Note: Descriptions are shown in the official language in which they were submitted.
CA 02356539 2001-05-23
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USE OF 17-ICETOSTEROID COMPOUNDS AND DERIVATIVES, METABOLTTES AND
PRECURSORS THEREOF IN THE TREATMENT OF MALARIA AND THE
TREATMENT OF AFRICAN AND AMERICAN TRYPANOSOMIASIS
BACKGROUND OF THE INVENTION
The present invention is directed to the use of 17-ketosteroid compounds, as
well as
derivatives, metabolites and precursors of such compounds, and
pharmaceutically acceptable salts
of any of these compounds, collectively defined herein as the "compounds of
the present
invention", optionally together with one or more additional administration
and/or treatment (as
I O described below) in the treatment of malaria, African Trypanosomiasis and
American
Trypanosomiasis. The present invention is further directed to the use of such
compounds (and
combinations) in the treatment of one or more kind of parasites and/or one or
more di~'eases caused
by such parasites, against one or more kind of Mycoplasma and/or one or more
diseases caused by
such Mycoplasmas andlor against one or more of the following indications or
infections: (a) hairy
Leukoplakia, (b) oral candidosis, (c) mouth ulcerations-
aphthous/herpetic/bacterial, (d) fungal
candida, (e) human papilloma virus, (f) molluscum contagiosum, (g) squamous
oral carcinoma, (h)
Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis,
(k) orafacial herpes
zoster, and (1) rotaviruses, as well as all other indications and infections
disclosed in U.S. Patent
No. 5,292,725, the entire disclosure of which is hereby incorporated by
reference, (in particular,
the disclosure from column 1, line 13 through column 4, line 25).
In the case of malaria, infection of man occurs by mosquito bites, during
which
"sporozoites" are introduced into the human bloodstream. Sporozoites travel
through the
bloodstream, and eventually reach and penetrate parenchyma) cells of the liver
(sporozoites can
remain in the blood for up to 1 hour).
The preerythrocytic cycle, the first cycle of growth and reproduction, lasts
about one week
and takes place in the liver. The preerythrocytic cycle liberates several
thousand merozoites, which
then pass into the blood.
The merozoites parasitize erythrocytes. Growth and reproduction take place in
red blood cells,
bringing about rhythmic fever attacks and other symptoms as the number of
parasites in the blood
increases. In the course of the erythrocytic growth-cycle, upon entering a red
cell, the parasite
assumes a ring form with a central vacuole. After about 6 hours, the vacuole
gradually disappears
as the parasite increases in size until it nearly fills the red cell. During
the last 12 hours of the
growth cycle, nuclear fission occurs from which, on average, about sixteen
merozoites are formed.
The growing parasite is termed a trophozoite and, after nuclear fission has
started, it is called a
schizont.
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The malaria parasite feeds on hemoglobin, utilizing the protein portion of the
molecule and
leaving the heme portion, which accumulates to form malarial pigment. It has
been shown by EM
that the parasite feeds by phagocytosis, engulfing red cell cytoplasm;
digestion takes place in food
vacuoles, where the pigment accumulates until it is released into the plasma
when the host cell
ruptures and the merozoites escape. Shortly after the parasite has started
reproducing in the blood,
the sexual forms or gametocytes begin to appear in the red cells. Gametocytes
may survive for
several days in the mammalian host, but they cannot develop further unless
they are ingested by a
suitable mosquito host. The single nucleus of the gametocyte distinguishes it
from the fully-grown
asexual forms.
Sleeping sickness is caused by T. gambiense and T. rhodesiense and is
transmitted from
man to man or from animals to man by tsetse flies (Glossing). In the mammalian
host, the
organisms inhabit the blood but may penetrate other organs where they occur in
intercellular
spaces. In a drop of blood, the trypanosomes appear as minute, wriggling
objects.
When a tsetse fly feeds, its toothed proboscis tears the skin, causing a small
hemorrhage to
form. If trypanosomes are present, they are sucked into the gut of the fly
with the blood drawn up
from this pool. For the first few days after an infective feed, the
trypanosomes are found in the
midgut. Then some travel forwards to the proventriculus. For development to be
successful, some
must pass right forward to near the tip of the proboscis where the opening of
the salivary duct is
located. They must then pass up the duct to the salivary glands where forms
develop which are
infective to the mammal. These are called metacyclic trypanosomes because they
appear at the end
of the developmental cycle. Reproduction takes place at all sites in the fly.
The time required for
this cycle is 2-3 weeks or even longer. Not only do the trypanosomes alter in
morphology in the
insect host, but they also differ physiologically from the blood stream forms.
If a tsetse fly harboring a mature infection bites man, metacyclic
trypanosomes may be
injected along with saliva. In the early stages of an infection, the parasites
may be found in the
blood. A characteristic feature is that the number of trypanosomes builds up
to a peak and then
declines, and these cycles are repeated. The trypanosomes stimulate the host
to produce antibody,
which agglutinates and lyses the organisms. Some of the trypanosomes become
resistant to
antibody and so a new population develops of different antigenic type; these
flourish until specific
antibody is again formed to destroy them. At later stages of the infection,
the trypanosomes
become scarce or absent from the blood but invade the central nervous system
to cause sleeping
sickness.
Trypanosomes can establish and develop in a wide range of mammalian species,
and have
been isolated from many species of African game animals. In these hosts, the
association seems to
be a benign one and the mammal remains in good health. But the same
trypanosomes in man or in
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CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
man's domestic animals are highly pathogenic. Trypanosomiasis of domestic
animals is an urgent
problem in large areas of Africa where stock cannot be reared because of the
piesence of tsetse
flies and game animals.
Chagas disease (American Trypanosomiasis) is caused by T. cruzi. It is
transmitted by
blood-feeding insects of the family Triatomidae.
After infection in man, the parasite soon leaves the blood and settles in
tissues, most
frequently in cardiac, striated or smooth muscle. Here they lose their
flagella and round up. Next,
they multiply and clusters of several hundred cells may be formed, displacing
muscle fibers. After
a time, the colony starts to disperse; the cells elongate, each develops a
flagellum and the new
trypanosomes enter the circulation. The trypanosomes remain in the circulation
for several days
and then again disappear into the tissues to undergo another reproductive
cycle. In chronic
infections, the tissue phase predominates, since the blood forms can rarely be
detected.
If an insect feeds on blood containing trypanosomes, these trypanosomes become
established first in the midgut. In the midgut, the trypanosomes multiply
rapidly and within a few
days some pass into the hindgut and infective forms begin to appear in the
feces. In contrast to the
African trypanosomes, where the infective forms are situated anteriorly in the
vector and are
introduced into man by inoculation, the infective forms of T. cruzi are
located at the posterior end
of the vector's gut and infection is by contamination. Triatomid insects
ingest a large amount of
blood relative to their body weight and the ingested blood is concentrated by
fluid excretion while
the insect feeds. In this way, infective trypanosomes are deposited on the
skin of the host. The
trypanosomes cannot penetrate unbroken skin but may gain entry through the
puncture wound.
Since the insects are nocturnal and feed in the facial areas, the trypanosomes
are commonly
smeared into eyes, mouth or nose where they penetrate mucous membranes.
T. cruzi develops in several species of insects, all of which function as
hosts. If ten insects
were allowed to feed on an infected person, all ten would probably become
infected. Laboratory
reared or "clean" insects are often used in diagnosis.
T. cruzi has been found in many species of wild animals and in reduviid
insects in Central
and South America and in some of the Southern states of the USA. Human
infection in the USA is
rare, but in parts of Central and South America, the incidence of infection in
man may be as high
as 20 per cent. It has been established that some 35 million people are at
risk to the infection. The
infection may be spread from man to man or from animals to man. Domestic dogs
and cats are
reservoirs in urban areas. Drugs that are effective against the African
trypanosomes have no action
on human infections with T. cruzi; no curative drugs have yet been discovered
that combat this
parasite.
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CA 02356539 2001-05-23
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A number of steroid compounds and their uses have been described. See, e.g.,
U.S. patent .
numbers 4956355, 5859000, 4268441, 4666898, 5837269, 5827841, 5811418,
5824313, 5686438, ,
5635496, 5587369, 5583126, 5562910, 5532230, 5518725, 5736537, 5843932,
5837700, 5824671,
5807849, 5798347, 5780460, 5776923, 5728688, 5610150, 5593981, 5372996,
5110810, 5807848,
5707983, 5641766, 5585371, 5506223, 5424463, 5296481, 5292730, 5776921,
5641768, 5559107,
- _ 5478566, 5461042, 540?684, 5387583, 5277907, 5206008, 5077284, 5162198,
5660835, 5527789,
5756482, 5709878, 5804576, 5744462, 5714481, 5700793, 5696106, 5656621,
5175154, 5157031,
5028631, 5001119, 4898694, 5824668, 5710143, 5795880, 5527788, 5591736,
5861390 and PCT
publication numbers WO 98/05338, WO 95/21617, WO 98/50040, WO 98/50041 and WO
97/48367, all of which are incorporated herein by reference.
A number of flavonoids, methods to obtain them and their uses have been
described. See,
e.g., J.A. Manthey and B.S. Buslig, editors, Flavonoids in the living system,
Advances in
experimental medicine and biology, volume 439, Plenum Press, New York, 1998,
chapter 15
(pages 191-225), chapter 16 (pages 227-235) and chapter 17 (pages 237-247),
which are
incorporated herein by reference.
SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention, it now has been
discovered that
surprisingly, Trypanosome parasites, e.g., the malaria parasites, may be
treated with compounds
(or pharmaceutically acceptable salts thereof) of the following formula 1
R~ R~ X
R~ Q6
R~ ;
12
R~ R~
R' 11 13 1716 pz
Q3 14 15
Gla~2~1
10 g ~R ~ ~ R~
RZ 3 5 7 ~ R~
4 . , 6~Q~
R~ p5
i
~~ R~ - 1
wherein
Q 1 is -C(R 1 )2- or -C(O)-;
Q2 is -C(R1)2-~ -C(R1)(Y)-~ -C(Y)- or -CH2-CH2-;
Q3 is -H or -C(R1)3-;
Q4 is -C(R1)2-, -C(O)-, hydroxyvinylidine (-CH(CH=CHOH)-) or methyl methylene
(-
CH(CH)3-);
4
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QS is -C(R1 )2- or -C(O)-;
X and Y independently are -OH, -H, lower alkyl (e.g., C 1 _6 alkyl), -O-C(O)-
R5,
-C(O)-ORS, halogen (i.e., -F, -CI, -Br or -I) or =O;
each R1 independently is -H, -F, -Cl, -Br, -I, -OH, C 1_6 alkoxy, or C 1_6
alkyl;
R2 is -H, -OH, -F, -CI, -Br, -I, C1-6 alkyl, C1-6 alkoxy, -OR3, an ester
(e.g., -O-C(O)-R4
- or -C(O)-O-R4), a thioester (e.g., -O-C(S)-R4 or -C(S)-O-R4), a thioacetal
(e.g., -S-C(O)-R4, or -
C(O)-S-R4), a sulfate ester (e.g., -O-S(O)(O)-O-R4), a sulfonate ester (e.g., -
O-S(O)-O-R4) or a
carbamate (e.g., -O-C(O)-NH-R4 or -NH-C(O)-O-R4) or R2, together with the R1
that is bonded to
the same carbon atom is =O;
R3 is -S(O)(O)-OM, -S(O)(O)-O-CH2-CH(O-C(O)-R6)-CH2-O-C(O)-R6,
-P(O)(O)-O-CH2-CH(O-C(O)-R7)-CH2-O-C(O)-R~, a glucuronide group of structure
(A)
COOH '
H3C Q
OH
OH
CH3 (A),
or R3 is C 1 _ 1 g alkyl, C2_ 1 g alkenyl, C2-1 g alkynyi, a C 1 _ 1 g ester
or a C 1 _ 1 g thioester, where any
of the foregoing C 1 _ I g or C2_ 1 g moieties are optionally substituted at
one or more hydrogen atoms
1 S with one or more independently selected -ORPR, (including -OH), -NHRPR,
(including -NH2) or -
SRPR, (including -SH) groups, or R3 is a C 1 _ 1 g fatty acid, C2_ 10 alkynyl,
(J)n-phenyl-C 1 _5-alkyl,
(~n-Phenyl-C2_5-alkenyl;
R4 is -H, a protecting group, optionally substituted C 1 _ 1 g alkyl,
optionally substituted C 1 _
1 g alkenyl, optionally substituted C 1 _ 1 g alkynyl, optional ly substituted
aryl, optionally substituted
aryl-Cl_(, alkyl, optionally substituted aryl-C2_6 aikenyl, optionally
substituted aryl-C2-6 alkynyl,
optionally substituted heterocycle-Cl_6 alkyl, optionally substituted C2-6
alkenyl-heterocycle,
optionally substituted C2_( alkynyl-heterocycle or an optionally substituted
heterocycle, where any
of the foregoing moieties are optionally substituted at one, two, three, four,
five or more carbon or
hydrogen atoms with one or more independently selected -O-, -S-, -NRPR-
(including -NH-), -NH-
C(O)-, -ORPR (including -OH), -NHRPR (including -NH2), -SRPR (including -SH),
=O, =S, =N-
OH, -CN, -N02, -F, -Cl, -Br or -I groups or atoms;
each RS independently is straight or branched C 1 _ 14 alkyl;
each R6 independently is straight or branched C 1 _ 14 alkyl;
each R~ independently is straight or branched C 1 _ 14 alkyl or a glucuronide
group of
structure (A);
each RPR independently is -H or an independently selected protecting group;
nis0, l,2or3;
5
CA 02356539 2001-05-23
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each J independently is -F, -CI, -Br, -I, C 1-4 alkyl, C 1-4 alkenyl, C 1 _4
alkoxy, carboxy,
nitro, sulfate, sulfonyl, a C 1 _6 carboxyl ester or a C 1 _6 sulfate ester;
M is hydrogen, sodium, -S(O)(O)-O-CH2-CH(O-C(O)-R6)-CH2-O-C(O)-R6, -P(O)(O)-O-
CH2-CH(O-C(O)-R7)-CH2-O-C(O)-R7 or a glucuronide group of structure (A);
the dotted lines in formula 1 represent an optional double bond, provided that
there are not
double bonds at both the 4-5 and 5-6 positions and provided that when a double
bond is present,
zero or 1 R1 group is bonded to carbon atoms at the 1-, 2-, 4-, S-, 6- or 17
positions so that these
carbon atoms are tetravalent; and
the salts, stereoisomers, positional isomers, metabolites, analogs,
precursors, hydrates,
tautomers, ionized forms and solvates thereof. The formula 1 compounds are
collectively referred
herein to as the "compounds of the invention" or the "compounds of the present
invention".
In addition, as discussed above, the present invention is directed to the
treatment of
sleeping sickness and the treatment of Chagas disease by administering one or
more of the
compounds of the present invention. Also, the present invention relates to the
use of the
compounds of the present invention in the treatment of one or more kind of
parasites andlor one or
more diseases caused by such parasites, against one or more kind of Mycoplasma
and/or one or
more diseases caused by such Mycoplasmas and/or against one or more of the
following
indications or infections: (a) hairy Leukoplakia, (b) oral candidosis, (c)
mouth ulcerations
(aphthous/herpetic/bacterial), (d) fungal candida, (e) human papilloma virus,
(f) molluscum
contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions,
(i) periodontitis, (j)
necrotizing gingivitis, (k) orafacial herpes zoster, and (I) rotaviruses, as
well as all other
indications and infections disclosed in U.S. Patent No. 5,292,725, which is
incorporated herein by
reference.
Accordingly, the present invention provides a method for treating these
parasitic infections
which comprises administering to an afflicted host a therapeutically effective
amount of a
compound (or a pharmaceutically acceptable salt thereof) having the structure
of Formula 1
(defined above), as well as derivatives, metabolites, and precursors thereof,
as defined herein.
The present invention is further directed to a method for treating any of the
conditions
described herein by administering a compound that inhibits glucose-6phosphate
dehydrogenase.
Another invention embodiment comprises a method to treat or prevent a
Trypanosome or
Plasmodium infection comprising administering to a subject a compound of the
invention
simultaneously or sequentially with a compound of formula 2A or 2B
6
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R.. R..
R10 X
R
8
4
_ Re X;
2A
Rp
Roc X
t
4
RB X;
2B
wherein a double or a single bond is present at the dotted line and, when a
double bond is present,
(i) the optionally substituted phenyl ring at the 2- or 3-position is present
and the Rg that is bonded
to the carbon is absent, and (ii) one Rg at the adjacent 2- or 3-position is
absent;
X 1 is -O- or -C(Rg)2-;
X2 is -C(O)- or -C(R11)2-~
each Rg independently is -H, -OH, halogen, C1_6 alkyl, C1-6 alkoxy,
glucuronide, a
C1_25 fatty acid, the residue of a formula 2A or 2B compound where a hydrogen
atom is removed
to form the formula 2A or 2B compound radical, -CH2CH=C(CH3)2, glucoside, a
group having
structure (B) or (C),
O O~
HO
HO
OH (B) or
_ (C)
Rl0 is CI_6 alkyl, CI_6 alkoxy, neohesperidoside, apioglucoside, rutinoside,
glucoside,
galactoside, rhamnoside, arabinoside, or a stereoisomer, hydrate, analog,
derivative or metabolite
of any of these moieties, any of which are optionally independently
substituted at one or more
hydrogen atoms with -OH, halogen, C 1 _6 alkyl, C 1 _6 alkoxy, glucuronide or
a C 1 _25 fatty acid or
R10 is -H, -OH or halogen; ,
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each RI 1 independently is -H, -OH, halogen, CI-6 alkyl, C1-6 alkoxy,
glucuronide, a C1_
25 fatty acid, or both R11 together are =O; and
the salts, stereoisomers, positional isomers, metabolites, analogs,
precursors, hydrates,
tautomers, ionized forms and solvates thereof.
DETAILED DESCRIPTION OF THE INVENTION
- As used herein and unless otherwise stated or implied by context, the
following terms have
the meanings defined here.
A "patient" or "subject" means a human or animal. Usually the animal is a
vertebrate such
as a primate, rodent, domestic animal or game animal. Primates include
chimpanzees,
cynomologous monkeys, spider monkeys, and macaques, e.g., RheSUS. Rodents
include mice, rats,
woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include
cows, horses, pigs,
deer, bison, buffalo, felines, e.g., domestic cat, canines, e.g., dog, avians,
e.g., chicken; emu,
ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject
includes any subset of the
foregoing, e.g., all of the above, but excluding one or more groups or species
such as humans,
primates or rodents.
"Alkyl" as used herein, unless stated to the contrary, is a C1-Clg hydrocarbon
containing
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms
in the form of normal,
secondary, tertiary, cyclic or mixed structures. Examples are -CH3, -CH2CH3, -
CH2CH2CH3, -
CH(CH3)2, -CH2CH2CH2CH3, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -C(CH3)3. -
CH2CH2CH2CH2CH3, -CH(CH3)CH2CH2CH3, -CH(CH2CH3)2, -C(CH3)2CH2CH3, -
CH(CH3)CH(CH3)2, -CH2CH2CH(CH3)2, -CH2CH(CH3)CH2CH3, -CH2C(CH3)3.
CH2CH2CH2CH2CH2CH3, -CH(CH3)CH2CH2CH2CH3, -CH(CH2CH3)(CH2CH2CH3), -
C(CH3)2CH2CH2CH3, -CH(CH3)CH(CH3)CH2CH3, -CH(CH3)CH2CH(CH3)2, -
C(CH3)(CH2CH3}2, -CH(CH2CH3)CH(CH3)2, -C{CH3)2CH(CH3)2, -CH(CH3)C(CH3)3,
cyclopropyl, cyclobutyl, cyclopropylmethyl, cyclopentyl, cyclobutylmethyl, 1-
cyclopropyl-1-ethyl,
2-cyclopropyl-1-ethyl, cyclohexyl, cyclopentylmethyl, I-cyclobutyl-I-ethyl, 2-
cyclobutyl-I-ethyl,
1-cyclopropyl-1-propyl, 2-cyclopropyl-1-propyl, 3-cyclopropyl-1-propyl, 2-
cyclopropyl-2-propyl,
and 1-cyclopropyl-2-propyl.
"Alkenyl" as used herein, unless stated to the contrary, is C2-C 1 g
hydrocarbon comprising
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms
in the form of normal,
secondary, tertiary, cyclic or mixed structures and comprising I, 2, 3 or more
double bonds
between adjacent carbon atoms. Examples are -CH=CH2, -CH=CHCH3, -CH2CH=CH2, -
C(=CH2)(CH3), -CH=CHCH2CH3, -CH2CH=CHCH3, -CH2CH2CH=CH2, -CH=C(CH3)2.
CH2C(=CH2)(CH3), -C(=CH2)CH2CH3, -C(CH3)=CHCH3, -CH(CH3)CH=CH2, -
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C=CHCH2CH2CH3, -CHCH=CHCH2CH3, -CHCH2CH=CHCH3, -CHCH2CH2CH=CH2, -
C(=CH2)CH2CH2CH3, -C(CH3)=CH2CH2CH3, -CH(CH3)CH=CHCH3, -
CH(CH3)CH2CH=CH2, -CH2CH=C(CH3)2, 1-cyclopent-I-enyl, 1-cyclopent-2-enyl, 1-
cyclopent-
3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and I-cyclohex-3-enyl.
"Alkynyl" as used herein, unless stated to the contrary, is C2-Clg hydrocarbon
comprising
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms
in the form of normal,
secondary, tertiary, cyclic or mixed structures and comprising 1, 2, 3 or more
triple bonds between
adjacent carbon atoms. Examples are -CCH, -CCCH3, -CH2CCH, -CCCH2CH3, -
CH2CCCH3, -
CH2CH2CCH, -CH(CH3)CCH, -CCCH2CH2CH3, -CH2CCCH2CH3, -CH2CH2CCCH3 and -
CH2CH2CH2CCH.
"Halogen" or "halo" means fluorine (-F), chlorine (-C1), bromine (-Br) or
iodine (-I) and if
more than one halogen is referred to (e.g., two or more variable groups may be
a halogen), each
halogen is independently selected.
"Steroid nucleus" means 4 fused rings having the formula 1 structure.
"PEG" means an ethylene glycol polymer that contains about 20 to about 2000000
linked
monomers, typically about 50-1000 linked monomers, usually about 100-300.
Polyethylene
glycols include PEGS containing various numbers of linked monomers, e.g.,
PEG20, PEG30,
PEG40, PEG60, PEG80, PEG100, PEG115, PEG 200, PEG 300, PEG400, PEG500, PEG600,
PEG
1000, PEG1500, PEG2000, PEG 3350, PEG4000, PEG4600, PEGS000, PEG6000, PEG8000,
PEG 1 I 000, PEG 12000, PEG2000000 and any mixtures thereof.
An "excipient" or a "carrier" means a component or an ingredient that is
acceptable in the
sense of being compatible with the other ingredients of compositions or
formulations as disclosed
herein and not overly deleterious to the patient or animal to which the
formulation is to be
administered. As used here, excipients and carriers include liquids, including
benzyl benzoate,
cottonseed oil, N,N-dimethylacetamide, a C2_12 alcohol (e.g., ethanol),
glycerol, peanut oil, a
PEG, vitamin E, poppyseed oil, propylene glycol, safflower oil, sesame oil,
soybean oil and
vegetable oil. Excipients, as used herein may exclude solvents such as
chloroform, dioxane or
DMSO. Excipients comprise one or more components typically used in the
pharmaceutical
formulation arts, e.g., fillers, binders, disintegrants and lubricants.
Unless otherwise specified, expressions that refer to "a formula 1
compounds)", a
"compound of the invention", a "formula 2A or 2B compound", a "plasma
concentration-
enhanching compound" and the like mean compositions or methods, e.g., methods
to treat a
Trypanosome infection as disclosed herein, where one or more than one fon~nula
1 or formula 2A
or 2B compound is present, typically 1, 2, 3 or 4, usually i.
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"Alcohol" as used herein, includes excipients, means an alcohol that comprises
a C 1 _ 12
alkyl moiety substituted at one or more hydrogen atoms with one or more
hydroxyl groups, usually
one, two or three. Alcohols include, e.g., ethanol, n-propanol, i-propanol, n-
butanol, i-butanol, s-
butanol, t-butanol, n-pentanol, i-pentanol, n-hexanol, cyclohexanol, n-
heptanol, n-octanol, n-
nonanol, n-decanol and benzyl alcohol. The carbon atoms in alcohols can be
straight, branched or
cyclic. Alcohol includes any subset of the foregoing, e.g., C2_4 alcohols
(alcohols having 2, 3 or 4
carbon atoms).
"Ester" means a moiety that comprises a -C(O)-O- structure. Typically, esters
as used here
comprise an organic moiety containing about I-50 carbon atoms (e.g., about 2-
12 carbon atoms)
and 0 to about 10 independently selected heteroatoms (e.g., O, S, N, P, Si),
where the organic
moiety is bonded to a formula 1 steroid nucleus at R2 through the -C(O)-O-
structure, e.g., organic
moiety-C(O)-O-steroid or organic moiety-O-C(O)-steroid. The organic moiety
usually comprises
one or more of any of the organic groups described above, e.g., C 1-20 alkyl
moieties, C2_20
alkenyl moieties, C2_20 alkynyl moieties, aryl moieties, C2_9 heterocycles or
substituted
derivatives of any of these, e.g., comprising 1, 2, 3, 4 or more substituents,
where each substituent
is independently chosen. Typical substitutions for hydrogen or carbon atoms in
these organic
groups include 1, 2, 3, 4 or more, usually 1, 2, or 3 -O-, -S-, -NRPR-
(including -NH-), -C(O)-, -0,
=S, -N(RPR)2 (including -NH2), -C(O)ORPR (including -C(O)OH), -OC(O)RPR
(including -O-
C(O)-H), -ORPR (including -OH), -SRPR (including -SH), -N02, -CN, -NHC(O)-, -
C(O)NH-, -
OC(O)-, -C(O)O-, -O-A8, -S-A8, -C(O)-A8, -OC(O)-A8, -C(O)O-A8, =N-, -N=, =N-
OH, -
OP03(RPR)2, -OS03H2 or halogen moieties or atoms, where each RPR is -H, an
independently
selected protecting group or both RPR together comprise a protecting group,
and A8 is C1_g alkyl,
C2_g alkenyl, C2_g alkynyi, C 1 _4 alkyl-aryl (e.g., benryl), aryl (e.g.
phenyl) or C0_4 alkyl-C2_9
heterocycle. Substitutions are independently chosen. The organic moiety
includes compounds
defined by the R4 variable. The organic moieties exclude obviousiy unstable
moieties, e.g., -O-O-,
except where such unstable moieties are transient species that one can use to
make a compound
with sufficient chemical stability for the one or more of the uses described
herein. The
substitutions listed above are typically substituents that one can use to
replace one or more carbon
atoms, e.g., -O- or -C(O)-, or one or more hydrogen atom, e.g., halogen, -NH2
or -OH.
"Thioester" means a moiety that comprises a -C(S)-O- structure. Typically,
thioesters as
used here comprise an organic moiety containing about 1-50 carbon atoms (e.g.,
about 2-12 carbon
atoms) and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si), where the organic
moiety is bonded to a
formula 1 steroid nucleus at R2 through the -C(S)-O- structure, e.g., organic
moiety-C(S)-O-steroid
or organic moiety-O-C(S)-steroid. The organic moiety usually comprises one or
more of any of
the organic groups described above, e.g., C1_20 alkyl moieties, C2_20 alkenyl
moieties, C2_20
alkynyl moieties, aryl moieties, C2_9 heterocycles or substituted derivatives
of any of these, e.g.,
comprising 1, 2, 3, 4 or more substituents, where each substituent is
independently chosen.
Typical substitutions for hydrogen or carbon atoms in these organic groups
include l, 2, 3, 4 or
CA 02356539 2001-05-23
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more, usually 1, 2, or 3 -O-, -S-, -NRPR- (including -NH-), -C(O)-, =O, =S, -
N(RPR)2 (including -
NH2), -C(O)ORPR (including -C(O)OH), -OC(O)RPR (including -O-C(O~H), -ORPR
(including -
OH), -SRPR (including -SH), -N02, -CN, -NHC(O}-, -C(O)NH-, -OC(O)-, -C(O)O-, -
O-A8, -S-
A8, -C(O)-A8, -OC(O)-A8, -C(O)O-A8, =N-, -N=, =N-OH, -OP03(RPR)2, -OS03H2 or
halogen
moieties or atoms, where each RPR is -H, an independently selected protecting
group or both RPR
together comprise a protecting group, and A8 is C1_g alkyl, C2_g alkenyl, C2_g
alkynyl, CI~
alkyl-aryl (e.g., benzyl), aryl (e.g. phenyl) or CO_4 alkyl-C2_9 heterocycle.
Substitutions are
independently chosen. The organic moiety includes compounds defined by the R4
variable. The
organic moieties exclude obviously unstable moieties, e.g., -O-O-, except
where such unstable
moieties are transient species that one can use to make a compound with
sufficient chemical
stability for the one or more of the uses described herein. The substitutions
listed above are
typically substituents that one can use to replace one or more carbon atoms,
e.g., -O- or -C(O)-, or
one or more hydrogen atom, e.g., halogen, -NH2 or -OH.
"Thioacetal" means a moiety that comprises a -C(O)-S- structure. Typically,
thioacetals as
IS used here comprise an organic moiety containing about 1-50 carbon atoms
(e.g., about 2-12 carbon
atoms) and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si), where the organic
moiety is bonded to a
formula 1 steroid nucleus at R2 through the -C(O)=S- structure, e.g., organic
moiety-C(O)-S-steroid
or organic moiety-S-C(O)-steroid. The organic moiety is as described above for
thioesters.
"Carbamate" means an organic moiety as described for ester that comprises 1,
2, 3, 4 or
more -O-C(O)NRPR- structures where RPR is -H, a protecting group or an organic
moiety as
described for ester. Typically, carbamate groups as used here comprise an
organic moiety
containing about 1-50 carbon atoms (e.g., about 2-12 carbon atoms) and 0 to
about 10 heteroatoms
(e.g., O, S, N, P, Si), where the organic moiety is bonded to a formula 1
steroid nucleus at R2
through the -O-C(O)-NRPR- structure, e.g., organic moiety-NRPR-C(O)-O-steroid
or organic
moiety-O-C(O)-NRPR-steroid. The organic moiety is as described above for
thioesters.
"Sulfate ester" means a moiety that comprises a -O-S(O)(O)-O- structure.
Typically,
sulfate esters as used here comprise an organic moiety containing about I-50
carbon atoms (e.g.,
about 2-12 carbon atoms) and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si),
where the organic
moiety is bonded to a formula 1 steroid nucleus at R2 through the -O-S(O)(O)-O-
structure, e.g.,
organic moiety-O-S(O)(O)-O-steroid. The organic moiety is as described above
for thioesters.
"Sulfite ester" means a moiety that comprises a -O-S(O)-O- structure.
Typically, sulfite
esters as used here comprise an organic moiety containing about 1-50 carbon
atoms (e.g., about 2-
12 carbon atoms) and 0 to about 10 heteroatoms (e.g., O, S, N, P, Si), where
the organic moiety is
bonded to a formula 1 steroid nucleus at R2 through the -O-S(O)-O- structure,
e.g., organic
moiety-O-S(O)-O-steroid. The organic moiety is as described above for
thioesters.
The compositions disclosed herein optionally comprise salts of the formula 1
and 2
compounds that comprise an ionizable moiety or a polar moiety. As used herein,
"salts" include
complexes that comprise moieties of opposite charge. Ionizable moieties
include -O-S(O)(O)-OH
or a -NH2 group at R2 and polar moieties include -OH. Salts include
pharmaceutically acceptable
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salts that comprise, for example, an uncharged moiety or a monovalent anion
moiety or a
monovalent cation moiety. Salts include compounds derived by combination of
appropriate anions
such as inorganic acids. Suitable acids include those having sufficient
acidity to form a stable salt,
preferably acids of low toxicity. For example, one may form invention salts
from acid addition of
S certain inorganic acids, e.g., HF, HC1, HBr, HI, H2S04~ H3P04, to basic
centers, typically amines
that may be present in formula 1, 2A or 2B compounds. Or one may use certain
organic acids, e.g.,
organic sulfonic acids, organic carboxylic acids in the same manner. Exemplary
organic sulfonic
acids include C6-16 aryl sulfonic acids, C6_16 heteroaryl sulfonic acids and
C~_16 alkyl sulfonic
acids such as phenyl, a-naphthyl, ~i-naphthyl, (,S~-camphor, methyl, ethyl, n-
propyl, i-propyl, n-
butyl, s-butyl, i-butyl, t-butyl, pentyl and hexyl sulfonic acids. Exemplary
organic carboxylic
acids include C I _ 16 alkyl, C6_ 16 aryl carboxylic acids and C4_ I 6
heteroaryl carboxylic acids such
as acetic, glycolic, lactic, pyruvic, malonic, glutaric, tartaric, citric,
fumaric, succinic, malic,
malefic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,
salicylic and 2-
phenoxybenzoic. Salts also include the invention compound salts with one or
more amino acids.
Many amino acids are suitable, especially the naturally occurring amino acids
found as protein
components, although the amino acid typically is one bearing a side chain with
a basic or acidic
group, e.g., lysine, arginine or giutamic acid, or a neutral group such as
glycine, serine, threonine,
alanine, isoleucine, or ieucine. Salts are usually biologically compatible or
pharmaceutically
acceptable or non-toxic, particularly for mammalian cells. Salts that are
biologically toxic are
generally used as synthetic intermediates for making other invention
compounds.
The neohesperidoside, rutinoside and glucoside groups have the structures
O CH20H
HO O O
p_.
CH3 OH
HO
OH OH ~ OH and OH
respectively
wherein one or more of the hydrogen atoms are optionally independently
substituted with hydroxy,
halogen, CI_6 alkyl, CI-6 alkoxy, glucuronide or a CI-25 fatty acid.
Heterocvcie. "Heterocycle" or "heterocyclic" includes by way of example and
not
limitation these heterocycles described in Paquette, Leo A.; "Principles of
Modern Heterocyclic
Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6,
7, and 9; "The
I2
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Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York,
1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am.
Chem. Soc. 1960,
82:5566; and U.S. patent 5763483, all of which are incorporated herein by
reference.
Examples of heterocycles include by way of example and not limitation pyridyl,
thiazolyl,
tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,
furanyl, thienyl, pyrrolyl,
pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl,
indolenyl, quinolinyl,
isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-
pyrrolidonyl, pyrrolinyl,
tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,
decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-
1,5,2dithiazinyl, thienyl,
thianthrenyl, pyranyl, isobenzofuranyl, chromenyI, xanthenyl, phenoxathiinyl,
2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-
indolyl, IH-indazoly,
purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,
quinazolinyl, cinnolinyl,
pteridinyl, 4aH-carbazolyl, carbazolyl, ~carbolinyl, phenanthridinyl,
acridinyl, pyrimidinyl,
phenanthrolinyi, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl,
isochromanyl, chromanyl,
imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl,
indolinyl, isoindolinyl,
quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl,
oxindolyl,
benzoxazoIinyl, and isatinoyl.
By way of example and not limitation, carbon bonded heterocycles are bonded at
position
2, 3, 4, 5, or 6 of a pyridine, position 3, 4, S, or 6 of a pyridazine,
position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a
furan, tetrahydrofuran,
thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an
oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole,
position 2 or 3 of an aziridine,
position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a
quinoline or position 1, 3, 4, 5,
6, 7, or 8 of an isoquinoline. Still more typically, carbon bonded
heterocycles include 2-pyridyl, 3-
pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-
pyridazinyl, 6-pyridazinyl,
2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-
pyrazinyl, S-pyrazinyl,
6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.
By way of example and not limitation, nitrogen bonded heterocycles are bonded
at position
1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline,
imidazole, imidazolidine,
2-imidazoline, 3-imidazoline, pyrazole, pyrazoline; 2-pyrazoline, 3-
pyrazoline, piperidine,
piperazine, indole, indoline, IH-indazole, position 2 of a isoindole, or
isoindoline, position 4 of a
morpholine, and position 9 of a carbazole, or (3-carboline. Typically,
nitrogen bonded heterocycies
include 1-aziridyl, I-azetedy(, 1-pyrrolyl, 1-imidazolyl, I-pyrazolyl, and 1-
piperidinyl.
"Heteroaryl" means an aromatic ring or two or more fused rings that contain
one or more
aromatic rings where the ring or fused rings comprise 1; 2, 3 or more
heteroatoms, usually oxygen
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(-O-), nitrogen (-NX-) or sulfur (-S-) where X is -H, a protecting group or C
1 _6 alkyl, usually -H.
Examples are as described for heterocycle.
Protectine rouos. Various groups that the formula 1, 2A or 2B compounds may
comprise
include, e.g., substituted alkyl groups, substituted alkenyl groups, esters or
substituted
heterocycles, which can contain one or more reactive moieties such as
hydroxyl, or thiol.
Intermediates used to make formula 1 or formula 2A or 2B compounds may be
protected as is
apparent in the art. Noncyclic and cyclic protecting groups and corresponding
cleavage reactions
are described in "Protective Groups in Organic Chemistry", Theodora W. Greene
(John Wiley &
Sons, Inc., New York, 1991, ISBN 0-471-62301-6) (hereafter "Greene"). In the
context of the
present invention, these protecting groups are groups that can be removed from
the molecule of the
invention without irreversibly changing the covalent bond structure or
oxidation/reduction state of
the remainder of the molecule. For example, the protecting group, -X, that is
bondedZo a -OX or -
NHX group can be removed to form -OH or -NH2, respectively, without affecting
other covalent
bonds in the molecule. At times, when desired, more than one protecting group
can be removed at
a time, or they can be removed sequentially. In compounds of the invention
containing more than
one protecting group, the protecting groups are the same or different.
Protecting groups are intended to be removed by known procedures, although it
will be
understood that the protected intermediates fall within the scope of this
invention. The removal of
the protecting group may be arduous or straightforward, depending upon the
economics and nature
of the conversions involved. In general, one will use a protecting group with
exocyclic amines or
with carboxyl groups during synthesis of a formula 1 compound. For most
therapeutic applications
amine groups should be deprotected. Protecting groups commonly are employed to
protect against
covalent modification of a sensitive group in reactions such as alkylation or
acylation. Ordinarily,
protecting groups are removed by, e.g. hydrolysis, elimination or aminolysis.
Thus, simple
functional considerations will suffce to guide the selection of a reversible
or an irreversible
protecting group at a given locus on the invention compounds. Suitable
protecting groups and
criteria for their selection are described in T.W. Greene and P.G.M. Wuts,
Eds. "Protective Groups
in Organic Synthesis" 2nd edition, Wiley Press, at pps. 10-142, 143-174, 175-
223, 224-276, 277-
308, 309-405 and 406-454, which is incorporated herein by reference.
