10. A composition according to claim 7 adapted for parenteral
administration.
11. Use of one or more triterpene saponins for the preparation of a
pharmaceutical composition for treating leishmaniases in hosts
infected by Leishmania species, characterized in that the saponin
has the formula 6a stereoisomeric form thereof or a
pharmaceutically acceptable addition salt thereof, wherein R.sub.1
is hydrogen, --(C.dbd.O)C.sub.1-5alkyl,
--(C.dbd.O)C.sub.2-5alkenyl, --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl, a monosaccharide group or an
oligosaccharide group; R.sub.2 is hydrogen, hydroxy,
--O(C.dbd.O)C.sub.1-5alkyl, --O(C.dbd.O)C.sub.2-5alken- yl,
--O(C.dbd.O)C.sub.6H.sub.5, or --O(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl; R.sub.3 is hydrogen, hydroxy,
--O(C.dbd.O)C.sub.1-5alkyl, --O(C.dbd.O)C.sub.2-5alkenyl,
--O(C.dbd.O)C.sub.6H.sub.5, or --O(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl; R.sub.4 is hydrogen, C.sub.1-6alkyl,
--(C.dbd.O)C.sub.1-5alk- yl, --(C.dbd.O)C.sub.2-5alkenyl,
--(C.dbd.O)C.sub.6H.sub.5, or --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl; R.sub.5 is CH.sub.3, CH.sub.2OH,
CH.sub.2OCH.sub.3, CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, COOH; or
R.sub.5 and R.sub.2 form a divalent radical of formula
--C(.dbd.O)--O--; R.sub.6 and R.sub.7 are hydrogen; or taken
together they form a bond; or R.sub.5 and R.sub.6 form a divalent
radical of formula --CH.sub.2--O-- (a), --CH(OR.sub.13)--O-- (b),
--C(.dbd.O)--O-- (c), wherein R.sub.13 is hydrogen, C.sub.1-6alkyl
or --(C.dbd.O)C.sub.1-5alkyl; R.sub.8.alpha. and R.sub.8.beta. each
independently represent CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, CH(OCH.sub.3).sub.2,
CH.dbd.NOH, COOH; R.sub.8.beta. and R.sub.3 form a divalent radical
of formula --C(.dbd.O)--O--; R.sub.8.beta. and R.sub.5 form a
divalent radical of formula --CH.sub.2O--CHOH--; R.sub.9 is
CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH; R.sub.10 is
CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH; R.sub.11, is
hydrogen, hydroxy or O--C(.dbd.O)C.sub.1-5alkyl; or R.sub.10 and
R.sub.11, form a divalent radical of formula --CH.sub.2O--; and
R.sub.12 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, CH.dbd.NOH, or COOH.
12. Use according to claim 11 wherein R.sub.1 is hydrogen,
--(C.dbd.O)C.sub.1-5alkyl, or an oligosaccharide group; R.sub.3 is
hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkle- nyl, --O(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl; R.sub.4 is hydrogen, C.sub.1-6alkyl,
--(C.dbd.O)C.sub.1-5alkyl, --(C.dbd.O)C.sub.2-5alkenyl; R.sub.5 is
CH.sub.2OH, CH.sub.2O--C(.dbd.O)CH.sub.3, CHO; and R.sub.6 and
R.sub.7 taken together form a bond; or R.sub.5 and R.sub.6 form a
divalent radical of formula --CH.sub.2--O-- (a),
--CH(OR.sub.13)--O-- (b), --C(.dbd.O)--O-- (c), wherein R).sub.13
is hydrogen, C.sub.1-6alkyl or --(C.dbd.O)C.sub.1-5alky- l; and
R.sub.7 is hydrogen; R.sub.8.beta. represents CH.sub.3, CH.sub.2OH,
CHO, CH(OCH.sub.3).sub.2, CH.dbd.NOH, COOH; R.sub.8.alpha.
represents CH.sub.3; R.sub.8.beta. and R.sub.3 form a divalent
radical of formula --C(.dbd.O)--O--; or R.sub.8.beta. and R.sub.5
form a divalent radical of formula --CH.sub.2O--CHOH--; R.sub.10 is
CH.sub.3, CH.sub.2OH; R.sub.11 is hydrogen, hydroxy or
O--C(.dbd.O)C.sub.1-5alkyl; or R.sub.10 and R.sub.11 form a
divalent radical of formula --CH.sub.2O--; and R.sub.12 is
CH.sub.3, CH.sub.2OH, CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, or
CH.dbd.NOH.
13. Use according to claim 12 wherein R.sub.1 is hydrogen or an
oligosaccharide group; R.sub.2 is hydrogen, hydroxy,
--O(C.dbd.O)C.sub.1-5alkyl, --O(C.dbd.O)C.sub.2-5alkenyl,
--O(C.dbd.O)C.sub.6H.sub.5, or --O(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl; R.sub.3 is hydrogen, hydroxy,
--O(C.dbd.O)C.sub.1-5alkyl, --O(C.dbd.O)C.sub.2-5alkenyl,
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl; R.sub.4 is
hydrogen, C.sub.1-6alkyl, --(C.dbd.O)C.sub.1-5alk- yl,
--(C.dbd.O)C.sub.2-5alkenyl, --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl; R.sub.5 is CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, COOH; and R.sub.6 and R.sub.7
taken together form a bond; or R.sub.5 and R.sub.6 form a divalent
radical of formula --CH.sub.2--O-- (a), --CH(OR.sub.13)--O-- (b),
--C(.dbd.O)--O-- (c), wherein R.sub.13 is hydrogen; and R.sub.7 is
hydrogen; R.sub.8.alpha. and R.sub.8.beta. both represent CH.sub.3;
R.sub.9 is CH.sub.3; R.sub.10 is CH.sub.3; R.sub.11 is hydrogen;
and R.sub.12 is CH.sub.3.
