U.S. patent application number 14/957745 was filed with the patent office on 2016-06-02 for compositions and methods for the treatment of progressive multifocal leukoencephalopathy (pml).
This patent application is currently assigned to Biogen MA Inc.. The applicant listed for this patent is Biogen MA Inc.. Invention is credited to Margot Brickelmaier, Leonid Gorelik, Alexey Lugovskoy.
Application Number | 20160151390 14/957745 |
Document ID | / |
Family ID | 40468203 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151390 |
Kind Code |
A1 |
Gorelik; Leonid ; et
al. |
June 2, 2016 |
COMPOSITIONS AND METHODS FOR THE TREATMENT OF PROGRESSIVE
MULTIFOCAL LEUKOENCEPHALOPATHY (PML)
Abstract
The invention relates to compositions, methods, and kits for
treating subjects infected by or at risk of infection with a DNA
virus (e.g., a JC Virus or a BK virus). Aspects of the invention
are useful to prevent or treat DNA virus associated conditions
(e.g., PML) in subjects that are immuno-compromised. Compositions
are provided that inhibit intracellular replication of DNA
viruses.
Inventors: |
Gorelik; Leonid; (Quincy,
MA) ; Brickelmaier; Margot; (Boxford, MA) ;
Lugovskoy; Alexey; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biogen MA Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Biogen MA Inc.
Cambridge
MA
|
Family ID: |
40468203 |
Appl. No.: |
14/957745 |
Filed: |
December 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12678011 |
Oct 7, 2010 |
9233107 |
|
|
PCT/US2008/010734 |
Sep 13, 2008 |
|
|
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14957745 |
|
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60993769 |
Sep 14, 2007 |
|
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Current U.S.
Class: |
424/133.1 ;
514/182 |
Current CPC
Class: |
A61K 31/58 20130101;
A61P 31/12 20180101; A61K 31/7048 20130101; A61K 31/44 20130101;
A61K 38/12 20130101; A61K 31/437 20130101; A61M 1/38 20130101; Y02A
50/467 20180101; A61K 31/196 20130101; A61K 31/4709 20130101; A61K
31/554 20130101; A61K 31/352 20130101; A61K 31/573 20130101; A61K
45/06 20130101; A61K 31/69 20130101; A61K 31/575 20130101; A61K
31/47 20130101; A61K 39/3955 20130101; C07K 16/2839 20130101; A61K
31/47 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/575 20060101
A61K031/575; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395 |
Claims
1-42. (canceled)
43. A method comprising administering a composition comprising
fusidic acid to a subject before, during or after the subject
receives an immunomodulatory therapy for a disease or
condition.
44. The method of claim 43, wherein the composition is administered
to the subject during the immunomodulatory therapy.
45. The method of claim 43, wherein the composition is administered
to the subject before the immunomodulatory therapy.
46. The method of claim 43, wherein the subject is known to have
been exposed to a polyomavirus infection.
47. The method of claim 46, wherein the subject is identified as
having been exposed to a polyomavirus infection by detecting one or
more serum antibodies against a polyomavirus molecule or by
detecting one or more polyomavirus molecules obtained from the
subject.
48. The method of claim 46, wherein the polyomavirus is JC virus or
BK virus.
49. The method of claim 46, wherein the subject has a polyomavirus
infection.
50. The method of claim 43, wherein the disease or condition is
multiple sclerosis, rheumatoid arthritis, myasthenia gravis,
systemic lupus erythematosus or an inflammatory bowel disease or
syndrome.
51. The method of claim 50, wherein the disease or condition is
multiple sclerosis.
52. The method of claim 50, wherein the disease or condition is
Crohn's disease.
53. The method of claim 43, wherein the immunomodulatory therapy
comprises administration of a VLA-4 antibody.
54. The method of claim 53, wherein the VLA-4 antibody is
natalizumab.
55. A method comprising administering a composition comprising
fusidic acid to a subject who has a weakened immune system.
56. The method of claim 55, wherein the subject has a polyomavirus
infection.
57. The method of claim 56, wherein the polyomavirus is JC virus or
BK virus.
58. A method comprising administering a composition comprising
fusidic acid to a subject having multiple sclerosis, wherein the
composition is administered to the subject before, during or after
the subject receives an immunomodulatory therapy for multiple
sclerosis.
59. The method of claim 58, wherein the subject has a JC virus
infection or a BK virus infection.
60. The method of claim 58, wherein the immunomodulatory therapy
comprises the administration of a VLA-4 antibody.
61. The method of claim 60, wherein the VLA-4 antibody is
natalizumab.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/678,011, filed Oct. 7, 2010, which is a national stage
filing under 35 U.S.C. .sctn.371 of PCT International application
PCT/US2008/010734 designating the United States of America, and
filed Sep. 13, 2008, which claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application Ser. No. 60/993,769,
filed Sep. 14, 2007, the entire contents of each of which are
herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for
treating subjects infected with a DNA virus. In particular, aspects
of the invention relate to subjects infected with JCV virus and
subjects having, or at risk for developing, progressive multifocal
leukoencephalopathy (PML).
BACKGROUND OF THE INVENTION
[0003] JC polyomavirus (JCV) is the causative agent of a
demyelinating disease of the central nervous system, progressive
multifocal leukoencephalopathy (PML). The incidence of PML can be
related to a weakened immune system or treatment with
immunosuppressants. Currently, there is no specific antiviral
therapy that has been proven effective for treatment of PML.
SUMMARY OF THE INVENTION
[0004] Aspects of the invention relate to compositions that inhibit
DNA virus activity, including viral proliferation (e.g., viral
replication), mutation rate and infectivity, and the use of such
compositions to treat or suppress conditions associated with DNA
virus activity in subjects that are infected with a DNA virus, or
the use of such compositions to lower the risk of infection with
the DNA virus. In some embodiments, the invention provides one or
more compositions that inhibit JCV activity. In some embodiments,
the invention provides one or more compositions that inhibit BK
virus (BKV) activity. Such compositions may be used to prevent DNA
viral infection (e.g., JCV infection or BKV infection), to prevent
an increase in DNA viral activity (e.g., active JCV infection of
the brain), to prevent DNA virus proliferation (e.g., JCV
proliferation or BKV proliferation) to prevent symptoms associated
with viral infection (e.g., JCV or BKV infection), to treat a
subject infected with a DNA virus (e.g., JCV or BKV), or treat a
subject at risk of infection with a DNA virus (e.g., JCV or BKV),
or to treat a subject that has developed a disease or condition
associated with infection by a DNA virus (e.g., PML). Compositions
of the invention also may be administered to a subject at risk of a
viral infection or at risk of an increase in viral activity (e.g.,
viral proliferation, for example in the brain or CNS), regardless
of whether the subject is actually known to have been exposed to,
or infected by, the virus.
[0005] In some embodiments, one or more compositions of the
invention may be administered to subjects that have a compromised
immune system. It should be appreciated that a subject's immune
system may be compromised due to treatment with an
immunosuppressive therapeutic agent and/or due to a disease or
condition that impacts the immune system. In some embodiments, one
or more compositions of the invention may be administered to a
subject that is at risk of PML due to a compromised immune system,
regardless of whether the subject is known to be infected with JCV
or known to have been exposed to JCV. Accordingly, compositions of
the invention may be administered to subjects that are receiving an
immunosuppressive treatment for a disease or condition. In some
embodiments, compositions of the invention may be administered to
multiple sclerosis (MS) patients that are being treated with one or
more immunosuppressive agents (e.g., natalizumab). However, in some
embodiments, compositions of the invention may be administered to
subjects that have a weakened immune system caused by a disease or
condition itself, rather than by an immunosuppressive treatment.
For example, subjects infected with an immuno-compromising pathogen
(e.g., a virus such as HIV) may be treated with one or more
compositions of the invention.
[0006] It should be appreciated that while the JCV status of a
subject need not be known, it may be useful to know the status in
some embodiments. In some embodiments, the efficacy of such
treatment or therapy may be monitored by detecting and/or
monitoring the presence of JCV in a subject.
[0007] In some embodiments, one or more compositions of the
invention may be administered to a subject before, during, and/or
after the subject receives and immunomodulatory therapy (e.g., a
treatment that inhibits the immune system of the subject).
Accordingly, in some embodiments one or more compounds described
herein as being effective to inhibit DNA virus replication may be
administered to a subject prior to initiation of an
immunomodulatory therapy. For example, a therapeutic regimen of one
or more compositions of the invention may be initiated prior to an
immunomodulatory treatment against a disease or in preparation for
a transplant in to prevent or reduce any risk of DNA virus
replication or proliferation associated with the immunomodulatory
treatment.
[0008] In some embodiments, one or more compositions of the
invention may be administered alone or in combination with other
compositions described herein or along with other therapeutic
agents (e.g., one or more immunosuppressive therapeutic agents).
Compositions of the invention may be provided (e.g., administered)
in pharmaceutical preparations. Compositions of the invention may
be provided in kits.
[0009] In some aspects, compositions of the invention may be used
to develop further anti-viral treatments (e.g., as starting
material for a medicinal chemistry study or as references in an in
vitro assay).