Determination of whether a group is a protecting group is made in the
conventional
manner, e.g., as illustrated by Kocienski, Philip J.; "Protecting Groups"
(Georg Thieme Verlag
Stuttgart, New York, 1994) (hereafter "Kocienski"}, Section 1.1, page 2, and
Greene Chapter 1,
pages 1-9; and U.S. patent 5763483, all of which are incorporated herein by
reference. In
particular, a group is a protecting group if when, based on mole ratio, 90% of
that protecting group
has been removed by a deprotection reaction, no more than 50%, preferably 25%,
more preferably
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WO 00/32201 PCT/US99/28079
I 0%, of the deprotected product molecules of the invention have undergone
changes to their
covalent bond structure or oxidation/reduction state other than those
occasioned by the removal of
the protecting group. When multiple protecting groups of the same type are
present in the
molecule, the mole ratios are determined when all of the groups of that type
are removed. When
multiple protecting groups of different types are present in the molecule,
each type of protecting
group is treated (and the mole ratios are determined) independently or
together with others
depending on whether the deprotection reaction conditions pertinent to one
type are also pertinent
to the other types present. In one embodiment of the invention, a group is a
protecting group if
when, based on mole ratio determined by conventional techniques, 90% of that
protecting group
has been removed by a conventional deprotection reaction, no more than 50%,
preferably 25%,
more preferably 10%, of the deprotected product molecules of the invention
have undergone
irreversible changes to their covalent bond structure or oxidation/reduction
state othefthaa those
occasioned by the removal of the protecting group. Irreversible changes
require chemical reactions
(beyond those resulting from aqueous hydrolysis, acid/base neutralization or
conventional
separation, isolation or purification) to restore the covalent bond structure
or oxidation/reduction
state of the deprotected molecule of the invention.
Protecting groups are also described in detail together with general concepts
and specific
strategies for their use in Kocienski, Philip J.; "Protecting Groups" (Georg
Thieme Verlag
Stuttgart, New York, 1994), which is incorporated by reference in its entirety
herein. In particular
Chapter 1, Protecting Groups: An Overview, pages 1-20, Chapter 2, Hydroxyl
Protecting Groups,
pages 21-94, Chapter 3, Diol Protecting Groups, pages 95-117, Chapter 4,
Carboxyl Protecting
Groups, pages 118-154, Chapter 5, Carbonyl Protecting Groups, pages 155-184,
Chapter 6, Amino
Protecting Groups, pages 185-243, Chapter 7, Epilog, pages 244-252, and Index,
pages 253-260,
are incorporated with specificity in the context of their contents. More
particularly, Sections 2.3
Silyl Ethers, 2.4 Alkyl Ethers, 2.5 Alkoxyalkyl Ethers (Acetals), 2.6 Reviews
(hydroxy and thiol
protecting groups), 3.2 Acetals, 3.3 Silylene Derivatives, 3.4 1,1,3,3-
Tetraisopropyldisiloxanylidene Derivatives, 3.5 Reviews (diol protecting
groups), 4.2 Esters, 4.3
2,6,7-Trioxabicyclo[2.2.2]octaves [OBO] and Other Ortho Esters, 4.4
Oxazolines, 4.5 Reviews
(carboxyl protecting groups), 5.2 O,O-Acetals, 5.3 S,S-Acetals, 5.4 O,S-
Acetals, S.5 Reviews
(carbonyl protecting groups), 6.2 N-Acyl Derivatives, 6.3 N-Sulfonyl
Derivatives, 6.4 N-Sulfenyl
Derivatives, 6.5 N-Alkyl Derivatives, 6.6 N-Silyl Derivatives, 6.7 Imine
Derivatives, and 6.8
Reviews (amino protecting groups), are each incorporated with specificity
where
protection/deprotection of the requisite functionalities is discussed. Further
still, the tables "Index
to the Principal Protecting Groups" appearing on the inside front cover and
facing page,
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"Abbreviations" at page xiv, and "reagents and Solvents" at page xv are each
incorporated in their
entirety herein at this location.
Typical hydroxy protecting groups are described in Greene at pages 14-118 and
include
Ethers (Methyl); Substituted Methyl Ethers (Methoxymethyl, Methylthiomethyl, t-
Butylthiomethyl, (Phenyldimethylsilyl)methoxymethyl, Benzyloxymethyl, p-
Methoxybenzyloxymethyl, (4-Methoxyphenoxy)methyl, Guaiacoimethyl, t-
Butoxymethyl, 4-
Pentenyloxymethyl, Siloxymethyl, 2-Methoxyethoxymethyl, 2,2,2-
Trichloroethoxymethyl, Bis(2-
chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl, Tetrahydropyranyl, 3-
Bromotetrahydropyranyl, Tetrahydropthiopyranyl, 1-Methoxycyclohexyl, 4-
methoxytetrahydropyranyl, 4-Methoxytetrahydrothiopyranyl, 4-
Methoxytetrahydropthiopyranyl
S,S-Dioxido, 1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl, 1,4-Dioxan-
2-yl,
Tetrahydrofuranyl, Tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-
trimeth~l-4,7-
methanobenzofuran-2-yl); Substituted Ethyl Ethers (1-Ethoxyethyl, 1-{2-
Chloroethoxy)ethyl, 1-
Methyl-1-methoxyethyl, 1-Methyl-1-benzyloxyethyl, 1-Methyl-1-benzyloxy-2-
fluoroethyl, 2,2,2-
Trichloroethyl, 2-Trimethylsilylethyl, 2-(Phenylselenyl)ethyl, t-Butyl, Allyl,
p-Chlorophenyl, p-
Methoxyphenyl, 2,4-Dinitrophenyl, Benzyl); Substituted Benzyl Ethers (p-
Methoxybenzyl, 3,4-
Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, p-Halobenzyl, 2,6-
Dichlorobenzyl, p-
Cyanobenzyl, p-Phenylbenzyl, 2- and 4-Picolyl, 3-Methyl-2-picolyl N-Oxido,
Diphenylmethyl, p,
p'-Dinitrobenzhydryl, 5-Dibenzosuberyl, Triphenylmethyl, alpha-
Naphthyldiphenylmethyl, p-
methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl)phenylmethyl, Trip-
methoxyphenyl)methyl,
4-(4'-Bromophenacyloxy)phenyldiphenylmethyl, 4,4', 4"-Tris(4,5-
dichlorophthalimidophenyl)methyl, 4,4', 4"-Tris(levulinoyloxyphenyl)methyl,
4,4', 4"-
Tris(benzoyloxyphenyl)methyl, 3-(Imidazol-1-ylmethyl)bis(4', 4"-
dimethoxyphenyl)methyl, 1,1-
Bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-Anthryl, 9-(9-Phenyl)xanthenyl, 9-(9-
Phenyl-10-
oxo)anthryl, 1,3-Benzodithiolan-2-yl, Benzisothiazolyl, S,S-Dioxido); Silyl
Ethers (Trimethylsilyl,
Triethylsilyl, Triisopropylsilyl, Dimethylisopropylsilyl,
Diethylisopropylsily, Dimethylthexylsilyl,
t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl, Tri-p-xylylsilyl,
Triphenylsilyl,
Diphenylmethylsilyl, t-Butylmethoxyphenylsilyl); Esters (Formate,
Benzoylfonmate, Acetate,
Choroacetate, Dichloroacetate, Trichloroacetate, Trifluoroacetate,
Methoxyacetate,
Triphenyfmethoxyacetate, Phenoxyacetate, p-Chlorophenoxyacetate, p-poly-
Phenylacetate, 3-
Phenylpropionate, 4-Oxopentanoate (Levulinate), 4,4-
(Ethylenedithio)pentanoate, Pivaloate,
Adamantoate, Crotonate, 4-Methoxycrotonate, Benzoate, p-Phenylbenzoate, 2,4,6-
Trimethyibenzoate (Mesitoate); Carbonates (Methyl, 9-Fluorenylmethyl, Ethyl,
2,2,2-
Trichloroethyl, 2-(Trimethylsilyl)ethyl, 2-(Phenylsulfonyl)ethyl, 2-
(Triphenylphosphonio)ethyl,
Isobutyl, Vinyl, Aliyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3,4-
Dimethoxybenzyl, o-
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Nitrobenryl, p-Nitrobenryl, S-Benryl Thiocarbonate, 4-Ethoxy-1-naphthyl,
Methyl
Dithiocarbonate); Groups With Assisted Cleavage (2-Iodobenzoate, 4-
Azidobutyrate, 4-Nitro-4-
methylpentanoate, o-(Dibromomethyl)benzoate, 2-Formylbenzenesulfonate, 2-
(Methylthiomethoxy)ethyl Carbonate, 4-(Methylthiomethoxy)butyrate, 2-
(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters (2,6-Dichloro-4-
methylphenoxyacetate, 2,6-Dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-Bis(1,1-
dimethylpropyl)phenoxyacetate, Chorodiphenylacetate, Isobutyrate,
Monosuccinoate, (E)-2-
Methyl-2-butenoate (Tigloate), o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, a-
Naphthoate,
Nitrate, Alkyl N,N,N', N'-Tetramethylphosphorodiamidate, N-Phenylcarbamate,
Borate,
Dimethylphosphinothioyl, 2,4-Dinitrophenylsulfenate); and Sulfonates (Sulfate,
Methanesulfonate
(Mesylate), Benryisulfonate, Tosylate).
More typically hydroxy protecting groups include subtituted methyl ethers,
substituted
benryl ethers, silyl ethers, and esters including sulfonic acid esters, still
more typically, triaikylsilyl
ethers, tosylates and acetates.
Typical 1,2- and 1,3-dioi protecting groups are described in Greene at pages
118-142 and
include Cyclic Acetals and Ketals (Methylene, Ethylidene, 1-t-Butylethylidene,
1-
Phenylethylidene, (4-Methoxyphenyl)ethylidene, 2,2,2-Trichloroethylidene,
Acetonide
(Isopropylidene), Cyclopentylidene, Cyclohexylidene, Cycloheptylidene,
Benrylidene, p-
Methoxybenrytidene, 2,4-Dimethoxybenrylidene, 3,4-Dimethoxybenrylidene, 2-
Nitrobenzylidene); Cyclic Ortho Esters (Methoxymethylene, Ethoxymethylene,
Dimethoxymethylene, 1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-
Dimethoxyethylidene, alpha-
Methoxybenrylidene, 1-(N,N-Dimethylamino)ethylidene Derivative, alpha-(N,N-
Dimethylamino)benrylidene Derivative, 2-Oxacyclopentyiidene); and Sily1
Derivatives (Di-t-
butylsilylene Group, 1,3-(1,1,3,3-Tetraisopropyldisiloxanylidene) Derivative,
Tetra-t-
butoxydisiloxane-1,3-diylidene Derivative, Cyclic Carbonates, Cyclic
Boronates, Ethyl Boronate,
Phenyl Boronate).
More typically, 1,2- and 1,3-diol protecting groups include epoxides and
acetonides.
Typical amino protecting groups are described in Greene at pages 315-385 and
include
Carbamates (Methyl and Ethyl, 9-Fluorenyimethyl, 9(2-Sulfo)fluoroenylmethyl, 9-
(2,7-
Dibromo)fluorenylmethyl, 2,7-Di-t-buthyl-[9-(10;10-dioxo-10,10,10,10-
tetrahydrothioxanthyl)]-
methyl, 4-Methoxyphenacyl); Substituted Ethyi (2,2,2-Trichoroethyl, 2-
Trimethylsilylethyl, 2-
Phenylethyl, 1-(1-Adamantyi)-1-methylethyl, l,l-Dimethyl-2-hatoethyl, 1,1-
Dimethyl-2,2-
dibromoethyl, 1,1-Dimethyl-2,2,2-trichloroethyl, 1-Methyl-1-(4-
biphenylyl)ethyl, 1-(3,5-Di-t-
butylphenyl)-1-methylethyl, 2-(2'- and 4'-Pyridyl)ethyl, 2-(N,N-
Dicyclohexylcarboxamido)ethyl, t-
Butyl, 1-Adamantyl, Vinyl, Allyl, 1-Isopropylallyl, Cirinamyl, 4-
Nitrocinnamyl, 8-Quinolyl, N-
17
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Hydroxypiperidinyl, Alkyldithio, Benzyl, p-Methoxybenzyl, p-Nitrobenzyl, p-
Bromobenzyl, p-
Chorobenzyl, 2,4-Dichlorobenzyl, 4-Methylsulfmylbenzyl, 9-Anthrylmethyl,
Diphenylmethyl);
Groups With Assisted Cleavage (2-Methylthioethyl, 2-Methylsulfonylethyl, 2-(p-
Toluenesulfonyl)ethyl, [2-(1,3-Dithianyl)]methyl, 4-Methylthiophenyl, 2,4-
DimethylthiophenyI, 2-
Phosphonioethyl, 2-Triphenylphosphonioisopropyl, 1,1-Dimethyl-2-cyanoethyl, m-
Choro-p-
acyloxybenzyl, p-(Dihydroxyboryl)benzyl, 5-Benzisoxazolylmethyl, 2-
(Trifluoromethyl)-6-
chromonylmethyl); Groups Capable of Photolytic Cleavage (m-Nitrophenyl, 3,5-
Dimethoxybenzyl, o-Nitrobenzyl, 3,4-Dimethoxy-6-nitrobenzyl, Phenyl(o-
nitrophenyl)methyl);
Urea-Type Derivatives (Phenothiazinyl-(10)-carbonyl Derivative, N'-p-
Toluenesulfonylaminocarbonyl, N'-Phenylaminothiocarbonyl); Miscellaneous
Carbamates (t-
Amyl, S-Benzyl Thiocarbamate, p-Cyanobenzyl, Cyclobutyl, Cyclohexyl,
Cyclopentyl,
Cyclopropylmethyl, p-Decyloxybenzyl, Diisopropylmethyl, 2,2-
Dimethoxycarbonylvinyl, o-(N,N-
Dimethylcarboxamido)benzyl, 1,1-Dimethyl-3-(N,N-dimethylcarboxamido)propyl,
1,1-
Dimethylpropynyl, Di(2-pyridyl)methyl, 2-Furanylmethyl, 2-Iodoethyl,
Isobornyl, Isobutyl,
Isonicotinyl, p-(p'-Methoxyphenyiazo)benzyl, 1-Methylcyclobutyl, 1-
Methylcyciohexyl, 1-Methyl-
1-cyclopropylmethyl, 1-Methyl-1-(3,5-dimethoxyphenyi)ethyl, 1-Methyl-1-(p-
phenylazophenyl)ethyl, 1-Methyl-1-phenylethyl, 1-Methyl-1-(4-pyridyl)ethyl,
Phenyl, p-
(Phenylazo)benzyl, 2,4,6-Tri-t-butylphenyl, 4-(Trimethylammonium)benzyl, 2,4,6-
Trimethylbenzyl); Amides (N-Formyl, N-Acetyl, N-Choroacetyl, N-Trichoroacetyl,
N-
Trifluoroacetyl, N-Phenylacetyl, N-3-Phenylpropionyl, N-Picolinoyl, N-3-
Pyridylcarboxamide, N-
Benzoylphenylalanyl Derivative, N-Benzoyl, N-p-Phenylbenzoyl); Amides With
Assisted
Cleavage (N-o-Nitrophenylacetyl, N-o-Nitrophenoxyacetyl, N-Acetoacetyl, (N'-
Dithiobenzyloxycarbonylamino)acetyl, N-3-(p-Hydroxyphenyl)propionyl, N-3-(o-
Nitrophenyl)propionyl, N-2-Methyl-2-(o-nitrophenoxy)propionyl, N-2-Methyl-2-(0-
phenylazophenoxy)propionyl, N-4-Chlorobutyryl, N-3-Methyl-3-nitrobutyryl, N-o-
Nitrocinnamoyl, N-Acetylmethionine Derivative, N-o-Nitrobenzoyl, N-o-
(Benzoyloxymethyl)benzoyl, 4,5-biphenyl-3-oxazolin-2-one); Cyclic Imide
Derivatives (N-
Phthalimide, N-Dithiasuccinoyl, N-2,3-Diphenylmaleoyl, N-2,5-Dimethylpyrrolyl,
N-1,1,4,4-
Tetramethyldisilylazacyclopentane Adduct, 5-Substituted 1,3-Dimethyl-1,3,5-
triazacyclohexan-2-
one, 5-Substituted 1,3-Dibenzyl-1,3,5-triazacyclohexan-2-one, 1-Substituted
3,5-Dinitro-4-
pyridonyl); N-Alkyl and N-Aryl Amines (N-Methyl, N-Allyl, N-[2-
(Trimethylsilyl)ethoxy]methyl,
N-3-Acetoxypropyl, N-(1-Isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl), Quaternary
Ammonium Salts,
N-Benzyl, N-Di(4-methoxyphenyl)methyl, N-5-Dibenzosuberyl, N-Triphenylmethyl,
N-(4-
Methoxyphenyl)diphenylmethyl, N-9-Phenylfluorenyl, N-2,7-Dichloro-9-
fluorenylmethylene, N-
Ferrocenylmethyl, N-2-Picolylamine N'-Oxide); Imine Derivatives (N-1,1-
Dimethylthiomethylene,
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N-Benzylidene, N-p-methoxybenylidene, N-Diphenylmethyiene, N-[(2-
Pyridyl)mesityi]methylene,
N,(N',N'-Dimethylaminomethylene, N,N'-Isopropylidene, N-p-Nitrobenzylidene,-N-
Salicylidene,
N-5-Chlorosalicylidene, N-(5-Chloro-2-hydroxyphenyl)phenylmethyiene, N-
Cyciohexylidene);
Enamine Derivative (N-(5,5-Dimethyl-3-oxo-1-cyclohexenyl)); N-Metal
Derivatives (N-Borane
Derivatives, N-Diphenylborinic Acid Derivative, N-
[Phenyl(pentacarbonylchromium- or -
' tungsten)]carbenyl, N-Copper or N-Zinc Chelate); N-N Derivatives (N-Nitro, N-
Nitroso, N-
Oxide); N-P Derivatives (N-Diphenylphosphinyl, N-Dimethylthiophosphinyl, N-
Diphenylthiophosphinyl, N-Diaikyl Phosphoryl, N-Dibenryl Phosphoryl, N-
biphenyl Phosphoryl);
N-Si Derivatives; N-S Derivatives; N-SulfenyI Derivatives (N-Benzenesulfenyl,
N-o-
Nitrobenzenesulfenyl, N-2,4-Dinitrobenzenesulfenyl, N-
Pentachlorobenzenesulfenyl, N-2-nitro-4-
methoxybenzenesulfenyl, N-Triphenylmethylsulfenyl, N-3-Nitropyridinesulfenyl);
andN-Sulfonyl
Derivatives (N-p-Toluenesulfonyl, N-Benzenesulfonyl, N-2,3,6-Trimethyl-4- '
methoxybenzenesuifonyl, N-2,4,b-Trimethoxybenzenesulfonyl, N-2,6-Dimethyl-4-
methoxybenzenesulfonyl, N-Pentamethylbenzenesulfonyl, N-2,3,5,6,-Tetramethyl-4-
methoxybenzenesulfonyl, N-4-methoxybenzenesulfonyl, N-2,4,6-
Trimethylbenzenesulfonyl, N-
2,6-Dimethoxy-4-methylbenzenesulfonyl, N-2,2,5,7,8-Pentamethylchroman-6-
sulfonyl, N-
Methanesulfonyl, N-.beta.-Trimethylsilyethanesulfonyl, N-9-Anthracenesulfonyl,
N-4-(4', 8'-
Dimethoxynaphthylmethyl)benzenesulfonyl, N-Benzylsulfonyl, N-
Trifluoromethylsulfonyl, N-
Phenacylsulfonyl).
More typically, amino protecting groups include carbamates and amides, still
more
typically, N-acetyl groups.
Stereoisomers. The formula 1 and fonmula 2A or 2B compounds include enriched
or
resolved optical isomers at any or all asymmetric atoms as are apparent from
the depictions. Both
racemic and diasteromeric mixtures, as well as the individual optical isomers
can be isolated or
synthesized so as to be substantially free of their enantiomeric or
diastereomeric partners, and
these are all within the scope of the invention. Chiral centers may be found
in invention
compounds at, for example, RI, R2 or R4.
One or more of the following methods are used to prepare the enantiomerically
enriched
or pure isomers herein. The methods are listed in approximately their order of
preference, i.e.; one
ordinarily should employ stereospecific synthesis from chiral precursors
before chromatographic
resolution before spontaneous crystallization.
Stereospecific synthesis is described in the examples. Methods of this type
conveniently
are used when the appropriate chiral starting material is available and
reaction steps are chosen that
do not result in undesired racemization at chiral sites. One advantage of
stereospecific synthesis is
that it does not produce undesired enantiomers that must be removed from the
final product,
19
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thereby lowering overall synthetic yield. In general, those skilled in the art
would understand what
starting materials and reaction conditions should be used to obtain the
desired enantiomerically
enriched or pure isomers by stereospecific synthesis.
If a suitable stereospecific synthesis cannot be empirically designed or
determined with
S routine experimentation then those skilled in the art would turn to other
methods. One method of
' general utility is chromatographic resolution of enantiomers on chiral
chromatography resins.
These resins are packed in columns, commonly called Pirkle columns, and are
commercially
available. The columns contain a chiral stationary phase. The racemate is
placed in solution and
loaded onto the column, and thereafter separated by HPLC. See for example,
Proceedings
Chromatographic Society - International Symposium on Chiral Separations, Sept.
3-4, 1987, which
is incorporated herein by reference. Examples of chiral columns that could be
used to screen for
the optimal separation technique would include Diacel Chriacel OD, Regis
Pirkle Co4alent D-
phenylglycine, Regis Pirkle Type 1 A, Astec Cyclobond II, Astec Cyclobond III,
Serva Chiral D-
DL=Daltosil 100, Bakerbond DNBLeu, Sumipax OA-1000, Merck Cellulose Triacetate
column,
Astec Cyciobond I-Beta, or Regis Pirkle Covalent D-Naphthylalanine. Not all of
these columns
are likely to be effective with every racemic mixture. However, those skilled
in the art understand
that a certain amount of routine screening may be required to identify the
most effective stationary
phase. When using such columns it is desirable to employ embodiments of the
compounds of this
invention in which the charges are not neutralized, e.g., where acidic
functionalities such as
carboxyl are not esterified or amidated.
Another method entails converting the enantiomers in the mixture to
diasteriomers with
chiral auxiliaries and then separating the conjugates by ordinary column
chromatography. This is a
very suitable method, particularly when the embodiment contains a free
hydroxyl that will form a
salt or covalent bond to a chiral auxiliary. Chirally pure amino acids,
organic acids or
organosulfonic acids are all worthwhile exploring as chiral auxiliaries, all
of which are well known
in the art. Salts with such auxiliaries can be formed, or they can be
covalently (but reversibly)
bonded to the functional group.
Enzymatic resolution is another method of potential value. In such methods one
prepares
covalent derivatives of the enantiomers in the racemic mixture, generally
lower alkyl esters, and
then exposes the derivative to enzymatic cleavage; generally hydrolysis. For
this method to be
successful an enzyme must be chosen that is capable of stereospecific
cleavage, so it is frequently
necessary to routinely screen several enzymes. If esters are to be cleaved,
then one selects a group
of esterases, phosphatases, and lipases and determines their activity on the
derivative. Typical
esterases are from liver, pancreas or other animal organs, and include porcine
liver esterase.
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If the enatiomeric mixture separates from solution or a melt as a
conglomerate, i.e., a
mixture of enantiomerically pure crystals, then the crystals can be
mechanically separated, thereby
producing the enantiomerically enriched preparation. This method, however, is
not practical for
large-scale preparations and is of limited value for true racemic compounds.
Asymmetric synthesis is another technique for achieving enantiomeric
enrichment. For
example, a chiral protecting group is reacted with the group to be protected
and the reaction
mixture allowed to equilibrate. If the reaction is enantiomerically specific
then the product will be
enriched in that enantiomer.
Further guidance in the separation of enantiomeric mixtures can be found, by
way of
example and not limitation, in "Enantiomers, Racemates, and resolutions", Jean
Jacques, Andre
Collet, and Samuel H. Wilen (Krieger Publishing Company, Malabar, FL, 1991,
ISBN 0-89464-
618-4): Part 2, Resolution of Enantiomer Mixture, pages 217-435; more
particularly, Sbction 4,
Resolution by Direct Crystallization, pages 217-251, section 5, Formation and
Separation of
Diastereomers, pages 251-369, section 6, Crystallization-Induced Asymmetric
Transformations,
pages 369-378, and section 7, Experimental Aspects and Art of Resolutions,
pages 378-435; still
more particularly, section 5.1.4, Resolution of Alcohols, Transformation of
Alcohols into Salt-
Forming Derivatives, pages 263-266, section 5.2.3, Covalent Derivatives of
Alcohols, Thiols, and
Phenols, pages 332-335, section 5.1.1, Resolution of Acids, pages 257-259,
section 5.1.2,
Resolution of Bases, pages 259-260, section 5.1.3, Resolution of Amino Acids,
page 261-263,
section 5.2.1, Covalent Derivatives of Acids, page 329, section 5.2.2,
Covalent derivatives of
Amines, pages 330-331, section 5.2.4, Covalent Derivatives of Aldehydes,
Ketones, and
Sulfoxides, pages 335-339, and section 5.2.7, Chromatographic Behavior of
Covalent
Diastereomers, pages 348-354, all of which are incorporated herein by
reference.
Embodiments include compositions that transiently occur when a method step or
operation
is performed. For example, when a formula 1 compound is contacted with an
excipient, e.g.,
water, a cyciodextrin, a PEG, an alcohol, propylene glycol, benzyl alcohol or
benzyl benzoate, the
composition before addition of one ingredient with another is a non-homogenous
mixture. As the
ingredients are contacted, the mixture's homogeneity increases and the
proportion of ingredients
relative to each other approaches a desired value. Thus, some compositions as
disclosed herein,
optionally contain less than about 3% w/w water, e.g., less than 0.5% w/w
water, can comprise
about 0.0001-99% w/w of a formula 1 compound such as l6oc-bromoepiandrosterone
and one or
more excipients. These transient compositions are intermediates that
necessarily arise when one
makes an invention composition or formulation and they are included in
invention embodiments to
the extent that they are patentable.
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Formula 1 compounds. The formula 1 compounds, or the "compounds of the
invention",
are useful to treat a subject having, or prevent infection of a subject with,
one or more
Trypanosome parasites.
For preferred formula 1 compounds, the R2 moiety bonded to the steroid ring is
generally
in the (3-configuration, two R1 are bonded to Q2 and X is a double bonded
oxygen moiety (~).
Typically, one of the R1 bonded to Q2 is hydrogen in the (configuration, the
other R1 bonded to
Q2 is hydrogen or a halogen, usually bromine, in the a-configuration and a
double bond is present
at the 5-6 positions. Such preferred compounds include dehydroepiandrosterone
("DHEA") and
16a-bromodehydroepiandrosterone ("Br-DHEA").
Other preferred formula 1 compounds include 17-ketosteriods of formula 1
where a double bond is present at the 5-6 positions, X is =O, Q2 is -CH2- or -
CHBr-, R2 is -H, -
S(O)(O)-OH, -S(O)(O)-ONa, -S(O)(O)-O-CH2-CH(O-C(O)-R6)-CH2-O-C(O)-R6 (where R6
independently is C1-14 straight or branched alkyl), -P(O)(O)-O-CH2-CH(O-
C(O)R7)-CH2-O-
C(O)-R7 (where R7 independently is a glucuronide group or C 1 _ 14 straight or
branched alkyl) or
R2 is a glucuronide group. Other preferred compounds include compound having
the structures
20-43
R~ R~ X Rt R~ X
R~ R' Q6 R~ R R~ ~s
R~ ~ R
R, R~~ ~ R~ i~ R. R1~ ~ R,
/ Qz R ~ n_ R
R' ~R~ R~ R~
R2 R~ R~ R2 .R~.
R~ ~ ~ ~ ~ 'R~ R~ ~ ~ ~ ~~R~
R~ R~ R~ R~
R~ R~ R~ R~ R~ R~ R~ R~
21
Rt R~ X R~ R~ X
R~ Qs R~ R~ Qs
R~ Y R~
R~ R~ ~ ~~ R~~ ~ R~ Y
R~ R? nR
wR .~ 1 _R~ ..~ \ '~~ ~ ~R~
RZ ~ R~ R2 R' R~
Rt ~ ~ ~ ~ ,R~ R~ ~ I ~ ~~R~
R~ R~ R~ R~
20 Rt R~ R~ R~ R~ R~ R~ R~
22 23
22
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WO 00/32201 PCTNS99/28079
Rt Rt X R~
Rt Qs Rt Rt R~
X
Qs
Rt R~
Rt Rt Y R~ Rt Y
Rt Rt
Rt Rt ~3
Rt Rt ~Rt Rt
_
Rt
~Rt
_ Rz Rt Rt R2 Rt
Rt
Rt ~ I ~ Rt ~ ~ ~
~ Rt ,Rt
Rt Rt Rt Rt
Rt Rt ~ Rt
Rt Rt
Rt Rt
Rt
24 25
R
Rt t X Rt
Rt Ds R Rt
t X
Rt Rt a s
R Rt Rt
Rt t R Rt
Rt t
Rt Q3 R R Rt
Rt ~
t
Q R
Rt Rt ~ s
R Rt Rt Rt
R2 t Rt ~
R
Rt
Rt ~ ~ Rz Rt
~ ~Rt ~ t
~ Rt Rt R~
Rt Rt Rt
Rt Rt Rt
Rt
26 2~
Rt Rt X
Rt Qs R Rt Rt
t X
Rt Rt ~ s
R Y Rt
Rt t Rt Y
Rt
Rt Q R R Rt ~
Rt
t
Q3 R
Rt R~ ~
R Rt Rt Rt
Rz t Rt ~
Rt
Rt ~ ~ W Rz ~ Rt
Rt R
t
~ Rt Rt ~ '
~ 'Rt
Rt Rt Rt
Rt Rt Rt
Rt
28 29
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2001-05-23
WO 00/32 201 PCT/US99/28079
RtRt X Rt Rt
Rt Ds Rt Rt ( ~s_X
.
Rt Rt
Rt Rt Rt Rt
Rt Rt
Rt Q3 Y Rt G~3 Y
Rt Rt ~ Rt R t ~
Rt R
Rz Rt R R ,Rt ~
~ t 2 ~ Rt
Rt ~ \ Rt ~
Rt ~ ~Rt
~ Rt ~ Rt
Rt Rt Rt Rt
Rt R
t
30 31
RtRt X .. o
Rt DsRt
Rt
Rt Rt t
Rt Rt
Rt
Q3 R R
Rt R i ~ Rt
R2 _R~ R R2
Rt
t
Rt ~ R ~ ~~O R
~
Rt Rt Rt
Rt
32 33
RtRt X Rt Rt X
Rt Q s Rt Rt ~ s
Rt Rt
R Rt Y R Rt Y
R R
R t t R t t
t G73 R t Q R
I 3
Rt R ~ Rt R
R Rt t Rt
R2 ~ R R Rt
t 2 Rt
Rt ~ ~ \O Rt ~
R
Rt t
Rt Rt Rt Rt Rt Rt
Rt Rt
34 35
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R~ R~ X R~ R~
R1 Os R1 R' X
Qs_
R~ R~
R, R' R' Y R R, Y
R' R, R'
Q3 ~ 3
R~ R~ '\ Ri
w R~ \
R~
R'
_ Rz R' R~ Rz R'
R~
I w0 ~ ( w0
R~ ~ ~ R ~ \
~
R~ R~ R~ R1 R~
Ri R~
R~ R~
R~
36 37
R~ R~ X R~ R~
R~ Ds R~ Rt X
Ds
R~ Ri
R~ Ri R R~ R~ R
R~ R~
R, 03 R~ R Q3 R~
R~ R' ~R~ R' R~
~R
-
Rz \ R~ R1 Rz \ R~
R
R~ O R~ O
Ri R~ R~ R~ R~
R~
38 39
R~ R~ X R~ Ri
R~ Ds R~ R~ X
Ds
R~ R~
R~ R' Y R~ R~ Y
R~ R~
R1 Q3 \ R R Q3 \
R
R~ R~ ~ R~ R'
v R~ \
R~
Rz ~ R~ R~ R2 ~ R~
~ R~
R~ ~ ~ \ R~ ~ \
O ~ O
R~ Rt R~ R~ R~
R~
40 41
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WO 00/32201 PCT/US99/28079
R
R~ R~ ~ afi X R, R' R~ R~ ~s _ X
R~ R~
Rt o . .., R, ,.,
R~ _ r R~
Q3
R~ R' ~ R R'
R2 ~ 'R~ R~ R2w
R, ~ ~ ~ O R,/
R~ R~ R~ Ff~ R~ R~
42 43
wherein for each of structures 20-43
Q3 and Q6 are each -C(Rl)3 wherein each Rl is independently selected;
X and Y independently are -OH, -H, lower alkyl (e.g., C1_6 alkyl), -C(O)-O-Rs,
-O-C(Or
R5, halogen or, X and Y together with the R1 at the same position
independently are a ketone
(=o)
each R1 is independently selected and has the definition given above; and
R2 and RS have the definitions given above.
In some embodiments, the formula 1 compound has the structure 20-43 and 2, 3,
4, 5 or 6
R1 groups independently are -OH, halogen or alkoxy, and the remaining R1 are
all hydrogen; R2 is
-OH, an ester a thioester or a carbamate, or R2, together with the R1 at the 3-
position comprises
=O; Y is -H, -OH, a halogen or -O-C(O)-RS, or Y, together with the Rl at the
16-position
comprises =O; X is -OH or -O-C(O)-R5, or X, together with the R1 at the 17-
position comprises
=O; and Q3 and Q6 independently are -CH3 or -CH20H. Such embodiments include
structure 20-
43 compounds where two -OH are present at the 3-position, the 16-position or
at the 17-position.
Preferred invention embodiments include compounds having the formula 44
Y
44
wherein Y is hydrogen or bromine, R44 is -H, -S(O)(O)-OH, -S(O)(O)-ONa, -
S(O)(O)-O-CH2-
CH(O-C(O)-R6)-CH2-O-C(O)-R6, -P(O)(O)-O-CH2-CH(O-C(O)-R7)-CH2-O-C(O)-R7 or a
glucuronide group of structure (A). In other preferred embodiments, Y and R44
in formula 44 are
both hydrogen. An especialiy preferred compound is dehydroepiandrosterone (Y
and R44 in
formula 44 are both hydrogen and the double bond at the 5-6 position is
present). In other
embodiments, the compound is epiandrosterone (Y and R44 in formula 44 are both
hydrogen and
26
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WO 00/32201 PCT/US99/28079
the double bond at the 5-6 position is absent). A 16-haloepiandrosterone with
a F, Cl, Br or I at the
16 position can also be used as an antiviral agent, e.g., 16a-
bromoepiandrosterone. Other
preferred compounds are (i) 16a-bromodehydroepiandrosterone, (ii)
dehydroepiandrosterone-3-
sulfate (Y is -H and R44 is -S(O)(O)-OM in formula 44 are both hydrogen and
the double bond at
the 5-6 position is present) and (iii) 5(3-androstan-3(3-0l-17-one. Related
embodiments comprise
' compounds related to formula 44 compounds comprise the formula 44 compounds
wherein 1, 2, 3,
4, 5 or 6 hydrogen atoms that are bonded to the steroid nucleus are
substituted with independently
selected -OH, -Br, -Cl, -F, -I, -OCH3 or -OC2H5 atoms or groups.
In other embodiments, the 17-ketosteroids of formula 1 are dehydro-
epiandrosterone where
R44 in formula 44 is a -S(O)(O)-O-CH2-CH(O-C(O)-R6)-CH2-O-C(O)-RS, -P(O)(O)-O-
CH2-
CH(O-C(O)-R7)-CH2-O-C(O)-R7 or a glucuronide group of structure (A), Y is
hydrogen and the
S-6 double bond is present. Other formula 44 compounds include conjugates of
dehydroepiandrosterone wherein Y is hydrogen, a double bond is present at the
5-6 position and
R44 is hexyl sulfate, dodecyl sulfate, octadecyl sulfate, octadecanoyl
sulfate, O-
dihexadecylglycerol sulfate, hexadecane sulfonate, dioctadecanoylglycerol
phosphate or O-
hexadecylglycerol phosphate.
In another preferred aspect of the invention, the steroid of formula 1 is a
compound of
formula 45
Rs~
~so 45
wherein Rsp is -H, -OH or =O; RS 1 is -Br, -Cl, -F or -I; R52 is -OH or =O;
R49 is -H, -OH, or -
OR53; and R53 is C 1 _ 1 g alkyl, C2_ 1 g alkenyl, C2_ 1 g alkynyl, a C 1 _ 1
g ester, a C 1 _ 1 g thioester,
wherein any of the foregoing C 1 _ 1 g or C2_ 1 g groups is substituted at one
or more hydrogen atoms
with one or more independently selected -O-, -S-, -OH, -NH2, -SH or =O groups
or R53 is
thioacetal, a sulfate ester, a sulfonate ester, a carbamate or a thioester. In
one preferred aspect, R49
is -O-C(O)-CH2-CH2-CH(R54)-CH(R55)-CH2R56 wherein R54 is -NH2, -OH, -SH, -O-
P03, -
S03 or -OS03; R55 is -H, -NH2, -OH, -SH, -O-P03, -S03 or -OS03; and R56 is C 1
_ 1 g alkyl, C2_
1 g aIkenyl, C2_ 1 g aikynyl, a C 1 _ 1 g ester, a C 1 _ 1 g thioester,
wherein any of the foregoing C 1 _ 1 g or
C2_lg groups is substituted at one or more hydrogen atoms with one or more
independently
selected -OH, -NH2 or -SH groups, and the precursors, metabolites and analogs
thereof. Related
embodiments comprise compounds related to formula 44 compounds comprise the
formula 45
27
r.u R~~
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
compounds wherein 1, 2, 3, 4, 5 or 6 hydrogen atoms that are bonded to the
steroid nucleus are
substituted with independently selected -OH, -Br, -C1, -F, -I, -OCH3 or -OC2H5
atoms or groups.
In other preferred embodiments, the formula 1 compounds have the formula 1B or
IC
X
1B or - IC
wherein each RI independently is -H, -OH, a halogen, -CHCH2, -CHCHCH3, -CCH, -
CCCH3, or,
or, the other moiety that is bonded to the same carbon atom is absent and RI
is =O; R2 is -H, -OH,
a halogen, C 1-g alkoxy, -S-C(O)-(CH2)m-R4, -C(O)-S-(CH2)m-R4, -O-S(O)(O)-
(CI~)m-R4, -O-
S(O)(O)-O-(CH2)m-R4~ -O-C(O)-NH-(CH2)m-R4~ -NH-C(O)-O-(CH2)m-R4, -O-C(S)-(CH2~-
R4, -C(S)-O-(CH2)m-R4~ -O-C(O)-(CH2)m-R4 or -C(O)-O-(CH2)m-R4~ R4 is -H, -CH3,
-C2H5
-C3H~, -C2H40H, -C3H60H, -CH2-CH2-O-CH3, -CH2-CH2-O-CH2-CH3, -CH2-CH2-O-CH2-
CH20H, a C3_6 alkenyi group, a C3_6 alkynyl group, benzyl or phenyl, wherein
the phenyl or
benzyl groups are optionally substituted with 1, 2, or 3 independently
selected halogen, C 1 ~
alkoxy, -OH, -SH, -O- or -NH- moieties; and Q3 and Q6 independently are -H, -
CH3 or -CH20H;
and Q2 is -C(RI)2- or -CH2-CH2-. In these embodiments, Q3 and Q6 are usually
both in the /3-
IS configuration, typically they are -CH3, Q2 usually comprises -CH2-, -C(O)-,
-CH(Br)-, -CH(I)-, or
-CH(OH)- with the Br, I or OH moieties in the a.-configuration, or Q2
comprises =O, and RI at the
7-position is -H, -OH or =O. Related embodiments comprise compounds related to
formula 44
compounds comprise the formula lA or IB compounds wherein 1, 2, 3, 4, 5 or 6
hydrogen atoms
that are bonded to the steroid nucleus are substituted with independently
selected -OH, -Br, -C1, -F,
-I, -OCH3 or -OC2H5 atoms or groups.
