U.S. patent application number 12/897459 was filed with the patent office on 2011-04-14 for tetracycline compounds for treatment of cryptosporidium parvum related disorders.
This patent application is currently assigned to Trustees of Tufts College. Invention is credited to Stuart B. Levy, Mark L. Nelson.
Application Number | 20110086821 12/897459 |
Document ID | / |
Family ID | 22652851 |
Filed Date | 2011-04-14 |
United States Patent
Application |
20110086821 |
Kind Code |
A1 |
Levy; Stuart B. ; et
al. |
April 14, 2011 |
Tetracycline Compounds for Treatment of Cryptosporidium Parvum
Related Disorders
Abstract
Methods and compositions for treating Cryptosporidium parvum
related disorders in a mammal are discussed. Several novel
tetracycline compounds useful for treating Cryptosporidium parvum
related disorders are also included.
Inventors: |
Levy; Stuart B.; (Boston,
MA) ; Nelson; Mark L.; (Wellesley, MA) |
Assignee: |
Trustees of Tufts College
Medford
MA
|
Family ID: |
22652851 |
Appl. No.: |
12/897459 |
Filed: |
October 4, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11728346 |
Mar 26, 2007 |
7807660 |
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12897459 |
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10982728 |
Nov 4, 2004 |
7202235 |
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11728346 |
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09768189 |
Jan 23, 2001 |
6833365 |
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10982728 |
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60178519 |
Jan 24, 2000 |
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Current U.S.
Class: |
514/152 ; 549/25;
552/203; 552/205 |
Current CPC
Class: |
A61P 1/12 20180101; C07C
2601/10 20170501; C07C 237/26 20130101; A61K 31/65 20130101; Y02A
50/488 20180101; A61P 33/00 20180101; Y02A 50/30 20180101; A61P
33/02 20180101 |
Class at
Publication: |
514/152 ;
552/203; 552/205; 549/25 |
International
Class: |
A61K 31/65 20060101
A61K031/65; C07C 237/26 20060101 C07C237/26; C07D 335/04 20060101
C07D335/04; A61P 33/00 20060101 A61P033/00 |
Claims
1. A method for controlling Cryptosporidium parvum in a mammal,
comprising administering to said mammal an effective amount of a
tetracycline compound, such that Cryptosporidium parvum is
controlled in said mammal.
2. A pharmaceutical composition comprising an effective amount of a
tetracycline compound to treat a Cryptosporidium parvum related
disorder in a mammal and a pharmaceutically acceptable carrier.
3. A compound selected from the group consisting of: ##STR00016##
and pharmaceutically acceptable salts thereof.
4. The compound of claim 3, wherein said compound is: ##STR00017##
or a pharmaceutically acceptable salt thereof.
5. The compound of claim 3, wherein said compound is: ##STR00018##
or a pharmaceutically acceptable salt thereof.
6. The compound of claim 3, wherein said compound is: ##STR00019##
or a pharmaceutically acceptable salt thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/728,346, filed Mar. 26, 2007; which is a
continuation of U.S. patent application Ser. No. 10/982,728, filed
Nov. 4, 2004; which is a continuation of U.S. patent application
Ser. No. 09/768,189, filed Jan. 23, 2001; which claims priority to
U.S. Provisional Application No. 60/178,519, filed on Jan. 24,
2000; the entire contents of each of the aforementioned
applications are hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Cryptosporidium parvum (or C. parvum) is an enteric protozoa
of the phylum Apicomplexa. It is a major cause of diarrhea in
humans and certain domestic animals (Tzipori, Advances in
Parasitology (1988) 27:63-129). It is responsible for sporadic
cases and major waterborne outbreaks of self-limiting diarrhea in
immunocompetent humans (Current, W. L. et al., Clinical
Microbiology Reviews, (1991) 4:325). C. parvum is one of several
important opportunistic infections (OI) associated with diarrhea
and wasting in patients with AIDS. Depending on location in the
United States, some 10 to 15% of individuals with AIDS contract the
disease (Peterson, Clinical Infectious Diseases, (1992) 15:903).
The infection in the immunodeficient host often becomes persistent,
causing life-threatening, profound, unremitting watery diarrhea and
wasting. A prolonged course of infection often leads to a spread of
infection into the hepatobiliary (HB) tract causing serious
complications (Flanigan, Progress in Clinical Parasitology (1993)
3:1). Of the OI affecting patients with AIDS, C. parvum is one of
only a few infections against which there is no consistently
effective treatment. There had been only a few reports of
successful treatment of individual AIDS patients with hyperimmune
bovine colostrums (Tzipori, Lancet. (1986) ii:344; Ungar,
Gastroenterology (1990) 98:486) and with paromomycin (PRM)
(Fitchenbaum, Clinical Infectious Diseases (1993) 16:298). Since
none of the available antimicrobial agents are consistently
effective, a search for novel therapeutic agents against C. parvum
is necessary. With increased survival time of patients with AIDS
due to much improved patient care, the incidence of the disease in
this population is likely to continue to rise.
[0003] The lifecycle of C. parvum is similar to that of other
coccidian which infect mammals. The lifecycle can be divided into
six major developmental events (Current, Journal of Protozoology,
(1986) 33:98); excystation, the release of infective sporozoites;
merogony, the asexual multiplication within host cells; gametogony,
the formation of micro and macrogametes; fertilization, the union
of micro and macrogametes; oocyst wall formation, to produce an
environmentally resistant stage that transmits infection from one
host to another; and sporogony, the formation of infective
sporozoites within the oocyst wall. Each intracellular stage of C.
parvum resides within a parasitophorous vacuole confined to the
microvillous region of the host cell, whereas comparable stages of
Toxoplasma gondii, Eimeria, or Isopora to which C. parvum is
closely related, occupy parasitophorous vacuoles deep within the
host cytoplasm. Oocysts of C. parvum undergo sporogony while they
are within the host cells and are infective when released in the
feces. Approximately 20% of the oocysts of C. parvum are thin
walled and discharge their sporozoites within the lumen of the same
host, while 80% form a thick two-layered environmentally resistant
oocyst wall, and are discharged in the feces. The four sporozoites
emerging from the thin-walled oocysts and repeated cycles of
schizogeny contribute to the persistence of the infection in the
immunodeficient host known as autoinfection.