[0010] In some aspects, the invention provides methods of
inhibiting viral replication, the methods comprising contacting a
cell comprising a DNA virus with a composition comprising
chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, (R)-angolensin,
ptaeroxylin, dipyridamole, nabumetone, rosiglitazone, diltiazem
hydrochloride, betamethasone, ichthynone, amcinonide, riluzole,
flufenamic acid, chrysin, dictamnine, piplartine, peucenin,
methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,
mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,
methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,
meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic
acid, acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin
b sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol
20-acetate, or any combination thereof.
[0011] In some aspects, the invention provides methods of
inhibiting viral replication, the methods comprising contacting a
cell comprising a DNA virus with a composition comprising
endosulfan, candesartan cilextil, mefenamic acid, fusidic acid,
tolfenamic acid, mefloquine, isotretinon, diclofenac sodium,
diltiazem hydrochloride, miconazole nitrate, flunixin meglumine,
propanil, dehydroabietamide, diffractic acid, harmane, xanthone,
methoxyvone, or any combination thereof.
[0012] In some aspects, the invention provides methods of
inhibiting viral replication, the methods comprising contacting a
cell comprising a DNA virus with a composition comprising
mefloquine.
[0013] In some aspects, the invention provides methods of
inhibiting viral replication, the methods comprising contacting a
cell comprising a DNA virus with a composition comprising
R*,S*-mefloquine.
[0014] In some embodiments of the methods described herein, the
composition further comprises a pharmaceutically acceptable
carrier.
[0015] In some embodiments of any of the methods described herein,
the DNA virus is a herpes virus, pox virus, parvovirus, or
polyomavirus. In some embodiments, the DNA virus is a polyomavirus.
In some embodiments, the polyomavirus is JC virus. In some
embodiments, the polyomavirus is BK virus.
[0016] In some embodiments of the methods described herein, a
composition is contacted to a virus-infected cell in a subject. In
some embodiments, the cell is a brain cell, a neuron, a kidney
cell, or any other cell in a subject. Accordingly, in some
embodiments a composition is administered to a subject. In some
embodiments, the subject is suspected of having a viral infection
(e.g., in at least one cell or tissue type). A subject suspected of
having a viral infection may be a subject that has been identified
as having, or known to have, a DNA virus infection (e.g., a JCV or
BKV infection). In addition, or alternatively, a subject suspected
of having a viral infection may be a subject that has been
identified as being at risk of, or is known to be at risk of, a DNA
virus infection (e.g., a JCV or BKV infection). It should be
appreciated, that a subject may be identified as having an
infection by directly detecting one or more viral molecules (e.g.,
RNA, DNA, protein, etc., or any combination thereof). However, a
subject may be identified as having an infection indirectly by
detecting one or more indicia of an infection (e.g., one or more
symptoms, one or more serum antibodies against a viral molecule,
etc., or any combination thereof). In some embodiments, one or more
symptoms of a viral infection may be used to identify a subject as
being at risk of a viral infection. A subject also may be
identified as being at risk of a viral infection if the subject has
a reduced or suppressed immune system (e.g., due to a disease,
condition, or treatment, or a combination thereof as described in
more detail herein). In some embodiments, the subject has been
identified as having a JC virus infection in the CNS. In some
embodiments, the subject has been identified as having a BK virus
infection in the kidney. It should be appreciated that one or more
of the compositions described herein may be administered to a
subject on the basis that the subject is suspected of having a DNA
virus infection. For example, a composition may be administered to
a subject, because the subject had been identified as having, or
known to have, or identified as being at risk of having, or known
to be at risk of having, a DNA virus infection. Accordingly, in
some embodiments a subject is evaluated to determine whether the
subject has, or is at risk of having, a DNA virus infection (e.g.,
a JCV or BKV infection), and a composition described herein is
administered to the subject if they are found to have, or be at
risk of having, the DNA virus infection. In some embodiments, a
composition described herein is not administered to a subject that
is identified or known to be virus free and/or risk free.
[0017] Accordingly, in some embodiments of the invention a subject
is evaluated (e.g., monitored) to determine whether the subject has
a sign or symptom of, or is at risk for, a DNA virus infection or
proliferation. In some embodiments, this evaluation involves
determining whether the subject has a symptom of a disease or
condition associated with DNA virus activation (e.g., a symptom of
PML). If the subject is identified as having a sign or symptom of,
or as being at risk for, a DNA virus (e.g., JCV or BKV) infection
or proliferation, the subject is treated with one or more compounds
or compositions of the invention. In some embodiments, a
composition comprising chloroacetoxyquinoline, demethylnobiletin,
propanil, aminoethoxydiphenylborane,
5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, (R)-angolensin,
ptaeroxylin, dipyridamole, nabumetone, rosiglitazone, diltiazem
hydrochloride, betamethasone, ichthynone, amcinonide, riluzole,
flufenamic acid, chrysin, dictamnine, piplartine, peucenin,
methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,
mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,
methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,
meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic
acid, acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin
b sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,
4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol
20-acetate, or any combination thereof, is administered to the
subject. In some embodiments, a composition comprising candesartan
cilextil, mefenamic acid, fusidic acid, tolfenamic acid,
mefloquine, isotretinon, diclofenac sodium, diltiazem
hydrochloride, miconazole nitrate, flunixin meglumine, propanil,
dehydroabietamide, diffractic acid, harmane, xanthone, or
methoxyvone, or any combination thereof, is administered to the
subject.
[0018] In some embodiments, the subject (e.g., a subject that has
been identified and treated based on the identification) is
evaluated (e.g., monitored) for one or more signs or symptoms of
DNA virus infection or proliferation after receiving a compound or
composition of the invention. For example, in some embodiments of
the invention a subject may be evaluated to determine or confirm
the reduction of at least one symptom of DNA virus (e.g., JCV or
BKV) infection or proliferation. In certain embodiments, a subject
may be evaluated to determine or confirm that no signs or symptoms
of DNA virus (e.g., JCV or BKV) infection or proliferation have
developed in the subject. In some embodiments, a subject may be
evaluated to determine or confirm that any detectable signs or
symptoms of DNA virus (e.g., JCV or BKV) infection or proliferation
have been maintained at a stable level, or that their development
has been slowed or reversed. In some embodiments, an
immunomodulatory treatment or therapy may be altered (e.g.,
increased, decreased, substituted, or discontinued) based on the
evaluation (e.g., monitoring) of the subject. In some embodiments,
a treatment or therapy with one or more compounds or compositions
of the invention may be altered (e.g., increased, decreased,
substituted, or discontinued) based on the evaluation (e.g.,
monitoring) of the subject before, after, or during, an
immunomodulatory therapy.
[0019] In some aspects, the invention provides methods of
inhibiting viral replication in a subject, the methods comprising
administering a composition to a subject suspected of having a DNA
virus infection, wherein the composition comprises
chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, (R)-angolensin,
ptaeroxylin, dipyridamole, nabumetone, rosiglitazone, diltiazem
hydrochloride, betamethasone, ichthynone, amcinonide, riluzole,
flufenamic acid, chrysin, dictamnine, piplartine, peucenin,
methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,
mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,
methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,
meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic
acid, acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin
b sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,
4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol
20-acetate, or any combination thereof, in an amount sufficient to
inhibit DNA viral replication in the subject.
[0020] In some aspects, the invention provides methods of
inhibiting viral replication in a subject, the methods comprising
administering a composition to a subject suspected of having a DNA
virus infection, wherein the composition comprises endosulfan,
candesartan cilextil, mefenamic acid, fusidic acid, tolfenamic
acid, mefloquine, isotretinon, diclofenac sodium, diltiazem
hydrochloride, miconazole nitrate, flunixin meglumine, propanil,
dehydroabietamide, diffractic acid, harmane, xanthone, methoxyvone,
or any combination thereof, in an amount sufficient to inhibit DNA
viral replication in the subject.
[0021] In some embodiments of the methods presented herein, the DNA
virus infection is a herpes virus, pox virus, parvovirus, or
polyomavirus infection. In some embodiments of the methods
presented herein the DNA virus infection is polyomavirus infection.
In some embodiments of the methods presented herein the
polyomavirus is JC virus. In some embodiments of the methods
presented herein the polyomavirus is BK virus.
[0022] In some embodiments of the methods presented herein, the
subject has been identified as having a JC virus infection of the
CNS. In some embodiments of the methods presented herein, the
subject has been identified as having a BK virus infection of the
kidney.
[0023] In some embodiments of the methods presented herein the
subject is identified as being at risk of a DNA virus infection. In
some embodiments of the methods presented herein, the subject is
undergoing, or has been undergoing, an immunomodulatory treatment.
In some embodiments of the methods presented herein, the
immunomodulatory treatment comprises the administration of a VLA-4
antibody. In some embodiments of the methods presented herein, the
VLA-4 antibody is natalizumab.
[0024] In some aspects, the invention provides methods of reducing
the risk of a DNA virus infection in a subject, the methods
comprising administering to the subject a composition comprising
chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, (R)-angolensin,
ptaeroxylin, dipyridamole, nabumetone, rosiglitazone, diltiazem
hydrochloride, betamethasone, ichthynone, amcinonide, riluzole,
flufenamic acid, chrysin, dictamnine, piplartine, peucenin,
methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,
mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,
methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,
meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic
acid, acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin
b sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol
20-acetate, or any combination thereof in an amount sufficient to
inhibit DNA viral infection.