The formula 1 compounds can exist in a crystalline or polymorphic form.
Metabolites. Also falling within the scope of this invention are the in vivo
metabolites of
the compounds of the invention, to the extent such products are novel and
unobvious over the prior
art. Such products may result for example from the oxidation, reduction,
hydrolysis, amidation,
esterification and the like of the administered formula 1 compound, due to
enzymatic or chemical
processes. Accordingly, the invention includes novel and unobvious compounds
produced by a
process comprising contacting a compound of this invention with a subject,
e.g., a human, rodent
or a primate, for a period of time sufficient to yield a metabolic product
thereof. Such products
typically are identified by preparing a radiolabelled (e.g. C 14 or H3)
compound of the invention,
administering it parenterally or orally in a detectable dose (e.g. greater
than about 0.5 mg/kg) to an
animal such as rat, mouse, guinea pig, primate, or to a human, allowing
sufficient time for
28
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
metabolism to occur (typically about 30 seconds to about 30 hours) and
isolating its conversion
products from the urine, blood or other biological samples. These products are
easily isolated since
they are labeled (others are isolated by the use of antibodies capable of
binding epitopes surviving
in the metabolite). The metabolite structures are determined in conventional
fashion, e.g. by
S HPLC, MS or NMR analysis. In general, analysis of metabolites is done in the
same way as
' conventional drug metabolism studies well-known to those skilled in the art.
The conversion
products, so long as they are not otherwise found in vivo, are useful in
diagnostic assays for
therapeutic dosing of the compounds of the invention even if they possess no
therapeutic activity
of their own.
The following description exemplifies embodiments of the formula 1 compounds.
Group 1. Exemplary embodiments include the formula I compounds named in table
B
based on the compound structure designations defined in table A. Each compound
nafned in Table
B is depicted as a compound of formula 4
Rz
4
I S where Q3 and Q6 are both -CH3, Q4 is -CH2- and R2, R1 A, and Y and X have
the structures
designated in Table A. The compounds named according to Tables A and B are
referred to as
"group 1" compounds.
Compounds named in Table B are designated by numbers assigned to R2, R1A, Y
and X
according to the following compound naming convention, R2.RIA.Y.X, using the
numbered
chemical structures depicted in Table A. As shown in formula 4, R2 is in the
3Gi-position and
hydrogen fills the remaining valence or R2 is double bonded to the 3 carbon,
R1A is an R1 group
at the 7~i-position or R1A is an R1 group double bonded to the 7 carbon, Y is
in the 16a.-position
and hydrogen fills the remaining valence or R2 is double bonded to the 16
carbon and X is in the
17(3-position and hydrogen fills the remaining valence or X is double bonded
to the 17 carbon.
When R2, R1A, Y or X is a divalent moiety, e.g., =O, the hydrogen at the
corresponding position is
absent. Thus, the group 1 compound named 1.2.1.1 specifies a formula 4
structure with a (3-
hydroxyl bonded to carbons at the 3- and 7-positions (the variable groups R2
and R1A
29
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
respectively), an oc-bromine bonded to carbon 16 (the variable group Y) and
double bonded oxygen
(=O) at carbon 17 (the variable group X), i.e., having the structure shown
below:
H
1.2.1.1
TABLE A
R1A
1 -OH I -H
2 =O 2 -OH
3 -O-P(O)(O)-OH 3 =O
4 -O-P(O)(O)-O-CH2-CH(O-C(O)-CH3)-CH2-O-C(O)CH34 -CH3
5 -O-S(O)(O)-OH 5 -OCH3
6 -O-S(O)(O)-O-Na+ 6 -OC2H5
7 -O-S(O)(O)-OC2H5 7 -OCH2CH2CH3
8 -O-S(O)(O)-O-CH2-CH(O-C(O)-CH3)-CH2-O-C(O)CH38 -OCH(CH3)CH3
9 -O-S(O)(O)-OCH2CH2CH2CH3 9 -OCH2CH2CH2CH3
10 -O-S(O)(O)-OC(CH3)3 10 -OC(CH3)3
I' X
1 -Br 1 =O
2 -Ci 2 -OH
3 -I 3 -H
4 -F 4 -F
5 -H 5 -Cl
6 -OH 6 -Br
7 =O 7 -I
8 -O-C(O)-CH3 8 -O-C(O)-CH3
9 -O-C(O)-CH2CH3 9 -O-C(O)-CH2CH3
10 -O-Cl0)-CH2CH C~,-I3 1 0 -O-C(O)-CH2CH2CH3
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
TABLE B
1.1.1.1, 1.1.1.2, 1.1.1.3, 1.1.1.4, 1.1.1.5, 1.1.1.6, 1.1.1.7, 1.1.1.8,
1.1.1.9, 1.1.1.1 0, 1.1.2.1, 1.1.2.2,
1.1.2.3, 1.1.2.4, 1.1.2.5, 1.1.2.6, 1.1.2.7, 1.1.2.8, 1.1.2.9, 1.1.2.1 0,
1.1.3.1, 1.1.3.2, 1.1.3.3, 1.1.3.4,
1.1.3.5, 1.1.3.6, 1.1.3.7, 1.1.3.8, 1.1.3.9, 1.1.3.10, 1.1.4.1, 1.1.4.2,
1.1.4.3, 1.1.4.4, 1.1.4.5, 1.1.4.6,
1.1.4.7, 1.1.4.8, 1.1.4.9, 1.1.4.10, 1.1.5.1, 1.1.5.2, 1.1.5.3, 1.1.5.4,
1.1.5.5, 1.1.5.6, 1.1.5.7, 1.1.5.8,
- 1.1.5.9, 1.1.5.10, 1.1.6.1, 1.1.6.2, 1.1.6.3, 1.1.6.4, 1.1.6.5, 1.1.6.6,
1.1.6.7, 1.1.6.8, 1.1.6.9, 1.1.6.10,
1.1.7.1,1.1.7.2,1.1.7.3, 1.1.7.4, 1.1.7.5, 1.1.7.6, 1.1.7.7,1.1.8.1,1.1.8.2,
1.1.7.8, 1.1.7.9, 1.1.7.10,
1.1.8.3,1.1.8.4,1.1.8.5, 1.1.8.6, 1.1.8.7, 1.1.8.8, 1.1.8.9,1.1.9.3,1.1.9.4,
1.1.8.10, 1.1.9.1, 1.1.9.2,
1.1.9.5,1.1.9.6,1.1.9.7,1.1.9.8,1.1.9.9,1.1.9.10,1.1.10.1,1.1.10.2,1.1.10.3,1.1
.10.4,1.1.10.5,
1.1.10.6,1.1.10
.7,1.1.10.8,1.1.10.9,1.1.10.10,1.2.1.1,1.2.1.2,1.2.1.3,1.2.1.4,1.2.1.5,1.2.1.6,
1.2.1.7,1.2.1.8,1.2.1.9, 1.2.1.10, 1.2.2.1, 1.2.2.2, 1.2.2.7,1.2.2.8,
1.2.2.3, 1.2.2.4, 1.2.2.5, 1.2.2.6,
1.2.2.9,1.2.2.10, 1.2.3.9,1.2.3.10,
1.2.3.1,
1.2.3.2,
1.2.3.3,
1.2.3.4,
1.2.3.5,
1.2.3.6,
1.2.3.7,
1.2.3.8,
1.2.4.1,1.2.4.2,1.2.4.3, 1.2.4.4, 1.2.4.5, 1.2.4.6, 1.2.4.7,1.2.5.1,1.2.5.2,
1.2.4.8, 1.2.4.9, 1.2.4.10,
1.2.5.3,1.2.5.4,1.2.5.5, 1.2.5.6, 1.2.5.7, 1.2.5.8, 1.2.5.9,1.2.6.3,1.2.6.4,
1.2.5.1 0, 1.2.6.1, 1.2.6.2,
1.2.6.5,1.2.6.6,1.2.6.7, 1.2.6.8, 1.2.6.9, 1.2.6.10, 1.2.7.5,1.2.7.6,
1.2.7.1, 1.2.7.2, 1.2.7.3, 1.2.7.4,
1.2.7.7,1.2.7.8,1.2.7.9, 1.2.7.10, 1.2.8.1, 1.2.8.2, 1.2.8.7,1.2.8.8,
1.2.8.3, 1.2.8.4, 1.2.8.5, 1.2.8.6,
1.2.8.9,1.2.8.10, 1.2.9.9,1.2.9.10,
1.2.9.1,
1.2.9.2,
1.2.9.3,
1.2.9.4,
1.2.9.5,
1.2.9.6,
1.2.9.7,
1.2.9.8,
1.2.10.1,
1.2.10.2,
1.2.10.3,
1.2.10.4,
1.2.10.5,
1.2.10.6,
1.2.10.7,
1.2.10.8,
1.2.10.9,
1.2.10.10,
1.3.1.1,1.3.1.2,1.3.1.3, 1.3.1.4, 1.3.1.5, 1.3.1.6, 1.3.1.7,1.3.2.1,1.3.2.2,
1.3.1.8, 1.3.1.9, 1.3.1.10,
1.3.2.3,1.3.2.4,1.3.2.5, 1.3.2.6, 1.3.2.7, 1.3.2.8, 1.3.2.9,1.3.3.3,1.3.3.4,
1.3.2.10, 1.3.3.1, 1.3.3.2,
1.3.3.5,1.3.3.6,1.3.3.7, 1.3.3.8, 1.3.3.9, 1.3.3.10, 1.3.4.5,1.3.4.6,
1.3.4.1, 1.3.4.2, 1.3.4.3, 1.3.4.4,
1.3.4.7,1.3.4.8,1.3.4.9, 1.3.4.10, 1.3.5.1, 1.3.5.2, 1.3.5.7,1.3.5.8,
1.3.5.3, 1.3.5.4, 1.3.5.5, 1.3.5.6,
1.3.5.9,1.3.5.10, 1.3.6.1, 1.3.6.2, 1.3.6.3, 1.3.6.4, 1.3.6.9,1.3.6.10,
1.3.6.5, 1.3.6.6, 1.3.6.7, 1.3.6.8,
1.3.7.1,1.3.7.2,1.3.7.3, 1.3.7.4, 1.3.7.5, 1.3.7.6, 1.3.7.7,1.3.8.1,1.3.8.2,
1.3.7.8, 1.3.7.9, 1.3.7.10,
2 1.3.8.3,1.3.8.4,1.3.8.5, 1.3.8.6, 1.3.8.7, 1.3.8.8,
1.3.8.9,1.3.9.3,1.3.9.4,
5 1.3.8.10, 1.3.9.1, 1.3.9.2,
1.3.9.5,1.3.9.6,1.3.9.7,1.3.9.8,1.3.9.9,1.3.9.10,1.3.10.1,1.3.10.2,1.3.10.3,1.3
.10.4,1.3 .10.5,
1.3.10.6,1.3.10.7,1.3.10.8,1.3.10.9,1.3.10.10,1.4.1.1,1.4.1.2,1.4.1.3,1.4.1.4,1
.4.1.5,1.4.1.6,
1.4.1.7,1.4.1.8,1.4.1.9, 1.4.1.10, 1.4.2.1, 1.4.2.2, 1.4.2.7,1.4.2.8,
1.4.2.3, 1.4.2.4, 1.4.2.5, 1.4.2.6,
1.4.2.9,1.4.2.10, 1.4.3.1, 1.4.3.2, 1.4.3.3, 1.4.3.4, 1.4.3.9,1.4.3.10,
1.4.3.5, 1.4.3.6, 1.4.3.7, 1.4.3.8,
3 1.4.4.1,1.4.4.2,1.4.4.3, 1.4.4.4, 1.4.4.5, 1.4.4.6,
1.4.4.7,1.4.5.1,1.4.5.2,
0 1.4.4.8, 1.4.4.9, 1.4.4.10,
1.4.5.3,1.4.5.4,1.4.5.5, 1.4.5.6, 1.4.5.7, 1.4.5.8, 1.4.5.9,1.4.6.3,1.4.6.4,
1.4.5.10, 1.4.6.1, 1.4.6.2,
1.4.6.5,1.4.6.6,1.4.6.7, 1.4.6.8, 1.4.6.9, 1.4.6.10, 1.4.7.5,1.4.7.6,
1.4.7.1, 1.4.7.2, 1.4.7.3, 1.4.7.4,
1.4.7.7,1.4.7.8,1.4.7.9, 1.4.7.10, 1.4.8.1, 1.4.8.2, 1.4.8.7,1.4.8.8,
1.4.8.3, 1.4.8.4, 1.4.8.5, 1.4.8.6,
1.4.8.9,1.4.8.10, 1.4.9.1, 1.4.9.2, 1.4.9.3, 1.4.9.4, 1.4.9.9,1.4.9.10,
1.4.9.5, 1.4.9.6, 1.4.9.7, 1.4.9.8,
35 1.4.10.1, 2, 1.4.10.3, 1.4.10.4, 1.4.10.5, 1.4.10.6, .10,
1.4.10. 1.4.10.7, 1.4.10.8, 1.4.10.9, 1.4.10
1.5.1.1,1.5.1.2,1.5.1.3, 1.5.1.4, 1.5.1.5, 1.5.1.6, 1.5.1.7,1.5.2.1,1.5.2.2,
1.5.1.8, 1.5.1.9, 1.5.1.10,
31
CA 02356539 2001-05-23
wo oo~3a2o1 PCT/US99/28079
1.5.2.3, 1.5.2.4, 1.5.2.5, 1.5.2.6, 1.5.2.7, 1.5.2.8, 1.5.2.9, 1.5.2.10,
1.5.3.1, 1.5.3.2, 1.5.3.3, 1.5.3.4,
1.5.3.5, 1.5.3.6, 1.5.3.7, 1.5.3.8, 1.5.3.9, 1.5.3.10, 1.5.4.1, 1.5.4.2,
1.5.4.3, 1.5.4.4; 1.5.4.5, 1.5.4.6,
1.5.4.7, 1.5.4.8, 1.5.4.9, 1.5.4.10, 1.5.5.1, 1.5.5.2, 1.5.5.3, 1.5.5.4,
1.5.5.5, 1.5.5.6, 1.5.5.7, 1.5.5.8,
1.5.5.9, 1.5.5.1 0, 1.5.6.1, 1.5.6.2, 1.5.6.3, 1.5.6.4, 1.5.6.5, 1.5.6.6,
1.5.6.7, 1.5.6.8, 1.5.6.9, 1.5.6.10,
1.5.7.1, 1.5.7.2, 1.5.7.3, 1.5.7.4, 1.5.7.5, 1.5.7.6, 1.5.7.7, 1.5.7.8,
1.5.7.9, 1.5.7.10, 1.5.8.1, 1.5.8.2,
1.5.8.3,1.5.8.4,1.5.8.5, 1.5.8.6, 1.5.8.7, 1.5.8.8,1.5.9.2,1.5.9.3,1.5.9.4,
1.5.8.9, 1.5.8.1 0, 1.5.9.1,
1.5.9.5,1.5.9.6,I .5.9.7, 1.5.9.8, 1.5.9.9, 1.5.9.10, 10.4,
1.5.10.1, 1.5.10.2, 1.5.10.3, 1.5. 1.5.10.5,
1.5.10.6,1.5.10
.7,1.5.10.8,1.5.10.9,1.5.10.10,1.6.1.1,1.6.1.2,1.6.1.3,1.6.1.4,1.6.1.5,1.6.1.6,
1.6.1.7,1.6.1.8,1.6.1.9, 1.6.1.10, 1.6.2.1, 1.6.2.2,1.6.2.6,1.6.2.7,1.6.2.8,
1.6.2.3, 1.6.2.4, 1.6.2.5,
1.6.2.9,1.6.2.10,1.6.3.1,1.6.3.2,1.6.3.3,1.6.3.4,1.6.3.5,1.6.3.6,1.6.3.7,
1.6.3.8,1.6.3.9,1.6.3.10,
1.6.4.1,1.6.4.2,1.6.4.3, 1.6.4.4, 1.6.4.5, 1.6.4.6,.6.4.10,1.6.5.1,1.6.5.2,
1.6.4.7, 1.6.4.8, 1.6.4.9, 1
1.6.5.3,1.6.5.4,1.6.5.5, 1.6.5.6, 1.6.5.7, I.6.5.8,1.6.6.2,1.6.3,1.6.6.4,
1.6.5.9, 1.6.5.10, 1.6.6.1,
1.6.6.5,1.6.6.6,1.6.6.7, 1.6.6.8, 1.6.6.9, 1.6.6.10,1.6.7.4,1.6.7.5,1.6.7.6,
1.6.7.1, 1.6.7.2, 1.6.7.3,
1.6.7.7,1.6.7.8,1.6.7.9, 1.6.7.10, 1.6.8.1, 1.6.8.2,1.6.8.6,1.6.8.7,1.6.8.8,
1.6.8.3, 1.6.8.4, 1.6.8.5,
1.6.8.9,1.6.8.10, 1.6.9.8,1.6.9.9,1.6.9.10,
1.6.9.1,
1.6.9.2,
1.6.9.3,
1.6.9.4,
1.6.9.5,
1.6.9.6,
1.6.9.7,
t.6.10.1,1.6.10.2,1.6.10.3,1.6.10.4,1.6.10.5,1.6.10.6,1.6.10.7,1.6.10.8,
1.6.10.9,1.6.10.10,
1.7.1.1,1.7.1.2,1.7.1.3, 1.7.1.4, 1.7.1.5, 1.7.1.6,.7.1.11.7.2.1,1.7.2.2,
1.7.1.7, 1.7.1.8, 1.7.1.9, 1 0,
1.7.2.3,1.7.2.4,1.7.2.5, 1.7.2.6, 1.7.2.7, 1.7.2.8,1.7.3.2,1.7.3.3,1.7.3.4,
1.7.2.9, 1.7.2.1 0, 1.7.3.1,
1.7.3.5,1.7.3.6,1.7.3.7, 1.7.3.8, 1.7.3.9, 1.7.3.10,1.7.4.4,1.7.4.5,1.7.4.6,
1.7.4.1, 1.7.4.2, 1.7.4.3,
1.7.4.7,1.7.4.8,1.7.4.9, 1.7.4.10, 1.7.5.1, 1.7.5.2,1.7.5.6,1.7.5.7,1.7.5.8,
1.7.5.3, 1.7.5.4, 1.7.5.5,
1.7.5.9,1.7.5.10, 1.7.6.8,I.7.6.9,1.7.6.10,
1.7.6.1,
1.7.6.2,
1.7.6.3,
1.7.6.4,
1.7.6.5,
1.7.6.6,
1.7.6.7,
1.7.7.1,1.7.7.2,1.7.7.3, 1.7.7.4, 1.7.7.5, 1.7.7.6,.7.7.I0,1.7.8.1,1.7.8.2,
1.7.7.7, 1.7.7.8, 1.7.7.9, 1
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1.7.8.9, 1.7.8.10, 1.7.9.1,
1.7.9.5,1.7.9.6,1.7.9.7, 1.7.9.8, 1.7.9.9, 1.7.9.10, .10.5,
1.7.10.1, 1.7.10.2, 1.7.10.3, 1.7.10.4,
1.7
1.7.10.6, 7, 1.7.10.8, 1.7.10.9, 1.7.10.10, 8.1.4,8.1.5,8.1.6,
1.7.10.1.8.1.1, 1.8.1.2, 1.8.1.3, 1. 1. 1.
1.8.1.7,1.8.1.8,1.8.1.9, 1.8.1.10, 1.8.2.1, 1.8.2.2,1.8.2.6,1.8.2.7,1.8.2.8,
1.8.2.3, 1.8.2.4, 1.8.2.5,
1.8.2.9,1.8.2.10, 1.8.3.1, 1.8.3.2, 1.8.3.3, 1.8.3.4,1.8.3.8,1.8.3.9,1.8.3.10,
1.8.3.5, 1.8.3.6, 1.8.3.7,
1.8.4.1,1.8.4.2,1.8.4.3, 1.8.4.4, 1.8.4.5, 1.8.4.6,.8.4.10,1.8.5.1,1.8.5.2,
1.8.4.7, 1.8.4.8, 1.8.4.9, 1
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1.8.5.9, 1.8.5.10, 1.8.6.1,
3 0 1.8.6.5,1.8.6.6,1.8.6.7, 1.8.6.8, 1.8.6.9,
1.8.6.10,1.8.7.4,1.8.7.5,1.8.7.6,
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1.8.7.7,1.8.7.8,1.8.7.9, 1.8.7.1 0, 1.8.8.1, 1.8.8.2,1.8.8.6,1.8.8.7,1.8.8.8,
1.8.8.3, 1.8.8.4, 1.8.8.5,
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1.8.9.5, 1.8.9.6, 1.8.9.7,
1.8.10.1, 2, 1.8.10.3, 1.8.10.4, 1.8.10.5, 1.8.10.9, .10,
1.8.10. 1.8.10.6, 1.8.10.7, 1.8.10.8, 1.8.10
1.9.1.1,1.9.1.2,1.9.1.3, 1.9.1.4, 1.9.1.5, 1.9.1.6,.9.1.10,1.9.2.1,1.9.2.2,
1.9.1.7, 1.9.1.8. 1.9.1.9, 1
3 5 1.9.2.3,1.9.2.4,1.9.2.5, 1.9.2.6, 1.9.2.7,
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1.9.2.9, 1.9.2.10, 1.9.3.1,
32
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1.9.3.5, 1.9.3.6, 1.9.3.7, 1.9.3.8, 1.9.3.9, 1.9.3.10, 1.9.4.1, 1.9.4.2,
1.9.4.3, 1.9.4.4, 1.9.4.5, 1.9.4.6,
1.9.4.7,1.9.4.8,1.9.4.9,1.9.4.10,1.9.5.1,1.9.5.2,1.9.5.3,1.9.5.4,1.9.5.5,1.9.S.
ii,1.9.5.7,1.9.5.8,
1.9.5.9, 1.9.5.1 0, 1.9.6.1, 1.9.6.2, 1.9.6.3, 1.9.6.4, 1.9.6.5, 1.9.6.6,
1.9.6.7, 1.9.6.8, 1.9.6.9, 1.9.6.10,
1.9.7.1, 1.9.7.2, 1.9.7.3, 1.9.7.4, 1.9.7.5, 1.9.7.6, 1.9.7.7, 1.9.7.8,
1.9.7.9, 1.9.7.1 0, 1.9.8.1, 1.9.8.2,
1.9.8.3, 1.9.8.4, 1.9.8.5, 1.9.8.6, 1.9.8.7, 1.9.8.8, 1.9.8.9, 1.9.8.1 0,
1.9.9.1, 1.9.9.2, 1.9.9.3, 1.9.9.4,
' 1.9.9.5, 1.9.9.6, 1.9.9.7, 1.9.9.8, 1.9.9.9, 1.9.9.10, 1.9.10.1, 1.9.10.2,
1.9.10.3, 1.9.10.4, 1.9.10.5,
1.9.10.6, 1.9.10.7, 1.9.10.8, 1.9.10.9, 1.9.10.10, 1.10.1.1, 1.10.1.2,
1.10.1.3, 1.10.1.4, 1.10.1.5,
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.4,1.10.7.5,
1.10.7.6, 1.10.7.7, 1.10.7.8, 1.10.7.9, 1.10.7.10, 1.10.8.1, 1.10.8.2,
1.10.8.3, 1.10.8.4, 1.10.8.5,
1.10.8.6, 1.10.8.7, 1.10.8.8, 1.10.8.9, 1.10.8.10, 1.10.9.1, 1.10.9.2,
1.10.9.3, 1.10.9.4, 1.10.9.5,
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0.10.4,
1.10.10.5, 1.10.10.6, 1.10.10.7, 1.10.10.8, 1.10.10.9, 1.10.10.10, 2.1.1.1,
2.1.1.2, 2.1.1.3, 2.1.1.4,
2.1.1.5, 2.1.1.6, 2.1.1.7, 2.1.1.8, 2.1.1.9, 2.1.1.10, 2.1.2.1, 2.1.2.2,
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2.1.3.9,2.1.3.10,2.1.4.1,2.1.4.2,2.1.4.3,2.1.4.4,2.1.4.5,2.1.4.6,2.1.4.7,2.1.4.
8,2.1.4.9,2.1.4.10,
2.1.5.1, 2.1.5.2, 2.1.5.3, 2.1.5.4, 2.1.5.5, 2.1.5.6, 2.1.5.7, 2.1.5.8,
2.1.5.9, 2.1.5.10, 2.1.6.1, 2.1.6.2,
2.1.6.3, 2.1.6.4, 2.1.6.5, 2.1.6.6, 2.1.6.7, 2.1.6.8, 2.1.6.9, 2.1.6.1 0,
2.1.7.1, 2.1.7.2, 2.1.7.3, 2.1.7.4,
2.1.7.5, 2.1.7.6, 2.1.7.7, 2.1.7.8, 2.1.7.9, 2.1.7.10, 2.1.8.1, 2.1.8.2,
2.1.8.3, 2.1.8.4, 2.1.8.5, 2.1.8.6,
2.1.8.7, 2.1.8.8, 2.1.8.9, 2.1.8.10, 2.1.9.1, 2.1.9.2, 2.1.9.3, 2.1.9.4,
2.1.9.5, 2.1.9.6, 2.1.9.7, 2.1.9.8,
2.1.9.9, 2.1.9.10, 2.1.10.1, 2.1.10.2, 2.1.10.3, 2.1.10.4, 2.1.10.5, 2.1.10.6,
2.1.10.7, 2.1.10.8,
2.1.10.9,2.1.10.10,2.2.1.1,2.2.1.2,2.2.1.3,2.2.1.4,2.2.1.5,2.2.1.6,2.2.1.7,2.2.
1.8,2.2.1.9,
2.2.1.10, 2.2.2.1, 2.2.2.2, 2.2.2.3, 2.2.2.4, 2.2.2.5, 2.2.2.6, 2.2.2.7,
2.2.2.8, 2.2.2.9, 2.2.2.10, 2.2.3.1,
2.2.3.2, 2.2.3.3, 2.2.3.4, 2.2.3.5, 2.2.3.6, 2.2.3.7, 2.2.3.8, 2.2.3.9,
2.2.3.10, 2.2.4.1, 2.2.4.2, 2.2.4.3,
2.2.4.4, 2.2.4.5, 2.2.4.6, 2.2.4.7, 2.2.4.8, 2.2.4.9, 2.2.4.10, 2.2.5.1,
2.2.5.2, 2.2.5.3, 2.2.5.4, 2.2.5.5,
3 0 2.2.5.6, 2.2.5.7, 2.2.5.8, 2.2.5.9, 2.2.5.10, 2.2.6.1, 2.2.6.2, 2.2.6.3,
2.2.6.4, 2.2.6.5, 2.2.6.6, 2.2.6.7,
2.2.6.8, 2.2.6.9, 2.2.6.1 0, 2.2.7.1, 2.2.7.2, 2.2.7.3, 2.2.7.4, 2.2.7.5,
2.2.7.6, 2.2.7.7, 2.2.7.8, 2.2.7.9,
2.2.7.1 0, 2.2.8.1, 2.2.8.2, 2.2.8.3, 2.2.8.4, 2.2.8.5, 2.2.8.6, 2.2.8.7,
2.2.8.8, 2.2.8.9, 2.2.8.10, 2.2.9.1,
2.2.9.2,2.2.9.3,2.2.9.4,2.2.9.5,2.2.9.6,2.2.9.7,2.2.9.8,2.2.9.9,2.2.9.10,2.2.10
.1,2.2.10.2,
2.2.10.3,2.2.10.4,2.2.10.5,2.2.10.6,2.2.10.7,2.2.10.8,2.2.10.9,2.2.10.10,2.3.1.
1,2.3.1.2,
2.3.1.3,2.3.1.4,2.3.1.5,2.3.1.6,2.3.1.7,2.3.1.8,2.3.1.9-
,2.3.1.10,2.3.2.1,2.3.2.2,2.3.2.3,2.3.2.4,
33
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2.3.2.5, 2.3.2.6, 2.3.2.7, 2.3.2.8, 2.3.2.9, 2.3.2.10, 2.3.3.I, 2.3.3.2,
2.3.3.3, 2.3.3.4, 2.3.3.5, 2.3.3.6,
2.3.3.7, 2.3.3.8, 2.3.3.9, 2.3.3.1 0, 2.3.4.1, 2.3.4.2, 2.3.4.3, 2.3.4.4,
2.3.4.5, 2.3.4.6; 2.3.4.7, 2.3.4.8,
2.3.4.9, 2.3.4.1 0, 2.3.5.1, 2.3.5.2, 2.3.5.3, 2.3.5.4, 2.3.5.5, 2.3.5.6,
2.3.5.7, 2.3.5.8, 2.3.5.9, 2.3.5.10,
2.3.6.1, 2.3.6.2, 2.3.6.3, 2.3.6.4, 2.3.6.5, 2.3.6.6, 2.3.6.7, 2.3.6.8,
2.3.6.9, 2.3.6.10, 2.3.7.1, 2.3.7.2,
2.3.7.3, 2.3.7.4, 2.3.7.5, 2.3.7.6, 2.3.7.7, 2.3.7.8, 2.3.7.9, 2.3.7.10,
2.3.8.1, 2.3.8.2, 2.3.8.3, 2.3.8.4,
2.3.8.5,23.8.6,2.3.8.7,2.3.8.8,2.3.8.9,2.3.8.10,2.3.9.1,2.3.9.2,2.3.9.3,2.3.9.4
,2.3.9.5,2.3.9.6,
2.3.9.7,2.3.9.8,2.3.9.9,2.3.9.10,2.3.10.1,2.3.I0.2,2.3.I0.3,2.3.10.4,2.3.10.5,2
.3.I0.6,2.3.10.7,
2.3.10.8, .9, 2.3.10.10, 2.4.1.1, 2.4.1.2, 1.6, 1.7, 2.4.1.8,
2.3.10 2.4.1.3, 2.4.1.4, 2.4.1.5, 2.4. 2.4.
2.4.1.9,2.4.1.1 2.4.2.8,2.4.2.9,
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2.4.2.7,
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2.4.3.7, 2.4.3.8, 2.4.3.9, 2.4.3.10, 2.4.4.2,
2.4.4.3,2.4.4.4,2.4.4.5, 2.4.4.6, 2.4.4.7, 2.4.4.8,2.4.5.2,2.4.5.3,
2.4.4.9, 2.4.4.10, 2.4.5.1, 2.4.5.4,
2.4.5.5,2.4.5.6,2.4.5.7, 2.4.5.8, 2.4.5.9, 2.4.5.10,2.4.6.4,2.4.'6.5,
2.4.6.1, 2.4.6.2, 2.4.6.3, 2.4.6.6,
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2.4.7.3, 2.4.7.4, 2.4.7.5, 2.4.7.8,
2.4.7.9,2.4.7.10, 2.4.8.8,2.4.8.9,
2.4.8.1, 2.4.8.10,
2.4.8.2,
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2.4.9.1,2.4.9.2,2.4.9.3,2.4.9.4,2.4.9.5,2.4.9.6,2.4.9.7,2.4.9.8,2.4.9.9,2.4.9.1
0, 2.4.10.1,
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10,2.5.1.1,
2.5.1.2,2.5.1.3,2.5.1.4, 2.5.1.5, 2.5.1.6, 2.5.1.7,2.5.2.1,2.5.2.2,
2.5.1.8, 2.5.1.9, 2.5.1.10, 2.5.2.3,
2.5.2.4,2.5.2.5,2.5.2.6, 2.5.2.7, 2.5.2.8, 2.5.2.9,2.5.3.3,2.5.3.4,
2.5.2.10, 2.5.3.1, 2.5.3.2, 2.5.3.5,
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2.5.4.8,2.5.4.9,2.5.4.1 0, 2.5.5.1, 2.5.5.2, 2.5.5.3,2.5.5.7,2.5.5.8,
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5 .10.5,2.5.10.6,
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0 2.6.3.12.6.3.6, 2.6.3.7, 2.6.3.8, 2.6.4.1,
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9,2.6.10.10,
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2.7.2.3,2.7.2.4,2.7.2.5,2.7.2.6,2.7.2.7,2.7.2.8,2.7.2.~,2.7.2.10,2.7.3.1,2.7.3.
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34
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
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2.7.5.5, 2.7.5.6, 2.7.5.7, 2.7.5.8,
2.7.5.9, 2.7.5.10, 2.7.6.1, 2.7.6.2, 2.7.6.3, 2.7.6.4, 2.7.6.5, 2.7.6.6,
2.7.6.7, 2.7.6.8, 2.7.6.9, 2.7.6.1 0,
2.7.7.1, 2.7.7.2, 2.7.7.3, 2.7.7.4, 2.7.7.5, 2.7.7.6, 2.7.7.7, 2.7.7.8,
2.7.7.9, 2.7.7.10, 2.7.8.1, 2.7.8.2,
S 2.7.8.3, 2.7.8.4, 2.7.8.5, 2.7.8.6, 2.7.8.7, 2.7.8.8, 2.7.8.9, 2.7.8.10,
2.7.9.1, 2.7.9.2, 2.7.9.3, 2.7.9.4,
-
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.10.4,2.7.10.5,
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2.8.2.9, 2.8.2.10, 2.8.3.1, 2.8.3.2, 2.8.3.3, 2.8.3.4, 2.8.3.5, 2.8.3.6,
2.8.3.7, 2.8.3.8, 2.8.3.9, 2.8.3.10,
2.8.4.1, 2.8.4.2, 2.8.4.3, 2.8.4.4, 2.8.4.5, 2.8.4.6, 2.8.4.7, 2.8.4.8,
2.8.4.9, 2.8.4.10, 2.8.5.1, 2.8.5.2,
2.8.5.3, 2.8.5.4, 2.8.5.5, 2.8.5.6, 2.8.5.7, 2.8.5.8, 2.8.5.9, 2.8.5.10,
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2.8.6.5, 2.8.6.6, 2.8.6.7, 2.8.6.8, 2.8.6.9, 2.8.6.10, 2.8.7.1, 2.8.7.2,
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2.8.7.7, 2.8.7.8, 2.8.7.9, 2.8.7.10, 2.8.8.1, 2.8.8.2, 2.8.8.3, 2.8.8.4,
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2.8.8.9, 2.8.8.10, 2.8.9.1, 2.8.9.2, 2.8.9.3, 2.8.9.4, 2.8.9.5, 2.8.9.6,
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2.8.10.1,2.8.10.2,2.8.10.3,2.8.10.4,2.8.10.5,2.8.10.6,2.8.10.7,2.8.10.8,2.8.10.
9,2.8.10.10,
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2.9.1.9, 2.9.1.10, 2.9.2.1, 2.9.2.2,
2.9.2.3, 2.9.2.4, 2.9.2.5, 2.9.2.6, 2.9.2.7, 2.9.2.8, 2.9.2.9, 2.9.2.10,
2.9.3.1, 2.9.3.2, 2.9.3.3, 2.9.3.4,
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2.9.5.9,2.9.5.10,2.9.6.I,2.9.6.2,2.9.6.3,2.9.6.4,2.9.6.5,2.9.6.6,2.9.6.7,2.9.6.
8,2.9.6.9,2.9.6.10,
2.9.7.1, 2.9.7.2, 2.9.7.3, 2.9.7.4, 2.9.7.5, 2.9.7.6, 2.9.7.7, 2.9.7.8,
2.9.7.9, 2.9.7.10, 2.9.8.1, 2.9.8.2,
2.9.8.3, 2.9.8.4, 2.9.8.5, 2.9.8.6, 2.9.8.7, 2.9.8.8, 2.9.8.9, 2.9.8.10,
2.9.9.1, 2.9.9.2, 2.9.9.3, 2.9.9.4,
2.9.9.5,2.9.9.6,2.9.9.7,2.9.9.8,2.9.9.9,2.9.9.10,2.9.10.1,2.9.10.2,2.9.10.3,2.9
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2.9.10.6, 2.9. I 0.7, 2.9.10.8, 2.9.10.9, 2.9.10.10, 2.10. i .1, 2.10.1.2,
2.10.1.3, 2.10.1.4, 2.10.1.5,
2.10.1.6, 2.10.1.7, 2.10.1.8, 2.10.1.9, 2.10.1.10, 2.10.2.1, 2.10.2.2,
2.10.2.3, 2.10.2.4, 2.10.2.5,
2.10.2.6,2.10.2.7,2.10.2.8,2.10.2.9,2.10.2.10,2.10.3.1,2.10.3.2,2.10.3.3,2.10.3
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2.10.3.6,2.10.3.7,2.10.3.8,2.10.3.9,2.10.3.10,2.10.4.1,2.10.4.2,2.10.4.3,2.10.4
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2.10.4.6,2.10.4.7,2.10.4.8,2.10.4.9,2.10.4.10,2.10.5.1,2.10.5.2,2.10.5.3,2.10.5
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2.10.6.6, 2.10.6.7, 2.10.6.8, 2.10.6.9, 2.10.6.10, 2:10.7.1, 2.10.7.2,
2.10.7.3, 2.10.7.4, 2.10.7.5,
2.10.7.6, 2.10.7.7, 2.10.7.8, 2.10.7.9, 2.10.7.10, 2.10.8.1, 2.10.8.2,
2.10.8.3, 2.10.8.4, 2.10.$.5,
2.10.8.6,2.10.8.7,2.10.8.8,2.10.8.9,2.10.8.10,2.10.9.1,2.10.9.2,2.10.9.3,2.10.9
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2.10.9.6,2.10.9.7,2.10.9.8,2.10.9.9,2.10.9.10,2.10.10.1,2.10.10.2,2.10.10.3,2.1
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2.10.10.5,2.10.10.6,2.10.10.7,2.10.10.8,2.10.10.9,2.10.10.10,3.1.1.1,3.1.1.2,3.