SUMMARY OF THE INVENTION
[0004] In one embodiment, the invention pertains to a method for
controlling Cryptosporidium parvum in a mammal, by administering to
the mammal an effective amount of a tetracycline compound. Examples
of tetracycline compounds of the invention include compounds of
formula I:
##STR00001##
[0005] wherein: [0006] X is CHC(R.sup.13Y'Y), CHR.sup.6, S,
NR.sup.6, or O; [0007] R.sup.2, R.sup.4 and R.sup.4' are each
hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety; [0008] R.sup.2',
R.sup.3, R.sup.10, R.sup.11 and R.sup.12 are each hydrogen or a
pro-drug moiety; [0009] R.sup.5 is hydroxy, hydrogen, thiol,
alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,
alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, or an arylalkyl; [0010] R.sup.6, R.sup.7, R.sup.8 and
R.sup.9 are each independently hydrogen, hydroxyl, halogen, thiol,
alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; [0011] R.sup.13 is
hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; [0012]
Y' and Y are each independently hydrogen, halogen, hydroxyl, cyano,
sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; [0013]
and pharmaceutically acceptable salts thereof.
[0014] The invention also pertains to a method for treating a
Cryptosporidium parvum related disorder in a mammal, by
administering to the mammal an effective amount of a tetracycline
compound. In an embodiment, the tetracycline compound is of formula
(I). In another advantageous embodiment, the mammal is
immunocompromised, e.g., suffering from AIDS or undergoing
chemotherapy. Preferably, the mammal is a human.
[0015] In another embodiment, the invention pertains to
pharmaceutical compositions containing an effective amount of a
tetracycline compound to treat a Cryptosporidium parvum related
disorder in a mammal and a pharmaceutically acceptable carrier.
[0016] In yet another embodiment, the invention features a
tetracycline compound of the formula:
##STR00002##
DETAILED DESCRIPTION OF THE INVENTION
[0017] The invention pertains, at least in part to methods for
controlling Cryptosporidium parvum in a mammal, by administering to
the mammal an effective amount of a tetracycline compound.
[0018] Cryptosporidium is a coccidian protozoan parasite that has
gained much attention in the last 20 years as a clinically
important human pathogen. For several decades, Cryptosporidium was
thought to be a rare, opportunistic animal pathogen, but the first
case of human cryptosporidiosis in 1976 involved a 3-year-old girl
from rural Tennessee who suffered severe gastroenteritis for two
weeks (Flanigan Prog Clin Parasitol (1993) 1). Electron microscopic
examination of the intestinal mucosa led to the discovery that
Cryptosporidium parvum was the infectious species in humans. In the
early 1980s, the strong association between cases of
cryptosporidiosis and immunodeficient individuals (such as those
with AIDS--acquired immunodeficiency syndrome) brought
Cryptosporidium to the forefront as a ubiquitous human pathogen.
Presently, the increasing population of immunocompromised patients
and the various outbreaks of cryptosporidiosis through infection by
water-borne Cryptosporidium oocysts (often in drinking water) have
created world wide interest in this pathogen. Unlike other
intestinal pathogens, Cryptosporidium can infect several different
hosts, can survive most environments for long periods of time
(Keusch, et al. Schweiz Med Wochenschr, (1995) 125(18):899), and
inhabit all climates and locales.
[0019] The terms "tetracycline" or "tetracycline derivative"
compounds include tetracycline and other tetracycline family
members such as, chlortetracycline, oxytetracycline,
demeclocycline, methacycline, doxycycline, minocycline, and
sancycline. Additional tetracycline compounds can be found, for
example, in U.S. patent application Ser. No. 09/234,847, and U.S.
Pat. Nos. 5,834,450; 5,532,227; 5,789,395; 5,639,742 and German
patents DE 28 14.974 and DE 28 20 983. The entire contents of the
aforementioned applications and patents are hereby expressly
incorporated herein by reference.
[0020] More recent research efforts have focused on developing new
tetracycline antibiotic compositions effective under varying
therapeutic conditions and routes of administration; and for
developing new tetracycline analogues which might prove to be equal
or more effective than the originally introduced tetracycline
families beginning in 1948. Representative of such developments
include U.S. Pat. Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331;
3,062,717; 3,557,280; 4,018,889; 4,024,272; 4,126,680; 3,454,697;
and 3,165,531. These issued patents are merely representative of
the range of diversity of investigations seeking tetracycline and
tetracycline analogue compositions which are pharmacologically
active, and the contents of each are expressly incorporated by
reference.
[0021] Historically, soon after their initial development and
introduction, the tetracyclines, regardless of specific formulation
or chemical structure, were found to be highly effective
pharmacologically against rickettsiae, a number of gram-positive
and gram-negative bacteria, and the agents responsible for
lymphogranuloma venereum, including conjunctivitis, and
psittacosis. Hence, tetracyclines became known as "broad spectrum"
antibiotics. With the subsequent establishment of their in vitro
antimicrobial activity, effectiveness in experimental infections,
and pharmacological properties, the tetracyclines as a class
rapidly became widely used for therapeutic purposes. However, this
widespread use of tetracyclines for both major and minor illnesses
and diseases led directly to the emergence of resistance to these
antibiotics even among highly susceptible bacterial species both
commensal and pathogenic (e.g., pneumococci and Salmonella). The
rise of tetracycline-resistant organisms has resulted in a general
decline in use of tetracyclines and tetracycline analogue
compositions as antibiotics of choice.