[0025] In some aspects, the invention provides methods of reducing
the risk of a DNA virus infection in a subject, the methods
comprising administering to the subject a composition comprising
endosulfan, candesartan cilextil, mefenamic acid, fusidic acid,
tolfenamic acid, mefloquine, isotretinon, diclofenac sodium,
diltiazem hydrochloride, miconazole nitrate, flunixin meglumine,
propanil, dehydroabietamide, diffractic acid, harmane, xanthone,
methoxyvone or combinations thereof in an amount sufficient to
inhibit DNA viral infection.
[0026] In some aspects, the invention provides methods of reducing
the risk of PML, the methods comprising administering to a subject
having PML, or at risk of PML, a composition comprising
chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, (R)-angolensin,
ptaeroxylin, dipyridamole, nabumetone, rosiglitazone, diltiazem
hydrochloride, betamethasone, ichthynone, amcinonide, riluzole,
flufenamic acid, chrysin, dictamnine, piplartine, peucenin,
methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,
mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,
methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,
meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic
acid, acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin
b sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol
20-acetate, or any combination thereof in an amount sufficient to
inhibit DNA viral infection.
[0027] In some aspects, the invention provides methods of reducing
the risk of PML, the methods comprising administering to a subject
having PML, or at risk of PML, a composition comprising endosulfan,
candesartan cilextil, mefenamic acid, fusidic acid, tolfenamic
acid, mefloquine, isotretinon, diclofenac sodium, diltiazem
hydrochloride, miconazole nitrate, flunixin meglumine, propanil,
dehydroabietamide, diffractic acid, harmane, xanthone, methoxyvone
or combinations thereof in an amount sufficient to inhibit DNA
viral infection.
[0028] In some aspects, the invention provides methods of reducing
the risk of PML, the methods comprising administering to a subject
having PML, or at risk of PML, a composition comprising an
arylalkanoic acid.
[0029] In some embodiments of the methods presented herein, the
composition is administered in a dosage sufficient to reduce the
number of JCV infected cells in an in vitro assay by more than 25%,
more than 40%, more than 50%, more than 75%, or more than 80%.
[0030] In some embodiments of the methods presented herein, the
composition comprises a compound having an anti-JCV IC.sub.50<20
.mu.M and a therapeutic index IC.sub.50/TC.sub.50<0.5.
[0031] In some embodiments of the methods presented herein, the
methods further comprise administering an antiviral agent.
[0032] In some embodiments of the methods presented herein, the
methods further comprise performing a plasma exchange.
[0033] In some aspects, the invention provides a pharmaceutical
composition and/or a kit comprising natalizumab and one or more
compounds selected from the group consisting of
chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, (R)-angolensin,
ptaeroxylin, dipyridamole, nabumetone, rosiglitazone, diltiazem
hydrochloride, betamethasone, ichthynone, amcinonide, riluzole,
flufenamic acid, chrysin, dictamnine, piplartine, peucenin,
methoxyvone, isotretinoin, chloroxylenol, tomatine, primuletin,
mefenamic acid, diethylstilbestrol, chloramphenicol palmitate,
methylxanthoxylin, 1-alaninol, diclofenac sodium, flunixin,
meglumine, dehydroabietamide, pachyrrhizin, dicumarol, diffractic
acid, acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin
b sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine, 2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,
4-estren-3-beta 17-beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate, or 4-pregnen-3-beta 20-beta-diol
20-acetate, and any combination thereof.
[0034] In some aspects, the invention provides a pharmaceutical
composition and/or a kit comprising natalizumab and one or more
compounds selected from the group consisting of endosulfan,
candesartan cilextil, mefenamic acid, fusidic acid, tolfenamic
acid, mefloquine, isotretinon, diclofenac sodium, diltiazem
hydrochloride, miconazole nitrate, flunixin meglumine, propanil,
dehydroabietamide, diffractic acid, harmane, xanthone, methoxyvone
and combinations thereof.
[0035] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising chloroacetoxyquinoline, demethylnobiletin,
propanil, aminoethoxydiphenylborane,
5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,
4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two
or more thereof.
[0036] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising endosulfan, candesartan cilextil, mefenamic
acid, fusidic acid, tolfenamic acid, mefloquine, isotretinon,
diclofenac sodium, diltiazem hydrochloride, miconazole nitrate,
flunixin meglumine, propanil, dehydroabietamide, diffractic acid,
harmane, xanthone, methoxyvone or combinations thereof.
[0037] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising mefloquine.
[0038] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising R,S-mefloquine.
[0039] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising a compound with the same mode of action as
endosulfan, candesartan cilextil, mefenamic acid, fusidic acid,
tolfenamic acid, mefloquine, isotretinon, diclofenac sodium,
diltiazem hydrochloride, miconazole nitrate, flunixin meglumine,
propanil, dehydroabietamide, diffractic acid, harmane, xanthone,
methoxyvone or any combination thereof.
[0040] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising a compound comprising an adenosine, guanine,
estren, or pregnan component.
[0041] In some embodiments, the invention provides methods of
treatment comprising administering to a subject having PML, or at
risk of PML, a therapeutically effective amount of a pharmaceutical
composition comprising a compound comprising an arylalkanoic
acid.
[0042] In some embodiments, the invention provides methods of
treating a subject infected with a DNA virus, or suspected of being
infected with a DNA virus, the method comprising administering to a
subject a therapeutically effective amount of a pharmaceutical
composition comprising chloroacetoxyquinoline, demethylnobiletin,
propanil, aminoethoxydiphenylborane,
5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,
4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two
or more thereof.
[0043] In some embodiments, the invention provides methods of
treatment for a subject infected with a DNA virus, or suspected of
being infected with a DNA virus, wherein the DNA virus is selected
from the group consisting of herpes virus, pox virus, parvovirus
and polyomavirus. In some embodiments, the invention provides
methods of treatment for a subject infected with JC virus, or
suspected of being infected with JC virus. In some embodiments, the
invention provides methods of treatment for a subject infected with
BK virus, or suspected of being infected with BK virus. In some
embodiments, the invention provides methods of treatment for a
subject infected with a JC virus, wherein JC virus infection is
characterized by the presence of JC virus in the central nervous
system (CNS). In some embodiments, the invention provides methods
of treatment for a subject infected with a BK virus, wherein BK
virus infection is characterized by the presence of BK virus in the
kidney.
[0044] In some embodiments, the invention provides methods of
preventing or suppressing PML, the method comprising administering
to a subject a therapeutically effective amount of a pharmaceutical
composition comprising chloroacetoxyquinoline, demethylnobiletin,
propanil, aminoethoxydiphenylborane,
5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,
4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two
or more thereof.
[0045] In some embodiments, the invention provides methods of
treatment of a subject having PML, or suspected of having PML, the
method comprising administering to a subject a therapeutically
effective amount of a pharmaceutical composition comprising a
compound in a dosage sufficient to reduce the number of JCV
infected cells in an in vitro assay by more than 40%.
[0046] In some embodiments, the invention provides methods of
treatment of a subject having PML, or suspected of having PML,
comprising administering to a subject a therapeutically effective
amount of a pharmaceutical composition comprising a compound,
wherein the compound has an anti-JCV IC.sub.50<20 .mu.M and a
therapeutic index IC.sub.50/TC.sub.50<0.5.
[0047] In some embodiments, the invention provides methods of
treatment for a subject infected with a DNA virus, or suspected of
being infected with a DNA virus, or of a subject having PML, or
suspected of having PML, wherein the subject is undergoing, or has
been undergoing, immunomodulatory treatment.
[0048] In some embodiments, the invention provides methods of
preventing viral infection in a person undergoing immunomodulatory
treatment, the method comprising administering to a subject a
therapeutically effective amount of a pharmaceutical composition
comprising chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,
4-pregnen-3-beta 20-beta-diol 20-acetate, or any combination of two
or more thereof.
[0049] In some embodiments, the immunomodulatory treatment
comprises the administration of a VLA-4 antibody. In some
embodiments, the immunomodulatory treatment comprises the
administration of natalizumab.
[0050] In some embodiments, the invention provides methods of
treatment for a subject infected with a DNA virus, or suspected of
being infected with a DNA virus, or of a subject having PML, or
suspected of having PML comprising administering one or more
compounds of Table 1 and an antiviral therapeutic.
[0051] In some embodiments, the invention provides methods of
treatment for a subject infected with a DNA virus, or suspected of
being infected with a DNA virus, or of a subject having PML, or
suspected of having PML, comprising administering one or more
compounds of Table 1 and an adjuvant.
[0052] In some embodiments, the invention provides methods of
treatment for a subject infected with a DNA virus, or suspected of
being infected with a DNA virus, or of a subject having PML, or
suspected of having PML, comprising administering multiple
compounds selected from the group consisting of
chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, and
4-pregnen-3-beta 20-beta-diol 20-acetate
[0053] In some embodiments, the invention provides methods of
treatment for a subject infected with a DNA virus, or suspected of
being infected with a DNA virus, or of a subject having PML, or
suspected of having PML, comprising administering one or more
compounds of Table 1 and performing a plasma exchange.
[0054] In some embodiments, the invention provides a pharmaceutical
composition comprising natalizumab and one or more compounds
selected from the group consisting of chloroacetoxyquinoline,
demethylnobiletin, propanil, aminoethoxydiphenylborane,
5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate and
4-pregnen-3-beta 20-beta-diol 20-acetate.