1.1.3,3.1.1.4,
3 5 3.1.1.5, 3.1.1.6, 3.1.1.7, 3.1.1.8, 3.1.1.9, 3.1.1.10, 3.1.2:1, 3.1.2.2,
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CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
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3.1.3.9, 3.1.3.10, 3.1.4.1, 3.1.4.2, 3.1.4.3, 3.1.4.4, 3.1.4.5, 3.1.4.6,
3.1.4.7, 3.1.4.$, 3.1.4.9, 3.1.4.10,
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3.1.5.9, 3.1.5.10, 3.1.6.1, 3.1.6.2,
3.1.6.3, 3.1.6.4, 3.1.6.5, 3.1.6.6, 3.1.6.7, 3.1.6.8, 3.1.6.9, 3.1.6.10,
3.1.7.1, 3.1.7.2, 3.1.7.3, 3.1.7.4,
3.1.7.5, 3.1.7.6, 3.1.7.7, 3.1.7.8, 3.1.7.9, 3.1.7.1 0, 3.1.8.1, 3.1.8.2,
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3 5 3.5.2.4, 3.5.2.5, 3.5.2.6, 3.5.2.7, 3.5.2.8, 3.5.2.9, 3.5.2.18, 3.5.3.1,
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36
CA 02356539 2001-05-23
WO 00/32201 PGT/US99/28079
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3.5.8.4, 3.5.8.5, 3.5.8.6, 3.5.8.7, 3.5.8.8, 3.5.8.9, 3.5.8.10, 3.5.9.I,
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3.6.1.8, 3.6.1.9, 3.6.1.10, 3.6.2.1, 3.6.2.2, 3.6.2.3, 3.6.2.4, 3.6.2.5,
3.6.2.6, 3.6.2.7, 3.6.2.8, 3.6.2.9,
3.6.2.1 0, 3.6.3.1, 3.6.3.2, 3.6.3.3, 3.6.3.4, 3.6.3.5, 3.6.3.6, 3.6.3.7,
3.6.3.8, 3.6.3.9, 3.6.3.10, 3.6.4.1,
3.6.4.2, 3.6.4.3, 3.6.4.4, 3.6.4.5, 3.6.4.6, 3.6.4.7, 3.6.4.8, 3.6.4.9,
3.6.4.1 0, 3.6.5.1, 3.6.5.2, 3.6.5.3,
3.6.5.4, 3.6.5.5, 3.6.5.6, 3.6.5.7, 3.6.5.8, 3.6.5.9, 3.6.5.10, 3.6.6.1,
3.6.6.2, 3.6.6.3, 3.6.6.4, 3.6.6.5,
3.6.6.6, 3.6.6.7, 3.6.6.8, 3.6.6.9, 3.6.6.10, 3.6.7.1, 3.6.7.2, 3.6.7.3,
3.6.7.4, 3.6.7.5, 3.6.'1.6, 3.6.7.7,
3.6.7.8, 3.6.7.9, 3.6.7.I 0, 3.6.8.1, 3.6.8.2, 3.6.8.3, 3.6.8.4, 3.6.8.5,
3.6.8.6, 3.6.8.7, 3.6.8.8, 3.6.8.9,
3.6.8.1 0, 3.6.9.1, 3.6.9.2, 3.6.9.3, 3.6.9.4, 3.6.9.5, 3.6.9.6, 3.6.9.7,
3.6.9.8, 3.6.9.9, 3.6.9.10,
3.6.10.1,3.6.10.2,3.6.10.3,3.6.10.4,3.6.10.5,3.6.10.6,3.6.10.7,3.6.10.8,3.6.10.
9,3.6.10.10,
3.7.1.1, 3.7.1.2, 3.7.1.3, 3.7.1.4, 3.7.1.5, 3.7.1.6, 3.7.1.7, 3.7.1.8,
3.7.1.9, 3.7.1.1 0, 3.7.2.1, 3.7.2.2,
3.7.2.3, 3.7.2.4, 3.7.2.5, 3.7.2.6, 3.7.2.7, 3.7.2.8, 3.7.2.9, 3.7.2.10,
3.7.3.1, 3.7.3.2, 3.7.3.3, 3.7.3.4,
3.7.3.5, 3.7.3.6, 3.7.3.7, 3.7.3.8, 3.7.3.9, 3.7.3.10, 3.7.4.I, 3.7.4.2,
3.7.4.3, 3.7.4.4, 3.7.4.5, 3.7.4.6,
3.7.4.7, 3.7.4.8, 3.7.4.9, 3.7.4.10, 3.7.5.1, 3.7.5.2, 3.7.5.3, 3.7.5.4,
3.7.5.5, 3.7.5.6, 3.7.5.7, 3.7.5.8,
3.7.5.9,3.7.5.10,3.7.6.1,3.7.6.2,3.7.6.3,3.7.6.4,3.7.6.5,3.7.6.6,3.7.6.7,3.7.6.
8,3.7.6.9,3.7.6.10,
3.7.7.1, 3.7.7.2, 3.7.7.3, 3.7.7.4, 3.7.7.5, 3.7.7.6, 3.7.7.7, 3.7.7.8,
3.7.7.9, 3.7.7.10, 3.7.8.1, 3.7.8.2,
3.7.8.3, 3.7.8.4, 3.7.8.5, 3.7.8.6, 3.7.8.7, 3.7.8.8, 3.7.8.9, 3.7.8.10,
3.7.9.1, 3.7.9.2, 3.7.9.3, 3.7.9.4,
3.7.9.5,3.7.9.6,3.7.9.7,3.7.9.8,3.7.9.9,3.7.9.10,3.7.10.1,3.7.10.2,3.7.10.3,3.7
.10.4,3.7.10.5,
3.7.10.6,3.7.10.7,3.7.10.8,3.7.10.9,3.7.10.10,3.8.1.1,3.8.1.2,3.8.1.3,3.8.1.4,3
.8.1.5,3.8.1.6,
2 5 3.8.1.7, 3.8.1.8, 3.8.1.9, 3.8.1.10, 3.8.2.1, 3.8.2.2, 3.8.2.3, 3.8.2.4,
3.8.2.5, 3.8.2.6, 3.8.2.7, 3.8.2.8,
3.8.2.9, 3.8.2.10, 3.8.3.1, 3.8.3.2, 3.8.3.3, 3.8.3.4, 3.8.3.5, 3.8.3.6,
3.8.3.7, 3.8.3.8, 3.8.3.9, 3.8.3.10,
3.8.4.1, 3.8.4.2, 3.8.4.3, 3.8.4.4, 3.8.4.5, 3.8.4.6, 3.8.4.7, 3.8.4.8,
3.8.4.9, 3.8.4.10, 3.8.5.1, 3.8.5.2,
3.8.5.3, 3.8.5.4, 3.8.5.5, 3.8.5.6, 3.8.5.7, 3.8.5.8, 3.8.5.9, 3.8.5.10,
3.8.6.1, 3.8.6.2, 3.8.6.3, 3.8.6.4,
3.8.6.5, 3.8.6.6, 3.8.6.7, 3.8.6.8, 3.8.6.9, 3.8.6.1 0, 3.8.7.1, 3.8.7.2,
3.8.7.3, 3.8.7.4, 3.8.7.5, 3.8.7.6,
3 0 3.8.7.7, 3.8.7.8, 3.8.7.9, 3.8.7.10, 3.8.8.1, 3.8.8.2, 3.8.8.3, 3.8.8.4,
3.8.8.5, 3.8.8.6, 3.8.8.7, 3.8.8.8,
3.8.8.9, 3.8.8.10, 3.8.9.1, 3.8.9.2, 3.8.9.3, 3.8.9.4, 3.8.9.5, 3.8.9.6,
3.8.9.7, 3.8.9.8, 3.8.9.9, 3.8.9.10,
3.8.10.1,3.8.10.2,3.8.10.3,3.8.10.4,3.8.10.5,3.8.10.6,3.8.10.7,3.8.10.8,3.8.10.
9,3.8.10.10,
3.9.1.1, 3.9.1.2, 3.9.1.3, 3.9.1.4, 3.9.1.5, 3.9.1.6, 3.9.1.7, 3.9.1.8,
3.9.1.9, 3.9.1.1 0, 3.9.2.1, 3.9.2.2,
3.9.2.3, 3.9.2.4, 3.9.2.5, 3.9.2.6, 3.9.2.7, 3.9.2.8, 3.9.2.9, 3.9.2.10,
3.9.3.1, 3.9.3.2, 3.9.3.3, 3.9.3.4,
3 5 3.9.3.5, 3.9.3.6, 3.9.3.7, 3.9.3.8, 3.9.3.9, 3.9.3.10, 3.9.4:1, 3.9.4.2,
3.9.4.3, 3.9.4.4, 3.9.4.5, 3.9.4.6,
37
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
3.9.4.7, 3.9.4.8, 3.9.4.9, 3.9.4.10, 3.9.5.1, 3.9.5.2, 3.9.5.3, 3.9.5.4,
3.9.5.5, 3.9.5.6, 3.9.5.7, 3.9.5.8,
3.9.5.9, 3.9.5.1 0, 3.9.6.1, 3.9.6.2, 3.9.6.3, 3.9.6.4, 3.9.6.5, 3.9.6.6,
3.9.6.7, 3.9.6:8, 3.9.6.9, 3.9.6.10,
3.9.7.1, 3.9.7.2, 3.9.7.3, 3.9.7.4, 3.9.7.5, 3.9.7.6, 3.9.7.7, 3.9.7.8,
3.9.7.9, 3.9.7.10, 3.9.8.1, 3.9.8.2,
3.9.8.3, 3.9.8.4, 3.9.8.5, 3.9.8.6, 3.9.8.7, 3.9.8.8, 3.9.8.9, 3.9.8.10,
3.9.9.1, 3.9.9.2, 3.9.9.3, 3.9.9.4,
3.9.9.5,3.9.9.6,3.9.9.7,3.9.9.8,3.9.9.9,3.9.9.10,3.9.10.1,3.9.10.2,3.9.10.3,3.9
.10.4,3.9.10.5,
' _
3.9.10.6,3.9.10.7,3.9.10.8,3.9.10.9,3.9.10.10,3.10.1.1,3.10.1.2,3.10.1.3,3.10.1
.4,3.10.1.5,
3.10.1.6,3.10.1.7,3.10.1.8,3.10.1.9,3.10.1.10,3.10.2.I,3.10.2.2,3.10.2.3,3.10.2
.4,3.10.2.5,
3.10.2.6,3.10.2.7,3.10.2.8,3.10.2.9,3.10.2.10,3.10.3.1,3.10.3.2,3.10.3.3,3.10.3
.4,3.10.3.5,
3.10.3.6,3.10.3.7,3.10.3.8,3.10.3.9,3.10.3.10,3.10.4.1,3.10.4.2,3.10.4.3,3.10.4
.4,3.10.4.5,
3.10.4.6, 3.10.4.7, 3.10.4.8, 3.10.4.9, 3.10.4.10, 3.10.5.1, 3.10.5.2,
3.10.5.3, 3.10.5.4, 3.10.5.5,
3.10.5.6, 3.10.5.7, 3.10.5.8, 3.10.5.9, 3.10.5.10, 3.10.6.1, 3.10.6.2,
3.10.6.3, 3.10.6.4, 3.10.6.5,
3.10.6.6, 3.10.6.7, 3.10.6.8, 3.10.6.9, 3.10.6.10, 3.10.7.1, 3.10.7.2,
3.10.7.3, 3.10.7.4, 3.10.7.5,
3.10.7.6,3.10.7.7,3.10.7.8,3.10.7.9,3.10.7.10,3.10.8.1,3.10.8.2,3.10.8.3,3.10.8
.4,3.10.8.5,
3.10.8.6,3.10.8.7,3.10.8.8,3.10.8.9,3.10.8.10,3.10.9.1,3.10.9.2,3.10.9.3,3.10.9
.4,3.10.9.5,
3.10.9.6,3.10.9.7,3.10.9.8,3.10.9.9,3.10.9.10,3.10.10.1,3.10.I0.2,3.10.10.3,3.1
0.10.4,
3.10.10.5,3.10.10.6,3.10.10.7,3.10.10.8,3.10.10.9,3.10.10.10,4.1.1.1,4.1.1.2,4.
1.1.3,4.1.1.4,
4.1.1.5, 4.1.1.6, 4.1.1.7, 4.1.1.8, 4.1.1.9, 4.1.1.10, 4.1.2.1, 4.1.2.2,
4.1.2.3, 4.1.2.4, 4.1.2.5, 4.1.2.6,
4.1.2.7, 4.1.2.8, 4.1.2.9, 4.1.2.10, 4.1.3.1, 4.1.3.2, 4.1.3.3, 4.1.3.4,
4.1.3.5, 4.1.3.6, 4.1.3.7, 4.1.3.8,
4.1.3.9, 4.1.3.1 0, 4.1.4.1, 4.1.4.2, 4.1.4.3, 4.1.4.4, 4.1.4.5, 4.1.4.6,
4.1.4.7, 4.1.4.8, 4.1.4.9, 4.1.4.10,
4.1.5.1, 4.1.5.2, 4.1.5.3, 4.1.5.4, 4.1.5.5, 4.1.5.6, 4.1.5.7, 4.1.5.8,
4.1.5.9, 4.1.5.10, 4.1.6.1, 4.1.6.2,
4.1.6.3, 4.1.6.4, 4.1.6.5, 4.1.6.6, 4.1.6.7, 4.1.6.8, 4.1.6.9, 4.1.6.10,
4.1.7.1, 4.1.7.2, 4.1.7.3, 4.1.7.4,
4.1.7.5, 4.1.7.6, 4.1.7.7, 4.1.7.8, 4.1.7.9, 4.1.7.10, 4.1.8.1, 4.1.8.2,
4.1.8.3, 4.1.8.4, 4.1.8.5, 4.1.8.6,
4.1.8.7, 4.1.8.8, 4.1.8.9, 4.1.8.10, 4.1.9.1, 4.1.9.2, 4.1.9.3, 4.1.9.4,
4.1.9.5, 4.1.9.6, 4.1.9.7, 4.1.9.8,
4.i.9.9,4.1.9.10,4.1.10.i,4.1.10.2,4.1.10.3,4.1.10.4,4.1.10.5,4.1.10.6,4.1.10.7
,4.1.10.8,
4.1.10.9,4.1.10.10,4.2.1.1,4.2.1.2,4.2.1.3,4.2.1.4,4.2.1.5,4.2.1.6,4.2.1.7,4.2.
1.8,4.2.1.9,
4.2.1.10, 4.2.2.1, 4.2.2.2, 4.2.2.3, 4.2.2.4, 4.2.2.5, 4.2.2.6, 4.2.2.7,
4.2.2.8, 4.2.2.9, 4.2.2.10, 4.2.3.1,
4.2.3.2, 4.2.3.3, 4.2.3.4, 4.2.3.5, 4.2.3.6, 4.2.3.7, 4.2.3.8, 4.2.3.9,
4.2.3.10, 4.2.4.1, 4.2.4.2, 4.2.4.3,
4.2.4.4, 4.2.4.5, 4.2.4.6, 4.2.4.7, 4.2.4.8, 4.2.4.9, 4.2.4.10, 4.2.5.1,
4.2.5.2, 4.2.5.3, 4.2.5.4, 4.2.5.5,
4.2.5.6, 4.2.5.7, 4.2.5.8, 4.2.5.9, 4.2.5.10, 4.2.6.1, 4.2.6.2, 4.2.6.3,
4.2.6.4, 4.2.6.5, 4.2.6.6, 4.2.6.7,
3 0 4.2.6.8, 4.2.6.9, 4.2.6.10, 4.2.7.1, 4.2.7.2, 4.2.7.3, 4.2.7.4, 4.2.7.5,
4.2.7.6, 4.2.7.7, 4.2.7.8, 4.2.7.9,
4.2.7.10, 4.2.8.1, 4.2.8.2, 4.2.8.3, 4.2.8.4, 4.2.8.5, 4.2.8.6, 4.2.8.7,
4.2.8.8, 4.2.8.9, 4.2.8.10, 4.2.9.1,
4.2.9.2,4.2.9.3,4.2.9.4,4.2.9.5,4.2.9.6,4.2.9.7,4.2.9.8,4.2.9.9,4.2.9.10,4.2.10
.1,4.2.10.2,
4.2.10.3,4.2.10.4,4.2.10.5,4.2.10.6,4.2.10.7,4.2.10.8,4.2.10.9,4.2.10.10,4.3.1.
1,4.3.1.2,
4.3.1.3, 4.3.1.4, 4.3.1.5, 4.3.1.6, 4.3.1.7, 4.3.1.8, 4.3.1.9, 4.3.1.10,
4.3.2.1, 4.3.2.2, 4.3.2.3, 4.3.2.4,
3 5 4.3.2.5, 4.3.2.6, 4.3.2.7, 4.3.2.8, 4.3.2.9, 4.3.2.10, 4.3.3:1, 4.3.3.2,
4.3.3.3, 4.3.3.4, 4.3.3.5, 4.3.3.6,
38
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
4.3.3.7, 4.3.3.8, 4.3.3.9, 4.3.3.10, 4.3.4.1, 4.3.4.2, 4.3.4.3, 4.3.4.4,
4.3.4.5, 4.3.4.6, 4.3.4.7, 4.3.4.8,
4.3.4.9, 4.3.4.1 0, 4.3.5.1, 4.3.5.2, 4.3.5.3, 4.3.5.4, 4.3.5.5, 4.3.5.6,
4.3.5.7, 4.3.5.8; 4.3.5.9, 4.3.5.10,
4.3.6.1, 4.3.6.2, 4.3.6.3, 4.3.6.4, 4.3.6.5, 4.3.6.6, 4.3.6.7, 4.3.6.8,
4.3.6.9, 4.3.6.10, 4.3.7.1, 4.3.7.2,
4.3.7.3, 4.3.7.4, 4.3.7.5, 4.3.7.6, 4.3.7.7, 4.3.7.8, 4.3.7.9, 4.3.7.1 0,
4.3.8.1, 4.3.8.2, 4.3.8.3, 4.3.8.4,
4.3.8.5, 4.3.8.6, 4.3.8.7, 4.3.8.8, 4.3.8.9, 4.3.8.10, 4.3.9.1, 4.3.9.2,
4.3.9.3, 4.3.9.4, 4.3.9.5, 4.3.9.6,
4.3.9.7,4.3.9.8,4.3.9.9,4.3.9.10,4.3.10.1,4.3.10.2,4.3.10.3,4.3.10.4,4.3.10.5,4
.3.10.6,4.3.10.7,
4.3.10.8,4.3.10.9,4.3.10.10,4.4.1.1,4.4.1.2,4.4.1.3,4.4.1.4,4.4.1.5,4.4.1.6,4.4
.1.7,4.4.1.8,
4.4.1.9, 4.4.1.10, 4.4.2.1, 4.4.2.2, 4.4.2.3, 4.4.2.4, 4.4.2.5, 4.4.2.6,
4.4.2.7, 4.4.2.8, 4.4.2.9, 4.4.2.10,
4.4.3.1, 4.4.3.2, 4.4.3.3, 4.4.3.4, 4.4.3.5, 4.4.3.6, 4.4.3.7, 4.4.3.8,
4.4.3.9, 4.4.3.10, 4.4.4.1, 4.4.4.2,
4.4.4.3, 4.4.4.4, 4.4.4.5, 4.4.4.6, 4.4.4.7, 4.4.4.8, 4.4.4.9, 4.4.4.10,
4.4.5.1, 4.4.5.2, 4.4.5.3, 4.4.5.4,
4.4.5.5, 4.4.5.6, 4.4.5.7, 4.4.5.8, 4.4.5.9, 4.4.5.10, 4.4.6.1, 4.4.6.2,
4.4.6.3, 4.4.6.4, 4.4.b.5, 4.4.6.6,
4.4.6.7, 4.4.6.8, 4.4.6.9, 4.4.6.10, 4.4.7.1, 4.4.7.2, 4.4.7.3, 4.4.7.4,
4.4.7.5, 4.4.7.6, 4.4.7.7, 4.4.7.8,
4.4.7.9, 4.4.7.1 0, 4.4.8.1, 4.4.8.2, 4.4.8.3, 4.4.8.4, 4.4.8.5, 4.4.8.6,
4.4.8.7, 4.4.8.8, 4.4.8.9, 4.4.8.10,
4.4.9.1, 4.4.9.2, 4.4.9.3, 4.4.9.4, 4.4.9.5, 4.4.9.6, 4.4.9.7, 4.4.9.8,
4.4.9.9, 4.4.9.10, 4.4.10.1,
4.4.10.2,4.4.10.3,4.4.10.4,4.4.10.5,4.4.10.6,4.4.10.7,4.4.10.8,4.4.10.9,4.4.10.
10,4.5.1.1,
4.5.1.2, 4.5.1.3, 4.5.1.4, 4.5.1.5, 4.5.1.6, 4.5.1.7, 4.5.1.8, 4.5.1.9,
4.5.1.10, 4.5.2.1, 4.5.2.2, 4.5.2.3,
4.5.2.4, 4.5.2.5, 4.5.2.6, 4.5.2.7, 4.5.2.8, 4.5.2.9, 4.5.2.10, 4.5.3.1,
4.5.3.2, 4.5.3.3, 4.5.3.4, 4.5.3.5,
4.5.3.6, 4.5.3.7, 4.5.3.8, 4.5.3.9, 4.5.3.10, 4.5.4.1, 4.5.4.2, 4.5.4.3,
4.5.4.4, 4.5.4.5, 4.5.4.6, 4.5.4.7,
4.5.4.8, 4.5.4.9, 4.5.4.10, 4.5.5.1, 4.5.5.2, 4.5.5.3, 4.5.5.4, 4.5.5.5,
4.5.5.6, 4.5.5.7, 4.5.5.8, 4.5.5.9,
4.5.5.10,4.5.6.1,4.5.6.2,4.5.6.3,4.5.6.4,4.5.6.5,4.5.6.6,4.5.6.7,4.5.6.8,4.5.6.
9,4.5.6.10,4.5.7.1,
4.5.7.2, 4.5.7.3, 4.5.7.4, 4.5.7.5, 4.5.7.6, 4.5.7.7, 4.5.7.8, 4.5.7.9,
4.5.7.1 0, 4.5.8.1, 4.5.8.2, 4.5.8.3,
4.5.8.4, 4.5.8.5, 4.5.8.6, 4.5.8.7, 4.5.8.8, 4.5.8.9, 4.5.8.1 0, 4.5.9.1,
4.5.9.2, 4.5.9.3, 4.5.9.4, 4.5.9.5,
4.5.9.6, 4.5.9.7, 4.5.9.8, 4.5.9.9, 4.5.9.10, 4.S.1U.1, 4.5.10.2, 4.5.10.3,
4.5.10.4, 4.5.10.5, 4.5.10.6,
4.5.10.7,4.5.10.8,4.5.10.9,4.5.10.10,4.6.1.1,4.6.1.2,4.6.1.3,4.6.1.4,4.6.1.5,4.
6.1.6,4.6.1.7,
4.6.1.8, 4.6.1.9, 4.6.1.10, 4.6.2.1, 4.6.2.2, 4.6.2.3, 4.6.2.4, 4.6.2.5,
4.6.2.6, 4.6.2.7, 4.6.2.8, 4.6.2.9,
4.6.2.1 0, 4.6.3.1, 4.6.3.2, 4.6.3.3, 4.6.3.4, 4.6.3.5, 4.6.3.6, 4.6.3.7,
4.6.3.8, 4.6.3.9, 4.6.3.10, 4.6.4.1,
4.6.4.2, 4.6.4.3, 4.6.4.4, 4.6.4.5, 4.6.4.6, 4.6.4.7, 4.6.4.8, 4.6.4.9,
4.6.4.10, 4.6.5.1, 4.6.5.2, 4.6.5.3,
4.6.5.4, 4.6.5.5, 4.6.5.6, 4.6.5.7, 4.6.5.8, 4.6.5.9, 4.6.5.1 0, 4.6.6.1,
4.6.6.2, 4.6.6.3, 4.6.6.4, 4.6.6.5,
4.6.6.6, 4.6.6.7, 4.6.6.8, 4.6.6.9, 4.6.6.10, 4.6.7.1, 4.6.7.2, 4.6.7.3,
4.6.7.4, 4.6.7.5, 4.6.7.6, 4.6.7.7,
3 0 4.6.7.8, 4.6.7.9, 4.6.7.10, 4.6.8.1, 4.6.8.2, 4.6.8.3, 4.6.8.4, 4.6.8.5,
4.6.8.6, 4.6.8.7, 4.6.8.8, 4.6.8.9,
4.6.8.10, 4.6.9.1, 4.6.9.2, 4.6.9.3, 4.6.9.4, 4.6.9.5, 4.6.9.6, 4.6.9.7,
4.6.9.8, 4.6.9.9, 4.6.9.10,
4.6.10.1,4.6.10.2,4.6.10.3,4.6.10.4,4.6.10.5,4.6.10.6,4.6.10.7,4.6.10.8,4.6.10.
9,4.6.10.10,
4.7.1.1, 4.7.1.2, 4.7.1.3, 4.7.1.4, 4.7.1.5, 4.7.1.6, 4.7.1.7, 4.7.1.8,
4.7.1.9, 4.7.1.10, 4.7.2.1, 4.7.2.2,
4.7.2.3, 4.7.2.4, 4.7.2.5, 4.7.2.6, 4.7.2.7, 4.7.2.8, 4.7.2.9, 4.7.2.10,
4.7.3.1, 4.7.3.2, 4.7.3.3, 4.7.3.4,
3 5 4.7.3.5, 4.7.3.6, 4.7.3.7, 4.7.3.8, 4.7.3.9, 4.7.3.10, 4.7.4.1, 4.7.4.2,
4.7.4.3, 4.7.4.4, 4.7.4.5, 4.7.4.6,
39
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
4.7.4.7, 4.7.4.8, 4.7.4.9, 4.7.4.10, 4.7.5.1, 4.7.5.2, 4.7.5.3, 4.7.5.4,
4.7.5.5, 4.7.5.6, 4.7.5.7, 4.7.5.8,
4.7.5.9, 4.7.5.10, 4.7.6.1, 4.7.6.2, 4.7.6.3, 4.7.6.4, 4.7.6.5, 4.7.6.6,
4.7.6.7, 4.7.6.8, 4.7.6.9, 4.7.6.10,
4.7.7.1, 4.7.7.2, 4.7.7.3, 4.7.7.4, 4.7.7.5, 4.7.7.6, 4.7.7.7, 4.7.7.8,
4.7.7.9, 4.7.7.1 0, 4.7.8.1, 4.7.8.2,
4.7.8.3, 4.7.8.4, 4.7.8.5, 4.7.8.6, 4.7.8.7, 4.7.8.8, 4.7.8.9, 4.7.8.10,
4.7.9.1, 4.7.9.2, 4.7.9.3, 4.7.9.4,
S
4.7.9.5,4.7.9.6,4.7.9.7,4.7.9.8,4.7.9.9,4.7.9.10,4.7.10.1,4.7.10.2,4.7.10.3,4.7
.10.4,4.7.10.5,
4.7.10.6,4.7.10.7,4.7.10.8,4.7.10.9,4.7.10.10,4.8.1.1,4.8.1.2,4.8.1.3,4.8.1.4,4
.8.1.5,4.8.1.6,
4.8.1.7, 4.8.1.8, 4.8.1.9, 4.8.1.10, 4.8.2.1, 4.8.2.2, 4.8.2.3, 4.8.2.4,
4.8.2.5, 4.8.2.6, 4.8.2.7, 4.8.2.8,
4.8.2.9, 4.8.2.10, 4.8.3.1, 4.8.3.2, 4.8.3.3, 4.8.3.4, 4.8.3.5, 4.8.3.6,
4.8.3.7, 4.8.3.8, 4.8.3.9, 4.8.3.10,
4.8.4.1, 4.8.4.2, 4.8.4.3, 4.8.4.4, 4.8.4.5, 4.8.4.6, 4.8.4.7, 4.8.4.8,
4.8.4.9, 4.8.4.10, 4.8.5.1, 4.8.5.2,
4.8.5.3, 4.8.5.4, 4.8.5.5, 4.8.5.6, 4.8.5.7, 4.8.5.8, 4.8.5.9, 4.8.5.10,
4.8.6.1, 4.8.6.2, 4.8.6.3, 4.8.6.4,
4.8.6.5, 4.8.6.6, 4.8.6.7, 4.8.6.8, 4.8.6.9, 4.8.6.10, 4.8.7.1, 4.8.7.2,
4.8.7.3, 4.8.7.4, 4.8.7.5, 4.8.7.6,
4.8.7.7, 4.8.7.8, 4.8.7.9, 4.8.7.10, 4.8.8.1, 4.8.8.2, 4.8.8.3, 4.8.8.4,
4.8.8.5, 4.8.8.6, 4.8.E.7, 4.8.8.8,
4.8.8.9, 4.8.8.1 0, 4.8.9.1, 4.8.9.2, 4.8.9.3, 4.8.9.4, 4.8.9.5, 4.8.9.6,
4.8.9.7, 4.8.9.8, 4.8.9.9, 4.8.9.10,
4.8.10.1,4.8.10.2,4.8.10.3,4.8.10.4,4.8.10.5,4.8.10.6,4.8.10.7,4.8.10.8,4.8.10.
9,4.8.10.10,
4.9.1.1, 4.9.1.2, 4.9.1.3, 4.9.I.4, 4.9.1.5, 4.9.1.6, 4.9.1.7, 4.9.1.8,
4.9.1.9, 4.9.1.10, 4.9.2.1, 4.9.2.2,
4.9.2.3, 4.9.2.4, 4.9.2.5, 4.9.2.6, 4.9.2.7, 4.9.2.8, 4.9.2.9, 4.9.2.10,
4.9.3.1, 4.9.3.2, 4.9.3.3, 4.9.3.4,
4.9.3.5, 4.9.3.6, 4.9.3.7, 4.9.3.8, 4.9.3.9, 4.9.3.10, 4.9.4.1, 4.9.4.2,
4.9.4.3, 4.9.4.4, 4.9.4.5, 4.9.4.6,
4.9.4.7, 4.9.4.8, 4.9.4.9, 4.9.4.10, 4.9.5.1, 4.9.5.2, 4.9.5.3, 4.9.5.4,
4.9.5.5, 4.9.5.6, 4.9.5.7, 4.9.5.8,
4.9.5.9, 4.9.5.1 0, 4.9.6.1, 4.9.6.2, 4.9.6.3, 4.9.6.4, 4.9.6.5, 4.9.6.6,
4.9.6.7, 4.9.6.8, 4.9.6.9, 4.9.6.10,
4.9.7.1, 4.9.7.2, 4.9.7.3, 4.9.7.4, 4.9.7.5, 4.9.7.6, 4.9.7.7, 4.9.7.8,
4.9.7.9, 4.9.7.1 0, 4.9.8.1, 4.9.8.2,
4.9.8.3, 4.9.8.4, 4.9.8.5, 4.9.8.b, 4.9.8.7, 4.9.8.8, 4.9.8.9, 4.9.8.10,
4.9.9.1, 4.9.9.2, 4.9.9.3, 4.9.9.4,
4.9.9.5,4.9.9.6,4.9.9.7,4.9.9.8,4.9.9.9,4.9.9.10,4.9.10.1,4.9.10.2,4.9.10.3,4.9
.10.4,4.9.10.5,
4.9.10.6,4.9.10.7,4.9.10.8,4.9.10.9,4.9.10.10,4.10.1.1,4.10.1.2,4.10.1.3,4.10.1
.4,4.10.1.5,
4.10.1.6,4.10.1.7,4.10.1.8,4.10.1.9,4.10.1.10,4.10.2.1,4.10.2.2,4.10.2.3,4.10.2
.4,4.10.2.5,
4.10.2.6,4.10.2.7,4.10.2.8,4.10.2.9,4.10.2.10,4.10.3.1,4.10.3.2,4.10.3.3,4.10.3
.4,4.10.3.5,
4.10.3.6,4.10.3.7,4.10.3.8,4.10.3.9,4.10.3.10,4.10.4.1,4.10.4.2,4.10.4.3,4.10.4
.4,4.10.4.5,
4.10.4.6,4.10.4.7,4.10.4.8,4.10.4.9,4.10.4.10,4.10.5.1,4.10.5.2,4.10.5.3,4.10.5
.4,4.10.5.5,
4.10.5.6,4.10.5.7,4.10.5.8,4.10.5.9,4.10.5.10,4.10.6.1,4.10.6.2,4.10.6.3,4.10.6
.4,4.10.6.5,
4.10.6.6,4.10.6.7,4.10.6.8,4.10.6.9,4.10.6.10,4.10.7.1,4.10.7.2,4.10.7.3,4.10.7
.4,4.10.7.5,
4.10.7.6,4.10.7.7,4.10.7.8,4.10.7.9,4.10.7.10,4.10.8.1,4.10.8.2,4.10.8.3,4.10.8
.4,4.10.8.5,
4.10.8.6,4.10.8.7,4.10.8.8,4.10.8.9,4.10.8.10,4.10.9.1,4.10.9.2,4.10.9.3,4.10.9
.4,4.10.9.5,
4.10.9.6,4.10.9.7,4.10.9.8,4.10.9.9,4.10.9.10,4.10.10.1,4.10.10.2,4.10.10.3,4.1
0.10.4,
4.10.10.5, 4.10.10.6, 4.10.10.7, 4.10.10.8, 4.10.10.9, 4.10.10.10, 5.1.1.1,
5.1.1.2, 5.1.1.3, 5.1.1.4,
5.1.1.5, 5.1.1.6, 5.1.1.7, 5.1.1.8, 5.1.1.9, 5.1.1.10, 5.1.2.1, 5.1.2.2,
5.1.2.3, 5.1.2.4, 5.1.2.5, 5.1.2.6,
3 5 5.1.2.7, 5.1.2.8, 5.1.2.9, 5.1.2.10, 5.1.3.1, 5.1.3.2, 5.1.3.3, 5.1.3.4,
5.1.3.5, 5.1.3.6, 5.1.3.7, 5.1.3.8,
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
5.1.3.9, 5.1.3.1 0, 5.1.4.1, 5.1.4.2, 5.1.4.3, 5.1.4.4, 5.1.4.5, 5.1.4.6,
5.1.4.7, 5.1.4.8, 5.1.4.9, 5.1.4.10,
S.I.S.1, 5.1.5.2, S.1.S.3, S.I.S.4, S.1.S.S, S.I.S.6, 5.1.5.7, 5.1.5.8,
S.1.S.9, 5.1.5.1 0, 5.1.6.1, 5.1.6.2,
5.1.6.3, 5.1.6.4, 5.1.6.5, 5.1.6.6, 5.1.6.7, 5.1.6.8, 5.1.6.9, 5.1.6.10,
5.1.7.1, S.I.7.2, 5.1.7.3, 5.1.7.4,
5.1.7.5, 5.1.7.6, 5.1.7.7, 5.1.7.8, 5.1.7.9, 5.1.7.10, 5.1.8.1, 5.1.8.2,
5.1.8.3, 5.1.8.4, 5.1.8.5, 5.1.8.6,
5.1.8.7, 5.1.8.8, 5.1.8.9, 5.1.8.10, 5.1.9.1, 5.1.9.2, 5.1.9.3, 5.1.9.4,
5.1.9.5, 5.1.9.6, 5.1.9.7, 5.1.9.8,
-
5.1.9.9,5.1.9.10,5.1.10.1,5.1.10.2,5.1.10.3,5.1.10.4,5.1.10.5,5.1.10.6,5.1.10.7
,5.1.10.8,
5.1.10.9,5.1.10.10,5.2.1.1,5.2.1.2,5.2.1.3,5.2.1.4,5.2.1.5,5.2.1.6,5.2.1.7,5.2.
1.8,5.2.1.9,
5.2.1.10, 5.2.2.1, 5.2.2.2, 5.2.2.3, 5.2.2.4, 5.2.2.5, 5.2.2.6, 5.2.2.7,
5.2.2.8, 5.2.2.9, 5.2.2.10, 5.2.3.1,
5.2.3.2, 5.2.3.3, 5.2.3.4, 5.2.3.5, 5.2.3.6, 5.2.3.7, 5.2.3.8, 5.2.3.9,
5.2.3.10, 5.2.4.1, 5.2.4.2, 5.2.4.3,
IO 5.2.4.4, 5.2.4.5, 5.2.4.6, 5.2.4.7, 5.2.4.8, 5.2.4.9, 5.2.4.10, 5.2.5.1,
5.2.5.2, 5.2.5.3, S.2.S.4, S.2.S.S,
S.2.S.6, 5.2.5.7, S.2.S.8, 5.2.5.9, S.2.S.10, 5.2.6.1, 5.2.6.2, 5.2.6.3,
5.2.6.4, 5.2.6.5, 5.2.6.6, 5.2.6.7,
5.2.6.8, 5.2.6.9, 5.2.6.10, 5.2.7.1, 5.2.7.2, 5.2.7.3, 5.2.7.4, 5.2.7.5,
5.2.7.6, 5.2.7.7, 5.2.7.8, 5.2.7.9,
5.2.7.1 0, 5.2.8.1, 5.2.8.2, 5.2.8.3, 5.2.8.4, 5.2.8.5, 5.2.8.6, 5.2.8.7,
5.2.8.8, 5.2.8.9, 5.2.8.10, 5.2.9.1,
5.2.9.2,5.2.9.3,5.2.9.4,5.2.9.5,5.2.9.6,5.2.9.7,5.2.9.8,5.2.9.9,5.2.9.10,5.2.10
.1,5.2.10.2,
5.2.10.3,5.2.10.4,5.2.10.S,S.2.10.6,5.2.10.7,5.2.10.8,5.2.10.9,5.2.10.10,5.3.1.
1,5.3.1.2,
5.3.1.3, 5.3.1.4, 5.3.1.5, 5.3.1.6, 5.3.1.7, 5.3.1.8, 5.3.1.9, 5.3.1.10,
5.3.2.1, 5.3.2.2, 5.3.2.3, 5.3.2.4,
5.3.2.5, 5.3.2.6, 5.3.2.7, 5.3.2.8, 5.3.2.9, 5.3.2.10, 5.3.3.1, 5.3.3.2,
5.3.3.3, 5.3.3.4, 5.3.3.5, 5.3.3.6,
5.3.3.7, 5.3.3.8, 5.3.3.9, 5.3.3.10, 5.3.4.1, 5.3.4.2, 5.3.4.3, 5.3.4.4,
5.3.4.5, 5.3.4.6, 5.3.4.7, 5.3.4.8,
5.3.4.9, 5.3.4.10, S.3.S.1, S.3.S.2, 5.3.5.3, 5.3.5.4, S.3.S.S, 5.3.5.6,
S.3.S.7, 5.3.5.8, 5.3.5.9, 5.3.5.10,
5.3.6.1, 5.3.6.2, 5.3.6.3, 5.3.6.4, 5.3.6.5, 5.3.6.6, 5.3.6.7, 5.3.6.8,
5.3.6.9, 5.3.6.I0, 5.3.7.1, 5.3.7.2,
5.3.7.3, 5.3.7.4, 5.3.7.5, 5.3.7.6, 5.3.7.7, 5.3.7.8, 5.3.7.9, 5.3.7.10,
5.3.8.1, 5.3.8.2, 5.3.8.3, 5.3.8.4,
5.3.8.5, 5.3.8.6, 5.3.8.7, 5.3.8.8, 5.3.8.9, 5.3.8.10, 5.3.9.1, 5.3.9.2,
5.3.9.3, 5.3.9.4, 5.3.9.5, 5.3.9.6,
5.3.9.7,5.3.9.8,5.3.9.9,5.3.9.10,5.3.10.1,5.3.10.2,5.3.10.3,5.3.10.4,5.3.10.5,5
.3.10.6,5.3.10.7,
5.3.10.8,5.3.10.9,5.3.10.10,5.4.1.1,5.4.1.2,5.4.1.3,5.4.1.4,5.4.1.5,5.4.1.6,5.4
.1.7,5.4.1.8,
5.4.1.9,5.4.i.10,S.4.2.1,5.4.2.2,5.4.2.3,5.4.2.4,5.4.2.5,5.4.2.6,5.4.2.7,5.4.2.