[0022] In one embodiment, the tetracycline compound of the
invention inhibits more than 70% of Cryptosporidium parvum at a
concentration less than 100 .mu.g/ml, less than 50 .mu.g/ml, less
than 20 .mu.g/ml, less than 10 .mu.g/ml, or less than 1 .mu.g/ml.
The inhibition of Cryptosporidium parvum can be tested using the
assay described in Example 2.
[0023] Tetracycline compounds of the invention include compounds of
Formula I:
##STR00003##
[0024] wherein: [0025] X is CHC(R.sup.13Y'Y), CHR.sup.6, S,
NR.sup.6, or O; [0026] R.sup.2, R.sup.4 and R.sup.4' are each
hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,
heterocyclic, heteroaromatic or a prodrug moiety; [0027] R.sup.2',
R.sup.3, R.sup.10, R.sup.11 and R.sup.12 are each hydrogen or a
pro-drug moiety; [0028] R.sup.5 is hydroxy, hydrogen, thiol,
alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,
alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,
alkylamino, or an arylalkyl; [0029] R.sup.6, R.sup.7, R.sup.8 and
R.sup.9 are each independently hydrogen, hydroxyl, halogen, thiol,
alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, or an arylalkyl; [0030] R.sup.13 is
hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; [0031]
Y' and Y are each independently hydrogen, halogen, hydroxyl, cyano,
sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulfinyl, alkylsulfonyl, alkylamino, or arylalkyl; and
pharmaceutically acceptable salts thereof.
[0032] In one further embodiment, R.sup.2', R.sup.3, R.sup.10,
R.sup.11, and R.sup.12 are each hydrogen or a prodrug moiety.
Furthermore, R.sup.4 and R.sup.4' can be alkyl, e.g., lower alkyl,
e.g., methyl, ethyl, propyl, or butyl. In another embodiment,
R.sup.5 is hydroxyl, hydrogen, or alkanoyl, e.g., an ester,
advantageously, a propanoic ester. In yet another embodiment, X is
S or CHR.sup.6. Examples of R.sup.6 include alkyl groups, e.g.,
methyl, ethyl, propyl, or halogens or hydroxyl groups.
Advantageously, R.sup.6 may comprise a heteroatom, such as, for
example, a sulfur atom. For example, R.sup.6 may be a thioether,
e.g., a cyclopentylthio ether. Advantageous examples of R.sup.9
include hydrogen atoms, and alkyl (e.g., t-butyl) and alkenyl
(e.g., cyclopentenyl) groups.
[0033] Tetracycline compounds of the invention include, for
example, compounds of the formulae:
##STR00004## ##STR00005##
[0034] Other examples of preferred tetracycline compounds of the
invention include, for example,
5-propionyl-6-cyclopentylsulfanylmethyl doxycycline;
thiatetracycline; 9-cyclopent-1-enyl-doxycycline;
5-propionyl-9-tert-butyl-doxycycline; doxycycline; 9-tert-butyl
doxycycline; 9-cyclohex-1-enylethynyl minocycline; and
6-cyclopentylsulfanylmethyl doxycycline.
[0035] The tetracycline compounds of the invention can be
synthesized using the methods described in Example 1. Scheme 1
depicts a general synthesis of a thiol ether from methacycline.
##STR00006##
[0036] 13-substituted thiols can be synthesized by the method
outlined in Scheme 1, above. The synthesis of the compounds is
described in greater detail in Example 1. Generally, 13-substituted
thiol ethers (1B) can be synthesized by heating a tetracycline salt
(such as methacycline hydrochloride, 1A), AIBN
(2,2'-azobisisobutyronitrile), and a thiol in ethanol at reflux for
six hours under an inert atmosphere.
[0037] 9-substituted tetracyclines such as 9-cyclopentenyl
doxycycline can be synthesized by the method shown in Scheme 2. As
in Scheme 2,9-substituted tetracycline compounds can be synthesized
by treating a tetracycline compound (e.g., doxycycline, 2A), with
sulfuric acid and sodium nitrate. The resulting product is a
mixture of the 7-nitro and 9-nitro isomers (2B and 2C,
respectively). The 7-nitro (2B) and 9-nitro (2C) derivatives are
treated by hydrogenation using hydrogen gas and a platinum catalyst
to yield amines 2D and 2E. The isomers are separated at this time
by conventional methods. To synthesize 9-substituted alkenyl
derivatives, the 9-amino tetracycline compound (2E) is treated with
HONO, to yield the diazonium salt (2F). The salt (2F) is treated
with an appropriate halogenated reagent (e.g., R.sup.9Br, wherein
R.sup.9 is an aryl, alkenyl, or alkynyl moiety) to yield the
desired compound (e.g., in Scheme 2,9-cyclopent-1-enyl doxycycline
(2G)).
##STR00007##
[0038] The term "alkenyl" includes unsaturated aliphatic groups,
including straight-chain alkenyl groups, branched-chain alkenyl
groups, cycloalkenyl (alicyclic) groups, alkenyl substituted
cycloalkyl or cycloalkenyl groups, and cycloalkenyl substituted
alkyl or alkenyl groups. The term alkenyl further includes alkenyl
groups, which can further include oxygen, nitrogen, sulfur or
phosphorous atoms replacing one or more carbons of the hydrocarbon
backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In
preferred embodiments, a straight chain or branched chain alkenyl
group has 10 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.10 for straight chain, C.sub.3-C.sub.10 for branched
chain), and more preferably 6 or fewer. Likewise, preferred
cycloalkenyl groups have from 4-7 carbon atoms in their ring
structure, and more preferably have 5 or 6 carbons in the ring
structure, e.g., cyclopentene or cyclohexene.