14. A method of alleviating clinical manifestations of, and curing
disorders known as leishmaniases attributable to infection by
protozoan parasites of the genus Leishmania in both men and
animals, comprising administering to an infected host a
therapeutically effective amount of a compound of formula: 7a
stereoisomeric form thereof or a pharmaceutically acceptable
addition salt thereof, wherein R.sub.1 is hydrogen,
--(C.dbd.O)C.sub.1-5alkyl, --(C.dbd.O)C.sub.2-5alkenyl,
--(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl, a
monosaccharide group or an oligosaccharide group; R.sub.2 is
hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5, or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl; R.sub.3 is
hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5, or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl; R.sub.4 is
hydrogen, C.sub.1-6alkyl, --(C.dbd.O)C.sub.1-5alkyl,
--(C.dbd.O)C.sub.2-5alkenyl, --(C.dbd.O)C.sub.6H.sub.5, or
--(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl; R.sub.5 is
CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, COOH; or R.sub.5 and R.sub.2
form a divalent radical of formula --C(.dbd.O)--O--; R.sub.6 and
R.sub.7 are hydrogen; or taken together they form a bond; or
R.sub.5 and R.sub.6 form a divalent radical of formula
--CH.sub.2--O-- (a), --CH(OR.sub.13)--O-- (b), --C(.dbd.O)--O--
(c), wherein R.sub.13 is hydrogen, C.sub.1-6alkyl or
--(C.dbd.O)C.sub.1-5alkyl; R.sub.8.alpha. and R.sub.8.beta. each
independently represent CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, CH(OCH.sub.3).sub.2,
CH.dbd.NOH, COOH; R.sub.8.beta. and R.sub.3 form a divalent radical
of formula --C(.dbd.O)--O--; R.sub.8.beta. and R.sub.5 form a
divalent radical of formula --CH.sub.2O--CHOH--; R.sub.9 is
CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH; R.sub.10 is
CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH; R.sub.11 is
hydrogen, hydroxy or O--C(.dbd.O)C.sub.1-5alkyl; or R.sub.10 and
R.sub.11 form a divalent radical of formula --CH.sub.2O--; and
R.sub.12 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, CH.dbd.NOH, or COOH.
Description
[0001] The present invention is concerned with a process for the
isolation of antiprotozoal saponins from plants belonging to the
family Myrsinaceae and the use of said saponins for preparing a
medicament for treating hosts, both men and animals, infected by
protozoan parasites of the genus Leishmania, and for alleviating
clinical manifestations of, and curing disorders known as
leishmaniases in said hosts.
[0002] Leishmaniases present a large variety of disease
manifestations differing markedly in their severity and health
impact. Primarily, leishmaniases are debilitating conditions caused
by any of several species of Leishmania and are transmitted by
several Phlebotomine sandflies. The leishmaniases appear to be far
more abundant and of greater public health importance than has been
previously recognized. Control of leishmanial infections is
complicated because many species of sandfly are potential vectors,
because many animal species can act as reservoir hosts and because
diagnostic procedures (clinical, serological, parasitological) are
not always applicable or have limited acceptable diagnostic
value.
[0003] The manifestations may be visceral, mucocutaneous and/or
cutaneous and the strain of the infecting organism and the
immunologic status of the host can influence the clinical
manifestations and outcome of the parasitic disease. Treatment of
leishmaniases is complex and prolonged systemic treatment is
imperative. The objectives of treatment are to cure the human or
animal patient of an intracellular parasitic infection, to prevent
relapse, to avoid development of unresponsiveness and to keep
hospitalisation and overall treatment costs to a minimum. To
achieve these objectives, appropriate drugs must be given at
adequate dose levels and frequency for a suitable period of time.
Despite the extensive research in the search of effective and well
tolerated antileishmanial agents, only few agents have been
discovered and are available to the patient. Currrently, two
pentavalent antimony compounds that have to be administered by deep
intramuscular injection are commonly used as first-line drugs:
meglumine antimonate (Glucantim.TM., Farmitalia) and sodium
stibogluconate (Pentostam.TM., Wellcome). Second-line drugs are
amphothericin-B (in particular the liposomal formulations),
pentamidine and allopurinol. The currently available therapies are
not sufficiently effective and cause toxic side effects in the
patient. In addition, their spectrum of activity is not
sufficiently broad. For these reasons, the need for new medications
remains very high. The present identification of new active
principles will have use in the treatment of disorders caused by
protozoan parasites belonging to the genus Leishmania.
[0004] Unexpectedly, triterpene saponins having very potent
prophylactic as well as therapeutical activity against Leishmania
have been isolated from the plants Maesa balansae and M.
lanceolata, two species from the family Myrsilaceae, genus
Maesa.
[0005] The invention is directed to a process for the isolation of
triterpene saponins from plants belonging to the family
Myrsinaceae, characterized in that said process comprises the steps
of
[0006] (a) removing apolar material from the dried plant parts with
an apolar solvent,
[0007] (b) extracting said plant parts with an alcohol, and
[0008] (c) further purifying the saponins in the alcohol extract by
liquid-liquid extraction, filtration and chromatography.
[0009] In particular, the apolar solvent is a halogenated
hydrocarbon, e.g. dichloromethane or chloroform; and the alcohol is
methanol, ethanol, isopropanol, butanol, each optionally admixed
with water. Particularly useful is a mixture of methanol:water
(90:10) or a mixture of ethanol:water (70:30) for isolating the
fraction containing the saponins.