[0055] In some embodiments, the invention provides a kit comprising
natalizumab and one or more compounds selected from the group
consisting of chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, 06-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate, and
4-pregnen-3-beta 20-beta-diol 20-acetate, and instructions for
administering the compounds.
[0056] In some embodiments, the invention provides a method of
inhibiting (e.g., reducing or suppressing) replication of a DNA
virus in a cell comprising contacting a cell comprising a DNA virus
with a compound, wherein contacting the cell with the compound
results in the reduction or suppression of replication of the DNA
virus, wherein the compound is selected from the group consisting
of chloroacetoxyquinoline, demethylnobiletin, propanil,
aminoethoxydiphenylborane, 5-nitro-2-phenylpropylaminobenzoic acid,
3beta-hydroxyisoallospirost-9(11)-ene, leoidin,
picropodophyllotoxin, thiabendazole, harmane,
6,4'-dihydroxyflavone, gentiopicroside, R-angolensin, ptaeroxylin,
dipyridamole, nabumetone, rosiglitazone, diltiazem hydrochloride,
betamethasone, ichthynone, amcinonide, riluzole, flufenamic acid,
chrysin, dictamnine, piplartine, peucenin, methoxyvone,
isotretinoin, chloroxylenol, tomatine, primuletin, mefenamic acid,
diethylstilbestrol, chloramphenicol palmitate, methylxanthoxylin,
1-alaninol, diclofenac sodium, flunixin, meglumine,
dehydroabietamide, pachyrrhizin, dicumarol, diffractic acid,
acemetacin, ginkgolic acid, xanthone, fusidic acid, polymyxin b
sulfate, pyrantel pamoate,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one, miconazole nitrate,
candesartan cilextil, endosulfan, dioxybenzone, tolfenamic acid,
mefloquine
([2,8-bis(trifluoromethyl)quinolin-4-yl]-piperidin-2-ylmethanol),
2-methoxyxanthone, 3-hydroxy-4-(succin-2-yl)-caryolane
delta-lactone, 5,7-dihydroxyflavone, avocadanofuran,
benzo(a)pyrene, beta-dihydrogedunol, decahydrogambogic acid,
diosmetin, niloticin, pectolinarin, totarol acetate,
8-chloroadenosine, 3-deazaadenosine, O6-cyclohexylmethylguanine,
roscovitine, 4-estren-3-beta 17-beta-diol 17-acetate,
5-beta-pregnan-3-alpha 6-alpha 20-beta-triol 20-acetate,
4-pregnen-3-beta 20-beta-diol 20-acetate and any combination of two
or more thereof.
[0057] In some embodiments, the invention provides a method of
suppressing replication of a DNA virus in a cell, wherein the DNA
virus is selected from the group consisting of herpes virus, pox
virus, parvovirus, JC virus, and BK virus.
[0058] These and other aspects of the invention are described in
more detail herein and illustrated by the following non-limiting
figures and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 illustrates a non-limiting embodiment of a decrease
in the amount of inhibition with an increase in the amount of
neutralizing JCV antibody added;
[0060] FIG. 2 illustrates a non-limiting embodiment of the
correlation between infection rate and JCV virus concentration;
[0061] FIG. 3 illustrates a non-limiting embodiment of detection
and measurement of cellular infection with JCV, panel (A) shows
cells infected with JCV, panel (B) shows the number of infected
cells plotted against the dilution factor of the viral stock used
to infect the cells, panels (C) and (D) illustrate the inhibition
of JCV in the presence of various dilutions of JCV neutralizing
antiserum or cidofovir;
[0062] FIG. 4 illustrates a non-limiting embodiment of a flow chart
for compound screening;
[0063] FIG. 5 illustrates a non-limiting embodiment of the efficacy
of mefloquine against two different viral strains, panel (A)
illustrates SVG-A cells infected with Mad1/SVE.DELTA. JCV virus,
panel (B) illustrates primary human fetal astrocytes infected with
Mad1/SVE.DELTA. JCV, and panel (C) illustrates SVG-A cells infected
with JCV strain Mad-4;
[0064] FIG. 6 illustrates a non-limiting embodiment of the effect
of mefloquine on JCV DNA replication;
[0065] FIG. 7 illustrates a non-limiting embodiment of the efficacy
of mefloquine against JCV infection;
[0066] FIG. 8 illustrates non-limiting embodiments of the efficacy
of different isomers of mefloquine;
[0067] FIG. 9 illustrates non-limiting embodiments of the efficacy
of different isomers of mefloquine;
[0068] FIG. 10 shows that mefloquine anti-JCV activity is not
inhibited by cerebrospinal fluid (CSF);
[0069] FIG. 11 illustrates non-limiting embodiments of the dose
response of drugs with anti-JCV activity;
[0070] FIG. 12 illustrates non-limiting embodiments of arylalkanoic
acid NSAIDs and their anti-JCV activity;
[0071] FIG. 13 illustrates non-limiting embodiments of arylalkanoic
acid NSAIDs and their anti-JCV activity;
[0072] FIG. 14 illustrates non-limiting embodiments of modeling
studies with mefloquine and related compounds;
[0073] FIG. 15 illustrates non-limiting embodiments of structures
of JCV-inhibitor compounds; and,
[0074] FIG. 16 illustrates non-limiting embodiments of structures
of JCV-inhibitor compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Aspects of the invention relate to compositions identified
as having anti-viral activity and their use to prevent or treat
viral infection in subjects. In particular, aspects of the
invention relate to compositions found to inhibit DNA viral
activity. In some embodiments, one or more compositions that
inhibit JCV activity are provided and may be administered to
subjects infected or at risk of infection with JCV and/or to
subjects with PML, or at risk of developing PML.
[0076] Aspects of the invention are based, at least in part, on the
surprising discovery that the compounds described in Table 1 reduce
the percentage or number of JCV infected cells in a cell-based
assay. The compounds described in Table 1 were not previously known
to have anti-JCV activity or any anti-viral activity. Accordingly,
one or more compounds described in Table 1 may be used as described
herein.
TABLE-US-00001 TABLE 1 Therapeutic Name Reference Indication CAS
number CHLOROACETOXYQUINOLINE antifungal 10173-02-1
DEMETHYLNOBILETIN Biochem Biophys Res Commun. 2005 2174-59-6 Dec 2;
337(4):1330-6. Epub 2005 Oct. 10. PROPANIL U.S. Pat. No. 6,060,432
herbicide 709-98-8 AMINOETHOXYDIPHENYLBORANE J Neurophysiol. 2005
November; 94(5): 3069-80. Ca release 524-95-8 Epub 2005 Jul. 13.
inhibitor, angiogensin II inhibitor 5-NITRO-2- J Cell Physiol. 1995
January; 162(1): 15-25. chloride channel PHENYLPROPYLAMINOBENZOIC
blocker ACID [NPPB] 3beta-HYDROXYISOALLOSPIROST- J Nat Prod. 2007
July; 70(7): 1203-6. 9(11)-ENE Epub 2007 Jul. 13. LEOIDIN
105350-54-7 PICROPODOPHYLLOTOXIN Biol Pharm Bull. 2007 July; 30(7):
1340-3. antineoplastic; 477-47-4 10% cytotoxicity of
podophyllotoxin THIABENDAZOLE U.S. Pat. No. 5,840,324 anthelmintic
148-79-8 HARMANE intercalating agent, 486-84-0 sedative
6,4'-DIHYDROXYFLAVONE antihaemorrhagic 79786-40-6 GENTIOPICROSIDE
Yao Xue Xue Bao. 2007 May; 42(5): 566-70. antimalarial, larvicide
(R)-ANGOLENSIN 4842-48-2 PTAEROXYLIN Biochem Syst Ecol. 2000 Aug.
1; 28(7): 713-716. 14729-11-4 DIPYRIDAMOLE U.S. Pat. No. 7,253,155
coronary 58-32-2 vasodilator NABUMETONE U.S. Pat. No. 6,544,556
antiinflammatory 42924-53-8 ROSIGLITAZONE U.S. Pat. No. 6,673,815
antidiabetic 122320-73-4 DILTIAZEM HYDROCHLORIDE U.S. Pat. No.
5,578,321 Ca channel 33286-22-5 blocker, coronary vasodilator
BETAMETHASONE U.S. Pat. No. 6,878,518 glucocorticoid, 378-44-9
antiinflammatory ICHTHYNONE Vopr Pitan. 1995; (4): 13-6. piscicide
24340-62-3 AMCINONIDE U.S. Pat. No. 6,426,339 glucocorticoid,
51022-69-6 antiinflammatory RILUZOLE U.S. Pat. No. 6,660,757
anticonvulsant, 1744-22-5 glutamate release inhibitor FLUFENAMIC
ACID U.S. Pat. No. 5,968,551 antiinflammatory, 530-78-9 analgesic
CHRYSIN U.S. Pat. No. 6,607,755 diuretic 480-40-0 DICTAMNINE Planta
Med. 2006 August; 72(10): 941-3. 484-29-7 PIPLARTINE Phytomedicine.