[0039] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups, branched-chain alkyl groups,
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups,
and cycloalkyl substituted alkyl groups. The term alkyl further
includes alkyl groups, which can further include oxygen, nitrogen,
sulfur or phosphorous atoms replacing one or more carbons of the
hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous
atoms. In preferred embodiments, a straight chain or branched chain
alkyl has 10 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.10 for straight chain, C.sub.3-C.sub.10 for branched
chain), and more preferably 6 or fewer. Likewise, preferred
cycloalkyls have from 4-7 carbon atoms in their ring structure, and
more preferably have 5 or 6 carbons in the ring structure.
[0040] Moreover, the term alkyl includes both "unsubstituted
alkyls" and "substituted alkyls", the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. It will be understood by those
skilled in the art that the moieties substituted on the hydrocarbon
chain can themselves be substituted, if appropriate. Cycloalkyls
can be further substituted, e.g., with the substituents described
above. An "alkylaryl" moiety is an alkyl substituted with an aryl
(e.g., phenylmethyl (benzyl)).
[0041] The term "aryl" includes aryl groups, including 5- and
6-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, benzoxazole, benzothiazole, triazole,
tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,
and the like. Aryl groups also include polycyclio-fused aromatic
groups such as naphthyl, quinolyl, indolyl, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles", "heteroaryls" or
"heteroaromatics". The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, as
for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano,
amino (including alkyl amino, dialkylamino, arylamino, diarylamino,
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,
azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety. Aryl groups can also be fused or bridged with alicyclic or
heterocyclic rings which are not aromatic so as to form a polycycle
(e.g., tetralin).
[0042] The terms "alkenyl" and "alkynyl" include unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond, respectively. Examples of substituents of alkynyl
groups include, for example alkyl, alkenyl (e.g., cycloalkenyl,
e.g., cyclohenxenyl), and aryl groups.
[0043] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to three carbon atoms in its backbone structure.
Likewise, "lower alkenyl" and "lower alkynyl" have similar chain
lengths.
[0044] The terms "alkoxyalkyl", "polyaminoalkyl" and
"thioalkoxyalkyl" include alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or
sulfur atoms.
[0045] The terms "polycyclyl" or "polycyclic radical" refer to two
or more cyclic rings (e.g., cycloalkyls, cycloalkenyls;
cycloalkynyls, aryls and/or heterocyclyls) in which two or more
carbons are common to two adjoining rings, e.g., the rings are
"fused rings". Rings that are joined through non-adjacent atoms are
termed "bridged" rings. Each of the rings of the polycycle can be
substituted with such substituents as described above, as for
example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate, phosphonato, phosphinato, cyano, amino (including alkyl
amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
[0046] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0047] The term "alkylsulfinyl" include groups which have one or
more sulfinyl (SO) linkages, typically 1 to about 5 or 6 sulfinyl
linkages. Advantageous alkylsulfinyl groups include groups having 1
to about 12 carbon atoms, preferably from 1 to about 6 carbon
atoms.
[0048] The term "alkylsulfonyl" includes groups which have one or
more sulfonyl (SO.sub.2) linkages, typically 1 to about 5 or 6
sulfonyl linkages. Advantageous alkylsulfonyl groups include groups
having 1 to about 12 carbon atoms, preferably from 1 to about 6
carbon atoms.
[0049] The term "alkanoyl" includes groups having 1 to about 4 or 5
carbonyl groups. The term "aroyl" includes aryl groups, such as
phenyl and other carbocyclic aryls, which have carbonyl
substituents. The term "alkaroyl" includes aryl groups with
alkylcarbonyl substituents, e.g., phenylacetyl.
[0050] The structures of some of the tetracycline compounds of this
invention include asymmetric carbon atoms. The isomers arising from
the chiral atoms (e.g., all enantiomers and diastereomers) are
included within the scope of this invention, unless indicated
otherwise. Such isomers can be obtained in substantially pure form
by classical separation techniques and by stereochemically
controlled synthesis.
[0051] The invention also pertains to a pharmaceutical composition
containing an effective amount of a tetracycline compound to treat
or prevent a Cryptosporidium parvum related disorder in a mammal
and a pharmaceutically acceptable carrier. The pharmaceutical
composition may contain an effective amount of a supplementary
anti-Cryptosporidium parvum agent.
[0052] The language "pharmaceutically acceptable carrier" includes
substances capable of being coadministered with the tetracycline
compound(s), and which allows the tetracycline compounds to perform
their intended function, e.g., treating a Cryptosporidium parvum
related disorder or preventing a Cryptosporidium parvum related
disorder. Examples of such carriers include solutions, solvents,
dispersion media, delay agents, emulsions and the like. The use of
such media for pharmaceutically active substances are well known in
the art. Any other conventional carrier suitable for use with the
tetracycline compounds of the present invention are included.
[0053] For example, one or more compounds of the invention may be
administered alone to a subject, or more typically a compound of
the invention will be administered as part of a pharmaceutical
composition in mixture with conventional excipient, i.e.,
pharmaceutically acceptable organic or inorganic carrier substances
suitable for parenteral, oral or other desired administration and
which do not deleteriously react with the active compounds and are
not deleterious to the recipient thereof. Suitable pharmaceutically
acceptable carriers include but are not limited to water, salt
solutions, alcohol, vegetable oils, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
petroethral fatty acid esters, hydroxymethylcellulose,
polyvinylpyrrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously react with the active compounds.
[0054] At least many of the tetracycline compounds of the invention
suitably may be administered to a subject in a protonated and
water-soluble form, e.g., as a pharmaceutically acceptable salt of
an organic or inorganic acid, e.g., hydrochloride, sulfate,
hemi-sulfate, phosphate, nitrate, acetate, oxalate, citrate,
maleate, mesylate, etc. Also, where an appropriate acidic group is
present on a compound of the invention, a pharmaceutically
acceptable salt of an organic or inorganic base can be employed
such as an ammonium salt, or salt of an organic amine, or a salt of
an alkali metal or alkaline earth metal such as a potassium,
calcium or sodium salt.