[0010] The saponins of the alcohol extract are further purified
by
[0011] (c1) evaporating the extract to dryness,
[0012] (c2) partitioning the residue between butanol and water,
[0013] (c3) evaporating the organic layer to dryness,
[0014] (c4) washing the residue in a ketone and
[0015] (c5) filtering off the crude saponin mixture.
[0016] In step (c2), the water layer is preferably extracted
several times with n-butanol.
[0017] The invention is also directed to an alternative process for
the isolation of triterpene saponins from plants belonging to the
family Myrsinaceae, characterized in that said process comprises
the steps of
[0018] (a) extracting the dried plant parts with an alcohol and
concentrating the extract,
[0019] (b) removing the apolar fraction from the extract by
liquid-liquid extraction with an apolar solvent, and
[0020] (c) further purifying the saponins in the alcohol extract by
liquid-liquid extraction, filtration and chromatography.
[0021] In particular, the alcohol is methanol, ethanol,
isopropanol, butanol, each optionally admixed with water,
preferably a mixture of ethanol: water (70:30); the apolar solvent
is a hydrocarbon, e.g. hexane.
[0022] The saponins of the alcohol extract are further purified
by
[0023] (c6) extracting the aqueous fraction with butanol satured
with water,
[0024] (c7) evaporating the organic layer to dryness,
[0025] (c8) washing the residue in a ketone, and
[0026] (c9) filtering off the crude saponin mixture,
[0027] In step (c6), the water layer is preferably extracted
several times with n-butanol.
[0028] When the saponins are isolated from the plant genus Maesa,
the chromatography can comprise reversed-phase liquid
chromatography with gradient eluent system using
[0029] A: 0.5% ammonium acetate in water
[0030] B: methanol
[0031] C: acetonitrile
[0032] wherein at t=0, (A:B:C)=(60:20:20) and t=end,
(A:B:C)=(0:50:50), or straight-phase liquid chromatography on
silicagel.
[0033] These processes yield a mixture that consists essentially of
saponins. In many pharmacological experiments described in the
experimental part, this mixture of saponins was used. For the
purpose of structure elucidation, this mixture was separated into
the individual constituents by HPLC as described in the
experimental part.
[0034] The present invention thus also relates to one or more
triterpene saponins obtainable by the processes described herein,
whether as a mixture or as isolated products.
[0035] In particular, the invention concerns triterpene saponins
obtainable from the plant genus Maesa, by chromatography comprising
reversed-phase liquid chromatography with gradient eluent system
using
[0036] A: 0.5% ammonium acetate in water
[0037] B: methanol
[0038] C: acetonitrile
[0039] wherein at t=0, (A:B:C)=(60:20:20) and t=end,
(A:B:C)=(0:50:50), and wherein said saponin has the following
characteristics:
[0040] Compound 1: MW=1532, .lambda..sub.max=228.6 nm,
.lambda..sub.max2=273.3 nm; t.sub.R=8.97
[0041] Compound 2: MW=1510, .lambda..sub.max=223.9 nm,
.lambda..sub.max2=274.5 nm; t.sub.R=9.39
[0042] Compound 3: MW=1532, .lambda..sub.max=279.2 nm,
.lambda..sub.max2=223.9 nm; t.sub.R=9.68
[0043] Compound 4: MW=1510, .lambda..sub.max=280.4 nm,
.lambda..sub.max2=222.7 nm; t.sub.R=10.09
[0044] Compound 5: MW=1574, .lambda..sub.max=276.8 nm,
.lambda..sub.max2=225.0 nm; t.sub.R=10.87; and
[0045] Compound 6: MW=1552, .lambda..sub.max=279.2 nm,
.lambda..sub.max2=223.9 nm; t.sub.R=11.37.
[0046] The relative retention time t.sub.R is the mean value of 10
measurements versus the retention time of uracil on a column
Hypersil BDS C-18, 3 .mu.m, 100.times.4 mm.
[0047] Specifically, the present invention concerns triterpene
saponins having the formula 2
[0048] wherein R.sub.2 is --O(C.dbd.O)C.sub.6H.sub.5 or
--O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3,
[0049] R.sub.3 is (E) or (Z) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
and
[0050] R.sub.4 is hydrogen or --(C.dbd.O)CH.sub.3;
[0051] more in particular,
[0052] in compound 1,
[0053] R.sub.2 is --O(C.dbd.O)C.sub.6H.sub.5,
[0054] R.sub.3 is (Z) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
[0055] R.sub.4 is hydrogen;
[0056] in compound 2,
[0057] R.sub.2 is --O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3,
[0058] R.sub.3 is (Z) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
[0059] R.sub.4 is hydrogen;
[0060] in compound 3,
[0061] R.sub.2 is --O(C.dbd.O)C.sub.6H.sub.5,
[0062] R.sub.3 is (E) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
[0063] R.sub.4 is hydrogen;
[0064] in compound 4,
[0065] R.sub.2 is --O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3,
[0066] R.sub.3 is (E) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
[0067] R.sub.4 is hydrogen;
[0068] in compound 5,
[0069] R.sub.2 is --O(C.dbd.O)C.sub.6H.sub.5,
[0070] R.sub.3 is (E) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
[0071] R.sub.4 is --(C.dbd.O)CH.sub.3;
[0072] in compound 6,
[0073] R.sub.2 is --O(C.dbd.O)C(CH.sub.3).dbd.CHCH.sub.3,
[0074] R.sub.3 is (E) --O(C.dbd.O)CH.dbd.CH--C.sub.6H.sub.5,
[0075] R.sub.4 is --(C.dbd.O)CH.sub.3;
[0076] Preferred compounds for use in the pharmaceutical
compositions and methods of treatment of the present invention are
compounds 3 and 4, in particular compound 3.