2007 September; 14(9): 605-12. anti-asthma, 20069-09-4 Epub 2007
Mar. 30. antibronchitis PEUCENIN Chem Pharm Bull (Tokyo). 2006
January; 54(1): 44-7. 578-72-3 METHOXYVONE Bioorg Med Chem. 2007
Sep. 15; 15(18): 6089-95. anabolic Epub 2007 Jun. 26. ISOTRETINON
U.S. Pat. No. 6,936,267 antiacne, 4759-48-2 antineoplastic
CHLOROXYLENOL U.S. Pat. No. 4,902,501 antibacterial, 88-04-0
topical and urinary TOMATINE U.S. Pat. No. 6,673,357 antifungal,
antibacterial, antiinflammatory agent PRIMULETIN 491-78-1 MEFENAMIC
ACID U.S. Pat. No. 6,645,520 antiinflammatory, 61-68-7 analgesic
DIETHYLSTILBESTROL U.S. Pat. No. 6,040,306 estrogen 56-53-1
CHLORAMPHENICOL PALMITATE antibacterial, 530-43-8 tetratogen
METHYLXANTHOXYLIN Phytother Res. 2004 July; 18(7): 542-5.
23121-32-6 L-ALANINOL Microbiology. 2005 July; 151(Pt 7): 2385-92.
antiproliferative 2749-11-3 DICLOFENAC SODIUM U.S. Pat. No.
6,387,410 antiinflammatory 15307-79-6 FLUNIXIN MEGLUMINE U.S. Pat.
No. 6,924,273 analgesic, 42461-84-7 antiinflammatory
DEHYDROABIETAMIDE U.S. Pat. No. 4,755,523 PACHYRRHIZIN Trans R Soc
Trop Med Hyg. 2004 August; 98(8): 451-5. insecticide 10091-01-7
DICUMAROL U.S. Pat. No. 5,024,998 anticoagulant 66-76-2 DIFFRACTIC
ACID Inflamm Res. 2007 April; 56 Suppl 1: 521-2. ACEMETACIN U.S.
Pat. No. 7,109,176 antiinflammatory 53164-05-9 GINKGOLIC ACID
antibacterial, 22910-60-7 antitubercular XANTHONE U.S. Pat. No.
6,927,234 90-47-1 FUSIDIC ACID U.S. Pat. No. 6,462,182
antibacterial 6990-06-3 POLYMYXIN B SULFATE U.S. Pat. No. 5,648,397
antibacterial 1405-20-5 PYRANTEL PAMOATE U.S. Pat. No. 7,144,878
anthelmintic 22204-24-6 4-(3-BUTOXY-4- Eur J Pharmacol. 2003 Mar.
28; 465(1-2): 133-9. cAMP PDE METHOXYBENZYL)IMIDAZOLIDIN-
inhibitor, inhibits 2-ONE cellular adhesion and superoxide &
platelet aggregation MICONAZOLE NITRATE U.S. Pat. No. 6,001,864
antifungal (topical) 22832-87-7, 22916-47-8 [miconazole]
CANDESARTAN CILEXTIL angiotensin 1 170791-09-0 receptor antagonist
ENDOSULFAN U.S. Pat. No. 6,294,570 insecticide 115-29-7, 959-98-8
(alpha), 33213-65-9 (beta) DIOXYBENZONE U.S. Pat. No. 5,916,544
ultraviolet screen 131-53-3 TOLFENAMIC ACID U.S. Pat. No. 6,685,928
antiinflammatory, 13710-19-5 analgesia MEFLOQUINE U.S. Pat. No.
5,834,505 antimalarial 53230-10-7 2-METHOXYXANTHONE undetermined
1214-20-6 activity 3-HYDROXY-4-(SUCCIN-2-YL)- undetermined
CARYOLANE delta-LACTONE activity 5,7-DIHYDROXYFLAVONE undetermined
480-40-0 activity AVOCADANOFURAN undetermined activity
BENZO[a]PYRENE carcinogen, binds to DNA experimental 192-97-2
beta-DIHYDROGEDUNOL undetermined activity DECAHYDROGAMBOGIC ACID
undetermined activity DIOSMETIN undetermined 520-34-3 activity
NILOTICIN undetermined activity PECTOLINARIN undetermined
28978-02-1 activity TOTAROL ACETATE undetermined activity
8-CHLOROADENOSINE 34408-14-5 3-DEAZAADENOSINE 6736-58-9 O6-
CYCLOHEXYLMETHYLGUANINE ROSCOVITINE, (S)-ISOMER 4-ESTREN-3-BETA,
17-BETA-DIOL 17-ACETATE 5-BETA-PREGNAN-3-ALPHA, 6- ALPHA,
20-BETA-TRIOL 20- ACETATE 4-PREGNEN-3-BETA, 20-BETA- DIOL
20-ACETATE
[0077] In some embodiments, one or more of the compounds of Table 1
may be used (e.g., combinations of two or more, for example, 2, 3,
4, 5, 6, 7, 8, 9, 10, etc.). In some embodiments, a composition
comprises multiple compounds of Table 1. In some embodiments, the
treatment or prevention regimen comprises the use of multiple
compositions each comprising one or more compounds of Table 1.
[0078] In some embodiments, one or more compounds related to those
listed in Table 1 may be used as described herein. For example,
structurally related compounds, compounds with a similar mode of
action, and/or compounds that interact with one or more of the same
targets (e.g., viral or cellular targets, such as receptors,
intracellular pathway components or other targets) may be used to
treat or prevent DNA virus (e.g., JCV) infection and or
activity.
[0079] In some embodiments, the invention relates to the use of
mefloquine, and compounds with a similar mode of action as
mefloquine, to prevent or treat DNA viral infection or
proliferation (e.g., to reduce the risk of DNA viral infection or
proliferation). Mefloquine is a quinine related anti-malarial drug.
Non-limiting examples of drugs with a similar mode of action are
quinine, chloroquine and halofantrine. Quinines are lysosomotropic
drugs that selectively accumulate inside lysosomes. The uncharged
compound rapidly diffuses through the plasma and lysosomal
membranes, while once charged the compound becomes trapped inside
the acidic lysosomal compartment of the parasite. In some
embodiments, the invention relates to the treatment or prevention
of PML using lysosomotropic drugs.
[0080] In some embodiments, the invention relates to the use of
(+)-(R,S)-mefloquine to prevent or treat DNA viral infection or
proliferation. In some embodiments, the invention relates to the
use of (-)-(S,R)-mefloquine to prevent or treat DNA viral infection
or proliferation. In some embodiments, the invention relates to the
use of a racemic mixture of (+)-(R,S)-mefloquine and
(-)-(S,R)-mefloquine to prevent or treat DNA viral infection or
proliferation. In some embodiments, the invention relates to the
use of (R,R)-mefloquine to prevent or treat DNA viral infection or
proliferation. In some embodiments, the invention relates to the
use of (S,S)-mefloquine to prevent or treat DNA viral infection or
proliferation. In some embodiments, the invention relates to the
use of a mixture of (R,R)-mefloquine and (S,S)-mefloquine to
prevent or treat DNA viral infection or proliferation. In some
embodiments, the invention relates to the use of one or more of the
following mefloquine compounds: (+)-(R,S)-mefloquine,
(-)-(S,R)-mefloquine, (R,R)-mefloquine and (S,S)-mefloquine to
prevent or treat DNA viral infection or proliferation.
[0081] In some embodiments, the invention relates to the use of
compounds that are structurally related to mefloquine to prevent or
treat DNA viral infection or proliferation. In some embodiments,
compounds that are structurally related to mefloquine are compounds
that comprise the 2,8-bis-trifluoromethyl-quonolin-4-yl structural
group. In some embodiments, compounds that are structurally related
to mefloquine are are quinolines substituted on either or both
rings. In some embodiments, compounds that are structurally related
to mefloquine are substituted indoles. In some embodiments,
compounds that are structurally related to mefloquine are
substituted 3-indoleacetic acid derivatives. In some embodiments,
compounds that are structurally related to mefloquine are
substituted 6-amino purine derivatives. In some embodiments,
compounds that are structurally related to mefloquine are
substituted 2,6-diamino purine derivatives. In some embodiments,
compounds that are structurally related to mefloquine are
substituted imidazopyridin-4-amines. In some embodiments, compounds
that are structurally related to mefloquine are substituted
imidazopyridines. In some embodiments, compounds that are
structurally related to mefloquine are compounds that have a
similar shape as mefloquine. In some embodiments, compounds that
are structurally related to mefloquine are 3-deazaadenosine,
indomethacin, mefenamic acid, 8-chloroadenosine and
O6-cyclohexylmethylguanine (See FIGS. 13 and 14). In some
embodiments, a compound that is structurally related to mefloquine
is roscovitine. In some embodiments, a compound that is
structurally related to mefloquine is S-roscovitine. In some
embodiments, a compound that is structurally related to mefloquine
is R-roscovitine. The antiviral effects of R-roscovitine have been
described, for instance in Orba et al. (Virology 2008 Jan. 5;
370(1):173-83).
[0082] In some embodiments, the invention relates to the use of
tolfenamic acid, and compounds with the same mode of action as
tolfenamic acid, to prevent or treat DNA viral infection or
proliferation (e.g., to reduce the risk of DNA viral infection or
proliferation). Tolfenamic acid is a drug that belongs to the class
of NSAIDs (Non-Steroidal Anti-Inflammatory Drugs), and acts by
inhibiting isoforms of cyclo-oxygenase 1 and 2 (COX 1 and COX2). In
some embodiments, the invention relates to the treatment or
prevention of PML using NSAIDs. In some embodiments, the invention
relates to the treatment or prevention of PML using NSAID
arylalkanoic acids. In some embodiments, the invention relates to
the treatment or prevention of PML using arylalkanoic acids.