[0055] Therapeutic compounds can be administered to a subject in
accordance with the invention by any of a variety of routes.
Topical (including transdermal, buccal or sublingual), and
parenteral (including intraperitoneal, subcutaneous, intravenous,
intradermal or intramuscular injection) are generally
preferred.
[0056] For parenteral application, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Therapeutic compounds will be formulated in sterile form in
multiple or single dose formats such as being dispersed in a fluid
carrier such as sterile physiological saline or 5% saline dextrose
solutions commonly used with injectables.
[0057] For enteral application, particularly suitable are tablets,
dragees or capsules having talc and/or carbohydrate carrier binder
or the like, the carrier preferably being lactose and/or corn
starch and/or potato starch. A syrup, elixir or the like can be
used wherein a sweetened vehicle is employed. Sustained release
compositions can be formulated including those wherein the active
component is protected with differentially degradable coatings,
e.g., by microencapsulation, multiple coatings, etc.
[0058] For topical applications, the tetracycline compound(s) can
be suitably admixed in a pharmacologically inert topical carrier
such as a gel, an ointment, a lotion or a cream. Such topical
carriers include water, glycerol, alcohol, propylene glycol, fatty
alcohols, triglycerides, fatty acid esters, or mineral oils. Other
possible topical carriers are liquid petrolatum,
isopropylpalmitate, polyethylene glycol, ethanol 95%,
polyoxyethylene monolauriate 5% in water, sodium lauryl sulfate 5%
in water, and the like. In addition, materials such as
anti-oxidants, humectants, viscosity stabilizers and the like also
may be added if desired.
[0059] The actual preferred amounts of active compounds used in a
given therapy will vary according to the specific compound being
utilized, the particular compositions formulated, the mode of
application, the particular site of administration, etc. Optimal
administration rates for a given protocol of administration can be
readily ascertained by those skilled in the art using conventional
dosage determination tests conducted with regard to the foregoing
guidelines.
[0060] In general, compounds of the invention for treatment can be
administered to a subject in dosages used in prior tetracycline
therapies. See, for example, the Physicians' Desk Reference. For
example, a suitable effective dose of one or more compounds of the
invention will be in the range of from 0.01 to 100 milligrams per
kilogram of body weight of recipient per day, preferably in the
range of from 0.1 to 50 milligrams per kilogram body weight of
recipient per day, more preferably in the range of 1 to 20
Milligrams per kilogram body weight of recipient per day. The
desired dose is suitably administered once daily, or several
sub-doses, e.g. 2 to 5 sub-doses, are administered at appropriate
intervals through the day, or other appropriate schedule.
[0061] It will also be understood that normal, conventionally known
precautions will be taken regarding the administration of
tetracyclines generally to ensure their efficacy under normal use
circumstances. Especially when employed for therapeutic treatment
of humans and animals in vivo, the practitioner should take all
sensible precautions to avoid conventionally known contradictions
and toxic effects. Thus, the conventionally recognized adverse
reactions of gastrointestinal distress and inflammations, the renal
toxicity, hypersensitivity reactions, changes in blood, and
impairment of absorption through aluminum, calcium, and magnesium
ions should be duly considered in the conventional manner.
[0062] The language "effective amount" of the tetracycline compound
is that amount necessary or sufficient to control Cryptosporidium
parvum in a mammal, e.g., prevent the various morphological and
somatic symptoms of a Cryptosporidium parvum-related disorder. The
effective amount can vary depending on such factors as the size and
weight of the subject, the type of illness, or the particular
tetracycline compound. For example, the choice of the tetracycline
compound can affect what constitutes an "effective amount". One of
ordinary skill in the art would be able to study the aforementioned
factors and make the determination regarding the effective amount
of the tetracycline compound without undue experimentation. An in
vivo assay as described in Example 4 below or an assay similar
thereto (e.g., differing in choice of cell line or type of illness)
also can be used to determine an "effective amount" of a
tetracycline compound. The ordinarily skilled artisan would select
an appropriate amount of a tetracycline compound for use in the
aforementioned in vivo assay. Preferably, the effective amount of
the tetracycline is effective to treat a mammal suffering from a
Cryptosporidium parvum related disorder.
[0063] The term "mammal" includes animals which are capable of
having a Cryptosporidium parvum related disorder. Examples of
mammals include, but are not limited to, ruminants (e.g., cattle
and goats), mice, rats, hamsters, dogs, cats, horses, pigs, sheep,
lions, tigers, bears, monkeys, chimpanzees, and, in a preferred
embodiment, humans. The mammal may be immunocompetent or
immunocompromised, e.g., suffering from an immunodeficiency. For
example, the mammal may have AIDS or may have previously or
concurrently undergone chemotherapy. In another embodiment, the
mammal may be elderly or young. The mammal may or may not be
suffering from a Cryptosporidium parvum related disorder. The
tetracycline compounds may be administered to a mammal susceptible
to a Cryptosporidium parvum disorder to prevent the occurrence of
the disorder.
[0064] The language "Cryptosporidium parvum related disorder"
includes disorders which are related to the infection or the
presence of Cryptosporidium parvum in a mammal. Examples of
Cryptosporidium parvum related disorders include diarrhea and
cryptosporidiosis.
[0065] In another embodiment, the invention pertains to a method
for treating a Cryptosporidium parvum related disorder in a mammal,
by administering to the mammal an effective amount of a
tetracycline compound such that said mammal is treated for the
disorder.