8,5.4.2.9,5.4.2.10,
5.4.3.1, 5.4.3.2, 5.4.3.3, 5.4.3.4, 5.4.3.5, 5.4.3.6, 5.4.3.7, 5.4.3.8,
5.4.3.9, 5.4.3.10, 5.4.4.1, 5.4.4.2,
5.4.4.3, 5.4.4.4, 5.4.4.5, 5.4.4.6, 5.4.4.7, 5.4.4.8, 5.4.4.9, 5.4.4.10,
S.4.S.1, S.4.S.2, S.4.S.3, 5.4.5.4,
5.4.5.5, 5.4.5.6, S.4.S.7, S.4.S.8, 5.4.5.9, S.4.S.10, 5.4.6.1, 5.4.6.2,
5.4.6.3, 5.4.6.4, 5.4.6.5, 5.4.6.6,
5.4.6.7, 5.4.6.8, 5.4.6.9, 5.4.6.10, 5.4.7.1, 5.4.7.2, 5.4.7.3, 5.4.7.4,
5.4.7.5, 5.4.7.6, 5.4.7.7, 5.4.7.8,
5.4.7.9, 5.4.7.10, 5.4.8.1, 5.4.8.2, 5.4.8.3, 5.4.8.4; 5.4.8.5, 5.4.8.6,
5.4.8.7, 5.4.8.8, 5.4.8.9, 5.4.8.10,
5.4.9.1, 5.4.9.2, 5.4.9.3, 5.4.9.4, 5.4.9.5, 5.4.9.6, 5.4.9.7, 5.4.9.8,
5.4.9.9, 5.4.9.10, 5.4.10.1,
5.4.10.2,S.4.I0.3,5.4.10.4,5.4.10.5,5.4.10.6,5.4.10.7,5.4.10.8,5.4.10.9,5.4.10.
10,5.5.1.1,
5.5.1.2, 5.5.1.3, S.S.1.4, S.S.1.S, S.S.1.6, 5.5.1.7, S.S.1.8, S.S.1.9,
5.5.1.10, S.S.2.1, 5.5.2.2, S.S.2.3,
5.5.2.4, 5.5.2.5, S.S.2.6, 5.5.2.7, 5.5.2.8, 5.5.2.9, S.S.2.1 0, 5.5.3.1,
S.S.3.2, S.S.3.3, 5.5.3.4, 5.5.3.5,
3 5 5.5.3.6, 5.5.3.7, 5.5.3.8, 5.5.3.9, 5.5.3.10, 5.5.4.1, 5.5.4.2, 5.5.4.3,
S.S.4.4, S.S.4.S, 5.5.4.6, S.S.4.7,
41
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
5.5.4.8, 5.5.4.9, 5.5.4.10, 5.5.5.1, S.S.S.2, 5.5.5.3, 5.5.5.4, 5.5.5.5,
5.5.5.6, 5.5.5.7, 5.5.5.8, 5.5.5.9,
5.5.5.1 0, 5.5.6.1, 5.5.6.2, 5.5.6.3, 5.5.6.4, 5.5.6.5, 5.5.6.6, 5.5.6.7,
S.S.6.8, 5.5.6.9, 5.5.6.10, 5.5.7.1,
5.5.7.2, 5.5.7.3, 5.5.7.4, 5.5.7.5, 5.5.7.6, 5.5.7.7, 5.5.7.8, 5.5.7.9,
5.5.7.10, 5.5.8.1, 5.5.8.2, 5.5.8.3,
5.5.8.4, 5.5.8.5, 5.5.8.6, 5.5.8.7, 5.5.8.8, 5.5.8.9, 5.5.8.1 0, 5.5.9.1,
5.5.9.2, 5.5.9.3, 5.5.9.4, 5.5.9.5,
S
5.5.9.6,5.5.9.7,5.5.9.8,5.5.9.9,5.5.9.10,5.5.10.1,5.5.10.2,5.5.10.3,5.5.10.4,5.
5.10.5,5.5.10.6,
'
S.5.10.7,5.5.10.8,5.5.10.9,5.5.10.10,5.6.1.1,5.6.1.2,5.6.1.3,5.6.1.4,5.6.1.5,5.
6.1.6,5.6.1.7,
5.6.1.8, 5.6.1.9, 5.6.1.10, 5.6.2.1, 5.6.2.2, 5.6.2.3, 5.6.2.4, 5.6.2.5,
5.6.2.6, 5.6.2.7, 5.6.2.8, 5.6.2.9,
5.6.2.10, 5.6.3.1, 5.6.3.2, 5.6.3.3, 5.6.3.4, 5.6.3.5, 5.6.3.6, 5.6.3.7,
5.6.3.8, 5.6.3.9, 5.6.3.1 0, 5.6.4.1,
5.6.4.2, 5.6.4.3, 5.6.4.4, 5.6.4.5, 5.6.4.6, 5.6.4.7, 5.6.4.8, 5.6.4.9,
5.6.4.10, 5.6.5.1, 5.6.5.2, 5.6.5.3,
5.6.5.4, 5.6.5.5, S.6.S.6, 5.6.5.7, 5.6.5.8, 5.6.5.9, 5.6.5.10, 5.6.6.1,
5.6.6.2, 5.6.6.3, 5.6.6.4, 5.6.6.5,
5.6.6.6, 5.6.6.7, 5.6.6.8, 5.6.6.9, 5.6.6.10, 5.6.7.1, 5.6.7.2, 5.6.7.3,
5.6.7.4, 5.6.7.5, 5.6.7.6, 5.6.7.7,
5.6.7.8, 5.6.7.9, 5.6.7.10, 5.6.8.1, 5.6.8.2, 5.6.8.3, 5.6.8.4, 5.6.8.5,
5.6.8.6, 5.6.8.7, 5.6.8.8, 5.6.8.9,
5.6.8.1 0, 5.6.9.1, 5.6.9.2, 5.6.9.3, 5.6.9.4, 5.6.9.5, 5.6.9.6, 5.6.9.7,
5.6.9.8, 5.6.9.9, 5.6.9.10,
5.6.10.1,5.6.10.2,5.6.10.3,5.6.10.4,5.6.10.5,5.6.10.6,5.6.10.7,5.6.10.8,5.6.10.
9,5.6.10.10,
5.7.I.1, 5.7.1.2, 5.7.1.3, 5.7.1.4, 5.7.1.5, 5.7.1.6, 5.7.1.7, 5.7.1.8,
5.7.1.9, 5.7.1.10, 5.7.2.1, 5.7.2.2,
5.7.2.3, 5.7.2.4, 5.7.2.5, 5.7.2.6, 5.7.2.7, 5.7.2.8, 5.7.2.9, 5.7.2.10,
5.7.3.1, 5.7.3.2, 5.7.3.3, 5.7.3.4,
5.7.3.5, 5.7.3.6, 5.7.3.7, 5.7.3.8, 5.7.3.9, 5.7.3.10, 5.7.4.1, 5.7.4.2,
5.7.4.3, 5.7.4.4, 5.7.4.5, 5.7.4.6,
5.7.4.7, 5.7.4.8, 5.7.4.9, 5.7.4.10, 5.7.5.1, 5.7.5.2, 5.7.5.3, 5.7.5.4,
5.7.5.5, 5.7.5.6, 5.7.5.7, 5.7.5.8,
5.7.5.9, 5.7.5.10, 5.7.6.1, 5.7.6.2, 5.7.6.3, 5.7.6.4, 5.7.6.5, 5.7.6.6,
5.7.6.7, 5.7.6.8, 5.7.6.9, 5.7.6.10,
5.7.7.1, 5.7.7.2, 5.7.7.3, 5.7.7.4, 5.7.7.5, 5.7.7.6, 5.7.7.7, 5.7.7.8,
5.7.7.9, 5.7.7.10, 5.7.8.1, 5.7.8.2,
5.7.8.3, 5.7.8.4, 5.7.8.5, 5.7.8.6, 5.7.8.7, 5.7.8.8, 5.7.8.9, 5.7.8.10,
5.7.9.1, 5.7.9.2, 5.7.9.3, 5.7.9.4,
5.7.9.5,5.7.9.6,5.7.9.7,5.7.9.8,5.7.9.9,5.7.9.10,5.7.10.1,5.7.10.2,5.7.10.3,5.7
.10.4,5.7.10.5,
5.7.10.6,5.7.10.7,5.7.10.8,5.7.10.9,5.7.10.10,5.8.1.1,5.8.1.2,5.8.1.3,5.8.1.4,5
.8.1.5,5.8.1.6,
5.8.1.7, 5.8.1.8, 5.8.1.9, 5.8.1.10, 5.8.2.1, 5.8.2.2, 5.8.2.3, 5.8.2.4,
5.8.2.5, 5.8.2.6, 5.8.2.7, 5.8.2.8,
5.8.2.9,5.8.2.10,5.8.3.1,5.8.3.2,5.8.3.3,5.8.3.4,5.8.3.5,5.8.3.6,5.8.3.7,5.8.3.
8,5.8.3.9,5.8.3.10,
5.8.4.1, 5.8.4.2, 5.8.4.3, 5.8.4.4, 5.8.4.5, 5.8.4.6, 5.8.4.7, 5.8.4.8,
5.8.4.9, 5.8.4.10, 5.8.5.1, 5.8.5.2,
5.8.5.3, 5.8.5.4, 5.8.5.5, 5.8.5.6, 5.8.5.7, 5.8.5.8, 5.8.5.9, 5.8.5.10,
5.8.6.1, 5.8.6.2, 5.8.6.3, 5.8.6.4,
5.8.6.5, 5.8.6.6, 5.8.6.7, 5.8.6.8, 5.8.6.9, 5.8.6.10, 5.8.7.1, 5.8.7.2,
5.8.7.3, 5.8.7.4, 5.8.7.5, 5.8.7.6,
5.8.7.7, 5.8.7.8, 5.8.7.9, 5.8.7.10, 5.8.8.1, 5.8.8.2, 5.8.8.3, 5.8.8.4,
5.8.8.5, 5.8.8.6, 5.8.8.7, 5.8.8.8,
5.8.8.9,5.8.8.10,5.8.9.1,5.8.9.2,5.8.9.3,5.8.9.4,5.8.9.5,5.8.9.6,5.8.9.7,5.8.9.
8,5.8.9.9,5.8.9.10,
5.8.10.1,5.8.10.2,5.8.10.3,5.8.10.4,5.8.10.5,5.8.10.6,5.8.10.7,5.8.10.8,5.8.10.
9,5.8.10.10,
5.9.1.1, 5.9.1.2, 5.9.1.3, 5.9.1.4, 5.9.1.5, 5.9.1.6, 5.9.1.7, 5.9.1.8,
5.9.1.9, 5.9.1.10, 5.9.2.1, 5.9.2.2,
5.9.2.3, 5.9.2.4, 5.9.2.5, 5.9.2.6, 5.9.2.7, 5.9.2.8, 5.9.2.9, 5.9.2.10,
5.9.3.1, 5.9.3.2, 5.9.3.3, 5.9.3.4,
5.9.3.5, 5.9.3.6, 5.9.3.7, 5.9.3.8, 5.9.3.9, 5.9.3.10, 5.9.4.1, 5.9.4.2,
5.9.4.3, 5.9.4.4, 5.9.4.5, 5.9.4.6,
3 5 5.9.4.7, 5.9.4.8, 5.9.4.9, 5.9.4.10, 5.9.5.1, 5.9.5.2, 5.9.5.3, 5.9.5.4,
5.9.5.5, 5.9.5.6, 5.9.5.7, 5.9.5.8,
42
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
5.9.5.9, 5.9.5.1 0, 5.9.6.1, 5.9.6.2, 5.9.6.3, 5.9.6.4, 5.9.6.5, 5.9.6.6,
5.9.6.7, 5.9.6.8, 5.9.6.9, 5.9.6.10,
5.9.7.1, 5.9.7.2, 5.9.7.3, 5.9.7.4, 5.9.7.5, 5.9.7.6, 5.9.7.7, 5.9.7.8,
5.9.7.9, 5.9.7.10; 5.9.8.1, 5.9.8.2,
5.9.8.3, 5.9.8.4, 5.9.8.5, 5.9.8.6, 5.9.8.7, 5.9.8.8, 5.9.8.9, 5.9.8.10,
5.9.9.1, 5.9.9.2, 5.9.9.3, 5.9.9.4,
5.9.9.5,5.9.9.6,5.9.9.7,5.9.9.8,5.9.9.9,5.9.9.10,5.9.10.1,5.9.10.2,5.9.10.3,5.9
.10.4,5.9.10.5,
5.9.10.6,5.9.10.7,5.9.10.8,5.9.10.9,5.9.10.10,5.10.1.1,5.10.1.2,5.10.1.3,5.10.1
.4,5.10.1.5,
'
S.10.1.6,5.10.1.7,5.10.1.8,5.10.1.9,5.10.1.10,5.10.2.1,5.10.2.2,5.10.2.3,5.10.2
.4,5.10.2.5,
5.10.2.6, 5.10.2.7, 5.10.2.8, 5.10.2.9, 5.10.2.10, 5.10.3.1, 5.10.3.2,
5.10.3.3, 5.10.3.4, 5.10.3.5,
5.10.3.6,5.10.3.7,5.10.3.8,5.10.3.9,5.10.3.10,5.10.4.1,5.10.4.2,5.10.4.3,5.10.4
.4,5.10.4.5,
5.10.4.6,5.10.4.7,5.10.4.8,5.10.4.9,5.10.4.10,5.10.5.1,5.10.5.2,5.10.5.3,5.10.5
.4,5.10.5.5,
5.10.5.6,5.10.5.7,5.10.5.8,5.10.5.9,5.10.5.10,5.10.6.1,5.10.6.2,5.10.6.3,5.10.6
.4,5.10.6.5,
5.10.6.6,5.10.6.7,5.10.6.8,5.10.6.9,5.10.6.i0,5.10.7.1,5.10.7.2,5.10.7.3,5.10.7
.4,5.10.7.5,
5.10.7.6,5.10.7.7,5.10.7.8,5.10.7.9,5.10.7.10,5.10.8.1,5.10.8.2,5.10.8.3,5.10.8
.4,5.10.8.5,
5.10.8.6,5.10.8.7,5.10.8.8,5.I0.8.9,5.10.8.10,5.10.9.1,5.10.9.2,5.10.9.3,5.10.9
.4,5.10.9.5,
5.10.9.6,5.10.9.7,5.10.9.8,5.10.9.9,5.10.9.10,5.10.10.1,5.10.10.2,5.10.10.3,5.1
0.10.4,
5.10.10.5,5.10.10.6,5.10.10.7,5.10.10.8,5.10.10.9,5.10.10.10,6.1.1.1,6.1.1.2,6.
1.1.3,6.1.1.4,
6.1.1.5, 6.1.1.6, 6.1.1.7, 6.1.1.8, 6.1.1.9, 6.1.1.10, 6.1.2.1, 6.1.2.2,
6.1.2.3, 6.1.2.4, 6.1.2.5, 6.1.2.6,
6.1.2.7, 6.1.2.8, 6.1.2.9, 6.1.2.10, 6.1.3.1, 6.1.3.2, 6.1.3.3, 6.1.3.4,
6.1.3.5, 6.1.3.6, 6.1.3.7, 6.1.3.8,
6.1.3.9, 6.1.3.1 0, 6.1.4.1, 6.1.4.2, 6.1.4.3, 6.1.4.4, 6.1.4.5, 6.1.4.6,
6.1.4.7, 6.1.4.8, 6.1.4.9, 6.1.4.10,
6.1.5.1, 6.1.5.2, 6.1.5.3, 6.1.5.4, 6.1.5.5, 6.1.5.6, 6.1.5.7, 6.1.5.8,
6.1.5.9, 6.1.5.10, 6.1.6.1, 6.1.6.2,
6.1.6.3, 6.1.6.4, 6.1.6.5, 6.1.6.6, 6.1.6.7, 6.1.6.8, 6.1.6.9, 6.1.6.1 0,
6.1.7.1, 6.1.7.2, 6.1.7.3, 6.1.7.4,
6.1.7.5, 6.1.7.6, 6.1.7.7, 6.1.7.8, 6.1.7.9, 6.1.7.10, 6.1.8.1, 6.1.8.2,
6.1.8.3, 6.1.8.4, 6.1.8.5, 6.1.8.6,
6.1.8.7, 6.1.8.8, 6.1.8.9, 6.1.8.10, 6.1.9.1, 6.1.9.2, 6.1.9.3, 6.1.9.4,
6.1.9.5, 6.1.9.6, 6.1.9.7, 6.1.9.8,
6.1.9.9,6.1.9.10,6.1.10.1,6.1.10.2,6.1.10.3,6.1.10.4,6.1.10.5,6.1.10.6,6.1.10.7
,6.1.10.8,
6.1.10.9,6.i.10.10,6.2.1.1,6.2.1.2,6.2.1.3,6.2.1.4,6.2.1.5,6.2.1.6,6.2.1.7,6.2.
1.8,6.2.1.9,
6.2.1.10,6.2.2.1,6.2.2.2,6.2.2.3,6.2.2.4,6.2.2.5,6.2.2.6,6.2.2.7,6.2.2.8,6.2.2.
9,6.2.2.10,6.2.3.1,
6.2.3.2, 6.2.3.3, 6.2.3.4, 6.2.3.5, 6.2.3.6, 6.2.3.7, 6.2.3.8, 6.2.3.9,
6.2.3.10, 6.2.4.1, 6.2.4.2, 6.2.4.3,
6.2.4.4, 6.2.4.5, 6.2.4.6, 6.2.4.7, 6.2.4.8, 6.2.4.9, 6.2.4.10, 6.2.5.1,
6.2.5.2, 6.2.5.3, 6.2.5.4, 6.2.5.5,
6.2.5.6, 6.2.5.7, 6.2.5.8, 6.2.5.9, 6.2.5.10, 6.2.6.1, 6.2.6.2, 6.2.6.3,
6.2.6.4, 6.2.6.5, 6.2.6.6, 6.2.6.7,
6.2.6.8, 6.2.6.9, 6.2.6.10, 6.2.7.1, 6.2.7.2, 6.2.7.3, 6.2.7.4, 6.2.7.5,
6.2.7.6, 6.2.7.7, 6.2.7.8, 6.2.7.9,
6.2.7.10,6.2.8.1,6.2.8.2,6.2.8.3,6.2.8.4,6.2.8.5,6.2.8.6,6.2.8.7,6.2.8.8,6.2.8.
9,6.2.8.10,6.2.9.1,
6.2.9.2,6.2.9.3,6.2.9.4,6.2.9.5,6.2.9.6,6.2.9.7,6.2.9.8,6.2.9.9,6.2.9.10,6.2.10
.1,6.2.10.2,
6.2.10.3,6.2.10.4,6.2.10.5,6.2.10.6,6.2.10.7,6.2.10.8,6.2.10.9,6.2.10.10,6.3.1.
1,6.3.1.2,
6.3.1.3, 6.3.1.4, 6.3.1.5, 6.3.1.6, 6.3.1.7, 6.3.1.8, 6.3.1.9, 6.3.1.10,
6.3.2.1, 6.3.2.2, 6.3.2.3, 6.3.2.4,
6.3.2.5, 6.3.2.6, 6.3.2.7, 6.3.2.8, 6.3.2.9, 6.3.2.10, 6.3.3.1, 6.3.3.2,
6.3.3.3, 6.3.3.4, 6.3.3.5, 6.3.3.6,
6.3.3.7,6.3.3.8,6.3.3.9,6.3.3.10,6.3.4.1,6.3.4.2,6.3.4.3,6.3.4.4,6.3.4.5,6.3.4.
6,6.3.4.7,6.3.4.8,
43
_ CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
6.3.4.9, 6.3.4.10, 6.3.5.1, 6.3.5.2, 6.3.5.3, 6.3.5.4, 6.3.5.5, 6.3.5.6,
6.3.5.7, 6.3.5.8, 6.3.5.9, 6.3.5.10,
6.3.6.1, 6.3.6.2, 6.3.6.3, 6.3.6.4, 6.3.6.5, 6.3.6.6, 6.3.6.7, 6.3.6.8,
6.3.6.9, 6.3.6.1 0, 6.3.7.1, 6.3.7.2,
6.3.7.3, 6.3.7.4, 6.3.7.5, 6.3.7.6, 6.3.7.7, 6.3.7.8, 6.3.7.9, 6.3.7.10,
6.3.8.1, 6.3.8.2, 6.3.8.3, 6.3.8.4,
6.3.8.5, 6.3.8.6, 6.3.8.7, 6.3.8.8, 6.3.8.9, 6.3.8.10, 6.3.9.1, 6.3.9.2,
6.3.9.3, 6.3.9.4, 6.3.9.5, 6.3.9.6,
6.3.9.7,6.3.9.8,6.3.9.9,6.3.9.10,6.3.10.1,6.3.10.2,6.3.10.3,6.3.10.4,6.3.10.5,6
.3.10.6,6.3.10.7,
-
6.3.10.8,6.3.10.9,6.3.10.10,6.4.1.1,6.4.1.2,6.4.1.3,6.4.1.4,6.4.1.5,6.4.1.6,6.4
.1.7,6.4.1.8,
6.4.1.9, 6.4.1.10, 6.4.2.1, 6.4.2.2, 6.4.2.3, 6.4.2.4, 6.4.2.5, 6.4.2.6,
6.4.2.7, 6.4.2.8, 6.4.2.9, 6.4.2.10,
6.4.3.1, 6.4.3.2, 6.4.3.3, 6.4.3.4, 6.4.3.5, 6.4.3.6, 6.4.3.7, 6.4.3.8,
6.4.3.9, 6.4.3.10, 6.4.4.1, 6.4.4.2,
6.4.4.3, 6.4.4.4, 6.4.4.5, 6.4.4.6, 6.4.4.7, 6.4.4.8, 6.4.4.9, 6.4.4.10,
6.4.5.1, 6.4.5.2, 6.4.5.3, 6.4.5.4,
6.4.5.5, 6.4.5.6, 6.4.5.7, 6.4.5.8, 6.4.5.9, 6.4.5.10, 6.4.6.1, 6.4.6.2,
6.4.6.3, 6.4.6.4, 6.4.6.5, 6.4.6.6,
6.4.6.7, 6.4.6.8, 6.4.6.9, 6.4.6.10, 6.4.7.1, 6.4.7.2, 6.4.7.3, 6.4.7.4,
6.4.7.5, 6.4.7.6, 6.4.7.7, 6.4.7.8,
6.4.7.9, 6.4.7.1 0, 6.4.8.1, 6.4.8.2, 6.4.8.3, 6.4.8.4, 6.4.8.5, 6.4.8.6,
6.4.8.7, 6.4.8.8, 6.4.E.9, 6.4.8.10,
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S.I0.5,6.5.10.6,
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6.6.1.8, 6.6.1.9, 6.6.1.1 0, 6.6.2.1, 6.6.2.2, 6.6.2.3, 6.6.2.4, 6.6.2.5,
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6.6.4.2, 6.6.4.3, 6.6.4.4, 6.6.4.5, 6.6.4.6, 6.6.4.7, 6.6.4.8, 6.6.4.9,
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44
- CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
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6.7.10.6,6.7.10.7,6.7.10.8,6.7.10.9,6.7.10.10,6.8.1.1,6.8.1.2,6.8.1.3,6.8.1.4,6
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6.$.2.9, 6.8.2.10, 6.8.3.1, 6.8.3.2, 6.8.3.3, 6.8.3.4, 6.8.3.5, 6.8.3.6,
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CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
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2 5 7.4.3.1, 7.4.3.2, 7.4.3.3, 7.4.3.4, 7.4.3.5, 7.4.3.6, 7.4.3.7, 7.4.3.8,
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7.4.4.3, 7.4.4.4, 7.4.4.5, 7.4.4.6, 7.4.4.7, 7.4.4.8, 7.4.4.9, 7.4.4.10,
7.4.5.1, 7.4.5.2, 7.4.5.3, 7.4.5.4,
7.4.5.5, 7.4.5.6, 7.4.5.7, 7.4.5.8, 7.4.5.9, 7.4.5.10, 7.4.6.1, 7.4.6.2,
7.4.6.3, 7.4.6.4, 7.4.6.5, 7.4.6.6,
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7.4.7.5, 7.4.7.6, 7.4.7.7, 7.4.7.8,
7.4.7.9, 7.4.7.10, 7.4.8.1, 7.4.8.2, 7.4.8.3, 7.4.8.4, 7.4.8.5, 7.4.8.6,
7.4.8.7, 7.4.8.8, 7.4.8.9, 7.4.8.10,
7.4.9.1,7.4.9.2,7.4.9.3,7.4.9.4,7.4.9.5,7.4.9.6,7.4.9.7,7.4.9.$,7.4.9.9,7.4.9.1
0,7.4.10.1,
7.4.10.2,7.4.10.3,7.4.I0.4,7.4.10.5,7.4.10.6,7.4.10.7,7.4.10.8,7.4.10.9,7.4.10.
10,7.5.1.1,
7.5.1.2, 7.5.1.3, 7.5.1.4, 7.5.1.5, 7.5.1.6, 7.5.1.7, 7.5.1.8, 7.5.1.9,
7.5.1.10, 7.5.2.1, 7.5.2.2, 7.5.2.3,
7.5.2.4, 7.5.2.5, 7.5.2.6, 7.5.2.7, 7.5.2.8, 7.5.2.9, 7.5.2.10, 7.5.3.1,
7.5.3.2, 7.5.3.3, 7.5.3.4, 7.5.3.5,
7.5.3.6, 7.5.3.7, 7.5.3.8, 7.5.3.9, 7.5.3.10, 7.5.4.1, 7.5.4.2, 7.5.4.3,
7.5.4.4, 7.5.4.5, 7.5.4.6, 7.5.4.7,
3 5 7.5.4.8, 7.5.4.9, 7.5.4.10, 7.5.5.1, 7.5.5.2, 7.5.5.3, 7.5.5.4, 7.5.5.5,
7.5.5.6, 7.5.5.7, 7.5.5.8, 7.5.5.9,
46
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
7.5.5.10,7.5.6.1,7.5.6.2,7.5.6.3,7.5.6.4,7.5.6.5,7.5.6.6,7.5.6.7,7.5.6.8,7.5.6.
9,7.5.6.10,7.5.7.1,
7.5.7.2, 7.5.7.3, 7.5.7.4, 7.5.7.5, 7.5.7.6, 7.5.7.7, 7.5.7.8, 7.5.7.9,
7.5.7.10, 7.5.8.1; 7.5.8.2, 7.5.8.3,
7.5.8.4, 7.5.8.5, 7.5.8.6, 7.5.8.7, 7.5.8.8, 7.5.8.9, 7.5.8.1 0, 7.5.9.1,
7.5.9.2, 7.5.9.3, 7.5.9.4, 7.5.9.5,
7.5.9.6,7.5.9.7,7.5.9.8,7.5.9.9,7.5.9.10,7.5.10.1,7.5.10.2,7.5.10.3,7.5.10.4,7.
5.10.5,7.5.10.6,
7.5.10.7,7.5.10.8,7.5.10.9,7.5.10.10,7.6.1.1,7.6.1.2,7.6.1.3,7.6.1.4,7.6.1.5,7.
6.1.6,7.6.1.7,
7.6.1.8, 7.6.1.9, 7.6.1.1 0, 7.6.2.1, 7.6.2.2, 7.6.2.3, 7.6.2.4, 7.6.2.5,
7.6.2.6, 7.6.2.7, 7.6.2.8, 7.6.2.9,
7.6.2.1 0, 7.6.3.1, 7.6.3.2, 7.6.3.3, 7.6.3.4, 7.6.3.5, 7.6.3.6, 7.6.3.7,
7.6.3.8, 7.6.3.9, 7.6.3.10, 7.6.4.1,
7.6.4.2, 7.6.4.3, 7.6.4.4, 7.6.4.5, 7.6.4.6, 7.6.4.7, 7.6.4.8, 7.6.4.9,
7.6.4.10, 7.6.5.1, 7.6.5.2, 7.6.5.3,
7.6.5.4, 7.6.5.5, 7.6.5.6, 7.6.5.7, 7.6.5.8, 7.6.5.9, 7.6.5.10, 7.6.6.1,
7.6.6.2, 7.6.6.3, 7.6.6.4, 7.6.6.5,
7.6.6.6, 7.6.6.7, 7.6.6.8, 7.6.6.9, 7.6.6.10, 7.6.7.1, 7.6.7.2, 7.6.7.3,
7.6.7.4, 7.6.7.5, 7.6.7.6, 7.6.7.7,
7.6.7.8, 7.6.7.9, 7.6.7.10, 7.6.8.1, 7.6.8.2, 7.6.8.3, 7.6.8.4, 7.6.8.5,
7.6.8.6, 7.6.8.7, 7.6.8.8, 7.6.8.9,
7.6.8.10,7.6.9.1,7.6.9.2,7.6.9.3,7.6.9.4,7.6.9.5,7.6.9.6,7.6.9.7,7.6.9.8,7.6.9.
9,7.69.10,
7.6.10.1,7.6.10.2,7.6.10.3,7.6.10.4,7.6.10.5,7.6.10.6,7.6.10.7,7.6.10.8,7.6.10.
9,7.6.10.10,
7.7.1.1, 7.7.1.2, 7.7.1.3, 7.7.1.4, 7.7.1.5, 7.7.1.6, 7.7.1.7, 7.7.1.8,
7.7.1.9, 7.7.1.1 0, 7.7.2.1, 7.7.2.2,
7.7.2.3, 7.7.2.4, 7.7.2.5, 7.7.2.6, 7.7.2.7, 7.7.2.8, 7.7.2.9, 7.7.2.1 0,
7.7.3.1, 7.7.3.2, 7.7.3.3, 7.7.3.4,
7.7.3.5, 7.7.3.6, 7.7.3.7, 7.7.3.8, 7.7.3.9, 7.7.3.10, 7.7.4.1, 7.7.4.2,
7.7.4.3, 7.7.4.4, 7.7.4.5, 7.7.4.6,
7.7.4.7, 7.7.4.8, 7.7.4.9, 7.7.4.10, 7.7.5.1, 7.7.5.2, 7.7.5.3, 7.7.5.4,
7.7.5.5, 7.7.5.6, 7.7.5.7, 7.7.5.8,
7.7.5.9, 7.7.5.1 0, 7.7.6.1, 7.7.6.2, 7.7.6.3, 7.7.6.4, 7.7.6.5, 7.7.6.6,
7.7.6.7, 7.7.6.8, 7.7.6.9, 7.7.6.10,
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7.7.7.9, 7.7.7.10, 7.7.8.1, 7.7.8.2,
7.7.8.3, 7.7.8.4, 7.7.8.5, 7.7.8.6, 7.7.8.7, 7.7.8.8, 7.7.8.9, 7.7.8.10,
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.10.4,7.7.10.5,
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.8.1.5,7.8.1.6,
7.8.1.7, 7.8.1.8, 7.8.1.9, 7.8.1.10, 7.8.2.1, 7.8.2.2, 7.8.2.3, 7.8.2.4,
7.8.2.5, 7.8.2.6, 7.8.2.7, 7.8.2.8,
7.8.2.9, 7.8.2.10, 7.8.3.1, 7.8.3.2, 7.8.3.3, 7.8.3.4, 7.8.3.5, 7.8.3.6,
7.8.3.7, 7.8.3.8, 7.8.3.9, 7.8.3.10,
2 5 7.8.4.1, 7.8.4.2, 7.8.4.3, 7.8.4.4, 7.8.4.5, 7.8.4.6, 7.8.4.7, 7.8.4.8,
7.8.4.9, 7.8.4.10, 7.8.5.1, 7.8.5.2,
7.8.5.3, 7.8.5.4, 7.8.5.5, 7.8.5.6, 7.8.5.7, 7.8.5.8, 7.8.5.9, 7.8.5.10,
7.8.6.1, 7.8.6.2, 7.8.6.3, 7.8.6.4,
7.8.6.5, 7.8.6.6, 7.8.6.7, 7.8.6.8, 7.8.6.9, 7.8.6.10, 7.8.7.1, 7.8.7.2,
7.8.7.3, 7.8.7.4, 7.8.7.5, 7.8.7.6,
7.8.7.7, 7.8.7.8, 7.8.7.9, 7.8.7.10, 7.8.8.1, 7.8.8.2, 7.8.8.3, 7.8.8.4,
7.8.8.5, 7.8.8.6, 7.8.8.7, 7.8.8.8,
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9,7.8.10.10,
7.9.1.1, 7.9.1.2, 7.9.1.3, 7.9.1.4, 7.9.1.5, 7.9.1.6, 7.9.1.7, 7.9.1.8,
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8,7.9.6.9,7.9.6.10,
47
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
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7.10.1.6, 7.10.1.7, 7.10.1.8, 7.10.1.9, 7.10.1.10, 7.10.2.1, 7.10.2.2,
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8.3.3.3, 8.3.3.4, 8.3.3.5, 8.3.3.6,
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8,8.3.5.9,8.3.5.10,
48
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
8.3.6.1, 8.3.6.2, 8.3.6.3, 8.3.6.4, 8.3.6.5, 8.3.6.6, 8.3.6.7, 8.3.6.8,
8.3.6.9, 8.3.6.10, 8.3.7.1, 8.3.7.2,
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49
CA 02356539 2001-05-23
WO 00/32201 PCT/US99/28079
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SO
CA 02356539 2001-05-23
WO 00/32201 PCTNS99/28079
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9.3.3.7, 9.3.3.8, 9.3.3.9, 9.3.3.10, 9.3.4.1, 9.3.4.2, 9.3.4.3, 9.3.4.4,
9.3.4.5, 9.3.4.6, 9.3.4.7, 9.3.4.8,
9.3.4.9, 9.3.4.10, 9.3.5.1, 9.3.5.2, 9.3.5.3, 9.3.5.4, 9.3.5.5, 9.3.5.6,
9.3.5.7, 9.3.5.8, 9.3.5.9, 9.3.5.10,
9.3.6.1, 9.3.6.2, 9.3.6.3, 9.3.6.4, 9.3.6.5, 9.3.6.6, 9.3.6.7, 9.3.6.8,
9.3.6.9, 9.3.6.1 0, 9.3.7.1, 9.3.7.2,
9.3.7.3, 9.3.7.4, 9.3.7.5, 9.3.7.6, 9.3.7.7, 9.3.7.8, 9.3.7.9, 9.3.7.10,
9.3.8.1, 9.3.8.2, 9.3.8.3, 9.3.8.4,
9.3.8.5, 9.3.8.6, 9.3.8.7, 9.3.8.8, 9.3.8.9, 9.3.8.10, 9.3.9.1, 9.3.9.2,
9.3.9.3, 9.3.9.4, 9.3.9.5, 9.3.9.6,
9.3.9.7,9.3.9.8,9.3.9.9,9.3.9.10,9.3.10.1,9.3.10.2,9.3.10.3,9.3.10.4,9.3.10.5,9
.3.10.6,9.3.10.7,
9.3.10.8, 9.3.10.9, 9.3.10.10, 9.4.1.1, 9.4.1.2, 9.4.1.3, 9.4.1.4, 9.4.1.5,
9.4.1.6, 9.4.1.7, 9.4.1.8,
9.4.1.9, 9.4.1.1 0, 9.4.2.1, 9.4.2.2, 9.4.2.3, 9.4.2.4, 9.4.2.5, 9.4.2.6,
9.4.2.7, 9.4.2.8, 9.4.2.9, 9.4.2.10,
9.4.3.1, 9.4.3.2, 9.4.3.3, 9.4.3.4, 9.4.3.5, 9.4.3.6, 9.4.3.7, 9.4.3.8,
9.4.3.9, 9.4.3.10, 9.4.4.1, 9.4.4.2,
9.4.4.3, 9.4.4.4, 9.4.4.5, 9.4.4.6, 9.4.4.7, 9.4.4.8, 9.4.4.9, 9.4.4.10,
9.4.5.1, 9.4.5.2, 9.4.5.3, 9.4.5.4,
9.4.5.5, 9.4.5.6, 9.4.5.7, 9.4.5.8, 9.4.5.9, 9.4.5.10, 9.4.6.1, 9.4.6.2,
9.4.6.3, 9.4.6.4, 9.4.6.5, 9.4.6.6,
9.4.6.7, 9.4.6.8, 9.4.6.9, 9.4.6.10, 9.4.7.1, 9.4.7.2, 9.4.7.3, 9.4.?.4,
9.4.7.5, 9.4.7.6, 9.4.7.7, 9.4.7.8,
9.4.7.9, 9.4.7.1 0, 9.4.8.1, 9.4.8.2, 9.4.8.3, 9.4.8.4, 9.4.8.5, 9.4.8.6,
9.4.8.7, 9.4.8.8, 9.4.8.9, 9.4.8.10,
9.4.9.1, 9.4.9.2, 9.4.9.3, 9.4.9.4, 9.4.9.5, 9.4.9.6, 9.4.9.7, 9.4.9.8,
9.4.9.9, 9.4.9.10, 9.4.10.1,
9.4.10.2,9.4.10.3,9.4.10.4,9.4.10.5,9.4.10.6,9.4.10.7,9.4.10.8,9.4.10.9,9.4.10.
10,9.5.1.1,
9.5.1.2, 9.5.1.3, 9.5.1.4, 9.5.1.5, 9.5.1.6, 9.5.1.7, 9.5.1.8, 9.5.1.9,
9.5.1.10, 9.5.2.1, 9.5.2.2, 9.5.2.3,
9.5.2.4, 9.5.2.5, 9.5.2.6, 9.5.2.7, 9.5.2.8, 9.5.2.9, 9.5.2.10, 9.5.3.1,
9.5.3.2, 9.5.3.3, 9.5.3.4, 9.5.3.5,
9.5.3.6, 9.5.3.7, 9.5.3.8, 9.5.3.9, 9.5.3.10, 9.5.4.1, 9.5.4.2, 9.5.4.3,
9.5.4.4, 9.5.4.5, 9.5.4.6, 9.5.4.7,
9.5.4.8, 9.5.4.9, 9.5.4.10, 9.5.5.1, 9.5.5.2, 9.5.5.3, 9.5.5.4, 9.5.5.5,
9.5.5.6, 9.5.5.7, 9.5.5.8, 9.5.5.9,
9.5.5.10,9.5.6.1,9.5.6.2,9.5.6.3,9.5.6.4,9.5.6.5,9.5.6:6,9.5.6.7,9.5.6.8,9.5.6.
9,9.5.6.10,9.5.7.1,
51
CA 02356539 2001-05-23
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9.5.7.2, 9.5.7.3, 9.5.7.4, 9.5.7.5, 9.5.7.6, 9.5.7.7, 9.5.7.8, 9.5.7.9,
9.5.7.10, 9.5.8.1, 9.5.8.2, 9.5.8.3,
9.5.8.4, 9.5.8.5, 9.5.8.6, 9.5.8.7, 9.5.8.8, 9.5.8.9, 9.5.8.10, 9.5.9.1,
9.5.9.2, 9.5.9.3; 9.5.9.4, 9.5.9.5,
9.5.9.6,9.5.9.7,9.5.9.8,9.5.9.9,9.5.9.10,9.5.10.1,9.5.10.2,9.5.10.3,9.5.10.4,9.
5.10.5,9.5.10.6,
9.5.10.7,9.5.10.8,9.5.10.9,9.5.10.10,9.6.1.1,9.6.1.2,9.6.1.3,9.6.1.4,9.6.1.5,9.