[0077] Specifically, the present invention concerns the use of one
or more triterpene saponins for the preparation of a pharmaceutical
composition for treating leishmaniases in hosts infected by
Leishmania species, characterized in that the saponin has the
formula (I) 3
[0078] a stereoisomeric form thereof or a pharmaceutically
acceptable addition salt thereof, wherein
[0079] R.sub.1 is hydrogen, --(C.dbd.O)C.sub.1-5alkyl,
--(C.dbd.O)C.sub.2-5alkenyl, --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl, a monosaccharide group or an
oligosaccharide group;
[0080] R.sub.2 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5, or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl;
[0081] R.sub.3 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5, or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl;
[0082] R.sub.4 is hydrogen, C.sub.1-6alkyl,
--(C.dbd.O)C.sub.1-5alkyl, --(C.dbd.O)C.sub.2-5alkenyl,
--(C.dbd.O)C.sub.6H.sub.5, or --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl;
[0083] R.sub.5 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, COOH; or
[0084] R.sub.5 and R.sub.2 form a divalent radical of formula
--C(.dbd.O)--O--;
[0085] R.sub.6 and R.sub.7 are hydrogen; or taken together they
form a bond; or
[0086] R.sub.5 and R.sub.6 form a divalent radical of formula
--CH.sub.2--O-- (a),
--CH(OR.sub.13)--O-- (b),
--C(.dbd.O)--O-- (c),
[0087] wherein R.sub.13 is hydrogen, C.sub.1-6alkyl or
--(C.dbd.O)C.sub.1-5alkyl;
[0088] R.sub.8.alpha. and R.sub.8.beta. each independently
represent CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alk- yl, CHO, CH(OCH.sub.3).sub.2,
CH.dbd.NOH, COOH; or
[0089] R.sub.8.beta. and R.sub.3 form a divalent radical of formula
--C(.dbd.O)--O--; or
[0090] R.sub.8.beta. and R.sub.5 form a divalent radical of formula
--CH.sub.2O--CHOH--;
[0091] R.sub.9 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH;
[0092] R.sub.10 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH;
[0093] R.sub.11, is hydrogen, hydroxy or
O--C(.dbd.O)C.sub.1-5alkyl; or R.sub.10 and R.sub.11 form a
divalent radical of formula --CH.sub.2O--; and
[0094] R.sub.12 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, CH.dbd.NOH or COOH.
[0095] Preferred are the compounds of formula (I) wherein
[0096] R.sub.1 is hydrogen, --(C.dbd.O)C.sub.1-5alkyl, or an
oligosaccharide group;
[0097] R.sub.3 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl;
[0098] R.sub.4 is hydrogen, C.sub.1-6alkyl,
--(C.dbd.O)C.sub.1-5alkyl, --(C.dbd.O)C.sub.2-5alkenyl;
[0099] R.sub.5 is CH.sub.2OH, CH.sub.2O--C(.dbd.O)CH.sub.3, CHO;
and
[0100] R.sub.6 and R.sub.7 taken together form a bond; or
[0101] R.sub.5 and R.sub.6 form a divalent radical of formula
--CH.sub.2--O-- (a),
--CH(OR.sub.13)--O-- (b),
--C(.dbd.O)--O-- (c),
[0102] wherein R.sub.13 is hydrogen, C.sub.1-6alkyl or
--(C.dbd.O)C.sub.1-5alkyl; and
[0103] R.sub.7 is hydrogen;
[0104] R.sub.8.alpha. represents CH.sub.3;
[0105] R.sub.8.beta. represents CH.sub.3, CH.sub.2OH, CHO,
CH(OCH.sub.3).sub.2, CH.dbd.NOH, COOH; or
[0106] R.sub.8.beta. and R.sub.3 form a divalent radical of formula
--C(.dbd.O)--O--; or
[0107] R.sub.8.beta. and R.sub.5 form a divalent radical of formula
--CH.sub.2O--CHOH--;
[0108] R.sub.10 is CH.sub.3, CH.sub.2OH;
[0109] R.sub.11 is hydrogen, hydroxy or O--C(.dbd.O)C.sub.1-5alkyl;
or
[0110] R.sub.10 and R.sub.11 form a divalent radical of formula
--CH.sub.2O--; and
[0111] R.sub.12 is CH.sub.3, CH.sub.2OH,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, CH.dbd.NOH.
[0112] Especially preferred compounds are those of formula (I)
wherein
[0113] R.sub.1 is hydrogen or an oligosaccharide group;
[0114] R.sub.2 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5 or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl;
[0115] R.sub.3 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl;
[0116] R.sub.4 is hydrogen, C.sub.1-6alkyl,
--(C.dbd.O)C.sub.1-5alkyl, --(C.dbd.O)C.sub.2-5alkenyl,
--(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl;
[0117] R.sub.5 is CH.sub.2OH, CH.sub.2OCH.sub.3.
CH.sub.2O--C(.dbd.O)CH.su- b.3, CHO, COOH; and
[0118] R.sub.6 and R.sub.7 taken together form a bond; or
[0119] R.sub.5 and R.sub.6 form a divalent radical of formula
--CH.sub.2--O-- (a),
--CH(OR.sub.13)--O-- (b),
--C(.dbd.O)--O-- (c),
[0120] wherein R.sub.13 is hydrogen; and
[0121] R.sub.7 is hydrogen;
[0122] R.sub.8.alpha. and R.sub.8.beta. both represent CH.sub.3
[0123] R.sub.9 is CH.sub.3;
[0124] R.sub.10 is CH.sub.3;
[0125] R.sub.11 is hydrogen; and
[0126] R.sub.12 is CH.sub.3.