[0066] In a further embodiment, the invention includes the
administration of a supplementary anti-Cryptosporidium parvum agent
in combination with the tetracycline compound of the invention.
[0067] The language "in combination with" includes simultaneous
administration of the tetracycline compound of the invention and
the supplementary anti-Cryptosporidium parvum agent, administration
of the agent first, followed by the tetracycline compound and
administration of the tetracycline compound first, followed by the
agent. The invention also includes the administration of other
therapeutic agents in combination with the tetracycline compounds
of the invention. For example, the tetracycline compounds of the
invention may be administered in combination with drugs used in
AIDS therapy for AIDS patients.
[0068] The term "supplementary agent" includes compounds known in
the art to have anti-Cryptosporidium parvum activity such as, for
example, paromomycin and derivatives thereof.
[0069] The present invention is further illustrated by the
following examples. These examples are provided to aid in the
understanding of the invention and are not to be construed as
limitations thereof.
EXEMPLIFICATION OF THE INVENTION
Example 1
Synthesis of Tetracycline Compounds
[0070] The following example discusses methods of synthesizing the
tetracycline compounds of the invention.
Experimental
[0071] Melting points were taken on a Mel-Temp capillary melting
point apparatus and are uncorrected. Nuclear magnetic resonance
(.sup.1H NMR) spectra were recorded at 300 MHz on a Bruker Avance
spectrometer. The chemical shift values are expressed in .delta.
values (ppm) relative to tetramethylsilane or
3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt, as either
an internal or external standard using CDCl.sub.3, DMSO-d.sub.6, or
MeOH-d.sub.4 as the solvent. Column chromatography was performed
according to the method of Still using Baker "flash" grade silica
gel (40 .mu.m) that was treated with a saturated solution of
Na.sub.2EDTA, washed with water, filtered and dried in an oven at
130.degree. C. for three hours prior to use. Analytical TLC
separations employed the use of 0.25 mm silica gel plates with
florescence indicator obtained from J.T. Baker Chemical Co.,
Phillipsburg, N.J., that were pretreated by immersion into a
saturated solution of Na.sub.2EDTA for five minutes and reactivated
at 130.degree. C. for three hours. Solvent systems used were as
follows: 50:50:5 CHCl.sub.3/MeOH/5% Na.sub.2EDTA (lower phase) (1),
65:20:5, CHCl.sub.3/MeOH/Na.sub.2EDTA (lower phase) (II).
Visualization of TLC was accomplished by 0.5% aqueous Fast Blue BB
salt and heating at 130.degree. C. for 5 minutes. Analytical HPLC
was performed on a Waters Bondapak C18 reverse phase column by
using two Varian SD 100 HPLC pumps at a 1.6 mL/min flow rate
controlled by software. Detection was by UV absorption with Model
441 absorbance detector operating at 280 nm. Mobile phases used
followed a linear gradient from 30% to 100% methanol over 30
minutes at 1.6 mL/min flow rate followed by isocratic elution with
MeOH; solvent system A: 0.02 M Na.sub.2HPO.sub.4+0.001 M
Na.sub.2EDTA adjusted to pH 4.5 with H.sub.3PO.sub.3; solvent
system B: 100% MeOH. Semipreparative HPLC separations used a Waters
semipreparative C18 reverse-phase column at a flow rate of 6.4
mL/min. Low and high resolution mass spectra were performed on a PE
Mariner spectrometer (Nelson et al., J. Med. Chem. (1993)
36(3):374).
General Procedure for the Synthesis of 13-[(Substituted
phenyl)thio]-5-hydroxy-6-deoxytetracyclines
13-(Phenylthio)-5-hydroxy-6-.alpha.-deoxytetracycline
[0072] A mixture of methacycline hydrochloride (3.0 g, 6.2 mmol),
AIBN (250 mg), and thiophenol (1.32 g, 12.4 mmol) in ethanol (50
mL) was heated at reflux for 6 hours while under N.sub.2. The
reaction mixture was cooled, filtered to remove insolubles, and
concentrated to one-fifth volume under reduced pressure.
Precipitation of the resulting solution in cold Et.sub.2O led to
isolation of crude-product (2.17 g). The solid was dissolved in hot
H.sub.2O, and extracted into CHCl.sub.3 at pH 5.0. Removal of the
solid and treatment with activated charcoal in MeOH led to
isolation of the product (0.958 g, 27.1%): mp=164-171.degree. C.;
TLC R.sub.f=0.67 (I); HPLC t.sub.R=14.45 min; .sup.1H NMR
(CDCl.sub.3) .delta. 11.9 (br s), 9.3 (br s), 7.35 (m, 6H), 6.83
(d, 1H), 6.74 (d, 1H) 5.95 (br s, 1H), 4.10 (br s, 1H), 3.82 (s,
2H), 3.60 (br s, 1H), 3.10 (m, 2H), 2.60 (m, 1H), 2.48 (s, 6H); MS
(FAB) m/z 552, 553 ([M+H]+); 445 (M-C.sub.6H.sub.5--S+H).
13-(Cyclopentylthio)-5-hydroxy-6-.alpha.-deoxytetracycline
[0073] Methacycline hydrochloride (5.0 g, 10.4 mmol) was placed in
a round-bottomed flask and suspended in 100 mL of ethanol. Twenty
milliliters of cyclopentanethiol (0.0270 mol) and AIBN (250 mg)
were added, and the reaction mixture was refluxed with stirring for
12 hours while under N.sub.2. The mixture was reduced to one-fifth
volume by distillation and filtered. The filtrate was dripped
slowly into cold Et.sub.2O with stirring, resulting in the
formation of a yellow precipitate. The compound was purified
further by either column chromatography on EDTA-silica, by
extraction at pH 4.5 into CH.sub.2Cl.sub.2, or by HPLC
chromatography. An analytical sample was produced by HPLC as a
yellow solid in moderate yield (28.3%). Higher yields were obtained
by the extraction method and treatment with activated charcoal in
MeOH (32.1%). mp=132-139.degree. C.; TLC R.sub.f=0.80 (I); HPLC
t.sub.R=21.19 min; .sup.1H NMR (MeOH-d.sub.4) .delta. 7.38 (t, 1H),
7.02 (d, 1H), 6.72 (d, 1H), 4.10 (s, 2H), 2.70 (br s, 6H),
1.81-2.01 (br m, 2H), 1.28-1.75 (br m, 6H); HRMS (FAB) calculated
for C.sub.27H.sub.32N.sub.2O.sub.8S 545.1957 (M+1), found 545.1960
(M+1).