6.1.6,9.6.1.7,
9.6.1.8, 9.6.1.9, 9.6.1.1 0, 9.6.2.1, 9.6.2.2, 9.6.2.3, 9.6.2.4, 9.6.2.5,
9.6.2.6, 9.6.2.7, 9.6.2.8, 9.6.2.9,
9.6.2.10, 9.6.3.1, 9.6.3.2, 9.6.3.3, 9.6.3.4, 9.6.3.5, 9.6.3.6, 9.6.3.7,
9.6.3.8, 9.6.3.9, 9.6.3.10, 9.6.4.1,
9.6.4.2, 9.6.4.3, 9.6.4.4, 9.6.4.5, 9.6.4.6, 9.6.4.7, 9.6.4.8, 9.6.4.9,
9.6.4.10, 9.6.5.1, 9.6.5.2, 9.6.5.3,
9.6.5.4, 9.6.5.5, 9.6.5.6, 9.6.5.7, 9.6.5.8, 9.6.5.9, 9.6.5.10, 9.6.6.1,
9.6.6.2, 9.6.6.3, 9.6.6.4, 9.6.6.5,
9.6.6.6, 9.6.6.7, 9.6.6.8, 9.6.6.9, 9.6.6.10, 9.6.7.1, 9.6.7.2, 9.6.7.3,
9.6.7.4, 9.6.7.5, 9.6.7.6, 9.6.7.7,
9.6.7.8, 9.6.7.9, 9.6.7.10, 9.6.8.1, 9.6.8.2, 9.6.8.3, 9.6.8.4, 9.6.8.5,
9.6.8.6, 9.6.8.7, 9.6.8.8, 9.6.8.9,
9.6.8.10, 9.6.9.1, 9.6.9.2, 9.6.9.3, 9.6.9.4, 9.6.9.5. 9.6.9.6, 9.6.9.7,
9.6.9.8, 9.6.9.9, 9.6.9.10,
9.6.10.1,9.6.10.2,9.6.10.3,9.6.10.4,9.6.10.5,9.6.10.6,9.6.10.7,9.6.10.8,9.6.10.
9,9.6-10.10,
9.7.1.1, 9.7.1.2, 9.7.1.3, 9.7.1.4, 9.7.1.5, 9.7.1.6, 9.7.1.7, 9.7.1.8,
9.7.1.9, 9.7.1.1 0, 9.7.2.1, 9.7.2.2,
9.7.2.3, 9.7.2.4, 9.7.2.5, 9.7.2.6, 9.7.2.7, 9.7.2.8, 9.7.2.9, 9.7.2.10,
9.7.3.1, 9.7.3.2, 9.7.3.3, 9.7.3.4,
9.7.3.5, 9.7.3.6, 9.7.3.7, 9.7.3.8, 9.7.3.9, 9.7.3.10, 9.7.4.1, 9.7.4.2,
9.7.4.3, 9.7.4.4, 9.7.4.5, 9.7.4.6,
9.7.4.7, 9.7.4.8, 9.7.4.9, 9.7.4.10, 9.7.5.1, 9.7.5.2, 9.7.5.3, 9.7.5.4,
9.7.5.5, 9.7.5.6, 9.7.5.7, 9.7.5.8,
9.7.5.9, 9.7.5.1 0, 9.7.6.1, 9.7.6.2, 9.7.6.3, 9.7.6.4, 9.7.6.5, 9.7.6.6,
9.7.6.7, 9.7.6.8, 9.7.6.9, 9.7.6.10,
9.7.7.1, 9.7.7.2, 9.7.7.3, 9.7.7.4, 9.7.7.5, 9.7.7.6, 9.7.7.7, 9.7.7.8,
9.7;7.9, 9.7.7.10, 9.7.8.1, 9.7.8.2,
9.7.8.3, 9.7.8.4, 9.7.8.5, 9.7.8.6, 9.7.8.7, 9.7.8.8, 9.7.8.9, 9.7.8.10,
9.7.9.1, 9.7.9.2, 9.7.9.3, 9.7.9.4,
9.7.9.5,9.7.9.6,9.7.9.7,9.7.9.8,9.7.9.9,9.7.9.10,9.7.10.1,9.7.10.2,9.7.10.3,9.7
.10.4,9.7.10.5,
9.7.10.6,9.7.10.7,9.7.10.8,9.7.10.9,9.7.10.10,9.8.1.1,9.8.1.2,9.8.1.3,9.8.1.4,9
.8.1.5,9.8.1.6,
9.8.1.7, 9.8.1.8, 9.8.1.9, 9.8.1.10, 9.8.2.1, 9.8.2.2, 9.8.2.3, 9.8.2.4,
9.8.2.5, 9.8.2.6, 9.8.2.7, 9.8.2.8,
9.8.2.9, 9.8.2.1 0, 9.8.3.1, 9.8.3.2, 9.8.3.3, 9.8.3.4, 9.8.3.5, 9.8.3.6,
9.8.3.7, 9.8.3.8, 9.8.3.9, 9.8.3.10,
9.8.4.1, 9.8.4.2, 9.8.4.3, 9.8.4.4, 9.8.4.5, 9.8.4.6, 9.8.4.7, 9.8.4.8,
9.8.4.9, 9.8.4.10, 9.8.5.1, 9.8.5.2,
9.8.5.3, 9.8.5.4, 9.8.5.5, 9.8.5.6, 9.8.5.7, 9.8.5.8, 9.8.5.9, 9.8.5.10,
9.8.6.1, 9.8.6.2, 9.8.6.3, 9.8.6.4,
9.8.6.5, 9.8.6.6, 9.8.6.7, 9.8.6.8, 9.8.6.9, 9.8.6.10, 9.8.7.1, 9.8.7.2,
9.8.7.3, 9.8.7.4, 9.8.7.5, 9.8.7.6,
9.8.7.7, 9.8.7.8, 9.8.7.9, 9.8.7.10, 9.8.8.1, 9.8.8.2, 9.8.8.3, 9.8.8.4,
9.8.8.5, 9.8.8.6, 9.8.8.7, 9.8.8.8,
9.8.8.9, 9.8.8.1 0, 9.8.9.1, 9.8.9.2, 9.8.9.3, 9.8.9.4, 9.8.9.5, 9.8.9.6,
9.8.9.7, 9.8.9.8, 9.8.9.9, 9.8.9.10,
9.8.10.1,9.8.10.2,9.8.10.3,9.8.10.4,9.8.10.5,9.8.10.6,9.8.10.7,9.8.10.8,9.8.10.
9,9.8.10.10,
3 0 9.9.1.1, 9.9.1.2, 9.9.1.3, 9.9.1.4, 9.9.1.5, 9.9.1.6, 9.9.1.7, 9.9.1.8,
9.9.1.9, 9.9.1.10, 9.9.2.1, 9.9.2.2,
9.9.2.3, 9.9.2.4, 9.9.2.5, 9.9.2.6, 9.9.2.7, 9.9.2.8, 9.9.2.9, 9.9.2.10,
9.9.3.1, 9.9.3.2, 9.9.3.3, 9.9.3.4,
9.9.3.5, 9.9.3.6, 9.9.3.7, 9.9.3.8, 9.9.3.9, 9.9.3.10, 9.9.4.1, 9.9.4.2,
9.9.4.3, 9.9.4.4, 9.9.4.5, 9.9.4.6,
9.9.4.7, 9.9.4.8, 9.9.4.9, 9.9.4.10, 9.9.5.1, 9.9.5.2, 9.9.5.3, 9.9.5.4,
9.9.5.5, 9.9.5.6, 9.9.5.7, 9.9.5.8,
9.9.5.9, 9.9.5.1 0, 9.9.6.1, 9.9.6.2, 9.9.6.3, 9.9.6.4, 9.9.6.5, 9.9.6.6,
9.9.6.7, 9.9.6.8, 9.9.6.9, 9.9.6.10,
3 5 9.9.7.1, 9.9.7.2, 9.9.7.3, 9.9.7.4, 9.9.7.5, 9.9.7.6, 9.9.7.7, 9.9.7.8,
9.9.7.9, 9.9.7.10, 9.9.8.1, 9.9.8.2,
52
CA 02356539 2001-05-23
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9.9.8.3, 9.9.8.4, 9.9.8.5, 9.9.8.6, 9.9.8.7, 9.9.8.8, 9.9.8.9, 9.9.8.10,
9.9.9.1, 9.9.9.2, 9.9.9.3, 9.9.9.4,
9.9.9.5,9.9.9.6,9.9.9.7,9.9.9.8,9.9.9.9,9.9.9.10,9.9.10.1,9.9.10.2,9.9.10.3,9.9
:10.4,9.9.10.5,
9.9.10.6, 9.9.10.7, 9.9.10.8, 9.9.10.9, 9.9.10.10, 9.10.1.1, 9.10.1.2,
9.10.1.3, 9.10. I .4, 9.10.1.5,
9.10.1.6,9.10.1.7,9.10.1.8,9.10.1.9,9.10.1.10,9.10.2.1,9.10.2.2,9.10.2.3,9.10.2
.4,9.10.2.5,
9.10.2.6,9.10.2.7,9.10.2.8,9.10.2.9,9.10.2.10,9.10.3.1,9.10.3.2,9.10.3.3,9.10.3
.4,9.10.3.5,
9.10.3.6,9.10.3.7,9.10.3.8,9.10.3.9,9.10.3.10,9.10.4.1,9.10.4.2,9.10.4.3,9.10.4
.4,9.10.4.5,
9.10.4.6,9.10.4.7,9.10.4.8,9.10.4.9,9.10.4.10,9.10.5.1,9.10.5.2,9.10.5.3,9.10.5
.4,9.10.5.5,
9.10.5.6,9.10.5.7,9.10.5.8,9.10.5.9,9.10.5.10,9.10.6.1,9.10.6.2,9.10.6.3,9.10.6
.4,9.10.6.5,
9.10.6.6,9.10.6.7,9.10.6.8,9.10.6.9,9.10.6.10,9.10.7.1,9.10.7.2,9.10.7.3,9.10.7
.4,9.10.7.5,
9.10.7.6,9.10.7.7,9.10.7.8,9.10.7.9,9.10.7.10,9.10.8.1,9.10.8.2,9.10.8.3,9.10.8
.4,9.10.8.5,
9.10.8.6,9.10.8.7,9.10.8.8,9.10.8.9,9.10.8.10,9.10.9.1,9.10.9.2,9.10.9.3,9.10.9
.4,9.10.9.5,
9.10.9.6,9.10.9.7,9.10.9.8,9.10.9.9,9.10.9.10,9.10.10.1,9.10.10.2,9.10.10.3,9.1
0.10'4,
9.10.10.5, 9.10.10.6, 9.10.10.7, 9.10.10.8, 9.10.10.9, 9.10.10.10, 10.1.1.1,
10.1.1.2, 10.1.1.3,
I0.1.1.4,10.1.1.5,10.1.1.6,10.1.1.7,10.1.1.8,10.1.1.9,10.1.1.10,10.1.2.1,10.1.2
.2,10.1.2.3,
10.1.2.4, I 0. I .2.5, 10.1.2.6, 10.1.2.7, 10.1.2.8, I 0. i .2.9, 10.1.2.10,
10. I .3.1, 10.1.3.2, 10.1.3.3,
10.1.3.4, 10.1.3.5, 10.1.3.6, 10.1.3.7, 10.1.3.8, 10.1.3.9, 10.1.3.10,
10.1.4.1, 10.1.4.2, 10.1.4.3,
10.1.4.4, 10.1.4.5, 10.1.4.6, 10.1.4.7, 10.1.4.8, 10.1.4.9, 10.1.4.10,
10.1.5.1, 10.1.5.2, 10.1.5.3,
10.1.5.4, 10.1.5.5, 10.1.5.6, 10.1.5.7, 10.1.5.8, 10.1.5.9, 10.1.5.10,
10.1.6.1, 10.1.6.2, 10.1.6.3,
10.1.6.4, 10.1.6.5, 10.1.6.6, 10.1.6.7, 10.1.6.8, 10.1.6.9, 10.1.6.10,
10.1.7.1, 10.1.7.2, 10.1.7.3,
10.1.7.4, 10.1.7.5, 10.1.7.6, 10.1.7.7, 10.1.7.8, 10.1.7.9, 10.1.7.10,
10.1.8.1, 10.1.8.2, 10.1.8.3,
10.1.8.4, 10.1.8.5, 10.1.8.6, 10.1.8.7, 10.1.8.8, 10.1.8.9, 10.1.8.10,
10.1.9.1, 10.1.9.2, 10.1.9.3,
10.1.9.4, 10.1.9.5, 10.1.9.6, 10.1.9.7, 10.1.9.8, 10.1.9.9, 10.1.9.10,
10.1.10.1, 10.1.10.2, 10.1.10.3,
10.1.10.4, 10.1.10.5, 10.1.10.6, 10.1.10.7, 10.1.10.8, 10.1.10.9, 10.1.10.10,
10.2.1.1, 10.2.1.2,
10.2.1.3, 10.2.1.4, 10.2.1.5, 10.2.1.6, 10.2.1.7, 10.2.1.8, 10.2.1.9,
10.2.1.10, 10.2.2.1, 10.2.2.2,
10.2.2.3, 10.2.2.4, 10.2.2.5, 10.2.2.6, i 0.2.2.7, 10.2.2.8, 10.2.2.9,
10.2.2.10, 10.2.3.1, I 0.2.3.2,
10.2.3.3, 10.2.3.4, 10.2.3.5, 10.2.3.6, 10.2.3.7, 10.2.3.8, 10.2.3.9,
10.2.3.10, 10.2.4.1, 10.2.4.2,
10.2.4.3, 10.2.4.4, 10.2.4.5, 10.2.4.6, 10.2.4.7, 10.2.4.8, 10.2.4.9,
10.2.4.10, 10.2.5.1, 10.2.5.2,
10.2.5.3, 10.2.5.4, 10.2.5.5, 10.2.5.6, 10.2.5.7, 10.2.5.8, 10.2.5.9,
10.2.5.10, 10.2.6.1, I0.2.6.2,
10.2.6.3, 10.2.6.4, 10.2.6.5, 10.2.6.6, 10.2.6.7, 10.2.6.8, 10.2.6.9,
10.2.6.10, 10.2.7.1, 10.2.7.2,
10.2.7.3,10.2.7.4,10.2.7.5,10.2.7.6,10.2.7.7,1U.2_7.8.10.2.7.9,10.2.7.10,10.2.8
.1,10.2.8.2,
10.2.8.3, 10.2.8.4, 10.2.8.5, 10.2.8.6, 10.2.8.7, 10.2.8.8, 10.2.8.9,
10.2.8.10, 10.2.9.1, 10.2.9.2,
10.2.9.3, 10.2.9.4, 10.2.9.5, 10.2.9.6, 10.2.9.7, 10.2.9.8, 10.2.9.9,
10.2.9.10, 10.2.10.1, 10.2.10.2,
10.2.10.3, 10.2.10.4, 10.2.10.5, 10.2.10.6, 10.2.10.7, 10.2.10.8, 10.2.10.9,
10.2.10.10, 10.3.1.1,
10.3.1.2, 10.3.1.3, 10.3.1.4, 10.3.1.5, 10.3.1.6, 10.3.1.7, 10.3.1.8,
10.3.1.9, 10.3.1.10, 10.3.2.1,
10.3.2.2, 10.3.2.3, 10.3.2.4, 10.3.2.5, 10.3.2.6, 10.3.2.7, 1Ø3.2.8,
10.3.2.9, 10.3.2.10, 10.3.3.1,
53
CA 02356539 2001-05-23
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10.3.3.2,10.3.3.3,10.3.3.4,10.3.3.5,10.3.3.6,10.3.3.7,10.3.3.8,10.3.3.9,10.3.3.
10,10.3.4.1,
10.3.4.2, t 0.3.4.3, I 0.3.4.4, 10.3.4.5, I 0.3.4.6, 10.3.4.7, 10.3.4.8,
10.3.4.9, 10.3.4.10, 10.3.5.1,
'
10.3.5.2,10.3.5.3,10.3.5.4,10.3.5.5,10.3.5.6,10.3.5.7,10.3.5.8,10.3.5.9,10.3.5.
10,10.3.6.1,
10.3.6.2,10.3.6.3,10.3.6.4,10.3.6.5,10.3.6.6,10.3.6.7,10.3.6.8,10.3.6.9,10.3.6.
10,10.3.7.1,
10.3.7.2,10.3.7.3,10.3.7.4,10.3.7.5,10.3.7.6,10.3.7.7,10.3.7.8,10.3.7.9,10.3.7.
10,10.3.8.1,
_ 10.3.8.2, 10.3.8.3, 10.3.8.4, 10.3.8.5, 10.3.8.6, 10.3.8.7, 10.3.8.8,
10.3.8.9, 10.3.8.I0, 10.3.9.1,
10.3.9.2, 10.3.9.3, 10.3.9.4, 10.3.9.5, 10.3.9.6, 10.3.9.7, 10.3.9.8,
10.3.9.9, 10.3.9.10, 10.3.10.1,
10.3.10.2, 10.3.10.3, 10.3.10.4, 10.3.10.5, 10.3.10.6, 10.3.10.7, 10.3.10.8,
10.3.10.9, 10.3.10.10,
10.4.1.1, 10.4.1.2, 10.4.1.3, 10.4.1.4, 10.4.1.5, 10.4.1.6, 10.4.1.7,
10.4.1.8, 10.4.1.9, 10.4.1.10,
10.4.2.1, 10.4.2.2, 10.4.2.3, 10.4.2.4, 10.4.2.5, 10.4.2.6, 10.4.2.7,
10.4.2.8, 10.4.2.9, 10.4.2.10,
10.4.3.1,10.4.3.2,10.4.3.3,10.4.3.4,10.4.3.5,10.4.3.6,10.4.3.7,10.4.3.8,10.4.3.
9,10.4.3.10,
10.4.4.1, 10.4.4.2, 10.4.4.3, 10.4.4.4, 10.4.4.5, 10.4.4.6, 10.4.4.7,
10.4.4.8, 10.4.4.9, 10.44.10,
10.4.5.1,10.4.5.2,10.4.5.3,10.4.5.4,10.4.5.5,1U.4.5.6,10.4.5.7,10.4.5.8,10.4.5.
9,10.4.5.10,
10.4.6.1, 10.4.6.2, 10.4.6.3, 10.4.6.4, 10.4.6.5, 10.4.6.6, 10.4.6.7,
10.4.6.8, 10.4.6.9, 10.4.6.10,
10.4.7.1, 10.4.7.2, 10.4.7.3, 10.4.7.4, 10.4.7.5, 10.4.7.6, 10.4.7.7,
10.4.7.8, 10.4.7.9, 10.4.7.10,
10.4.8.1, 10.4.8.2, 10.4.8.3, 10.4.8.4, 10.4.8.5, 10.4.8.6, 10.4.8.7,
10.4.8.8, 10.4.8.9, 10.4.8.10,
10.4.9.1, 10.4.9.2, 10.4.9.3, 10.4.9.4, 10.4.9.5, 10.4.9.6, 10.4.9.7,
10.4.9.8, 10.4.9.9, 10.4.9.10,
10.4.10.1,I0.4.10.2,10.4.10.3,10.4.10.4,10.4.10.5,10.4.10.6,10.4.10.7,10.4.10.8
,10.4.10.9,
10.4.10.10,10.5.1.1,10.5.1.2,10.5.1.3,10.5.1.4,10.5.1.5,10.5.1.6,10.5.1.7,10.5.
1.8,10.5.1:9,
10.5.1.10,10.5.2.1,10.5.2.2,10.5.2.3,10.5.2.4,10.5.2.5,10.5.2.6,10.5.2.7,10.5.2
.8,10.5.2.9,
10.5.2.10,10.5.3.1,10.5.3.2,10.5.3.3,10.5.3.4,10.5.3.5,10.5.3.6,10.5.3.7,10.5.3
.8,10.5.3.9,
10.5.3.10, 10.5.4.1, 10.5.4.2, 10.5.4.3, 10.5.4.4, 10.5.4.5, 10.5.4.6,
10.5.4.7, 10.5.4.8, 10.5.4.9,
10.5.4.10, 10.5.5.1, 10.5.5.2, 10.5.5.3, 10.5.5.4, 10.5.5.5, 10.5.5.6,
10.5.5.7, 10.5.5.8, 10.5.5.9,
10.5.5.10, 10.5.6.1, 10.5.6.2, 10.5.6.3, 10.5.6.4, 10.5.6.5, 10.5.6.6,
10.5.6.7, 10.5.6.8, 10.5.6.9,
10.5.6.10, 10.5.7.1, 10.5.7.2, 10.5.7.3, 10.5.7.4, 10.5.7.5, 10.5.7.6,
10.5.7.7, 10.5.7.8, 10.5.7.9,
10.5.7.10, 10.5.8.1, 10.5.8.2, 10.5.8.3, 10.5.8.4, 10.5.8.5, 10.5.8.6,
10.5.8.7, 10.5.8.8, 10.5.8.9,
10.5.8.10, 10.5.9.1, 10.5.9.2, 10.5.9.3, 10.5.9.4, 10.5.9.5, 10.5.9.6,
10.5.9.7, 10.5.9.8, 10.5.9.9,
10.5.9.10,10.5.10.1,10.5.10.2,10.5.I0.3,10.5.10.4,10.5.10.5,10.5.10.6,10.5.10.7
,10.5.10.8,
10.5.10.9, 10.5.10.10, 10.6.1.1, 10.6.1.2, 10.6.1.3, 10.6.1.4, 10.6.1.5,
10.6.1.6, 10.6.1.7, 10.6.1.8,
10.6.1.9,10.6.1.10,10.6.2.1,10.6.2.2,10.6.2.3,10.6.2.4,10.6.2.5,10.6.2.6,10.6.2
.7,10.6.2.8,
10.6.2.9, 10.6.2.10, 10.6.3.1, 10.6.3.2, I 0.6.3.3, 10.6.3.4, 10.6.3.5,
10.6.3.6, 10.6.3.7, 10.6.3.8,
10.6.3.9, 10.6.3.10, 10.6.4.1, 10.6.4.2, 10.6.4.3, 10.6.4.4, 10.6.4.5,
10.6.4.6, 10.6.4.7, 10.6.4.8,
10.6.4.9, 10.6.4.10, 10.6.5.1, 10.6.5.2, 10.6.5.3, 10.6.5.4, 10.6.5.5,
10.6.5.6, 10.6.5.7, 10.6.5.8,
10.6.5.9, 10.6.5.10, 10.6.6.1, 10.6.6.2, 10.6.6.3, 10.6.6.4, 10.6.6.5,
10.6.6.6, 10.6.6.7, 10.6.6.8,
10.6.6.9, 10.6.6.10, 10.6.7.1, 10.6.7.2, 10.6.7.3, 10.6.7.4, 10.6.7.5,
10.6.7.6, 10.6.7.7, 10.6.7.8,
54
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I 0.6.7.9, 10.6.7.10, 10.6.8.1, 10.6.8.2, 10.6.8.3, l 0.6.8.4, 10.6.8.5,
10.6.8.6, 10.6.8.7, 10.6.8.8,
10.6.8.9, 10.6.8.10, 10.6.9.1, 10.6.9.2, 10.6.9.3, 10.6.9.4, 10.6.9.5,
10.6.9.6, 10.6.9.7, 10.6.9.8,
10.6.9.9,10.6.9.10,10.6.10.1,10.6.10.2,10.6.10.3,10.6.10.4,10.6.10.5,10.6.10.6,
10.6.10.7,
10.6.10.8, 10.6.10.9, 10.6.10.10, 10.7.1.1, 10.7.1.2, 10.7.1.3, 10.7.1.4,
10.7.1.5, 10.7.1.6, 10.7.1.7,
10.7.1.8,10.7.1.9,10.7.1.10,10.7.2.1,10.7.2.2,10.7.2.3,10.7.2.4,10.7.2.5,10.7.2
.6,10.7.2.7,
10.7.2.8, 10.7.2.9, 10.7.2.I0, 10.7.3.1, 10.7.3.2, 10.7.3.3, 10.7.3.4,
10.7.3.5, 10.7.3.6, 10.7.3.7,
10.7.3.8, 10.7.3.9, 10.7.3.10, 10.7.4.1, 10.7.4.2, 10.7.4.3, 10.7.4.4,
10.7.4.5, 10.7.4.6, 10.7.4.7,
10.7.4.8, 10.7.4.9, 10.7.4.10, 10.7.5.1, 10.7.5.2, 10.7.5.3, 10.7.5.4,
10.7.5.5, 10.7.5.6, 10.7.5.7,
10.7.5.8, 10.7.5.9, 10.7.5.10, 10.7.6.1, 10.7.6.2, 10.7.6.3, 10.7.6.4,
10.7.6.5, 10.7.6.6, 10.7.6.7,
10.7.6.8, 10.7.6.9, 10.7.6.10, 10.7.7.1, 10.7.7.2, 10.7.7.3, 10.7.7.4,
10.7.7.5, 10.7.7.6, 10.7.7.7,
10.7.7.8, 10.7.7.9, 10.7.7.10, 10.7.8.1, 10.7.8.2, 10.7.8.3, 10.7.8.4,
10.7.8.5, 10.7.8.6, 10.7.8.7,
10.7.8.8, 10.7.8.9, 10.7.8.10, 10.7.9.1, 10.7.9.2, 10.7.9.3, 10.7.9.4,
10.7.9.5, 10.7.9.6, 107.9.7,
10.7.9.8, 10.7.9.9, 10.7.9.10, 10.7.10.1, 10.7.10.2, 10.7.10.3, 10.7.10.4,
10.7.10.5, 10.7.10.6,
10.7.10.7, 10.7.10.8, 10.7.10.9, 10.7.10.10, 10.8.1.1, 10.8.1.2, 10.8.1.3,
10.8.1.4, 10.8.1.5, 10.8.1.6,
I S 10.8.1.7, 10.8.1.8, 10.8.1.9, 10.8.1.10, 10.8.2.1, 10.8.2.2, I 0.8.2.3,
10.8.2.4, 10.8.2.5, 10.8.2.6,
10.8.2.7, 10.8.2.8, 10.8.2.9, 10.8.2.10, 10.8.3.1, 10.8.3.2, 10.8.3.3,
10.8.3.4, 10.8.3.5, 10.8.3.6,
10.8.3.7, 10.8.3.8, 10.8.3.9, 10.8.3.10, 10.8.4.1, 10.8.4.2, 10.8.4.3,
10.8.4.4, 10.8.4.5, 10.8.4.6,
10.8.4.7, I 0.8.4.8, 10.8.4.9, 10.8.4.10, 10.8.5.1, 10.8.5.2, 10.8.5.3,
10.8.5.4, 10.8.5.5, 10.8.5.6,
10.8.5.7, 10.8.5.8, 10.8.5.9, 10.8.5.10, 10.8.6.1, 10.8.6.2, 10.8.6.3,
10.8.6.4, 10.8.6.5, 10.8.6.6,
10.8.6.7,I0.8.6.8,10.8.6.9,10.8.6.10,10.8.7.1,10.8.7.2,10.8.7.3,10.8.7.4,10.8.7
.5,10.8.7.6,
10.8.7.7,10.8.7.8,10.8.7.9,10.8.7.10,10.8.8.1,10.8.8.2,10.8.8.3,10.8.8.4,10.8.8
.5,10.8.8.6,
10.8.8.7, 10.8.8.8, 10.8.8.9, 10.8.8.10, 10.8.9.1, 10.8.9.2, 10.8.9.3,
10.8.9.4, 10.8.9.5, 10.8.9.6,
10.8.9.7, 10.8.9.8, 10.8.9.9, 10.8.9.10, 10.8.10. I , 10.8.10.2, 10.8.10.3,
10.8.10.4, 10.8.10.5,
10.8.10.6, 10.8.10.7, 10.8.10.8, 10.8.10.9, 10.8.10.10, 10.9.1.1, 10.9.1.2,
10.9.1.3, 10.9.1.4,
10.9.1.5, 10.9.1.6, 10.9.1.7, 10.9.1.8, 10.9.1.9, 10.9.1.10, 10.9.2.1,
10.9.2.2, 10.9.2.3, 10.9.2.4,
10.9.2.5, 10.9.2.6, 10.9.2.7, 10.9.2.8, 10.9.2.9, 10.9.2.10, 10.9.3 .1,
10.9.3.2, 10.9.3.3, 10.9.3.4,
10.9.3.5,10.9.3.6,10.9.3.7,10.9.3.8,10.9.3.9,10.9.3.10,10.9.4.1,10.9.4.2,10.9.4
.3,10.9.4.4,
10.9.4.5,10.9.4.6,10.9.4.7,10.9.4.8,10.9.4.9,10.9.4.10,10.9.5.1,10.9.5.2,10.9.5
.3,10.9.5.4,
10.9.5.5, 10.9.5.6, 10.9.5.7, 10.9.5.8, 10.9.5.9, 10.9.5.10, 10.9.6.1,
10.9.6.2, 10.9.6.3, 10.9.6.4,
10.9.6.5, 10.9.6.6, 10.9.6.7, 10.9.6.8, 10.9.6.9, 10.9.6.10, 10.9.7.1,
10.9.7.2, 10.9.7.3, 10.9.7.4,
10.9.7.5, 10.9.7.6, 10.9.7.7, 10.9.7.8, 10.9.7.9, 10.9.7.10, 10.9.8.1,
10.9.8.2, 10.9.8.3, 10.9.8.4,
10.9.8.5, 10.9.8.6, I 0.9.8.7, 10.9.8.8, 10.9.8.9, 10.9.8.10, I 0.9.9.1,
10.9.9.2, i 0.9.9.3, 10.9.9.4,
10.9.9.5, 10.9.9.6, 10.9.9.7, 10.9.9.8, 10.9.9.9, 10.9.9.10, 10.9.10.1,
10.9.10.2, 10.9.10.3, 10.9.10.4,
10.9.10.5, 10.9.10.6, 10.9.10.7, 10.9.10.8, 10.9.10.9, 10.9.10.10, 10.10.1.1,
10.10.1.2, 10.10.1.3,
10.10.1.4, 10.10.1.5, 10.10.1.6, 10.10.1.7, 10.10.1.8, 10.10.1.9, 10.10.1.10,
10.10.2.1, 10.10.2.2,
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10.10.2.3, 10.10.2.4, 10.10.2.5, 10.10.2.6, 10.10.2.7, 10.10.2.8, 10.10.2.9,
10.10.2.10, 10.10.3.1,
10.10.3.2, 10.10.3.3, 10.10.3.4, 10.10.3.5, 10.10.3.6, 10.10.3.7, 10.10.3.8,
10.10.3.9, 10.10.3.10,
'
10.10.4.1,10.10.4.2,10.10.4.3,10.10.4.4,10.10.4.5,10.10.4.6,10.10.4.7,10.10.4.8
,10.10.4.9,
10. I 0.4.10, 10.10.5.1, 10. I 0.5.2, 10.10.5.3, 10. I 0.5.4, 10.10.5.5,
10.10.5.6, 10.10.5.7, 10.10.5.8,
10.10.5.9, 10.10.5.10, 10.10.6.1, 10.10.6.2, 10.10.6.3, 10.10.6.4, 10.10.6.5,
10.10.6.6, 10.10.6.7,
' . 10.10.6.8, 10.10.6.9, 10.10.6.10, 10.10.7.1, 10.10.7.2, 10.10.7.3,
10.10.7.4, 10.10.7.5, 10.10.7.6,
10.10.7.7, 10.10.7.8, 10.10.7.9, 10.10.7.10, 10.10.8.1, 10.10.8.2, 10.10.8.3,
10.10.8.4, 10.10.8.5,
10.10.8.6, 10.10.8.7, 10.10.8.8, 10.10.8.9, 10.10.8.10, 10.10.9.1, 10.10.9.2,
10.10.9.3, 10.10.9.4,
10.10.9.5, 10.10.9.6, 10.10.9.7, 10.10.9.8, 10.10.9.9, 10.10.9.10, 10.10.10.1,
10.10.10.2,
10.10.10.3, 10.10.10.4 10.10.10.5 10.10.10.6 10.10.10.7 10.10.10.8 10.10.10.9
10.10.10.10
Additional exemplary formula 1 compound groups include the following groups as
disclosed below.
Group 2. Group 2 compounds are as named in Table B, i.e., R2, R1A, Y and X
substituents are as defined in Table A, but they are bonded to the steroid
nucleus shown in formula
5, which is the same as the formula 4 steroid nuclcus, except that the 5-6
double bond is absent and
hydrogen is present at the 5-position in the a-configuration
R2
Thus, the group 2 compound named 1.2.1.1 has the structure
r
' ' group 2, compound 1.2.1.1.
Group 3. Group 3 compounds are as named in Table 8, i.e., R2, R1A, Y and X
substitu;,nts are as defined in Table A, but they are bonded to the steroid
nucleus shown in formula
56
" 5.
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6, which is the same as the formula 4 steroid nucleus, except that the S-6
double bond is absent and
hydrogen is present at the 5-position in the (3-configuration
R2
" 6.
Thus, the group 3 compound named 1.2.1.1 has the structure
' ' group 3, compound 1.2.1. I .
Group 4. Group 4 compounds are as named in Table B, i.e., R2, R1A, Y and X
substituents are as defined in Table A, but they are bonded to the steroid
nucleus shown in formula
7, which is the same as the formula 4 steroid nucleus, except that Q3 is -
CH20H
R2
7.
Thus, the group 4 compound named 1.2.1.1 has the structure
H
group 4, compound 1.2.1.1.
57
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Group 5. Group 2 compounds are as named in Table B, i.e., R2, R1A, Y and X
substituents are as defined in Table A, but they are bonded to the steroid
nucleus Shown in formula
8, which is the same as the formula 4 steroid nucleus, except that the 5-6
double bond is absent and
hydrogen is present at the 5-position in the a-configuration and Q3 is -CH20H
Ds X H
CH20H ~ H
16 ._
R2 ~ s ~ RBA
Hue: _ ..iilH
H
8.
Thus, the group S compound named 1.2.1.1 has the structure
r~uO
Group 6. Group 6 compounds are as named in groups 1-5, except that Q6 in
formulas 4-8
is -CH20H instead of methyl. In group 6, there are 5 subgroups of group 6
compounds. The first
subgroup, subgroup 6-1, has the same steroid nucleus with the substituents as
defined for group I
compounds while the second, subgroup 6-2, has the same steroid nucleus with
the substituents as
defined for group 2 compounds. Subgroups 6-3 through 6-5 have the same steroid
nucleus with the
substituents as defined for group 3 through 5 respectively. Thus, for example,
the subgroup 6-1
compound named 1.2.1.1 has the structure
and the subgroup 6-2 compound named I .2.1.1 has the structure
58
_ CA 02356539 2001-05-23
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N
Group 7. Group 7 compounds are as named in groups 1-5, except that the Y
moiety in
formulas 4-8 is in the /3-configuration instead of in the a-configuration.
Group 7 comprises 5
subgroups, wherein the compounds are named essentially as described for group
6 compounds,
except that the Y group is in the (3-configuration.
Group 8. Group 8 compounds are as named in groups 1-5, except that the X
moiety in
formulas 4-8 is in the a-configuration instead of in the ~3-configuration.
Group 8 comprises 5
subgroups, wherein the compounds are named essentially as described for group
6 compounds,
except that the X group is in the a-configuration.
Group 9. Group 9 compounds are as named in groups 1-5, except that the R2
moiety in
formulas 4-8 is in the a,-configuration instead of in the (3-configuration.
Group 9 comprises S
subgroups, wherein the compounds are named essentially as described for group
6 compounds,
except that the R2 group is in the a.-configuration.
Group 10. Group 10 compounds are as named in groups 1-9, except that R2
moieties I
through 10 in Table A are replaced with the following moieties.
1 -S-C(O)-CH3
2 -S-C(O)-CH2-C6H5
3 -O-S(O)-O-CH3
4 -O-S(O)-O-CH2-C6H5
5 -O-S(O)(O)-O-CH3
6 -O-S(O)(O)-O-CH2-C6H5
7 -O-C(O)-NH-CH3
8 -O-C(O)-NH-C6H5
9 -O-C(S)-CH3
10 -O-C(S)-CH2-C6H5
Group 10 comprises 25 subgroups of compounds. The first, subgroup 10-1, has
the same
steroid nucleus with substituents as defined for group 1, except that the R2
moieties or groups
listed replace those in Table A above. The subgroup 10-1 compound named
1.2.1.1 has the
structure
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H3C S
O
the subgroup 10-2 compound named 1.2.1.1 has the structure
H3C" S
the subgroup 10-6-1 compound named 1.2.1.1 has the structure
H3C
O
and the subgroup 10-6-2 compound named 1.2.1.1 has the structure
-I
r
H3C" S
'COI
Group 11. Group i 1 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
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1 -S-C(O)-CH2CH2-O-CH2CH3
2 -S-C(O)-CH2-C6H40C113
3 -O-S(O)-O-CH2CH2-O-CH2CH3
4 -O-S(O)-O-CH2-C6H40CH3
5 -O-S(O)(O}-O-CH2CH2-O-CH2CH3
6 -O-S(O)(O)-O-CH2-C6H40CH3
7 -O-C(O)-NH-CH2CH2-O-CH2CH3
8 -O-C(O)-NH-C6H40CH3
9 -O-C(S)-CH2CH2-O-CH2CH3
10 -O-C(S)-CH2-C6H40CH3
Group 12. Group 12 compounds are as named in groups 1-9, except that R2
moieties I
through 10 in Table A are replaced with the following moieties.
1 -S-C(O)-CH2CH2-O-CH2C(O)OH
2 -S-C(O)-CH2-C6H4F
3 -O-S(O)-O-CH2CH2-O-CH2C(O)OH
4 -O-S(O)-O-CH2-C6H4F
5 -O-S(O)(O)-O-CH2CH2-O-CH2C(O)OH
6 -O-S(O)(O)-O-CH2-C6H4F
7 -O-C(O)-NH-CH2CH2-O-CH2C(O)OH
8 -O-C(O)-NH-C6H4F
9 -O-C(S)-CH2CH2-O-CH2C(O)OH
10 -O-C(S)-CH2-C6H4F
Group 13. Group 13 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
1 -S-C(O)-CH2CH2-O-CH2CH20H
2 -S-C(O)-CH2-C6H4CH3
3 -O-S(O)-O-CH2CH2-O-CH2CH20H
4 -O-S(O)-O-CH2-C6H4CH3
5 -O-S(O)(O)-O-CH2CH2-O-CH2CH20H
6 -O-S(O)(O)-O-CH2-C6H4CH3
7 -O-C(O)-NH-CH2CH2-O-CH2CH20H
8 -O-C(O)-NH-C6H4CH3
9 -O-C(S)-CH~CH2-O-CH2CH20H
10 -O-C(S)-CH2-C6H4CH3
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Group 14. Group 14 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
1 -S-C(O)-CH2CH2-O-CH2CH20RPR
2 -S-C(O)-CH2-C6H40RPR
S 3 -O-S(O)-O-CH2CH2-O-CH2CH20RPR
4 -O-S(O)-O-CH2-C6H40RPR
-O-S(O)(O)-O-CH2CH2-O-CH2CH20RPR
6 -O-S(O)(O)-O-CH2-C6H40RPR
7 -O-C(O)-NH-CH2CH2-O-CH2CH20RpR
8 -O-C(O)-NH-C6H40RPR
9 -O-C(S)-CH2CH2-O-CH2CH20RPR
10 -O-C(S)-CH2-C6H40RPR
Group 15. Group I S compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
1 -S-C(O)-CH2CH2-O-CH2CH2NHRPR
2 -S-C(O)-CH2-C6H3(ORPR)2
3 -O-S(O)-O-CH2CH2-O-CH2CH2NHRPR
4 -O-S(O)-O-CH2-C6H3(ORPR)2
5 -O-S(O)(O)-O-CH2CH2-O-CH2CH2NHRPR
6 -O-S(O)(O)-O-CH2-C6H3(ORPR)2
7 -O-C(O)-NH-CH2CH2-O-CH2CH2NHRPR
8 -O-C(O)-NH-C6H3(ORPR)2
9 -O-C(S)-CH2CH2-O-CH2CH2NHRPR
10 -O-C(S)-CH2-C6H3(ORpR)2
Group 16. Group 16 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
I -S-C(O)-(CH2)0-6-CH3
2 -S-C(O)-CH2-C6H5
3 -O-S(O)-O-(CH2)0-6-CH3
4 -O-S(O)-O-CH2-C6H5
5 -O-S(O)(O)-O-(CH2)0-6-CH3
6 -O-S(O)(O)-O-CH2-C6H5
7 -O-C(O)-NH-(CH2)0-6-CH3
8 -O-C(O)-NH-(CH2)0-6-C6H5
9 -O-C(S)-(CH2)0-6-CH3
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-O-C(S)-CH2-C6H5
Group 17. Group 17 compounds are as named in groups 1-9, except that R2
moieties I
through 10 in Table A are replaced with the following moieties.