[0127] The compounds of formula (I) may be converted into each
other through art-known processes. Particularly interesting
processes are saponification in basic media, transesterification in
acidic media, and enzymatic degradation of the oligosaccharide
moiety so as to produce aglycones, i.e. compounds of formula (I)
wherein R.sub.1 is hydrogen.
[0128] The present invention also concerns a method of alleviating
clinical manifestations of, and curing disorders known as
leishmaniases attributable to infection by protozoan parasites of
the genus Leishmania in both men and animals, comprising
administering to an infected host a therapeutically effective
amount of a compound of formula (I) 4
[0129] a stereoisomeric form thereof or a pharmaceutically
acceptable addition salt thereof, wherein
[0130] R.sub.1 is hydrogen, --(C.dbd.O)C.sub.1-5alkyl,
--(C.dbd.O)C.sub.2-5alkenyl, --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl, a monosaccharide group or an
oligosaccharide group;
[0131] R.sub.2 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5, or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl;
[0132] R.sub.3 is hydrogen, hydroxy, --O(C.dbd.O)C.sub.1-5alkyl,
--O(C.dbd.O)C.sub.2-5alkenyl, --O(C.dbd.O)C.sub.6H.sub.5, or
--O(C.dbd.O)C.sub.2-5alkenyl substituted with phenyl;
[0133] R.sub.4 is hydrogen, C.sub.1-6alkyl,
--(C.dbd.O)C.sub.1-5alkyl, --(C.dbd.O)C.sub.2-5alkenyl,
--(C.dbd.O)C.sub.6H.sub.5, or --(C.dbd.O)C.sub.2-5alkenyl
substituted with phenyl;
[0134] R.sub.5 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, COOH; or
[0135] R.sub.5 and R.sub.2 form a divalent radical of formula
--C(.dbd.O)--O--;
[0136] R.sub.6 and R.sub.7 are hydrogen; or taken together they
form a bond; or
[0137] R.sub.5 and R.sub.6 form a divalent radical of formula
--CH.sub.2--O-- (a),
--CH(OR.sub.13)--O-- (b),
--C(.dbd.O)--O-- (c),
[0138] wherein R.sub.13 is hydrogen, C.sub.1-6alkyl or
--(C.dbd.O)C.sub.1-5alkyl;
[0139] R.sub.8.alpha. and R.sub.8.beta. each independently
represent CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alk- yl, CHO, CH(OCH.sub.3).sub.2,
CH.dbd.NOH, COOH;
[0140] R.sub.8.beta. and R.sub.3 form a divalent radical of formula
--C(.dbd.O)--O--;
[0141] R.sub.8.beta. and R.sub.5 form a divalent radical of formula
--CH.sub.2O--CHOH--;
[0142] R.sub.9 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH;
[0143] R.sub.10 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)C.sub.1-5alkyl, CHO, COOH;
[0144] R.sub.11 is hydrogen, hydroxy or O--C(.dbd.O)C.sub.1-5alkyl;
or R.sub.10 and R.sub.11 form a divalent radical of formula
--CH.sub.2O--; and
[0145] R.sub.12 is CH.sub.3, CH.sub.2OH, CH.sub.2OCH.sub.3,
CH.sub.2O--C(.dbd.O)CH.sub.3, CHO, CH.dbd.NOH, or COOH.
[0146] For the purposes of treating leishmaniases, the compounds of
formula (I) may be administered orally, topically, parenterally, by
inhalation spray or rectally in dosage unit formulations containing
conventional nontoxic pharmaceutically acceptable carriers,
adjuvants and vehicles. The term parenteral as used herein includes
subcutaneous injection, intravenous, intramuscular, intrasternal
injection, intraarticular injection, or infusion techniques in
subjects susceptible to leishmania organism infection.
[0147] The pharmaceutical compositions containing the active
ingredient may be in a form suitable for oral use, for example, as
tablets, troches, lozenges, aqueous or oily solutions or
suspensions, dispersible powders or granules, emulsions, hard or
soft capsules, or syrups or elixirs. Compositions intended for oral
use may be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions and such
compositions may contain one or more agents selected from the group
consisting of sweetening agents, flavoring agents, coloring agents
and preserving agents in order to provide a pharmaceutically
elegant and palatable preparation. Tablets contain the active
ingredient in admixture with non-toxic pharmaceutically acceptable
excipients which are suitable for the manufacture of tablets
including but not limited to inert diluents, granulating and
disintegrating agents, and lubricating agents. Tablets may be
uncoated or they may be coated by known techniques to delay
disintegration and adsorption in the gastrointestinal tract thereby
providing a sustained action over a longer period; to mask an
unpleasant taste or mouthfeel; or to improve appearance and
recognizability.
[0148] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, dispersing or
wetting agents. The said aqueous suspensions may also contain one
or more preservatives, and oily suspensions may be formulated by
suspending the active ingredient in a suitable vegetable oil or in
a mineral oil such as liquid paraffin. The oily suspensions may
contain thickening agents, sweetening agents, and flavoring agents
to provide a palatable oral preparation. These compositions may be
preserved by the addition of an acceptable antioxidant.
[0149] Dispersible powders and granules suitable for preparation of
aqueous suspensions by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives.