[4S-(4.alpha.,12a.alpha.)]-9-(nitro)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12-
a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthaceneca-
rboxamide
[0074] To an ice cold solution of 1.0 g of doxycycline
hydrochloride in 10 ml of concentrated sulfuric acid was added
0.231 g of potassium nitrate. The reaction was stirred for 1 hour
under ambient atmosphere. The mixture was then poured over 150 g of
ice and the resulting solid was extracted with n-butanol and dried
to afford 0.9 g of the desired product as a yellow-green solid. MS
(FAB): m/z 490 (M+H). .sup.1H NMR (CD.sub.3OD): .delta. 7.50 (d,
1H, J=8.07 Hz, H-8); 6.86 (d, 1H, J=8.07 Hz, H-7); 4.44 (bs, 1H,
H-4); 3.62 (dd, 1H, J=11.42; 8.35 Hz, H-5); 2.95 (bs, 6H,
NMe.sub.2); 2.81 (d, 1H, J=11.45 Hz, H-4a); 2.71 (dq, 1H, J=12.41;
6.5 Hz, H-6); 2.53 (dd, 1H, J=12.23; 8.20 Hz, H-5a); 1.51 (d, 3H,
J=6.78 Hz, CH.sub.3).
[4S-(4.alpha.,12a.alpha.)]-9-(amino)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12-
a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthaceneca-
rboxamide
[0075] Into a 200 ml hydrogenation bottle is added 1.0 g of
compound 1, 40 ml of methanol, 1 ml of concentrated HCl, and 100 mg
of 10% palladium on carbon. Using a hydrogenation apparatus, the
mixture is subjected to 30 psi of hydrogen for 3 hours. The
catalyst is filtered off and the filtrate is dried to afford 0.9 g
of the dihydrochloride as a yellow solid. MS (FAB): m/z 460 (M+H).
.sup.1H NMR (CD.sub.3OD): .delta. 7.54 (d, 1H, J=8.08 Hz, H-8);
6.88 (d, 1H, J=8.08 Hz, H-7); 5.16 (dd, J=10.44; 7.94 Hz, H-5);
4.44 (bs, 1H, H-4); 3.74 (d, 1H, J=2.07 Hz, H-4); 3.04 (bs, 6H,
NMe.sub.2); 2.90 (dd, 1H, J=7.94; 2.07 Hz, H-4a); 2.72 (dq, 1H,
J=12.31; 6.56 Hz, H-6); 2.61 (dd, 1H, J=12.31; 10.44 Hz, H-5a);
2.54 (q, 2H, J=7.48 Hz, CH.sub.2--C); 1.44 (bs, 9H, CMe.sub.3);
1.29 (d, 3H, J=6.56 Hz, CH.sub.3); 1.20 (t, 3H, J=7.48 Hz,
C--CH.sub.3).
[4S-(4.alpha.,
12a.alpha.)]-9-(diazonium)-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahyd-
ro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide
[0076] A 10 ml roundbottom flask was charged with 100 mg of
compound 2 and dissolved in 4 ml of 0.1 N methanolic hydrochloric
acid. The solution was cooled to 0.degree. C. and 35 .mu.l of butyl
nitrite was added with stirring. After 1 hour, the bright red
reaction mixture was added dropwise to 100 ml of cold anhydrous
diethyl ether. The product was collected by filtration, washed with
ether, and dried in a vacuum dessicator to give 73 mg of the
diazonium salt as an orange solid. MS (FAB): m/z 472 (M+H). .sup.1H
NMR (CD.sub.3OD): .delta. 7.55 (d, 1H, J=8.08 Hz, H-8); 6.86 (d,
1H, J=8.08 Hz, H-7); 5.13 (dd, J=10.44; 7.94 Hz, H-5); 4.41 (bs,
1H, H-4); 3.72 (d, 1H, J=2.07 Hz, H-4); 3.04 (bs, 6H, NCH.sub.3);
2.90 (dd, 1H, J=7.94; 2.07 Hz, H-4a); 2.70 (dq, 1H, J=12.31; 6.56
Hz, H-6); 2.61 (dd, 1H, J=12.31; 10.44 Hz, H-5a); 2.2 (m, 6H,
J=7.48 Hz, Acetyl); 1.44 (bs, 9H, C(CH.sub.3).sub.3); 1.29 (d, 3H,
J=6.56 Hz, CH.sub.3); 1.20 (t, 3H, J=7.48 Hz, C--CH.sub.3).
General Procedure for Olefination.
[0077] To a solution of 0.1 g of a 9-diazonium compound in wet
methanol is added 0.05 equivalents of palladium acetate. The
reaction mixture is stirred for 5 minutes at room temperature, and
2 equivalents of the desired olefin is added. Stirring is continued
for 18 hours under ambient atmosphere or followed by HPLC. Upon
completion, the catalyst is filtered off and the filtrate dried to
give the crude product. The purified product is isolated by
preparative reverse-phase HPLC using methanol and phosphate buffer
gradient.
9-(1'-cyclopentenyl) minocycline
[0078] MS (FAB): m/z 511 (M+H).