[0083] In some embodiments, the invention relates to the use of
compounds that are structurally related to tolfenamic acid to
prevent or treat DNA viral infection or proliferation. In some
embodiments, compounds that are structurally related to tolfenamic
acid are compounds that comprise two aromatic rings linked by a
nitrogen. In some embodiments, compounds that are structurally
related to tolfenamic acid are arylalkanoic acids. In some
embodiments, arylalkanoic acids are secondary amines in which two
substituents are substituted and/or unsubstituted aryl groups.
Non-limiting examples of arylalkanoic acids are dicolfenac,
mefenamic acid, flufenamic acid and flunexin (See FIGS. 12 and
13).
[0084] In some embodiments, the invention relates to the use of
dioxybenzone (2,2'-Dihydroxy-4-methoxybenzophenone), and compounds
with the same mode of action as dioxybenzone, to prevent or treat
DNA viral infection or proliferation (e.g., to reduce the risk of
DNA viral infection or proliferation). In some embodiments, the
invention relates to the treatment or prevention of PML using
compounds that are structurally related to dioxybenzone.
[0085] In some embodiments, the invention relates to the use of
endosulfan, and compounds with the same mode of action as
endosulfan, to prevent or treat DNA viral infection or
proliferation (e.g., to reduce the risk of DNA viral infection or
proliferation). Endosulfan can act as a protein channel agonist. In
some embodiments, the invention relates to the treatment or
prevention of PML using protein channel antagonists. In some
embodiments, the invention relates to the treatment or prevention
of PML using compounds that are structurally related to
endosulfan.
[0086] In some embodiments, the invention relates to the use of
candesartan cilextil, and compounds with the same mode of action as
candesartan cilextil, to prevent or treat DNA viral infection or
proliferation (e.g., to reduce the risk of DNA viral infection or
proliferation). Candesartan cilextil can act as an angiotensin II
antagonist. In some embodiments, the invention relates to the
treatment or prevention of PML using angiotensin II antagonists. In
some embodiments, the invention relates to the treatment or
prevention of PML using compounds that are structurally related to
candesartan cilextil.
[0087] In some embodiments, the invention relates to the use of
fusidic acid, and compounds with the same mode of action as fusidic
acid, to prevent or treat DNA viral infection or proliferation
(e.g., to reduce the risk of DNA viral infection or proliferation).
In some embodiments, the invention relates to the treatment or
prevention of PML using antibiotics. In some embodiments, the
invention relates to the treatment or prevention of PML using
compound that interfere with bacterial protein synthesis. In some
embodiments, the invention relates to the treatment or prevention
of PML using compounds that are structurally related to fusidic
acid. In some embodiments, compounds that are structurally related
to fusidic acid are steroid derivatives. In some embodiments,
compounds that are structurally related to fusidic acid are
4-estren-3-beta 17 beta-diol 17-acetate, 5-beta-pregnan-3-alpha
6-alpha 20-beta-triol 20-acetate and 4-pregen-3-beta 20-beta-diol
20-acetate.
[0088] In some embodiments, the invention relates to the treatment
or prevention of PML using compounds that are structurally related
to candesartan cilextil.
[0089] In some embodiments, the invention relates to the use of
mefenamic acid, and compounds with the same mode of action as
mefenamic acid, to prevent or treat DNA viral infection or
proliferation (e.g., to reduce the risk of DNA viral infection or
proliferation). Mefenamic acid is a drug that belong to a class of
NSAIDs. In some embodiments, the invention relates to the treatment
or prevention of PML using NSAIDs.
[0090] In one aspect, the invention provides methods of treatment
of a subject infected with a DNA virus. In some embodiments, the
DNA virus is a JC virus. In some embodiments, the DNA virus is a BK
virus. In some embodiments, the invention provides methods of
treatment or prevention for subjects having, or at risk of
developing, progressive multifocal leukoencephalopathy (PML).
[0091] It should be appreciated that the inhibitory compounds or
compositions described herein may be used to reduce or suppress DNA
replication of DNA viruses (e.g., JC virus, BK virus, or any other
DNA virus).
[0092] The human genome is exposed to and may acquire many viruses
during the lifetime of an individual. One group of viruses the
genome is exposed to are DNA viruses. DNA viruses include papova
viruses and herpes viruses. Examples of papova viruses include, but
are not limited to SV40, human or bovine papilloma virus (HPV or
BPV), polyoma virus and human SV40-like viruses such as BK (BKV) or
JC (JCV). Examples of herpes viruses include, but are not limited
to herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr
virus (EBV), varicella or chickenpox virus, herpes zoster or
shingles virus. Exposure to a DNA virus is often asymptomatic and a
subject may not be aware that it has been exposed to a DNA virus.
Exposure to DNA viruses often leads to integration of the DNA virus
in the human genome. In a subject with a healthy immune system, the
immune system will generally suppress the proliferation of the
virus. However, in immuno-compromised subjects or in subjects
undergoing immune treatment, DNA viruses may be expressed and
proliferate resulting in the development of disease.
[0093] In one aspect, the current invention provides methods of
treating subjects infected with the JC polyomavirus (JCV). Primary
infection with JCV can occur asymptomatically during childhood
(Padgett et al., 1971 Lancet., 1257-1260). JCV is then disseminated
throughout the body, probably through viraemia (Ikegaya et al.,
2004, Archives of Virology 149, 1215-1220). It is thought that JCV
persists mostly in brain and renal tissue. While infection by JCV
is asymptomatic in most subjects, infection can result in serious
conditions (such as PML) and even death. PML is an extremely
debilitating demyelination disease of the central nervous system.
PML is generally characterized by neurological deficits that
progress rapidly, typically without signs of intracranial pressure,
including hemiparesis, cognitive disturbance, visual field
deficits, ataxia, aphasia, cranial nerve deficits and sensory
deficits. Patients who have PML typically deteriorate rapidly and
death commonly occurs within 6 months of diagnosis (Demeter L M. J
C, B K, and other polyomaviruses; progressive multifocal
leukoencephalopathy. In Mandell G L, Bennett J E, Dolin, eds.
Mandell, Douglas and Bennett's Principles and Practice of
Infectious Diseases, 4th edition, Vol. 2. New York, N.Y.: Churchill
Livingstone; 1995: 1400-1406). Subjects most susceptible to PML are
subjects that are immuno-compromised (e.g., AIDS patients) or
subjects undergoing treatment with immunosuppressants (for instance
after organ transplant or to treat an inflammation related
condition such as multiple sclerosis or rheumatoid arthritis).
[0094] PML has been reported to be associated with certain JCV
variants that have acquired sequence variations relative to a "wild
type" JCV sequence (Zheng et al., 2004, Microbes Infect., 6,
596-603). A "wild type" JCV sequence is used herein to refer to the
sequence of any of the archetypes of JCV found in healthy subjects
not having PML, and/or not being at risk for PML. In some
embodiments, a consensus "wild type" reference sequence may be an
average of sequences found in a group of healthy subjects.
Interestingly, in subjects having PML, a JCV with a number of
sequence variations was isolated from the brain, while a wild type
variant was isolated from the urine of the same individual (Yogo et
al., 2004, Rev Med Virol 14, 179-191). Accordingly, a subject may
be infected with several different versions of JCV concurrently. In
some embodiments of the present invention, a subject infected with
a JCV variant associated with PML is a subject at risk for
developing PML.
[0095] In one aspect, the current invention provides methods of
treating subjects infected with the BK virus. Infection with BK
virus is thought to be widespread but mostly asymptomatic. The
lung, eye, liver, brain and kidney are sites of BK virus-associated
disease. Infection in the kidney can lead to hemorrhage,
non-hemorrhagic cystitis, ureteric stenosis and nephritis.
Infection in the CNS has been associated with encephalitis and
Guillian-Barre syndrome. An overview of diseases associated with BK
virus can be found for instance in Reploeg et al. (Clinical
Infectious Diseases 2001; 33: 191-202), the contents of which are
incorporated herein by reference. In some embodiments, the current
invention provides methods of treating subjects at risk for
infection by BK virus. Subjects that are immuno-compromised or
receiving immunosuppressive agents are at an increased risk for BK
virus infection resulting in pathogenesis. Pathogenic BK virus
infection can be especially problematic in patients undergoing a
kidney transplant, which is often accompanied with the use of
immunosuppressive agents.
[0096] It should be appreciated that an immunosuppressive agent may
increase the susceptibility of a subject to the progression or
flare up of a latent microbial infection or to the contraction of a
new microbial infection. In some embodiments, the microbial
infection is infection by a DNA virus. In some embodiments, the
microbial infection is infection by JCV, which causes PML. In some
embodiments, the microbial infection is infection by BK virus. In
some embodiments, subjects that are immuno-compromised (e.g., AIDS
patients) or subjects undergoing treatment with immunosuppressants
are subjects art risk for PML. Subjects at risk for PML include
subjects that may receive or have received treatment with one or
more immunosuppressive agents (also called immunosuppressants). In
some cases, the immunosuppressive agent is administered to the
subject for treatment of a disease or condition, including one or
more of the following non-limiting examples: cancer, organ
transplant, tissue transplant, an inflammatory condition or
disease, multiple sclerosis (MS), arthritis, or any combination
thereof. In some embodiments, the immunosuppressive agent is an
anti-VLA-4 antibody (e.g., natalizumab). In some cases, an at-risk
subject tests positive for the presence of a JCV nucleic acid or a
JCV polypeptide. In other cases, the at-risk subject does not test
positive for the presence of a JCV nucleic acid or a JCV
polypeptide.