[0150] The pharmaceutical compositions of the present invention may
also be in the form of oil-in-water (o/w) or water-in-oil (w/o)
emulsions. The oily phase may be a pharmaceutically suitable
vegetable oil, arachis oils, or a mineral oil, containing suitable
emulsifying agents and antioxidants. The aqueous phase may contain
emulsifying agents, thickening agents and preservatives. The
emulsions may also contain sweetening, coloring and flavoring
agents.
[0151] Syrups and elixirs may be formulated with sweetening agents.
Such formulations may also contain a demulcent, a preservative and
flavoring and coloring agents.
[0152] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example as a sterile injectable
aqueous or oleaginous solution or suspension in a non-toxic
parenterally acceptable diluent or solvent. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides, fatty acids and
other suitable additives of injectables. Suspensions may be
formulated according to the known art using suitable dispersing or
wetting agents and suspending agents.
[0153] The compounds of formula (I) may also be administered in the
form of suppositories or other formulations such as solutions or
suspensions for rectal administration of the drug. Suppositories
can be prepared by mixing the drug with a suitable non-irritating
excipient which is solid at ordinary temperatures but liquid at the
rectal temperature to release the drug.
[0154] The daily dosage of the compounds of formula (I) may be
varied over a wide range, e.g. from 1.0 to 2,000 mg. Preferably,
the compound of formula (I) with a carrier in a pharmaceutical
composition, is administered in subdivided doses containing 5, 10,
25, 50, 100, 150, 250 or 500 mg of the active ingredient for the
appropriate dosage to the patient to be treated. An effective
amount of the drug is ordinarily supplied at a dosage level of from
about 0.01 mg to about 50 mg/kg of body weight Preferably, the
range is from about 0.1 mg to about 7 mg/kg of body weight.
[0155] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination and
the severity and organ systems affected and in need of therapy.
[0156] Experimental Part
[0157] ISOLATION OF TRITERPENE SAPONINS FROM Maesa balansae
[0158] The air-dried powdered leaves (3 kg) of Maesa balansae were
extracted with chloroform to remove apolar material, and then with
methanol:water (9:1). The alcoholic extract was evaporated under
reduced pressure and the residue was partitioned between n-butanol
(saturated with water) and water. The organic layer was evaporated
to dryness and the residue was washed with acetone and filtered.
The acetone insoluble part containing the saponins (10 g) was
purified by reversed-phase HPLC (stationary phase RP-18 HS BDS
Hyperprep 100 .ANG., 8 .mu.m; 200 g, .o slashed. column 5 cm) with
a gradient eluent system using:
[0159] A: 0.5% ammonium acetate in water,
[0160] B: methanol and
[0161] C: acetonitrile
[0162] at a flow rate of 80 ml/min with UV-detection at 235 nm.
Using the gradient eluent system (t=0 min) A:B:C (60:20:20) to
(t=50 min) A:B:C (0:50:50) a pure saponin mixture (5 g) is obtained
comprising six compounds.
[0163] Isolation of each of the six saponins was performed on the
same column under the same conditions using the above described
gradient solvent system. In order of elution, the following
compounds were obtained:
[0164] Compound 1: MW=1532, .lambda..sub.max=223.3 nm; was further
purified using isocratic solvent system A:B:C (33:64:03); yield 230
mg.
[0165] Compound 2: MW=1510, .lambda..sub.max=209.2 nm; gradient
elution system: (t=0 min) A:B:C (42:29:29) to (t=end) A:B:C
(24:38:38); yield 110 mg.
[0166] Compound 3: MW=1532, .lambda..sub.max=222.1 nm; isocratic
solvent system: A:B:C (40:30:30); yield 1,000 mg.
[0167] Compound 4: MW=1510, .lambda..sub.max=202.2 nm; isocratic
solvent system: A:B:C (59:00:41); yield 1,000 mg.
[0168] Compound 5: MW=1574, .lambda..sub.max=203.4 nm; and
isocratic solvent system: A:B:C (32:34:34); yield 220 mg.
[0169] Compound 6: MW=1552, .lambda..sub.max=216.3 nm; isocratic
solvent system: A:B:C (32:34:34) with recycling (4 times); yield
230 mg.
Evaluation of Antileishmania Activity
[0170] The test drug PX used in the following examples comprises
the mixture of saponins isolated from Maesa balansae.
[0171] 1. In Vitro Antileishmanial Activity
[0172] Methods for the in vitro growth of Leishmania organisms and
screening methodology are well documented in the international
literature. Testing protocols are flexible and may be adapted
according to the specific objectives and the characteristics of the
test compounds. Briefly, the following in vitro methodology has
been used:
[0173] Primary peritoneal macrophages derived from laboratory
rodents or macrophage cell lines were seeded in multiwell tissue
culture vessels and allowed to attach for about 24 hours.
Amastigotes of the Leishmania species (obtained from target tissues
of an infected donor animal or from amastigote-infected tissue
cultures) or promastigotes of the Leishmania species were added at
an appropriate infection ratio together with varying serially
diluted concentrations of the drug or test compound. The test drug
was solubilized in an appropriate solvent which was tolerated in
the in vitro test system (DMSO; water, alcohols, and the like work
equally well) and added to the tissue culture medium. The cultures
were incubated at 37.degree. C. in 5% CO.sub.2 for 5-15 days.