I -S-C(O)-CH2CH2-(O-CH2CH2)1-50-H
5 2 -S-C(O)-CH2-C6H40CH3
3 -O-S(O)-O-CH2CH2-(O-CH2CH2)I-50-H
4 -O-S(O)-O-CH2-C6H40CH3
5 -O-S(O)(O)-O-CHZCH2-(O-CH2CH2)1-50-H
6 -O-S(O)(O)-O-CH2-C6H40CH3
10 7 -O-C(O)-NH-CH2CH2-(O-CH2CH2)I-50-H
8 -O-C(O)-NH-C6H40CH3
9 -O-C(S)-CH2CH2-(O-CH2CH2)I-50-H -
10 -O-C(S)-CH2-C6H40CH3
Group 18. Group 18 compounds are as named in groups 1-9, except that R2
moieties 1
IS through 10 in Table A are replaced with the following moieties.
1 -O-C(O)-CH2CH2-(O-CH2CH2)1-50-H
2 -O-C(O)-CH2-C6H40CH3
3 -O-C(O)-(CH2)0-6-CH3
4 -O-C(O)-CH2-C6H4N02
S -O-C(O)-CH2CH2-(O-CH2CH2)1-SO-H
6 -O-C(O)-CH2-C6H5
7 -O-C(O)-CH2CH2-O-CH2CH3
8 -O-C(O)-C6H5
9 -O-C(O)-CH2CH2-S-CH2CH3
10 -O-C(O)-CH2-C6H4F
Group 19. Group 19 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
I -O-CH2CH2-(O-CH2CH2)1-50-H
2 -O-CH2-C6H40CH3
3 -O-(CH2)0-6-CH3 _
4 -O-CH2-C6H4N02
5 -O-CH2CH2-(O-CH2CH2)1-50-H
6 -O-CH2-C6H5
7 -O-CH2CH2-O-CH2CH3
8 -O-C6H5
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9 -O-CH2CH2-S-CH2CH3
-O-CH2-C6H4F
Group 20. Group 20 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
5 1 -C(O)-O-CH2CH2-(O-CH2CH2)1-50-H
' 2 -C(O)-O-CH2-C6H40CH3
3 -C(O)-O-(CH2)0-6-CH3
4 -C(O)-O-CH2-C6H4N02
5 -C(O)-O-CH2CH2-(O-CH2CH2)1-50-H
10 6 -C(O)-O-CH2-C6H5
7 -C(O)-O-CH2CH2-O-CH2CH3
8 -C(O)-~-C6H5
9 -C(O)-O-CH2CH2-S-CH2CH3
10 -C(O)-O-CH2-C6H4F
l5 Group 21. Group 21 compounds are as named in groups 1-9, except that R2
moieties 1
through 10 in Table A are replaced with the following moieties.
1 -C(O)-O-G12 (G12 is defined below)
2 -O-C(O)-G 12
3 -C(O)-S-G 12
4 -S-C(O)-G12
5 -C(S)-O-G 12
6 -O-C(S)-G 12
7 -O-C(O)-NH-G 12
8 -NH-C(O)-O-G 12
9 -C(O)-O-CH2-G 12
10 -O-C(O)-CH2-G12
Thus, the group 21-1 compound named 1.2.1.1 has the structure
G12 ~
O
while the group 21-2 compound named 1.2.1.1 has the structure
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r
G12
H
the group 21-3 compound named 1.2.1.1 has the structure
G12 '
H
the group 21-4 compound named 1.2.1.1 has the structure
G12
S
the group 21-6-1 compound named 1.2.1.1 has the structure
G12
the group 21-6-2 compound named 1.2.1.1 has the structure
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G12
H
i
f
and the group 21-6-3 compound named 1.2.1.1 has the structure
CH~OH
i
r
G12~
O
G12 in Group 21 is an organic moiety comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 10,
11 or 12
carbon atoms and 0, 1, 2, 3, 4, 5, 6, 7 or 8 independently selected O, S, N,
P, or Si atoms, but, if a
Si or P atom is present, only one Si or P is present, wherein the organic
moiety is optionally
selected from C 1 _ 12 alkyi, C2_ 12 alkenyl, C2_ 12 alkynyl, aryl, a C2_9
heterocycle or a substituted
derivative of any of these comprising 1, 2, 3, 4 or more substituents, wherein
each substituent is
independently chosen and is selected from -O-, -S-, -NRPR- (including -NH-), -
C(O)-, =O, =S, -
N(RPR)2 (including -NH2), -C(O)ORPR (including -C(O)OH), -OC(O)RPR (including -
O-C(O)-
H), -ORPR (including -OH), -SRPR (including -SH), -N02, -CN, -NHC(O)-, -C(O)NH-
, -OC(O)-,
-C(O)O-, -O-A8, -S-A8, -C(O)-A8, -OC(O)-A8, -C(O)O-A8, =N-, -N=, =N-OH, -
OP03(RPR)2,
OS03H2 and halogen moieties or atoms, where each RPR is -H, an independently
selected
protecting group or both RPR together comprise a protecting group, and A8 is
C1_g alkyl, C2_g
alkenyl, C2_g alkynyl, C1_4 alkyl-aryl (e.g., benzyl), aryl (e.g. phenyl) or
C1_4 alkyl-C2_9
heterocycle. G12 moieties include -CH3, -C2H5, -C3H~, -C4Hg, -C6H13, -
CH2_C6H5, -C2H4_
C6H5, -C3 H6-C6H5, -C6H5, -CH2-heterocycle, -CH2-CH2-heterocycle and a
heterocycle, and of
which are substituted with one, two, three or more independently selected -O-,
-S-, -F, -Cl, -Br, -I,
-NH-, =O, -CN, -OCH3, -OC2H5, -OC4H9, -N02, -NH2, -COOH, or -NH-C(O)-
moieties.
Other embodiments include the use of any formula 4 compound or genus of
formula 4
compounds that are named in any of the foregoing groups for any of the
therapeutic or other
applications described herein. This includes the use of any named formula 4
compound or genus
for any of those applications wherein (i) R2 is in the a-configuration, (ii)
Q4 is -CH(halogen)-, (iii)
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X is in the a-configuration and the -H at the 17-position is in the ~3-
configuration, (iv) Y is in the
~i-configuration and the -H at the 16-position is in the a-configuration or
(v) R1A is in the oc-
configuration and the -H at the 7-position is in the ~3-configuration.
Embodiments also include formula 1 compounds (e.g., formula 4 compounds)
wherein R4
is optionally substituted CI_g alkyl, optionally substituted C2_g alkenyl,
optionally substituted C2_
- . g alkynyl, optionally substituted aryl, optionally substituted
heterocycle, optionally substituted CI_
g alkyl-aryl, optionally substituted C1_g alkyl-heterocycle or optionally
substituted -CH2-CI_g
organic moiety (where the organic moiety is as described for esters), wherein
any of the foregoing
are independently substitued with 1, 2, 3, 4, 5 or 6 or more -O-, -S-, -NH-, -
NH-C(O)- (i.e., -NH-
C(O)- or -C(O)-NH-), =O, =NOH, -N02, -CN, -F, -Cl, -Br, -I, -OH, -SH, or -NH2.
Such R4
moieties include -CH2-C1_6 optionally substituted alkyl, -CH2-C2_6 optionally
substituted
alkenyl, -CH2-C 1 _6 -optionally substituted aryl and -CH2-C2_9 optionally
substituted~heterocycle.
Plasma concentration-enhancine compounds. An aspect of the invention comprises
administering an effective amount of a plasma concentration-enhancing
compound, e.g., a
compound of formula 2A or 2B compound with a formula I compound to facilitate
preventing or
treating one or more Trypanosome infections in a subject. In addition to the
formula 2A or 2B
compounds, the plasma concentration-enhancing compounds include bavachinin A,
didymin
(isosakuranetin-7-rutinoside or neoponcirin), flavanomarein (isookanine-7-
glucoside), flavanone
azine, flavanone diacetylhydrazone, flavanone hydrazone, silybin, which has
the structure
OH
O i H3
HO O \ O
OH / OH
OH O
hiral
H
H
isosilybin, which has the structure
67
silychristin, which has the structure
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OH O
__
HO~ ~ ~O~ ~ ~ i Ha
O
_ O ~ ~
OH
and a compound having the structure (E)
~3
CH3
(E).
Collectively, these compounds and the formula 2A and 2B compounds are referred
to as the
"plasma concentration-enhancing compounds".
The formula 2A and 2B compounds encompass a number of natural and synthetic
flavonoids, including certain flavones, flavans, and their iso analogs. The
presence of a formula
2A or 2B compound in compositions comprising a formula 1 compound has been
found to enhance
the systemic bioavailability of formulations that comprise a formula 1
compound. The presence of
a formula 2A or 2B compound, e.g., naringin or narineenin, results in enhanced
plasma
concentrations of the formula 1 compound. The formula 2A or 2B compound need
not be present
in a formulation that contains a formula 1 compound. The formula 2A or 2B can
also be
administered, e.g., about 1-4 hours, before or after, preferably before, the
formula 1 compound is
68
silandrin, which has the structure
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administered. In these embodiments, one will administer an oral or parenteral
formulation that
contains a formula 1 compound and a formula 2A or 2B compound.
The plasma concentration-enhancing compounds include compounds of formulas 50-
65
R8 B
Roc Rio
~8
Re R8
..n ,. ..
50 51
R8
R8 Re / Ra
R8 Re R8 I
Roc Rio
w
~a I ~ R
a
R8
Re Re / ReA
R8 R~~ R»
52 53
Ra Ra
Roc
~a
8
Re
Rs ~ . .o . _, ,
54 55
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Ra Ra
Rya Roc
~s ~a
_ . Re Re
,. ..~ ...."
56 57
Roc Ra Rio
Re ~a Ra
58 59
Ra Ra Ra Ra Ra R Ra Ra
Ra Ra 8 Ra Ra
Rio ~ / Ra Rio .\ / Ra
/ ~ /
Ra ~ ~ ~ ~Ro Ra ~ ~ ~ ~a_
O Ra Ra R» R~1 Ra
60 61
Ra
Rya Rio
Re Ra
Ra Ra
Ra Ra
62 63
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Rp Q_ Re Ra Ra Ra~
Rta Rio ~ Ra
Ra
R8A
Ra / Ra
Ra Ra ~ ~.
R~ t Rm I
_ 8
Ra Re ~ Ra
Ra
64 65
wherein
Rg at the 6-position independently are -H, -OH, -F, -C1, -Br, -I, C 1_6 alkyl,
C 1_6 aIkoxy,
giucuronide, a C 1 _25 fatty acid, glucoside, -CH2CH=C(CH3)2 or a group having
the structure (B);
Rg at the 8-position independently are -I-I, -OH, -F, -C1, -Br, -T, C 1 _6
alkyl, C I _~ alkoxy,
glucuronide, a C 1 _25 fatty acid, glucoside, -CH2CH=C(CH3 )2 or the residue
of a formula 50-65
compound where a hydrogen atom is removed to form the formula 50-65 radical;
R8A independently are -H, -OH, -F, -Cl, -Br, -I, C I _6 alkyl, C I _6 alkoxy,
glucuronide, a
C1-25 fatty acid, glucoside, -CH2CH=C(CH3)2 or a group having the structure
(C);
the remaining Rg independently are -H, -OH, -F, -C1, -Br, -I, C I _6 alkyl, C
1 _6 alkoxy,
glucuronide, a C I _25 fatty acid or -CH2-CH=C(CH3)2; and
RI 0 (i) is -OH or -F, -C1, -Br, -I, C 1 _6 alkyl, C 1 _6 alkoxy,
neohesperidoside,
apioglucoside, rutinoside, glucoside, galactoside, rhamnoside, arabinoside, or
a stereoisomer,
hydrate, analog, derivative or metabolite of any of these moieties, any of
which are optionally
independently substituted at one or more hydrogen atoms with -OH, -F, -C1, -
Br, -I, CI_6 alkyl,
C1_6 alkoxy, glucuronide or a C1_25 fatty acid, or (ii) Rlp is the radical of
bavachinin A, didymin,
flavanomarein, flavanone azine, flavanone diacetylhydrazone, flavanone
hydrazone, silybin,
silychristin, isosilybin, silandrin, a moiety of structure (E) or a
stereoisomer, hydrate, analog,
derivative or metabolite of any of these moieties.
Additional therapeutic embodiments. In accordance with another preferred
aspect of the
present invention, there is provided a method of treatment of one or more of
the conditions
described above, e.g., a Trypanosome infection, comprising administering a
combination therapy
including one or more of the compounds of the present invention administered
simultaneously or
sequentially with one or more macrophage stimulating factor (and optionally
further
co-administering one or more plasma concentration-enhancing compounds).
Macrophage
stimulating factors are well known to those of skill in the art, examples
including GM-CSF (see,
e.g., Callard et al., The Cytokine Facts Book, Academic Press, 1994, p. 139,
which is incorporated
herein) and Interleukin-4 (sold by Immunex as "Leukine': and by Schering
Plough as "Prokine").
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While not wishing to be bound by any theory, it is believed that compounds
that inhibit
glucose-6-phosphate dehydrogenase are surprisingly effective against malaria.
Accordingly, the
present invention also relates to the administration of a glucose-6-phosphate
dehydrogenase
inhibitor in the treatment of any of the conditions described herein,
particularly in the treatment of
malaria, optionally in a combination therapy with any other compounds of the
present invention or
any of the compounds described herein as being suitable for use in a
combination therapy. Those
of skill in the art are readily familiar with inhibitors of glucose-6-
phosphate dehydrogenase, and
can readily identify other material, which exhibits such inhibition.
In another preferred aspect of the present invention, to enhance destruction
of parasite
infected erythrocytes, the compounds of the present invention can be
coadministered with one or
more oxidation agent (optionally further together with a plasma concentration-
enhancing
compound and/or a macrophage stimulating factor), or the patient may be given
oxygectventilation
to increase oxidative steroids in the plasma.
The components of any of the combination therapies disclosed herein can be
administered
simultaneously (in a combination formulation), essentially simultaneously
(e.g., administration of
each compound a few minutes or a few hours apart), or can be administered
sequentially, e.g.,
several days apart, or more than a week apart. For example, a compound of the
present invention
and a plasma-concentration-enhancing compound (and/or a macrophage stimulating
factor) can be
administered together, or essentially simultaneously, e.g., administration of
each compound a few
minutes or a few hours apart, or can be administered sequentially, e.g.,
several days apart, or more
than a week apart (optionally together with simultaneous or sequential
administration of oxidating
agent or oxygen ventilation). All such variations in administration of the
combination therapy are
encompassed within the scope of the invention.
The invention also includes pharmaceutical formulations containing any such
combination
as described herein.
The invention also includes the use of combinations of compounds as disclosed
herein in
the manufacture of a medicament for use in the treatment of a condition
selected from malaria,
African Trypanosomiasis, American Trypanosomiasis, as well as one or more kind
of parasites
and/or one or more diseases caused by such parasites, against one or more kind
of Mycoplasma
and/or one or more diseases caused by such Mycoplasmas and/or against one or
more of the
foliowing indications or infections: (a) hairy Leukoplakia, (b) oral
candidosis, (c) mouth
ulcerations-aphthous/ herpetic/bacterial, (d) fungal candida, (e) human
papilloma virus, (f)
molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral
lesions, (i)
periodontitis, (j) necrotizing gingivitis, (k) orafacial herpes zoster, and
(1) rotaviruses, as well as all
other indications and infections disclosed in U.S. Patent No. 5,292,725.
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The present invention is also directed to the use of compounds of the present
invention in
the manufacture of a medicament for treatment as described herein.
The present invention is also directed to administering of compounds of the
present
invention to provide a prophylactic treatment of a patient against liver
parasites, e.g., Trypanosome
parasites.
- _ Articles of manufacture. The present invention also provides articles of
manufacture
comprising, for example, packaging material, at least one unit-dosage of a
compound of the present
invention (optionally together with one or more unit-dosage of a compound
which can be
administered in a combination therapy) and a label or package insert
indicating that the compound
can be used in a method disclosed herein.
In one embodiment, an article of manufacture comprises packaging material, at
least one
unit dose of a 17-ketosteroid compound (a formula 1 compound) and a label or
package insert
indicating that the 17-ketosteroid compound (a formula 1 compound) can be used
in a method as
described herein. The packaging material can be made from one or more
generally known
1 S materials, e.g., foam, cardboard, fiberboard, polystyrene and
polypropylene, and is of a size
suitable to contain the compounds) accompanying the packaging material. A
label or package
insert can be a tag or label secured to the packaging material, a label
printed on the packaging
material or a label inserted within the packaging material. The label
indicates that the
17-ketosteroid can be used in a therapy as disclosed herein, e.g., in
combination with a plasma
concentration-enhancing compound and/or a macrophage-stimulating factor. The
label can also
indicate that the compounds) have received approval from an official agency,
for example, the
U.S. Food and Drug Administration, for medical or veterinary use according to
the method. The
label may also indicate suitable administration routes, dosage regimen, and
the like. If desired, the
article may contain additional components such as at least one unit dose of a
plasma
concentration-enhancing compound or the macrophage-stimulating factor.
Methods of administration and formulations. The dosage for a particular
patient will vary
depending on factors such as the overall health of the patient, the method,
route and dose of
administration and the severity of side effects (if any). Determination of the
appropriate dose is
made by the clinician using parameters known in the art. Generally, the dose
begins with an
amount somewhat less than the optimum dose and_it is increased by small
increments thereafter
until the desired or optimum effect is achieved. The dosage of the compounds
of the invention is
suitably determined depending on the individual cases taking symptoms, age and
sex of the subject
and the like into consideration. With respect to the duration of treatment, it
is typica) for skilled
clinicians to monitor patients in order to determine when inhibition is
providing therapeutic
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benefit, and to determine whether to increase dosage, decrease dosage,
discontinue therapy, resume
therapy or alter therapy.
The therapeutically effective dosage of any specific compound will vary
somewhat from
compound to compound and patient to patient. As a general proposition, a
dosage in the range of
from about 0.1 to about 500 mglkg will have therapeutic efficacy. Typically, a
dosage in the range
of from about 0.5 mg/kg to about 500 mg/kg will be employed. A daily dosage of
a formula 1
compound will typically comprise about 10 to about 750 mg, usually about 20 to
about 400 mg,
which may be administered as a single dose or as two or more subdoses. Such
doses or subdoses
may be administered at one or more sites or by one or more than one route of
administration. The
duration for the treatment is usually once per day for a sufficient length of
time for the patient to
become asymptomatic, or for symptoms to abate noticeably. Depending upon the
severity of the
infection in the individual patient, this may last several days, weeks, or
longer.
With regard to the frequency and duration of treatment, it is well known that
it is within
the skill of the ordinary physician to monitor a patient's condition and to
make appropriate
decisions with regard to discontinuing, interrupting and resuming treatments.
The dosages used in accordance with the invention are suitably determined
depending on
the individual cases taking symptoms, age and sex of the subject and the like
into consideration. In
addition, it !is well known that it is within the skill of ordinary artisans
to determine suitable
dosages based on the above and other factors.
In accordance with the present method, a compound of the present invention may
be
administered orally, intramuscularly (IM), intravenously (IV), or
subcutaneously (SC), with
intravenous administration being especially preferred. Although other routes
of administration can
be used, it has been, found that intravenous administration provides
surprising effectiveness. For
oral administration, the use of a plasma concentration-enhancing compound may
be of great
importance. Alternatively, the compound or salt may also be administered
intravenously or
intramuscularly as a liposomal suspension. The administration may also be in a
cyclodextrin
formulation (given orally, SC, IV or IM). Compounds of the invention and their
pharmaceutically
or physiologically, acceptable salts, are thus administered by any route
suitable to the condition to
be treated, including oral, rectal, nasal, topical (including ocular, buccal
or sublingual), vaginal,
parenteral (including subcutaneous, intramuscular,_intravenous,
intraperitoneal, intraderma(,
intrathecal, intradural and epidural) and pulmonary by aerosol. Generally, the
compounds of the
invention are administered parenterally, orally or topically. If an embodiment
is not su~ciently
orally bioavailable it can be administered by the other routes noted above.
Embodiments include formulations that comprise a liposome or lipid complex
that
comprises a formula 1 compound. Such formulations are prepared according to
known methods,
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e.g., U.S. patents 4427649, 5043165, 5714163, 5744158. 5783211, 5795589,
5795987, 5798348,
5811118, 5820848, 5834016 and 5882678, all of which are incorporated herein by
reference. The
liposomes may optionally comprise an additional therapeutic or other agent(s),
e.g., a compound of
formula 2A or 2B. The liposomes can be delivered to a subject by any standard
route, e.g., oral,
aerosol or parenteral (e.g., SC, IV, IM).
Most often, the pharmaceutical compositions useful in the present invention
will comprise
a compound of Formula 1, or a pharmaceutically acceptable salt thereof, in any
pharmaceutically
acceptable carrier. If a solution is desired, water is the carrier of choice
with respect to
water-soluble compounds or salts. In other embodiments, an organic vehicle,
such as glycerol,
ethanol, propylene glycol, polyethylene glycol, DMSO, DMS02, vegetable,
mineral oils. ethanol,
benzyl benzoate, or mixtures thereof, may be suitable. In general, the
solutions in any instance
should be sterilized in a suitable manner, preferably by filtration through a
0.22 microttfilter. The
compositions useful in the practice of the present invention may be provided
in the form of vials,
ampoules, and the like.
In some embodiments, the formula 1 compound that is present in the
compositions or that
is used in the methods disclosed herein is completely dissolved in non-aqueous
excipients.
However, in some embodiments, e.g., transient compositions or some
formulations, the formula 1
compound is partially dissolved while the remaining portion is present as a
solid, which can be a
suspension or a colloid. In related embodiments, the formula 1 compound is
incompletely
dissolved and is present as a suspension or gel.
In addition to compounds of formula 1, or their salts, the pharmaceutical
compositions
may contain other additives, such as pH adjusting additives, in particular,
agents such as acids,
bases, or buffers, including sodium lactate, sodium acetate, and sodium
gluconate. Further, such
compositions may contain microbial preservatives, such as methylparaben,
propylparaben, benzyl
alcohol and benzyl benzoate. If a multiple use vial is supplied, the
pharmaceutical composition
should likewise include such a microbial preservative. The formulations may
be, of course,
lyophilized, using techniques well known in the art.
It has been found that with respect to the practice of the method of the
present invention,
treating malaria with a compound of formula 1, or a pharmaceutically
acceptable salt thereof,
certain compounds appear to possess superior efficacy to others.
The formulations include those suitable for the foregoing administration
routes. The
formulations may conveniently be presented in unit dosage form and may be
prepared by any of
the methods well known in the art of pharmacy. Techniques, excipients and
formulations generally
are found in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton, PA 1985,
17th edition, Nema et al., PDA J. Pharm. Sci. Tech. 1997 51:166-171, both of
which are
CA 02356539 2001-05-23
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incorporated herein by reference. Methods to make invention formulations
include the step of
bringing into association a formula 1 compound with one or more excipients or
carriers. In
general, the formulations are prepared by uniformly and intimately bringing
into association the
formula 1 compound with liquid excipients or finely divided solid excipients
or both, and then, if
appropriate, shaping the product.
Formulations of the present invention suitable for oral administration may be
presented as
discrete units such as capsules, cachets or tablets each containing a
predetermined amount of the
formula 1 or formula 2A or 2B compound; as a powder or granules; as solution
or a suspension in
an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil
liquid emulsion. The formula 1 or formula 2A or 2B compound may also be
presented as a bolus,
electuary or paste.
A tablet may be made by compression or molding, optionally with one or
mora~excipients.
Compressed tablets may be prepared by compressing in a suitable machine the
formula 1 or
formula 2A or 2B compound in a free-flowing form such as a powder or granules,
optionally
mixed with a binder, lubricant, inert diluent, preservative, surface active or
dispersing agent.
Molded tablets may be made by moulding in a suitable machine a mixture of the
powdered
compound moistened with an inert liquid diluent. The tablets may optionally be
coated or scored
and may be formulated so as to provide slow or controlled release of the
formula 1 or formula 2A
or 2B compound therein.
The oily phase of the emulsions of this invention may be constituted from
known
ingredients in a known manner. While the phase may comprise merely an
emulsifier (otherwise
known as an emulgent), it desirably comprises a mixture of at least one
emulsifier with a fat or an
oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is
included together with a
lipophilic emulsifier, which acts as a stabilizer. It is also preferred to
include both an oil and a fat.
Together, the emulsifiers) with or without stabilizers) make up the
emulsifying wax, and the wax
together with the oil and fat make up the emulsifying ointment base which
forms the oily dispersed
phase of the cream formulations. Emulgents and emulsion stabilizers suitable
for use in
formulations comprising a formula 1 or a formula 2A or 2B compound include
Tween~ 60, Span~
80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-
stearate and sodium lauryl
sulfate.
Formulations suitable for buccal administration include lozenges comprising a
formula 1
or formula 2A or 2B compound in a flavored basis, usually sucrose and acacia
or tragacanth;
pastilles comprising the formula 1 or formula 2A or 2B compound in an inert
basis such as gelatin
and glycerin, or sucrose and acacia.
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Formulations for rectal administration may be presented as a suppository with
a suitable
base comprising for example cocoa butter or a salicylate.
Formulations suitable for intrapulmonary or nasal administration will have a
particle size
for example in the range of 0.01 to 200 microns (including particle sizes in a
range between 0.01
S and 500 microns in increments of 0.1 microns such as 0.1, 0.2, 0.3, 0.4,
0.5, 1, 2, 5, 30 microns, 35
- _ microns, etc.), which is administered by inhalation through the nasal
passage or by inhalation
through the mouth so as to reach the various bronchi or alveolar sacs.
Formulations suitable for
aerosol or dry powder administration may be prepared according to conventional
methods and may
be delivered with other therapeutic agents such as compounds heretofore used
in the treatment or
prophylaxis of Trypanosome infections. Inhalation therapy is readily
administered by metered
dose inhalers.
Formulations suitable for vaginal administration may be presented as
pessaries; tampons,
creams, gels, pastes, foams or spray formulations containing in addition to
the formula I
compound such carriers or excipients as are known in the art to be
appropriate.
Formulations suitable for parenteral administration are sterile and include
aqueous and
non-aqueous injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes
which render the formulation isotonic with the blood of the intended
recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents and
thickening agents. The
formulations may be presented in unit-dose or mufti-dose containers, for
example sealed ampoules
and vials with elastomeric stoppers, and may be stored in a freeze-dried
(lyophilized) condition
requiring only the addition of the sterile liquid carrier, for example water
for injections,
immediately prior to use. Extemporaneous injection solutions and suspensions
may be prepared
from sterile powders, granules and tablets of the kind previously described.
Unit dosage
formulations will typically contain a daily dose or unit daily sub-dose, as
recited above, or an
appropriate fraction thereof, of a formula 1 or formula 2A or 2B compound.
In some embodiments, the formula 1 compounds will be administered on an
intermittent
basis. In these embodiments, the formula 1 compound, e.g., a dose that
comprises about 5-500 mg
of a formula 1 compound (typically about 50-400 mg), is administered to a
subject for at least one
day, followed by no dosing for at least one day (at least 24 hours),
optionally followed by at least
one more daily dose of, e.g., about 50-500 mg. Intermittent dosing methods may
comprise dosing
( 1, 2, 3 or 4 doses per week) based on a weekly schedule, e.g., dosing on
Monday, Wednesday and
Friday, or on Tuesday, Thursday Saturday for about i, 2, 3, 4, 6, 8 or more
weeks, followed by
periods of about 2, 3, 4, 5, 30, 45, 60, 90 or more days with no dosing,
optionally followed by
dosing again on Monday, Wednesday and Friday for about 1, 2, 3, 4, 6, 8 or
more weeks. Weekly
dosing methods may comprise administration of the forrpula 1 compound to a
subject I, 2, 3, 4 or 5
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times per week for I, 2, 3, 4, or more weeks.. In related embodiments, dosing
may be administered
to a subject daily for 2, 3, 4, 5, 6, 7 or more days, followed by a period of
about 1; 2, 3, 4, 5, 7, 14,
30, 45 60, 90 or more days, optionally followed by another course of daily
dosing. These
embodiments may further comprise treatment with a formula 2A or 2B compound or
another
treatment as described herein.
To the extent not already indicated, it will be understood by those of
ordinary skill in the
art that any one of the various specific embodiments herein described and
illustrated may be
further modified to incorporate features shown in any of the other embodiments
disclosed herein.
Therapeutic applications. For therapeutic applications, the compositions
disclosed herein
will typically comprise one or more compounds of formula 1, and, the methods
disclosed herein
will utilize such compositions, which will contain one, t<vo or more of such
compounds, usually
one. While it is possible for the compounds of the invention to be
administered as purecompounds
it is preferable to present them as pharmaceutical formulations. The
formulations of the present
invention comprise at least one formula 1 compound together with one or more
acceptable carriers
or excipients and optionally other therapeutic agents, e.g., a formula 2A or
2B compound(s),
chloroquine, a chlroquine analog, a macrophage stimulating factors) and/or an
oxidation agent(s).
The one or more carriers or excipients must be "acceptable" in the sense of
being compatible with
the other ingredients of the formulation and not deleterious to the patient.
In other embodiments, a dosing regimen for a formula I compound will comprise
the use
of a relatively high induction dose, e.g., about 150-750 mg per day or about
150-750 mg per day
using an intermittent dosing schedule (such as described herein), followed by
lower maintenance
dosing, e.g., about 50-Z50 mg per day or about 50-250 mg per day on an
intermittent dosing
schedule. These embodiments may further comprise treatment with a formula 2A
or 2B compound
or another treatment as described herein.
Parenteral formulations may comprise a cyclodextrin, e.g., an ac-cyciodextrin,
a ~i-
cyclodextrin (e.g., (3-hydroxypropylcyclodextrin) or a y-cyclodextrin, which
are typically employed
in aqueous formulations, which optionally comprise one or more of a buffer, a
salt (NaCI, etc.) to,
e.g., render the solution isotonic, a bacteriostat or other excipients as
known in the art and a
formula 1 compound at a concentration of, e.g., about 5-25 mg/mL, typically
about 10-20 mg/mL.
Parenteral formulations that comprise a formula 1 compound and one or more
excipients may be
diluted into, e.g., sterile saline and infeused into a subject. Parenteral
formulations are typically
administered by, e.g., intravenous, topical or oral delivery to a subject such
as a human. For non-
aqueous formulations, one or more solvents such as propylene glycol, a PEG,
e.g., PEG 300 or
PEG 400, ethanol, and benzyl benzoate may be employed. Typical aqueous and non-
aqueous
formulations will contain about S to about 400 mg/mL of a formula I compound,
usually about 10
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to about 200 mg/mL. Such parenteral formulations may be delivered orally, or
by intramuscular,
intravenous or subcutaneous injection.
In preparing compositions that comprise a formula 1 compound (and optionally
one or
more excipients), one may optionally mill or otherwise granulate the compound
to obtain a desired
particle size, before or after the formula 1 compound is contacted with one or
more excipients. For
' _ example, one may mill a formula I compound such as 16a.-
bromoepiandrosterone, to obtain an
average particle size (or diameter) of about 0.5-25 pM or about 1-10 p,M
(e.g., about 2, 5 or 10 1tM
average particle size or diameter) before contacting the milled formula 1
compound with a liquid
or solid excipient. Milled formula 1 compound is useful to facilitate
dissolution or suspension of
the formula 1 compound in one or more liquid excipients (e.g., a PEG such as
PEG 300, propylene
glycol or benzyl benzoate) or to facilitate uniformly distributing drug
substance when the milled
compound is contacted with one or more solid excipients (e.g., a filler, a
binder or a lubricant).
The compositions and formulations disclosed herein are useful in the treatment
of, or
ameliorate one or more symptoms associated with, the conditions or infections
disclosed herein.
These compositions and formulations may also be used to treat, or ameliorate
one or more
symptoms associated with, a retroviral infection such as a HIV 1 or H1V2
infection in humans. As
used herein, phrases such as "amelioration of one or more symptoms associated
with" means that
such compounds or formulations may be used to reduce replication of an
infectious agent or to
reduce the number of infectious agents that are present in a subject or to
ameliorate one or more
symptoms associated with, or caused by, the condition or infection (e.g.,
reduced fever, a shortened
duration of, or reduced level of, pain, or a noticeable reduction of or
elimination of diarrhea or
fatigue).
In addition to the ingredients particularly mentioned above the formulations
of this
invention may include other agents conventional in the art having regard to
the type of formulation
in question, for example those suitable for oral administration may include
flavoring or coloring
agents.
The present invention further provides veterinary compositions comprising at
least one
formula 1 or formula 2A or 2B compound together with a veterinary carrier
therefor. Also, the
formula 1 compound may be present in the animal's feed or water. Excipients
for veterinary
applications may include compounds, e.g., small amounts of chloroform, that
may not be generally
suitable for human use.
Veterinary carriers are materials useful for the purpose of administering the
composition to
cats, dogs, horses, mice, rats, hamsters, rabbits and other animals and may be
solid, liquid or
gaseous materials that are otherwise inert or acceptable in the veterinary art
and are compatible
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with the formula 1 or formula 2A or 2B compound. These veterinary compositions
may be
administered orally, parenterally or by any other desired route, e.g., as
described herein.
Embodiments of formula 1 compounds include or exclude any subset of compounds
within
the definition of formula I, provided that at least one compound remains. For
example, a subset of
formula 1 compounds that are generally preferred and are usually included, for
example are
_ aqueous or nonaqueous formulations comprising l6cc-bromoepiandrosterone. A
subset compounds
or applications for compounds that are optionally excluded from formula I
compounds or their
uses in any embodiment or claim herein comprises, e.g., the use of one or more
compounds (or
their use) that are disclosed in one or more prior art references or
publications, to the extent that
the disclosed compounds or uses renders any claim or embodiment unpatentable
for novelty,
obviousness and/or inventive step reasons.
In other embodiments, a formula 1 compound may be linked to an oligonucleotide
or an
oligonucleotide analog to facilitate delivery of the oligonucleotide or analog
into cells. Typically
the formula 1 compound will be linked to the steroid nucleus through a
terminal hydroxyl group at
a 5', 3' or 2' position of the oligonucleotide. Oligonucleotides and analogs
of oligonucleotides are
known and have been described, e.g., U.S. patents 472677, 4973679, 4997927,
4415732,
4458066, 5047524, 4959463, 5212295, 5386023, 5489677, 5594121, 5614622,
5624621; and PCT
publication Nos. WO 92/07864, WO 96/29337, WO 97/14706, WO 97/14709, WO
97/31009, WO
98/04585 and WO 98/04575 all of which are incorporated herein by reference.
Synthesis methods. In general, the compounds employed in the present invention
in
general may be synthesized in manners known and readily understood by those
skilled in the art.
Therefore, there is no need to explain in great detail the methodology used
for the synthesis of
most such compounds.
Formula 1 compounds that comprise a thioacecai moiety, sulfate ester, sulfite
ester,
carbamate or thioester moiety at R2 (the 3-position) are prepared essentially
according to methods
known in the art. Suitably protected intermediates will be used as is
apparent. See, for example,
U.S. patent 5198432; European patent publications EP 5769I5 and EP 576914; C.
Christians et al.,
J. Chem. Soc. Chem. Commun. 1991 vol 22, C. Christians et al., J. Chem. Soc.
Chem. Commun.
1991 19:1403-1405, H.N. Abramson et al., J. Pharm. Sci. 1977 66:602-603, E.J.
Corey et al., J.
Am. Chem. Soc. 1996 118:8765-8766, A.G.M. Barrett et al., J. Org. Chem. 1989
54:227, D.H.R.
Barton et al., J. Chem. Soc. Perkin Trans. 1 1976 19:21 12-2116, D.H.R. Barton
et al., J. Chem.
Soc. Perkin Trans. l 1975 16:1574-1585 and W.T. Smith et al., Trans.
KentuckyAcad. Sci. 1984
45:76-77, all of which are incorporated herein by reference.
Enumerated embodiments. Aspects of the invention include the following
enumerated
embodiments, which further illustrate the invention and preferred aspects
thereof or related subject
matter.
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1. A method of treating malaria or Trypanosomiasis in a patient in need of
such treatment,
comprising administering to said patient an effective amount of at least one
compound selected
from the group consisting of the compounds of the present invention.
2. A method as recited in embodiment l, further comprising administering to
said patient
at least one plasma concentration-enhancing compound.
- _ 3. A method as recited in embodiment 2, wherein said at least one compound
of the present
invention and said at least one plasma concentration enhancing compound are
administered
simultaneously.
4. A method as recited in embodiment 2, wherein said at least one compound of
the present
invention and said at least one plasma concentration-enhancing compound are
administered
sequentially.
5. A method as recited in embodiment 2, wherein said plasma concentration-
enhancing
compound is naringin and/or naringenin.
6. A method as recited in any one of embodiments 1 - S, further comprising
administering
to said patient at least one macrophage stimulating factor.
7. A method as recited in any one of embodiments I - G, further comprising
administering
to said patient one or morn oxidation agent and/'or oxygen ventilation.
8. A method as recited in any one of embodiments 1 - 7, wherein said patient
is a mammal.
9. A method as recited in embodiment 8, wherein said patient is a human.
10. A method as recited in any one of embodiments 1 - 9, wherein said
administering is by
injection.
1 I . A method as recited in any one of embodiments 1 - 9, wherein said
administering is by
infusion.
12. A method as recited in any one of embodiments 1 - 9, wherein said
administering is by
intravenous injection.
13. A method as recited in embodiment i, wherein said at least one compound is
selected
from the group consisting of compounds of formula 1, wherein R1 ~ is not
hydroxy.
14. A method as recited in embodiment l, wherein said at least one compound is
selected
from the group consisting of compounds of Formula 1, wherein Q2 is not CH2.
15. A method as recited in embodiment l, wherein said double bond is not
present.
16. A method as recited in embodiment 1, wherein said at least one compound is
selected
from the group consisting of compounds of Formula 1, wherein Q2 is a halogen.
17. A method as recited in embodiment 1, wherein said at least one compound is
not
DHEA.