[0097] Currently, there is no specific antiviral therapy that has
been proven effective for treatment of JCV infection, and current
treatment of immuno-compromised subjects infected with JCV, or at
risk for JCV infection, is primarily focused on improving the
function of the immune system in general. Similarly, in subjects
infected with JCV or at risk for JCV infection that are receiving
immuno-therapy, current treatment methods of the JCV infection are
limited to termination or decrease in dose of the immuno-therapy.
Therapies currently used to treat PML rely on enhancement of the
immune response, e.g., HAART (Highly Active Anti-Retroviral
Therapy) in HIV positive patients (e.g., Marzocchetti et al., 2005,
J Clin Microbiology 434: 4175-4177) or decrease in the amount of
immunosuppressive drugs in subjects receiving those drugs (e.g.,
transplant patients). Methods of treatment of JCV include, but are
not limited to, IFN-alpha, cytarabine, and cidofovir. 5HT2a
blockers may also be used as a treatment and are currently under
study.
[0098] In some embodiments, a subject receiving an
immunosuppressive agent may be treated with compositions comprising
one or more of the compounds of Table 1. In some embodiments, the
treatment is prophylactic, e.g., the subject receiving
immunosuppressants may be treated with compositions comprising one
or more compounds of Table 1, even if the subject has not been
shown to be infected with a DNA virus, including JCV or BKV, and
does not show any symptoms associated with PML.
[0099] In some embodiments, a subject may be treated by combining
immunosuppressive treatment with treatment against DNA virus
infection or the development of diseases associated with DNA virus
infection, including PML. In one aspect, the invention comprises
methods of treatment comprising the administration of one or more
compounds of Table 1 and an immunosuppressant. In one aspect, the
invention comprises methods of treatment comprising the
administration of one or more compounds of Table 1 and natalizumab.
In one aspect, the invention comprises methods of treatment
comprising the administration of a composition comprising one or
more compounds of Table 1 and an anti-VLA-4 antibody (e.g.,
natalizumab).
[0100] In some embodiments, administration of one or more compounds
of Table 1 may be combined with methods to remove or partially
remove an immunosuppressant from the bloodstream of a subject. In
some embodiments, the immunosuppressant may be removed from the
bloodstream prior to administration of one or more compounds of
Table 1. In some embodiments, the one or more compounds of Table 1
may be combined with a compound that can bind to an
immunosuppressant resulting in an increased clearance of the
immunosuppressant from the bloodstream. In some embodiments, the
method of removal or partial removal of the immunosuppressant from
the bloodstream of a subject is plasma exchange (PLEX). In plasma
exchange blood is taken from the body and plasma containing the
immunosuppressant is removed from the blood by a cell separator.
Blood can be removed from the body in batches or it can be removed
in a continuous flow mode, with the latter allowing for the
reintroduction of the processed blood into the body. The removed
plasma comprising the immunosuppressants will be discarded and the
patient will receive donor plasma or saline with added proteins in
return. In some embodiments, multiple rounds of plasma exchange may
be needed to remove the immunosuppressant from the blood or to
lower the level of the immunosuppressant in the blood to an
acceptable level. Methods of plasma exchange are well known in the
art and are described for instance in U.S. Pat. No. 6,960,178.
[0101] The term "immunomodulatory" treatment or therapy refers to
the administration of one or more compounds that modulate (e.g.,
upregulate or downregulate) one or more aspects of a subject's
immune system. In some embodiments, an immunomodulatory treatment
or therapy involves the administration of one or more
immunosuppressive agents to a subject. In some embodiments, an
immunomodulatory treatment or therapy downregulates the immune
system of a subject. Accordingly, in some embodiments, an
immunomodulatory therapy is an immunosuppressive therapy. In some
embodiments, an immunomodulatory treatment or therapy downregulates
the immune system of a subject. It should be appreciated that an
immunomodulatory treatment or therapy (e.g., an immunosuppressive
treatment or therapy) as used herein also may be referred to as an
immunotherapy in the context of methods described herein.
[0102] The term "immunosuppressive agent" as used herein refers to
substances that act to suppress or mask the immune system of a
subject being treated herein. Immunosuppressive agents may be
substances that suppress cytokine production, down-regulate or
suppress self-antigen expression, or mask the MHC antigens.
Examples of such agents include 2-amino-6-aryl-5-substituted
pyrimidines (see e.g., U.S. Pat. No. 4,665,077); nonsteroidal
anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,
glucocorticoids such as cortisol or aldosterone, anti-inflammatory
agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase
inhibitor, or a leukotriene receptor antagonist; purine antagonists
such as azathioprine or mycophenolate mofetil (MMF); alkylating
agents such as cyclophosphamide; bromocryptine; danazol; dapsone;
glutaraldehyde (which masks the MHC antigens, as described in U.S.
Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and
MHC fragments; cyclosporin A; steroids such as corticosteroids or
glucocorticosteroids or glucocorticoid analogs, e.g., prednisone,
methylprednisolone, and dexamethasone; dihydrofolate reductase
inhibitors such as methotrexate (oral or subcutaneous);
hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine
receptor antagonists including anti-interferon-alpha, -beta, or
-gamma antibodies, anti-tumor necrosis factor-alpha antibodies
(infliximab or adalimumab), anti-TNF-alpha immunoahesin
(etanercept), anti-tumor necrosis factor-beta antibodies,
anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;
anti-LFA-1 antibodies, including anti-CD11a and anti-CD18
antibodies; anti-CD20 antibodies (e.g., rituximab, for example
available under the trademark RITUXAN); anti-L3T4 antibodies;
anti-VLA-4 antibodies (e.g., natalizumab); heterologous
anti-lymphocyte globulin; pan-T antibodies, for example anti-CD3 or
anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3
binding domain (WO 90/08187 published Jul. 26, 1990);
streptokinase; TGF-beta; streptodornase; RNA or DNA from the host;
FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (Cohen
et al., U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner
et al., Science, 251: 430432 (1991); WO 90/11294; Ianeway, Nature,
341: 482 (1989); and WO 91/01133); and T cell receptor antibodies
(EP 340,109) such as T10B9. However, subjects receiving other
immunosuppressive agents are also encompassed by the invention.
[0103] Accordingly, immunosuppressive agents may be drugs that
inhibit or prevent certain aspects of the immune system.
Immunosuppressive agents may be drugs that are used in
immunomodulatory (e.g., immunosuppressive) therapy, for example, to
prevent the rejection of transplanted organs or tissues (e.g., bone
marrow, heart, kidney, liver), to treat autoimmune diseases or
diseases that are suspected of being associated with an autoimmune
reaction (e.g., rheumatoid arthritis, multiple sclerosis,
myasthenia gravis, systemic lupus erythematosus, an inflammatory
bowel disease or syndrome, including, for example, Crohn's disease,
ulcerative colitis, and pemphigus), or to treat a non-autoimmune
inflammatory disease or condition (e.g., for term allergic asthma
control).
[0104] Immunosuppressive agents may be classified into five general
categories: glucocorticoids, cytostatics, antibodies, drugs acting
on immunophilins, and other drugs. Glucocorticoids may include
drugs that suppress cell-mediated immunity (e.g., by inhibiting
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and/or TNF-.gamma..
However, glucocorticoids also may suppress humoral immunity.
Cytostatics may include drugs that inhibit cell division (e.g.,
agents that inhibit T cell and/or B cell proliferation). In some
embodiments, cytostatics may be alkylating agents such as nitrogen
mustards (e.g., cyclophosphamide), nitrosureas, platinum compounds,
and others. In certain embodiments, immunosuppressive agents may be
antimetabolites such as folic acid analogs (e.g., methotrexate),
purine analogs (e.g., azathiprine, mercaptopurine), pyrimidine
analogs, or protein synthesis inhibitors. An immunosuppressive
agent also may be a cytotoxic antibiotic such as dactinomycin, an
anthracycline, mytomicin C, bleomycin, or mithramycin. Cytostatic
antibodies may be polyclonal or monoclonal antibodies that inhibit
one or more aspects of the immune system (e.g., that inhibit T
lymphocytes). Non-limiting examples of immunosuppressive polyclonal
preparations include Atgam (R), obtained from horse serum, and
Thymoglobuline (R), obtained from rabbit serum. In some
embodiments, monoclonal antibodies may be IL-2 receptor (CD25)
and/or CD3 directed antibodies. Non-limiting examples of
immunosuppressive monoclonal antibodies include T-cell receptor
directed antibodies (e.g., murorab, an anti-CD3 antibody), and/or
IL-2 receptor directed antibodies (e.g., basiliximab (Simulect (R))
and daclizumab (Zenapax (R)). Cytostatic drugs acting on
immunophilins may include cyclosporin, tacrolimus (e.g., Prograf),
and/or sirolimus (e.g., Rapamune, or Rapamycin). Other cytostatic
drugs may include interferons, opioids, TNF binding proteins (e.g.,
infliximab (e.g., Remicade), etanercept (e.g., Embrel), or
adalimumab (e.g., Humira)), myophenolate, and/or small biological
agents (e.g., FTY720, or myriocin).