Treatment of uninfected control cultures was also included in order
to determine a selectivity index. Reference drug treated cultures
were included as well so as to determine the relative potency of
the test drug. Drug activity was determined in stained preparations
as the percentage reduction of the total parasite load or the
number of infected macrophages compared to the untreated control
cultures. Reading was performed microscopically and
EC.sub.50-values (effective concentration for 50% inhibition) were
determined. The percentage reduction and the EC.sub.50-value serve
as an indication for antileishmanial activity in vitro and provide
significant leads for clinically useful agents.
1TABLE I In vitro antileishmanial activity in primary mouse
macrophages EC.sub.50 (microgram/ml) Leishmania species PX
meglumine pentostam ampho-B itraconazole Visceral L. donovani 0.05
12 6 0.1 >12.5 L. infantum 0.05 12 6 0.1 >12.5 Cutaneous L.
mexicana 1 >50 25 0.1 nd L. major 5 >50 >50 0.2 nd
Mucocutaneous L. panamensis nd nd nd nd nd L. major nd nd nd nd nd
nd: not done
[0174] 2. In Vivo Antileishmanial Activity
[0175] Methods for in vivo maintenance of Leishmania organisms and
animals models are well documented in the international literature.
Balb-C mice and golden hamsters are the preferred laboratory animal
species for primary isolation, maintenance and use in artificial
infection models. Testing protocols are flexible and may be adapted
according to the specific objectives and characteristics of the
test compounds. Briefly, the following in vivo methodologies have
been used:
[0176] For visceral Leishmania species: Balb-C mice or young
hamsters were intravenously infected with about 10.sup.6 to
10.sup.7 amastigotes derived from the target organs (generally
spleen) of an infected donor animal or from an in vitro culture of
parasite forms. The animals were treated with the test compound at
different dose levels (dose range: 0.1 to 80 mg/kg in 100% DMSO or
any other acceptable vehicle), using different routes of
administration and treatment schedules. Initiation of treatment was
either at different times after infection (curatively), concomitant
with infection (prophylactically) or before infection (residual
activity). In the prophylactic study design, the first
administration of the test drug is given immediately before or
together with the artificial infection with the Leishmania species.
In the curative study design, the first administration of the test
drug is given several weeks after the artificial infection with the
Leishmania species (early curative=when the first clinical signs
appear; late curative=when the clinical symptoms are well
established or become chronic). Drug activity was evaluated by
determination of the total parasite burdens in the liver or any
other relevant target tissue/organ, compared to the tissue/organ
burdens in untreated control animals. The mean number of
amastigotes is enumerated quantitatively or semi-quantitatively on
stained impression smears or slides. The percentage reduction
serves as an indication for antileishmanial activity and provides
significant leads for clinically useful agents. The lowest active
dose (LAD) is defined as the lowest dose which reduces the parasite
burden in the primary target organ/tissue by at least 80%.
2Table II In vivo antileishmanial activity in mice and hamsters
against visceral Leishmania species % reduction of parasite load in
target organ after Animal dosing regimen parenteral dosing at
(mg/kg) species freq. timing 40 20 10 5 2.5 1.25 0.63 0.32 Mouse 5
.times. proph. 100 100 100 100 100 100 100 98 cur. 100 100 100 100
100 1 .times. proph. 100 100 100 100 100 cur. 98 90 80 Hamster 5
.times. proph. 100 100 100 cur. 100 100 1 .times. proph. 100 100
100 cur.
[0177]
3TABLE III In vitro and in vivo activity against visceral
Leishmania infantum Activity against L. infantum Result in vitro in
vivo Activity Product IC50 (ng/ml) LAD (mg/kg 1 .times.) score PX
50 0.4 +++ compound-1 70 0.8 ++ compound-2 50 >0.8 ++ compound-3
20 0.2 +++ compound-4 20 0.4 +++ compound-5 3400 >0.8 +
compound-6 700 >0.8 + Tri-OH derivative 10,000 >40 not active
Aglycon derivative >50,000 >40 not active
[0178] For cutaneous and mucocutaneous Leishmania species: Balb-C
mice or young hamsters were infected intradermally or
subcutaneously with about 10.sup.6 to 10.sup.7 amastigotes derived
from the target organs (generally skin lesion) of an infected donor
animal or from an in vitro culture of parasite forms. The animals
were treated with the test compound at different dose levels (mg/kg
in 100% DMSO or any other acceptable vehicle), using different
routes of administration and treatment schedules. Initiation of
treatment was either before infection (prophylactically) or at
different times after infection (curatively). In the prophylactic
study design, the first administration of the test drug is given
immediately before or together with the artificial infection with
the Leishmania species. In the curative study design, the first
administration of the test drug is given several weeks after the
artificial infection with the Leishmania species (early
curative=when the first dermal lesions appear; late curative=when
dermal lesions are well established or become chronic). Drug
activity was evaluated either by determination of the severity of
the lesion in the relevant target tissue/organ (primary parameter)
or of the parasite burdens in the relevant target tissue/organ
(secondary parameter), compared to untreated control animals. The
lesion size was assessed quantitatively using the method of J.
El-On. and A. D. Hamburger [Trans. Roy. Soc. Trop. Med. Hyg., 81,
734-737 (1987)]. The percentage reduction serves as an indication
for antileishmanial activity and provides significant leads for
clinically useful agents. The lowest active dose (LAD) is defined
as the lowest dose which prevents lesions to develop, stops further
evolution of the lesions or induces a clinical cure of the lesions
in the primary target organ or tissue.
4TABLE IV In vivo antileishmanial activity in mice and hamsters
against cutaneous and mucocutaneous Leishmania species A.