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18. A method of treating sleeping sickness in a patient in need of such
treatment,
comprising administering to said patient an effective amount of a compound of
the present
invention.
19. A method as recited in embodiment I8, further comprising administering to
said
patient at least one plasma concentration-enhancing compound.
' 20. A method as recited in embodiment 19, wherein said at least one compound
of the
present invention and said at least one plasma concentration-enhancing
compound are administered
simultaneously.
21. A method as recited in embodiment 19, wherein said at least one compound
of the 70
present invention and said at least one plasma concentration-enhancing
compound are administered
sequentially.
22. A method as recited in embodiment 19, wherein said plasma concentration-
cnhancing
compound is naringin and/or naringenin.
23. A method as recited in any one of embodiments l8 - 22, further comprising
administering to said patient at least one macrophage stimulating factor.
24. A method as recited in any one of embodiments 18 - 23, further comprising
administering to said patient one or more oxidation agent and/or oxygen
ventilation.
25. A method as recited in any one of embodiments 18 - 24, wherein said
patient is a
mammal.
26. A method as recited in embodiment 25, wherein said patient is a human.
27. A method as recited in any one of embodiments 18 - 26, wherein said
administering is
by injection.
28. A method as recited in any one of embodiments 18 - 26, wherein said
administering is
by infusion.
29. A method as recited in any one of embodiments 18 - 26, wherein said
administering is
by intravenous injection.
30. A method of treating Chagas disease in a patient in need of such
treatment, comprising
administering to said patient an effective amount of a compound of the present
invention.
31. A method as recited in embodiment 30, further comprising administering to
said
patient at least one plasma concentration-enhancing compound.
32. A method as recited in embodiment 31, wherein said at least one compound
of the 71
present invention and said at least one plasma concentration-enhancing
compound are administered
simultaneously.
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33. A method as recited in embodiment 31, wherein said at least one compound
of the
present invention and said at least one plasma concentration-enhancing
compound are administered
sequentially.
34. A method as recited in embodiment 31, wherein said plasma concentration-
enhancing
compound is naringin and/or naringenin.
' . 35. A method as recited in any one of embodiments 30 - 34, further
comprising
administering to said patient at least one macrophage stimulating factor.
36. A method as recited in any one of embodiments 30 - 35, further comprising
administering to said patient one or more oxidation agent and/or oxygen
ventilation.
37. A method as recited in any one of embodiments 30 - 36, wherein said
patient is a
mammal.
38. A method as recited in embodiment 37, wherein said patient is a human.
39. A method as recited in any one of embodiments 30 - 38, wherein said
administering is
by injection.
40. A method as recited in any one of embodiments 30 - 38, wherein said
administering is
by infusion.
41. A method as recited in any one of embodiments 30 - 38, wherein said
administering is
by intravenous injection.
42. A method of treating one or more kind of parasites and/or one or more
diseases caused
by such parasites, against one or more kind of Mycoplasma and/or one or more
diseases caused by
such Mycoplasmas and/or against one or more of the following indications or
infections: (a) hairy
Leukoplakia, (b) oral candidosis, (c) mouth ulcerations
(aphthous/herpetic/bacterial), (d) fungal
candida, (e) human papilloma virus, (f) moiluscum contagiosum, (g) squamous
oral carcinoma, (h)
Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis,
(k) orafacial herpes
zoster, and (1) rotaviruses in a patient in need of such treatment, comprising
administering to said
patient an effective amount of a compound of the present invention.
43. A method as recited in embodiment 42, further comprising administering to
said
patient at least one plasma concentration-enhancing compound.
44. A method as recited in embodiment 43, wherein said at least one compound
of the
present invention and said at least one plasma concentration-enhancing
compound are administered
simultaneously.
45. A method as recited in embodiment 43, wherein said at least one compound
of the
present invention and said at least one plasma concentration-enhancing
compound are administered
sequentially.
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46, A method as recited in embodiment 43, wherein said plasma concentration-
enhancing
compound is naringin and/or naringenin.
47. A method as recited in any one of embodiments 42 - 46, further comprising
administering to said patient at least one macrophage stimulating factor.
48. A method as recited in any one of embodiments 42 - 47, further comprising
- administering to said patient one or more oxidation agent and/or oxygen
ventilation.
49. A method as recited in any one of embodiments 42 - 48, wherein said
patient is a
mammal.
50. A method as recited in embodiment 49, wherein said patient is a human.
51. A method as recited in any one of embodiments 42 - 50, wherein said
administering is
by injection.
52. A method as recited in any one of embodiments 42 - 50, wherein said
administering is
by infusion.
53. A method as recited in any one of embodiments 42 - 50, wherein said
administering is
by intravenous injection.
54. A composition comprising at least one of the compounds of the present
invention, and
at least one plasma concentration-enhancing compound.
55. A composition as recited in embodiment 54, further comprising at least one
macrophage stimulating factor.
56. A composition as recited in embodiment 54 or 55, further comprising an
oxidation
agent.
57. A composition comprising at least one of the compounds of the present
invention, and
at least one macrophage stimulating factor.
58. A composition as recited in embodiment 57, further comprising at least one
oxidation
agent.
59. A composition comprising at least one of the compounds of the present
invention, and
at least one an oxidation agent.
60. A kit comprising unit dosages of at least one of the compounds of the
present
invention, and unit dosages of at least one plasma concentration-enhancing
compound.
61. A kit as recited in embodiment 60, further comprising unit dosages of at
least one
macrophage stimulating factor.
62. A kit as recited in embodiment 60 or 61, further comprising unit dosages
of an
oxidation agent.
63. A kit comprising unit dosages of at least one of the compounds of the
present
invention, and unit dosages of at least one macrophage stimulating factor.
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64. A kit as recited in embodiment 63, further comprising unit dosages of at
least one
oxidation agent.
65. A kit comprising unit dosages of at least one of the compounds of the
present
invention, and unit dosages of at least one oxidation agent.
66. The method, composition or kit of any of embodiments 1-65 wherein the
compound of
the invention is a formula 1 compound or a metabolite thereof.
67. The method of embodiment 66 wherein the formula 1 compound is a compound
named
in compound groups 1-21, or in any formula 1 (e.g., any formula 4) compound or
genus disclosed
or named herein, or a metabolite of any of these.
68. A composition comprising 16a-bromoepiandrosterone, and 2, 3, 4 or S
excipients
selected from polyethylene glycol, dehydrated ethanol, benzyl benzoate, benzyl
alcohol and
propylene glycol, wherein the composition comprises less than about 3% v/v, or
less than about
1 % v/v, or less than about 0.5% v/v of water, or less than about 0.1 % v/v of
water.
69. The composition of embodiment 68 wherein the composition comprises (i) 16a-
bromoepiandrosterone at a concentration of about 45-55 mg/mL, (ii) 20-30% v/v
polyethylene
glycol 300, polyethylene glycol 400 or a mixture of polyethylene glycol 300
and 400, (iii) 10-15%
v/v dehydrated ethyl alcohol, 2.5-7.5% vlv benzyl benzoate, and (iv) 55-60%
v/v propylene glycol.
70. The composition of embodiment 69 wherein the composition comprises 16a-
bromoepiandrosterone at a concentration of about 50 mg/mL, about 25% v/v
polyethylene glycol
300, about 12.5% v/v dehydrated ethyl alcohol, about 5% v/v benzyl benzoate,
about 57.5% v/v
propylene glycol and less than about 0.5% v/v water.
71. The composition of embodiment 68 wherein the composition comprises 16a-
bromoepiandrosterone at a concentration of about SO-105 mglmL, about 27-33%
w/w benryl
benzoate, about 27-33% w/w polyethylene glycol 300, about 25-30% w/w propylene
glycol and
about 1-3% w/w benzyl alcohol.
72. The composition of embodiment 71 wherein the composition comprises 16a-
bromoepiandrosterone at a concentration of about 100 mg/mL (about 10% w/w),
about 30.4% w/w
benzyl benzoate, about 30.7% w/w polyethylene glycol 300, about 28% w/w
propylene glycol and
benzyl alcohol about 1.9% w/w.
73. The use of a formula 1 compound for making a medicament for the treatment
of an
infection caused by one or more Trypanosoma or Plasmodium: parasites or a
Mycoplasma
bacterium in a subject, including one or more of Trypanosoma crud, Trypanosoma
brucei,
Trypanosoma gambie>tse, Trypanosorna rhodesiense, Trypanosoma brucei
rhodesiense,
Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovate
or
Plasmodium berghei, Mycoplasma jerme»tans, Mycoplasma genitalium or Mycoplasma
pneumoniae.
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74. The use of embodiment 73 wherein the formula 1 compound is a compound
named in
compound groups 1-21, or a metabolite thereof.
75. A method comprising administering an effective amount of a composition of
any of
embodiments 68-71 to a subject having, or susceptible to, a Trypanosoma, a
Plasmodium or a
Mycoplasma infection wherein the composition optionally comprises 16a-
bromoepiandrosterone
' and a pharmaceutically acceptable excipient.
76. A method to ameliorate or reduce one or more symptoms associated with a
Trypanosoma, a Plasmodium or a Mycoplasma infection in a subject, or to reduce
replication of a
Trypanosoma, a Plasmodium or a Mycoplasma in a subject infected with a
Trypanosoma, a
Plasmodium or a Mycoplasma, comprising administering to the subject an
effective amount of a
compound of fonmufa 1.
77. The method of embodiment 76 wherein the formula 1 compound is a compound
or
within a genus of compounds as disclosed herein, e.g., a compound or genus
named in compound
groups 1-21 or in the claims as originally filed, or the formula 1 compound is
present in a
composition comprising one or more pharmaceutical excipients, e.g., any of the
formulations
disclosed or described herein.
78. A composition comprising a formula 1 compound wherein the formula 1
compound is
a compound or within a genus of compounds as disclosed herein, e.g., a
compound or genus named
in compound groups 1-21 or in the claims as originally filed, and at least one
excipient and a local
anesthetic, wherein the local anaesthetic is optianally selected from
procaine, benzocaine and
lidocaine.
79. A product produced by the process of contacting a compound of formula 1,
e.g., any
compound named in compound groups 1-21, and a first excipient with a second
excipient wherein
the product optionally further comprises a local anesthetic, wherein the local
anaesthetic is
optionally selected from procaine, benzocaine and lidocaine.
80. A product produced by the process of contacting a compound of formula 1,
e.g., any
compound named in compound groups 1-21, and a first nonaqueous liquid
excipient with a second
nonaqueous liquid excipient wherein the product comprises less than about 3%
w/w water, or less
than about 0.5% w/w water, or less than about 0.1 % w/w water, and wherein the
first or the second
nonaqueous liquid excipient optionally excludes one or more of
dimethylsulfoxide, chloroform,
dioxane, a vegetable oil and olive oil, and wherein the product optionally
further comprises a local
anesthetic, wherein the local anaesthetic is optionally selected from
procaine, benzocaine and
lidocaine.
81. A method comprising administering an effective amount of the composition
of
embodiment 78 or the product of embodiments 79 or 80 to a subject having an
infection or
condition described herein, e.g., Malaria, African Trypanosomiasis or Chagas
disease, whereby the
infection or condition, or a symptom thereof, is eliminated, reduced, treated,
improved or
ameliorated.
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82. The method of embodiment 81 wherein the formula 1 compound is l6oc-
haloepiandrosterone or 16oc-halodehydroepiandrosterone.
EXAMPLES
The following examples further illustrate the invention and are not to be
construed as
limiting the invention.
Example i. l6oc-Bromoepiandrosterone formulation 1. Two lots of a non-aqueous
formulation was made at a 16a.-bromoepiandrosterone (''BrEA") concentration of
50 mg/mL in
25% polyethylene glycol 300, 12.5% dehydrated ethyl alcohol, 5% benzyl
benzoate, and 57.5%
propylene glycol, hereafter "formulation 1 ", as follows. BrEA was obtained
from Procyte, Inc.
The remaining excipients are shown below.
Excipient Specifica-tion Supplier Final Product
Lot No. Concentration
Propylene glycol USP Arco Chemical 57.5% (v:v)
HOC-61220-01104
Polyethylene NF Union Carbide 25% (v:v)
glycol 300 695752
Dehydrated alcohol USP McCormick Distilling 12.5% (v:v)
(ethanol) 97K10
Benzyl benzoate USP Spectrum S% (v:v)
Pharmaceuticals
MG025
The formulation was prepared by suspending BrEA in polyethylene glycol 300,
and
sequentially adding propylene glycol, benzyl benzoate, and dehydrated ethyl
alcohol to form a
solution, which was diluted to the final desired volume with additional
propylene glycol. The
procedure is described below.
The calculated amount of polyethylene glycol 300 was added to a compounding
vessel.
Then, while mixing, the calculated amount of BrEA was added to the vessel, and
mixed for at least
5 minutes to form a smooth, creamy liquid. Propylene glycol was added to the
vessel, and mixed
for a minimum of 5 minutes to form a uniform suspension. The calculated amount
of benzyl
benzoate is added to the vessel, and mixed for approximately S minutes to form
a translucent liquid
suspension. Dehydrated alcohol was added to the vessel, and mixed for
approximately 5 minutes
to form a clear, colorless solution. Propylene glycol was then added to
achieve the desired final
formulation, and mixed for approximately 5 minutes. The drug solution was
transferred to a
volume-dispensing device set to deliver 1.2 mL per vial. Under nitrogen
pressure, the solution was
filtered through two 0.2 um polyvinylidene fluoride filters in series before
dispensing. The vials
were capped with Teflon-coated, butyl-rubber stoppers and crimp sealed.
Materials used in the product vials are listed below.
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Material Source Product Code Description
Vial Wheaton 2702-BS1BA Tubing vial, 2 mL/13 mm,
glass, type 1 amber
Stopper Omniflex V9239 FM257/2 13 mm, Teflon coated, butyl
rubber stopper
- Seal West 4107 Flip seal, 13 mm, mist gray
bridge
Example Z. BrEA formulation 2. A formulation containing 100 mg/mL of HrEA, 10%
w/w, in benzyl benzoate (USP) 30.4% w/w, polyethylene glycol 300 (NF) 30.7%
w/w, propylene
glycol (USP), qs, about 28% w/w and benzyl alcohol (NF) 1.9% w/w, hereafter
"formulation 2",
was prepared as follows. A desired amount of BrEA ( I .0 kg) was suspended in
PEG 300 (about
3.0 L) in a compounding vessel and mixed for at least ~ minutes at room
temperature t0 form a
smooth creamy liquid. The needed amount of propylene glycol (about 1.5 L) was
then added and
mixing was continued for at least S minutes to form a uniform suspension.
Benzyl benzoate (about
3.0 L) was then added and the vessel contents were mixed for about Sminutes to
form a translucent
suspension. Benzyi alcohol (about 200 mL) was then added and the mixing was
continued for
about 5 minutes to form a clear, colorless solution. Propylene glycol was then
added to achieve the
desired final formulation volume (about 1.5 L) and mixing was continued for
about 5 minutes. The
drug solution was transferred to a volume-dispensing device, which was set to
deliver 1.2 mL per
vial (2 mL, glass, type 1 amber vials). The formulation was filtered under
nitrogen pressure (about
3 atm) through two 0.2 wm polyvinylidine fluoride filters in series. The vials
were capped using
Teflon-coated, butyl rubber stoppers and then crimp sealed essentially as
described in example 1.
The vials were stored in the dark at reduced temperature (about 2-8oC).
Example 3. In vitro testine. For in vitro antimalarial testing, micro-titer
plates were
used. The concentration of drugs were prepared as pMol/well according to WHO
standard
procedures (WHO, 1990). The test compound was dissolved in 1 S% DMSO in
sterile RPM 11640.
Both chloroquine sensitive (WS/97) and resistant (MN/97) isolates were used
throughout the
experiments.
A. Schizont inhibition assay: The micro-titer plates were predosed with
various
concentrations of the test compound. 50 ~L of parasitised erythrocyte
suspension in RPMI-1640
(0.2 mL erythrocyte + 0.3 mL serum + 4-5 ml RPMI-1640) were dispensed in
microtiter wells that
contained various concentrations of drug. Triplicate readings were made for
each concentration.
B. 3H-Hvnoxanthine incorporation assay: The testing was carried out according
to the
procedure of Desjardins et al.. 1979. After 30 hr culture at 37 degrees C, the
same microtiter plates
from schizont inhibition assays with another triplicate wells were pulsed with
3H-hypoxanthine for
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overnight. The cell suspensions were washed twice on milIipore glass fiber
filter with Millipore
filter apparatus. The filter discs were counted for DPM by a Beckman LS600U (3-
scintillation
counter. The activity of the drug was measured by plotting DPM against
concentration of drug.
Activity of compounds aeainst Chloro4uine sensitive T996/86 P. talciparum in
vitro.
Concentra-DHEA Bromine- Etienic AcidEtianic
_ tion (~cM)(% Inhibition)EpiandrosteroneMethyl EsterAcid
(% Inhibition)(% Inhibition)Methyl Ester
(% Inhibition)
30 65.6 98 60 61.5
15 44 60.1 45.7 47.4
7.5 38.3 SO 40.9 45.3
3.25 37.2 43.7 46 41.4
1.875 23.2 40.9 41 43.4
0.938 37.2 31.8 43.3 47.1 ' -
IC50 19.0pM 7.5pM 19.5pM 17.5~M
Concentration (nM) % Inhibition
Chloroquine Chloroquine
200 95.9
100 94.6
50 97.3
25 94.5
12.5 86.8
6.25 27.2
IC50 9.0 nM
The activity of 16-chloro-epiandrosterone and DHEA-Br against chloroquine
sensitive
T996.86 and chloroquine resistant ICI P. jalciparum in vitro is shown below.
T996.86 ICI
16-chloroepiandrosterone ICSp -9.25 pgmL-1 9.25 pgmL-1
DHEA-Br IC50 -25.0 pgmL-1 -25.0 p.gmL-1
Example 4. Four-day in vivo test protocol for P. berghei. The 4-day
suppressive test
has been widely used since it can be performed with a 1 -week period. The test
consists of the
inoculation of parasitised erythrocytes on Monday, the first day of the
experiment (DO), followed
by an injection of the test compound, which is repeatedly administered on D+1,
D+2, D+3. On
D+4 (Friday), blood films are taken and antimalarial activity is assessed
either by calculating
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parasitaemias, or by scoring parasite numbers on a predetermined scale (i.e.,
1-5). Peters (1970)
described a basic procedure using this 4-day test.
PROTOCOL
1 . 5 female TO mice per test group.
2. Parasites (P. berghei HP15 ANKA) were collected by cardiac puncture in a
heparinised syringe from a donor mouse harboring a 30+% parasitaemia.
3. Blood was diluted with diluting agent (50% HIFCS+50% sterile PBS) to a
final
concentration of 1% parasitaemia or iX107 infected erythrocytes per 0.2 mL
infecting
suspension.
4. Each mouse was inoculated intravenously, producing a more uniform infection
rate than an
intraperitoneal administration of parasites.
5. Test compounds were prepared at doses of 100 mg/kg in (16.7% DMSO + 83.3%
Celacol).
6. Test compounds were administered intraperitoneally 2 hours after parasite
inoculation.
7. The compounds were administered once a day starting on D0, and continued on
D+1, D+2 and D+3.
8. Blood films were made from tail blood on D+4, fixed with l00% methanol and
stained with 10% Giemsa.
9. Parasitaemias were scored on a scale of 0-S, where a 5 would be equal to
the
control.
EXPERIMENTAL PROTOCOL:
5 female mice/group (strain TO) were used and an inoculum consisting of 1
parasitaemia or 1x107 parasites/mL, 0.2 mL/mouse was delivered by intravenous
injection. Drug
administration commenced 2 hours after inoculation on Day 1 and continued for
3 days. Blood
films from all 20 mice were made on Day 5 and parasitaemias were assessed. The
results are
shown below.
Compound Treatment Parasitaemia Score (0 - Sl
Bromine-Epiandrosterone 100mg/kg x 4 days i.p.* 1
Etienic Acid 100mg/kg x 4 days i.p. 2
DHEA 100mg/kg x 4 days i.p. l
Chloroquine 3 mg/kg x 4 days i.p. 1
control N/A 5
* i.p. = intraperitoneal injection
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Example 5. Interim in vivo malaria study.
Basic Protocol: -
1. Infect mouse to 1 % parasitemia using a solution containing 1 x 107
erythrocytes/mL
by LV. injection.
2. Two hours later give drug preferably by LV. injection.
' 3. Drug Bromine-Epiandrosterone (Epi-Br) given (0.2 mL LV. or S.C.) once a
day for 4
days.
4. Tail snips to obtain blood after study.
Experimental results:
Day 0
T = 0 Mice were infected with P. berghei. Parasites were harvested from
cardiac mouse
blood, and mice were infected using 0.2 ml of blood with 14% parasitaemia pes
mouse
LV.
T = 2 hours First dose given: 100 mg/kg LV. or S.C.
T=4days Daily doses of 100 mg/kg LV. or S.C.
T = 7 days 2/S dead in the untreated control group
3/5 dead in the S.C. group.
T = 8 days 1/3 dead in the untreated control group.
No deaths occurred in the group receiving LV. drug at day 30, but all control
animals were
dead by day I0. All animals treated by S.C. delivery were dead by Day 11.
Example 6. Mouse in vitro and in vivo study. In the in vitro protocol the
parasite
(Plasmodium falciparum, chloroquine sensitive strain WT and chloroquine
resistant strain Dd2)
level is adjusted to I% and the hemocrit is adjusted 7% with medium. Using a
96 well plate, 50
ItL of parasite and 100 IzL of drug mixed with media are added to each well
and the procedure is
done in triplicate. The plate is placed in a chamber containing a
physiological gas mixture and
incubated at 37oC. The media/drug mixture is changed at 24, 48 and 72 hours.
On day S (96
hours) slides of each well are made, stained with Gemsia and 500 red blood
cells are counted for
each slide. The triplicates are averaged and data are reported in percent
inhibition.
In the in vivo protocol, Lewis rats weighing 80-85 grams were given a
standardized IP
injection of parasite (Plasmodium berghei). Rats were then intravenously
injected 2 hours later
with one of the treatments described in the table below, returned to their
housing, fed standard lab
chow and allowed free access to water. Animals were weighed and treated again
24, 48, and 72
hours after the first treatment and again returned to their housing and they
were allowed free access
to food and water. The animals were weighed again and then bled using a 26-
gauge needle on day
5, 1 1 and 28 post inoculation. Hemocrits were measured and blood smears are
prepared for each
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rat. The blood smears were then stained using Gemsia and the level of
parasitemia (defined as the
percent of red cells with parasites) were determined. Animals were again
returned to their housing
and observed twice daily for evidence of progressive disease, defined as
listlessness and or adverse
drug reaction, which is defined as a loss of 20% of original body weight, for
a total of 28 days. If
either progressive disease or drug reaction is noted, the animals are
euthanized.
' _ The PPB2 formulation comprised a sterile solution containing 15 mg/mL of
16a-
bromoepiandrosterone in 45% 43-cyclodextrin and 0.9% saline.
Group 1 Group 2 Group 3 Group 4
Control 0.9% Chloroquine PPB2 Low Dose PPB2 High Dose
Control
saline 40mg/kg (LD) 30 mg/kg (HD) 60 mg/kg
The intravenous injections were given on days 0, I, 2 and 3 and the results
are Shown
below. The results showed that treatment in vivo with a formulation comprising
16a-
bromoepiandrosterone reduced parasitemia to a level comparable to that seen
with the chloroquine
("Clq") control.
PPB2 In Vivo Run #1 Day 4
I
4o J
I
35 J
U 30
m
N 25 =
d
,NO 20 '
..
io
a
0 75
~o
5
0
92
Saline Clq LD PPB2 HD PPB2
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PPB2 In Vivo Run #1 (Day 11)
40
m 3C
d
..
R
0
5
0
Example 7. Human clinical study. Response to drug treatment was graded as per
World
Health Organization criteria (WHO 1973). Evaluation of therapeutic response
was determined
5 using the parasitic and fever clearance times. Parasite clearance was
expressed as three indices; the
time for the parasite count to fall by 50% of the pre-treatment (baseline)
value (PC50); to fall by 90
of the baseline value (PC9p) and to fall below the level of microscopic
detection (parasite
clearance time PCT) (White and Krishna 1989; White et al. 1992). The fever
clearance time was
defined as the time from drug administration till the oral or rectal
temperature fell to or below
10 37.2o C and remained so for at least 48 h.
Venous blood (SmL) was obtained from two patients before treatment and at 4,
6, 8, 12,
18, 20, 24, 30 and 36 h after treatment or at 4 or 6-hourly intervals after
treatment until there was
complete clearance of peripheral parasitemia. Blood was collected aseptically
and transferred to 10
mL syringes containing 2 mL of acid citrate dextrose (ACD) for in vitro
culture. Prior to
incubation, the plasma was separated from the red blood cells and the red
blood cells were washed
twice. Parasites were cultured by modification of standard in vitro culture
techniques (Trager and
Jensen 1976; Oduola et at. 1992). Samples were dispensed into sterile
centrifuge cubes within 10
min of collection and spun down. The supernatant plasma was stored while the
packed cells were
washed twice with culture medium (washing medium, RPMI 1640 medium, containing
25 mM
HEPES buffer and 25 mmol/L NaOH). The huffy coat was removed by vacuum
aspiration. A 1:10
fold dilution was done for each blood sample with complete washing medium [CMP
(washing
medium supplemented with 10 % human plasma)]. One milliliter each of the
sample was
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transferred into 2 wells of a 24 well micro culture plate. Cultures were
incubated at 37 degrees C in
an atmosphere of S% C02, S% 02 and 90% N2 premixed gas. The culture medium was
changed
daily and thin blood smears were prepared for microscopy at 24 and 48 h after
the culture has been
set up. The culture samples were diluted with unparasitized washed type A Rh-
positive red blood
S cells if the proportion of parasitized red blood cells was more than 2%.
Microscoov. During the in vivo study, thin and thick blood films were fixed
with
dehydrated methanol (100%) and heat, respectively, were stained with 10%
Giemsa for 20 min.
Parasitemia was quantified in thin films by counting 2000 red blood cells in
clear contiguous fields
and finding the proportion that was parasitized. In thick films, parasitemia
was quantified by
counting parasites against leukocytes. A film was declared negative if no
parasites were found after
examination of 200 microscope fields of a thick smear. During in vitro and ex
vivo study,
pretreatment thin and thick smears were, graded for ring stages by the method
of Jiang as modified
by Li et al. (Jiang et al, 1982; Silamut and White 1993; Li et al, 1994).
Approximately 5000
erythrocytes were counted in clear contiguous fields 24 and 48 h after
incubation of blood obtained
at each time point and graded for maturity into tiny rings, small rings, large
rings, pigmented
trophozoites and schizonts. Functional viability was estimated as the
percentage of asexual ring
forms capable of maturing to pigmented trophozoites or schizonts after 24-48 h
of in vitro culture
(Watkins et al. 1993).
Calculations of parameters. The Patients presented with acute symptomatic
severe
non-cerebral pure P. Falciparum malaria, they had oral fluid intolerance and
had body temperatures
greater than 39 degrees C, greater than 5000 parasites per micro liter of
blood and asexual
parasitemia and they had a negative urine test for antimalarial drugs. They
were administered 2S
mL intravenously every four hours with bromine epiandrosterone (16a,-
bromoepiandrosterone)
suspended in 45% beta cyctodextrin in saline at a concentration of 25 mg/mL.
This regimen was
2S continued for four days. Parasitemia quantification and clinical
examination were done once every
6 hours for the first 72 hours, followed by daily assessment of the parameters
up to day 7 ( 168 hrs)
and thereafter on day 14.
The blood films were Giemsa-stained and parasitemia quantification was done in
thick
films by counting 2000 parasites against leukocytes, and the thin films by
finding the proportion of
infected red blood cells. Response to drug treatment was graded according to
WHO criteria.
Evaluation of therapeutic response was done using the parasitic and fever
clearance times. Parasite
clearance was expressed as three indices: The time for the parasite count to
fall by SO% of the
pre-treatment (baseline) value (PC60); to fall by 90% of the baseline value
(PC90); and to fall
below the level of microscopic detection (parasite clearance time) PCT.
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The fever clearance time was defined as the time from drug administration
until the
oral/rectal temperature fell to below 37.2 degrees C and remained so for
greater than 48 hours.
LV. Bromine Eoiandrosterone Malaria Patient Trial
Patient A Patient B
Fever clearance time (H) 12 hrs 18 hrs
Parasite clearance times (H)
Time to 50% clearance 18 hrs 24 hrs
Time to 90% clearance 24 hrs 48 hrs
Time to 100% clearance 48 hrs 64 hrs
Results. The response consisted of parasite clearance in both patients, i.e.,
the clearance
rate at day 14 was 100%.
Example 8. Cellular studies in vitro. The effect of Bromine
Epiandrosterone~EPI, 16a,-
bromoepiandrosterone) on pentosephosphate shunt (PPS) activity in normal human
RBC was
examined using whole cells. Since glucose-6-phosphate dehydrogenase ("G6PD")
is the limiting
enzyme of the PPS, PPS flux measurement is considered to better reflect G6PD
activity in the
whole cell compared to G6PD activity measurement in a cell lysate. G6PD
activity measured in a
cell lysate is typically about 1100-fold higher than the PPS flux in whole
resting unstimulated RBC
(G6PD activity in cell lysate: 165; PPS flux 0.142 micromoles/hour/ml RBC).
PPS flux and G6PD
activity in the whole RBC depends on a number of factors (the concentration of
NADPH, NAD,
and ATP, and intracellular pH), which are kept constant if the measurement is
performed in the
lysate and may vary in the whole RBC. Levels of G6PD activity in cells is
considerably above
normal basal needs and inhibition of overall G6PD activity might have no or
minor consequence
on PPS flux in the whole cell. For example, RBC with the Mediterranean G6PD
mutant with about
1-3 percent residual activity compared with normal individuals have no
impairment in basal PPS
flux, but show impaired flux when flux through PPS is stimulated by methylene
blue addition. A
series of experiments were perfromed using varying amounts of EPI and PPS flux
was measured in
unstimulated basal RBC and in methylene-blue (MB)-stimulated RBC.
The data below shows PPS flux (micromoles/hour/ml RBC) in basal unstimulated,
and
MB-stimulated normal RBC. Different concentrations of EPI (0.3, 3.5 and 7
micromolar, final)
were supplemented to suspensions of washed RBC suspended in RPMI, pH 7.4 at
10% hematocrit,
whereby PPS flux was immediately measured without further incubation and
without further
washings. A minor inhibition of MB-stimulated PPS flux was observed with EPI
at 7 uM.
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PPS flux
control, unstimulated RBC 230
DMSO control, unstimulated ItBC 270
DMSO control, MB stimulated RBC 5090
' 0.3 uM EPI, unstimulated 250
0.3 p.M EPI, MB stimulated 5000
3.5 ~M EPI, unstimulated 270
3.5 ~M EPI, MB stimulated 4950
7 p.M EPI, unstimulated 295
7 ~M EPI, MB stimulated 4660
The data below shows average values of 3 experiments, where basal,
unstimulated, and
MB-stimulated PPS flux (micromoles/hour/ml RBC) was measured in normal RBC. In
these
experiments, different concentrations of EPI ~-0.8, 8 and 80 micromolar,
final) were supplemented
to suspensions of washed RBC suspended in R.PMI, pH 7.4 at 10% hematocrit.
After a 90-min
incubation at 37o C with and without EPI, PPS flux was measured. The results
showed a
dose-dependent inhibition of MB-stimulated PPS flux. Inhibition was 10% at 8
micromolar
(p=0.006 vs control+DMSO) and 25% at 80 micromolar (p=0.002 vs control+DMSO).
PPS flux
control, unstimulated RBC 430
control, MB stimulated RBC 5410
DMSO control, unstimulated ItBC 480
DMSO control, MB stimulated 4890
RBC
0.8 PM EPI, unstimulated 410
0.8 uM EPI, MB stimulated 4930
8 p.M EPI, unstimulated 450
8 ~M EPI, MB stimulated 4430
80 ~M EPI, unstimulated 450
80 uM EPI, MB stimulated 3660
Example 9. Inhibition of parasite growth. The effect of EPI (16a-bromo-
epiandrosterone) on parasite (Plasmodium falciparurn) growth was shown. EPI
was active at a
concentration of 1 ~M.
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Parasitemia after treatment
Time 24hrs 48hrs72hrs
0
control + DMSO 5% 5.40% 3.10% 5.20%
Epi 1 pM S% 5.70% 5,50% 1,60%
' Epi 10 EcM 5% 5,60% 0.90% 0
Epi 100 pM 5% 0 0 0
Epi 500 FtM 5% 0 0 0
control + DMSO2% 8.80% 11 %
8%
Epi 50 nM 2% 9.90% 9.20% 8.30%
Epi 1 EtM 2% 5.80% 6.10% 2.10%
Epi 2.5 uM 2% 7.30% 5.80% 3.20%
Epi 5 11.M 2% 5.40% 6% 1.80%
Epi 10 pM 2% 4.20% 3% 0
Epi 50 uM 2% 0 0 0
Parasitemias were determined by standard methods (microscopic inspection of at
least S00
cells, stained with Diff QuickTM (Baxter). Parasites were cultured under
standard conditions in
RPMI-1640 supplemented with Hepes/Glucose (10 mM), glutamine (0.3 g/liter) and
10% human
plasma. The hematocrit was 1 %.
Example 10. Stimulation of phagocytosis. The capacity of EPI (l6oc-bromo-
epiandrosterone) to influence phagocytosis of Plasmodium parasite infected RBC
is examined
using adherent human monocytes. The parasitemia level is about 8-10% and human
monocytes are
obtained from huffy coats from blood as follows. Peripheral blood mononuclear
cells are
separated from freshly collected platelet-poor huffy coats discarded from
blood samples of healthy
adult donors of both sexes. Separated cells are washed once with Juke-warm PBS
supplemented
with 10 mM glucose (PBS-G) and resuspended at 5 x 106 cells/mL in ice-cold
RPMI 1640 medium
supplemented with 23 mM NaHC03 and 25 mM Hepes, pH 7.4 (RMBH). Dynabeads M450
Pan B
and Pan T (Dynal) are added to cells in a 4:1 ratio for 20 min at 4oC. B-
lymphocytes and
T-lymphocytes are removed as specified by the manufacturer. The remaining
monocytes are
washed 2 times in RMBH, resuspended in AIM V cell culture medium (Gibco) at 1
x 106 cell/mL.
The monocyte layer is collected, washed with PBS-G at 37oC and resuspended in
AIM V medium
at 1 x 106 cells/mL. Purified cells are >90% monocytes as assessed by CD14
expression.
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Phagocytosis of opsonized parasitized RBC (PE) is determined as follows.
Phagocytosis
of fresh-serum opsonized PE is initiated by mixing 10 PE/monocyte. Suspensions
are briefly
centrifuged ( 150 x g for 5 sec at room temperature) to improve contact
between PE and monocytes.
To avoid attachement of monocytes after centrifugation and during the whole
incubation period,
cells are kept in
- suspension at S x 106 cells/5 mL AIM V medium in 6 cm diameter teflon bottom
dishes
(Heraeus) in a humidified incubator (95% air, 5% C02) at 37oC. On average, at
least 90%
of the monocytes phagocytose PE, as assessed by microscopic inspection.
Control cells are kept
under similar, conditions without phagocytosis. Quantitative assessment of
phagocytosis is
performed by a previously described bioluminescence method (E. Schwarzer, et
al., Br. J.
Haematol. 1994 88:740-745).
Erythrocyte treatments and parasite cultures are as follows. Fresh blood
(Ith+)~ts used to
isolate erythrocytes (RBC). Washed RBC are infected with schizont/trophozoite
parasite stages
(Palo Alto strain, mycoplasma-free). Stage specific parasites are isolated by
the Percoll-mannitol
method. Briefly, normal schizont-stage parasitized E (SPE) separated on
Percoll-mannitol
gradient (parasitemia > 95% SPE) are mixed with E suspended in growth medium
(RPMI 1640
medium containing 25 mmol/L Hepes, 20 mmol/L glucose, 2 mmol/L glutamine, 24
mmoUL
NaHC03, 32 mg/L gentamicin and 10% AB or A human serum, pH 7.30) to start
synchronous
cultures at selected hematocrit values. The inoculum parasitemia is adjusted
to 20% normal SPE
for isolation of ring parasitized ItBC (ItPE) and to 5% normal SPE for
isolation of
trophozoite-stage parasitized E (TPE). At 14-18 hours after inoculum parasites
are at ring-stage in
the first cycle; at 34-33 hours, parasites are at trophozoite-stage in the
first cycle; and at 40-44
hours after inoculum parasites are at schizont-stage in the first cycle. RPE,
TPE and SPE are
separated on Percoll-mannitol gradients. The parasitemia is usually 8-10% RPE,
and >95% TPE.
Nonparasitized and parasitized RBC are counted electronically. To assess total
parasitemia and
relative contribution of RPE, TPE and SPE, slides are prepared from cultures
at indicated times,
stained with Diff QuikTM parasite stain and 400-1000 cells are examined
microscopically.
The effect or EPI in parasitized RBC is examined using various concentrations
of EPI, e.g.,
0.5 ~M, I p,M, 10 p.M, 25 p.M and 50 ~M. Trophozoite-parasitized RBC, schizont-
parasitized
RBC or ring-parasitized RBC are examined as described.
Example 11. Human clinical trial. The clinical trial protocol that
incorporates about 15-
20 patients is established. For a phase I or I/II trial, the patients are
mildly infected with one or
more Plasmodium parasites and they are mildly symptomatic (less than about 8-
10% parasitemia
of RBC). Before treatment, the patients are optionally tested for infection
with HIV, HCV, TB,
and Cryptosporidium. Patients with one or morc co-infections are given
standard care for the
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coinfection. The patients are hospitalized for treatment for one week. Two or
more dose groups,
e.g., 25, 50 or 100 mg/day of l6oc-bromoepiandrosterone (BrEA), or an ester
thereof, administered
parenterally, e.g., by intramuscular or intravenous injection, on 3, 4 or S
days of the week when
patients are dosed. Dosing is on consecutive days or on an intermittent
schedule, e.g., 2, 3 or 4
doses with one dose administrered every other day. The formulation containing
BrEA is as
' described herein, e.g., the formulation of example 1 or 2. At day S-7, if
less than about 50%
reduction in parasitemia is observed, the patients are given standard care for
malaria (mefloquine).
During the week of treatment and for 1, 2 3, or more weeks theerafter, blood
samples are taken
periodically for evaluation of parasitemia, pharmacokinetics, plasma cytokines
(e.g., IL-2, IL-4,
IL-10, IGF1, yIFN, GM-CSF), and intracellular cytokines (e.g., IL-2, IL-4, IL-
10, IGFI, yIFN,
GM-CSF). The patients are optionally treated again at about 2 to 12 weeks
after the initial dosing,
using the same or a similar protocol as that used in the initial dosing
protocol.
To the extent not already indicated, it will be understood by those of
ordinary skill in the
art that any one of the various specific embodiments herein described and
illustrated may be
further modified to incorporate features shown in other of the specific
embodiments.
The foregoing detailed description has been provided for a better
understanding of the
invention only and no unnecessary limitation should be understood therefrom as
some
modifications will be apparent to those skilled in the art without deviating
from the spirit and scope
of the appended claims.
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