Example 2
Assay for the Ability of Tetracycline Compounds to Control
Cryptosporidium parvum Infection In Vitro
[0079] This assay is designed to test the ability of a tetracycline
compound to control Cryptosporidium parvum infection in vitro. The
results show that tetracycline compounds of the invention can be
used to control the growth of C. parvum.
[0080] MDCK cells were grown in 96-well microtiter plastic plates,
and were seeded with approximately 5.times.10.sup.4 cells per well,
using DMEM as the growth/maintenance medium with 10% FCS. Plates
were normally grown to confluence 2-3 days after having been seeded
with, bleached, less than four week-old, C. parvum oocysts. The C.
parvum isolate used most often was the human-derived,
calf-propagated GCH1 (Tzipori, Clin. Diagn. Lab. Immunol. (1994)
1:450; Tzipori, J. Infect. Dis. (1995) 172:1160). The DMEM medium
was also added to the negative control wells. The tetracycline
compounds and the oocysts were added to the wells concurrently. The
cells were subsequently monitored twice daily and any apparent
morphologic changes were recorded. The monolayers were fixed with
methanol after 48 hour incubation at 37.degree. C. in 8%
CO.sub.2.
[0081] Table 1 shows the Inhibition assay results of the
tetracycline compounds in MDBK infected cells. The tetracycline
compounds were used at various concentrations (0.1-2000 .mu.g/mL)
and were dissolved directly into the culture medium. A conventional
indirect immunofluorecensce (IF) assay was used to detect and
enumerate parasite forms after 48-hour incubation. For the purposes
of this assay, a polyclonal rabbit anti-sporozoite antibody was
produced. It was used at a dilution of 1:1000 and does not react
with oocyst shells. The secondary antibody, a
fluoroscein-conjugated goat anti-rabbit IgG, was used at a
concentration of 1:100, according the manufacturer's instructions.
The primary antibody was added after fixation for thirty minutes
and after vigorous washing, the secondary antibody was incubated
for another thirty minutes. The dried microtiter plate was viewed
with an inverted microscope (.times.10 mag.), under ultraviolet
light. For parasite counts, a semi-automated video imaging MCID
system was used to facilitate the enumeration and analysis of the
parasite data. In Table 1, good inhibition of C. parvum is
indicated by `*` (e.g., 70% inhibition at concentrations above 10
.mu.g/ml and above) and very good inhibition of C. parvum is
indicated by `**` (e.g., 70% inhibition at concentrations below 10
.mu.g/ml). Some compounds of the invention have 70% inhibition at
concentrations below 1 .mu.g/ml.
[0082] The study shows that all of the tetracycline compounds are
capable of inhibiting or decreasing the amount C. parvum in a
sample of cells.
TABLE-US-00001 TABLE 1 Compound Inhibition ##STR00008## **
##STR00009## ** ##STR00010## * ##STR00011## * ##STR00012## **
##STR00013## * ##STR00014## ** ##STR00015## **
Example 3
In Vitro Cytotoxicity Assay of Tetracycline Compounds
[0083] The following assay is designed to test the cytotoxicity of
the tetracycline compounds of the invention on MDBK cells.
Advantageous compounds of the invention are compounds with low
cytotoxicity.
[0084] Cytotoxicity of the tetracycline compound is measured by the
Cell Titer 96.TM. Aqueous, a non-radioactive cell proliferation
assay, available as a commercial kit. It is a colorimetric method
for determining the number of viable cells in proliferation or
chemosensitivity assays. The assay is performed by growing MDBK
cells in 96-well microliter plates, as in Example 2. Once
confluent, the media is aspirated and replaced with 200 .mu.L of
media containing the tetracycline compound concentrations which
were tested in Example 2. After 48 hour incubation, 40 .mu.L/well
of freshly prepared MTS/PMS solution is added. The plate is
incubated for two hours at 37.degree. C. and 8% CO.sub.2 and then
100 .mu.L of supernatant from each well is transferred to a new
96-well plate. The optical density is determined at 490 nm by an
ELISA plate reader and the results are recorded and analyzed.
Percent toxicity is calculated by subtracting the mean optical
density (OD) of the medium control supernatants (no tetracycline
compound) by the mean OD of the tetracycline compound supernatants
and dividing by the OD of the medium control and multiplying by
100.
Example 4
In Vivo Assay of Inhibition of Cryptosporidium parvum Infection
[0085] This study is designed to test the ability of a tetracycline
compound to control Cryptosporidium parvum infections in mice.
Advantageous compounds of the invention control the Cryptosporidium
parvum infection without killing the mice.
[0086] Three 4-week old C.B-17 SCID mice are randomized into six
groups of seven mice each. Each animal receives a single I.P.
injection of 1 mg of XMG1.2 mAb. Two hours later, mice in five of
the six groups are infected with 10.sup.7 GCH1 oocysts via oral
inoculation. Treatment with a tetracycline compounds begins on day
6, post infection, in two divided doses/day and continues for 10
days.
[0087] At the end of the experiment, all animals are necropsied and
sections are taken from the pyloric region of the stomach, mid
small intestine, terminal ileum, cecum, proximal colon, and
liver/gall bladder for histological analysis to determine the
extent of mucosal infection. Each site is assigned a score
depending upon the extent of the infection. In this system, scores
range from 0 (no infection) to 5 (extensive infection). Data is
presented as the mean total score of the five sites. Oocyst
shedding is monitored in all infected animals three times per week,
beginning on day 4 of infection. Body weights are determined once
per week throughout the course of study.
EQUIVALENTS
[0088] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of the present
invention and are covered by the following claims. The contents of
all references, issued patents, and published patent applications
cited throughout this application are hereby incorporated by
reference. The appropriate components, processes, and methods of
those patents, applications and other documents may be selected for
the present invention and embodiments thereof.
* * * * *