Prophylactic treatment In the prophylactic study design, the first
administration of the test drug is given immediately before the
artificial infection with the Leishmania species L. mexicana
(proph.) skin lesion size* at weeks post infection Group 0 1 2 3 4
5 6 7 8 9 10 11 12 Untreated control 0 0 0 0 0 1 1 10 33 59 86 108
98 Ampho-B 10 mg/kg 0 0 0 0 0 1 2 7 13 34 85 101 112 Pentostam 250
mg/kg 0 0 0 0 0 0 1 9 13 22 37 50 51 PX 10 mg/kg 0 0 0 0 0 0 0 0 0
0 0 0 0 PX 5 mg/kg 0 0 0 0 0 0 0 0 0 0 0 0 0 PX 2.5 mg/kg 0 0 0 0 0
0 0 0 0 0 0 0 0 L. major (proph.) skin lesion size* at weeks post
infection Group 0 1 2 3 4 5 6 7 8 Untreated control 0 0 1 1 17 20
27 72 113 Ampho-B 10 mg/kg 0 0 1 1 12 31 41 57 95 Pentostam 250
mg/kg 0 0 1 1 7 16 20 32 60 PX 10 mg/kg 0 0 0 0 0 0 0 0 0 PX 5
mg/kg 0 0 0 0 0 0 0 0 0 PX 2.5 mg/kg 0 0 0 0 0 0 0 0 0 L.
panamensis (proph.) skin lesion size* at weeks post infection Group
0 1 2 3 4 5 6 7 8 9 10 11 12 Untreated control 0 0 0 0 1 6 30 42 49
58 55 67 56 Ampho-B 10 mg/kg 0 0 0 0 1 9 15 30 30 42 42 43 39
Pentostam 250 mg/kg 0 0 0 0 0 0 0 0 0 0 0 0 0 PX 10 mg/kg 0 0 0 0 0
0 0 0 0 0 0 0 0 PX 5 mg/kg 0 0 0 0 0 0 0 0 0 0 0 0 0 PX 2.5 mg/kg 0
0 0 0 0 0 0 0 0 0 0 0 0 B. Curative treatment In the curative study
design, the first administration of the test drug is given several
weeks after the artificial infection with the Leishmania species
(generally when the first clinical signs appear). L. mexicana (cur)
Dose freq./ skin lesion size* at weeks post infection Group mg/kg
week 5** 6 7 8 9 Untreated control 1.7 3.6 10.7 17.7 32.8 Pentostam
250 2 .times. 2.5 4.9 8.5 13.3 19.2 PX 1 1 .times. 1.8 3.6 4.2 3.1
2.2 PX 1 2 .times. 2.2 3.7 3.6 2.4 2.3 PX 2 1 .times. 1.6 2.6 1.3
1.3 1.4 PX 2 2 .times. 1.3 1.1 1.8 0.7 0.6 L. major (cur) Dose
freq./ skin lesion size* at weeks post infection Group mg/kg week
2** 3 4 5 6 7 8 9 Untreated control 1 14 32 42 50 59 64 148
Pentostam 250 2 .times. 1 13 24 33 36 42 48 123 PX 1 1 .times. 1 14
27 34 40 46 59 64 PX 1 2 .times. 1 12 19 27 32 33 32 33 PX 2 1
.times. 1 16 25 30 33 38 42 42 PX 2 2 .times. 1 9 23 29 30 28 32 35
L. panamensis (cur) Dose freq./ skin lesion size* at weeks post
infection Group mg/kg week 5** 6 7 8 9 Untreated control 2.8 8.5
20.8 30.5 Pentostam 250 2 .times. 2.3 1.2 0.5 0.1 PX 1 1 .times.
2.2 2.1 3.5 3.7 PX 1 2 .times. 1.2 1.8 2.8 1.2 PX 2 1 .times. 1.9
4.0 4.8 2.6 PX 2 2 .times. 2.9 1.6 2.3 1.7 *using the formula:
[vertical diameter .times. horizontal diameter]/2 (mm.sup.2)
**initiation of treatment
[0179]
5TABLE V In vivo antileishmanial activity of the PX mixture against
different Leishmania species Lowest active dose (LAD) regimen in
Balb-C mice* Sodium stibogluconate Amphothericin-B Model PX
Pentostam .RTM. Fungizone .RTM. L. donovani prophylactic 0.4 mg/kg,
1 .times. 250 mg/kg, 1 .times. not effective curative early 1.6
mg/kg, 1 .times. 250 mg/kg, 1 .times. not effective curative late
nd nd nd residual activity 5 days after single nd not effective 2.5
mg/kg dose L. mexicana prophylactic <0.5 mg/kg, >>250
mg/kg, not effective 6 .times. (alternate days) 6 .times.
(alternate days) curative early <1 mg/kg, >>250 mg/kg, nd
4 .times. (in 4 weeks) 8 .times. (in 4 weeks) curative late <1
mg/kg, nd nd 2 .times./w for 4 weeks L. panamensis prophylactic
<0.5 mg/kg, <250 mg/kg, 6 .times. not effective 6 .times.
(alternate days) (alternate days) curative early 1 mg/kg, <250
mg/kg, nd 4 .times. (in 4 weeks) 8 .times. (in 4 weeks) curative
late <1 mg/kg, nd nd 2 .times./w for 4 weeks L. major
prophylactic 2 mg/kg, >>250 mg/kg, not effective 6 .times.
(alternate days) 6 .times. (alternate days) curative early 1 mg/kg,
>>250 mg/kg, nd 4 .times. (in 4 weeks) 8 .times. (in 4 weeks)
curative late 1 mg/kg, nd nd 22 .times. (in 11 weeks) *based on
amastigote burdens in the liver for visceral forms and on lesion
size for cutaneous forms
* * * * *