[0105] Immunosuppressive agents that may be used to reduce the risk
of transplant rejection include, but are not limited to:
calcineurin inhibitors (e.g., Cyclosporin or Tacrolimus), mTOR
inhibitors (e.g., Sirolimus or Everolimus), anti-proliferatives
(e.g., Azathioprine, Mycophenolic acid), corticosteroids (e.g.,
prednisolone or hydrocortisone), and/or antibodies (e.g.,
monoclonal anti-IL-2Ralpha receptor antibodies such a basiliximab
or daclizumab or polyclonal anti-T-cell antibodies such as
anti-thymocyte globulin (ATG) or anti-lymphocyte globulin
(ALG)).
[0106] In some embodiments, the immunosuppressant is a VLA-4
binding antibody like natalizumab (also known as TYSABRI.RTM.). In
some embodiments, a VLA-4 binding antibody is an IgG antibody
(e.g., an IgG4 antibody). In some embodiments, a VLA-4 binding
antibody is a polyclonal or monoclonal antibody. In some
embodiments, a VLA-4 binding antibody is a humanized version of a
murine antibody. Natalizumab and related VLA-4 binding antibodies
are described, e.g., in U.S. Pat. No. 5,840,299. mAb 21.6 and HP1/2
are exemplary murine monoclonal antibodies that bind VLA-4.
Natalizumab is a humanized version of murine mAb 21.6 (see, e.g.,
U.S. Pat. No. 5,840,299). A humanized version of HP1/2 has also
been described (see, e.g., U.S. Pat. No. 6,602,503). Several
additional VLA-4 binding monoclonal antibodies, such as HP2/1,
HP2/4, L25 and P4C2, are described (e.g., in U.S. Pat. No.
6,602,503; Sanchez-Madrid et al., 1986 Eur. J. Immunol.,
16:1343-1349; Hemler et al., 1987 J. Biol. Chem. 2:11478-11485;
Issekutz and Wykretowicz, 1991, J. Immunol., 147: 109 (TA-2 mab);
Pulido et al., 1991 J. Biol. Chem., 266(16):10241-10245; and U.S.
Pat. No. 5,888,507). Many useful VLA-4 binding antibodies interact
with VLA-4 on cells, e.g., lymphocytes, but do not cause cell
aggregation. However, other anti-VLA-4 binding antibodies have been
observed to cause such aggregation. HP1/2 does not cause cell
aggregation. The HP1/2 MAb (Sanchez-Madrid et al., 1986 Eur. J.
Immunol., 16:1343-1349) has an extremely high potency, blocks VLA-4
interaction with both VCAM1 and fibronectin, and has the
specificity for epitope B on VLA-4. This antibody and other B
epitope-specific antibodies (such as B1 or B2 epitope binding
antibodies; Pulido et al., 1991 J. Biol. Chem.,
266(16):10241-10245) represent one class of useful VLA-4 binding
antibodies.
[0107] An exemplary VLA-4 binding antibody has one or more CDRs,
e.g., all three HC CDRs and/or all three LC CDRs of a particular
antibody disclosed herein, or CDRs that are, in sum, at least 80,
85, 90, 92, 94, 95, 96, 97, 98, or 99% identical to natalizumab. In
one embodiment, the H1 and H2 hypervariable loops have the same
canonical structure as those of natalizumab. In some embodiments,
the L1 and L2 hypervariable loops have the same canonical structure
as natalizumab. In some embodiments, the amino acid sequence of the
HC and/or LC variable domain sequence is at least 70, 80, 85, 90,
92, 95, 97, 98, 99, or 100% identical to the amino acid sequence of
the HC and/or LC variable domain of natalizumab. The amino acid
sequence of the HC and/or LC variable domain sequence can differ by
at least one amino acid, but no more than ten, eight, six, five,
four, three, or two amino acids from the corresponding sequence of,
natalizumab. For example, the differences may be primarily or
entirely in the framework regions. The amino acid sequences of the
HC and LC variable domain sequences can be encoded by a sequence
that hybridizes under high stringency conditions to a nucleic acid
sequence described herein or one that encodes a variable domain or
to a nucleic acid encoding an amino acid sequence described herein.
In one embodiment, the amino acid sequences of one or more
framework regions (e.g., FR1, FR2, FR3, and/or FR4) of the HC
and/or LC variable domain are at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to corresponding framework regions of the
HC and LC variable domains of natalizumab. In some embodiments, one
or more heavy or light chain framework regions (e.g., HC FR1, FR2,
and FR3) are at least 70, 80, 85, 90, 95, 96, 97, 98, or 100%
identical to the sequence of corresponding framework regions from a
human germline antibody.
[0108] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) are
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). The optimal alignment is determined as
the best score using the GAP program in the GCG software package
with a Blossum 62 scoring matrix with a gap penalty of 12, a gap
extend penalty of 4, and a frameshift gap penalty of 5. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences. The skilled artisan will realize that
conservative amino acid substitutions may be made in VLA-4 binding
antibodies to provide functionally equivalent variants of these
antibodies. As used herein, a "conservative amino acid
substitution" refers to an amino acid substitution that does not
alter the relative charge or size characteristics of the protein in
which the amino acid substitution is made. Variants can be prepared
according to methods for altering polypeptide sequence known to one
of ordinary skill in the art such as are found in references that
compile such methods, e.g., Molecular Cloning: A Laboratory Manual,
J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N. Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. Exemplary functionally equivalent
variants of VLA-4 binding antibodies include conservative amino
acid substitutions of in the amino acid sequences of proteins
disclosed herein. Conservative substitutions of amino acids include
substitutions made amongst amino acids within the following groups:
(a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f)
Q, N; and (g) E, D.
[0109] In some embodiments, the methods of treatment or prevention
comprise administering a composition comprising one or more
compounds of Table 1 and one or more known or putative anti-viral
compounds or compounds displaying anti-viral activity. Known or
putative anti-viral compounds are compounds that suppress or
inhibit viral infection, viral proliferation and/or the development
of disease associated with viral infection. Anti-viral drugs can be
classified as targeting one of the life cycle stages of the virus.
One category of anti-viral drugs are based on interfering with
viral entry. A virus binds to a specific receptor to infiltrate a
target cell. Viral entry can be suppressed by blocking of the viral
entry way. Anti-viral drugs that have this mode of action are
anti-receptor antibodies, natural ligands of the receptor and small
molecules that can bind to the receptor. A second category of
antiviral drugs are compounds that suppress viral synthesis.
Antiviral drugs that have this mode of action are nucleoside
analogues that are similar to the DNA and RNA building blocks but
deactivate the protein machinery (e.g., reverse transcriptase or
DNA polymerase) used to replicate the virus. Other drugs are
targeted at blocking the transcription factors of viral DNA,
ribozymes, which can interfere with the production of viral DNA.
Other drugs target viral RNA for destruction, including siRNAs and
antisense nucleic acids against viral nucleic acid sequences. Yet
another class of antiviral drugs are drugs that can interfere with
the function of virus specific proteins. This class includes the
HIV protease inhibitors. Antiviral drugs also include drugs
directed at the release stage if the virus. This category of drugs
include compounds that interfere with the proteins necessary to
build the viral particles. Another class of antiviral drugs are
drugs that stimulate the immune system in targeting viral
infection. Drugs that fall in this class are interferons, which
inhibit viral synthesis in infected cells. and antibodies that can
target an infected cell for destruction by the immune system. Other
anti-viral agents are described in U.S. Pat. Nos. 6,130,326, and
6,440,985, and published US patent application 20020095033.
Accordingly, it should be appreciated that compounds identified
herein have antiviral activity and may act through any antiviral
mechanism described above. In some embodiments, compounds
identified herein inhibit or suppress viral replication (e.g.,
viral DNA replication).
[0110] The anti-viral activity of a compound may be assayed in an
in vitro cell based assay. Anti-viral activity may result from i)
the interaction of a compound with the virus to prevent infection
of a cell or to prevent replication, development, and/or
proliferation of the virus after infection, ii) the effect of a
compound on a cell to prevent infection by the virus or to prevent
replication, development, and/or proliferation of the virus after
infection, or iii) any other mechanism, or any combination thereof.
Regardless of the mode of action, a composition of the invention
may have anti-viral activity if it reduces the percentage or number
of infected cells in a cell-based assay. In some embodiments, a
compound (or a combination of two or more compounds) has anti-viral
activity when it reduces the percentage or number of infected cells
by at least 20%, at least 30%, at least 40%, at least 50%, or more
(e.g., in a cell-based assay). In some embodiments, a compound has
anti-viral activity when it reduces the amount of viral nucleic
acids within a cell. In certain embodiments, a compound inhibits
the replication of viral nucleic acids within a cell (e.g., a
compound reduces the amount of viral replication by about 5%, about
10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, about 90%, or higher or lower or intermediate
percentages of reduction). It should be appreciated that a
reduction in viral replication may be measured using a cellular
assay and measuring the amount of viral DNA or the rate of viral
DNA replication over time (or any other measure of viral
replication) in the presence of a compound and comparing it to the
viral replication in the absence of the compound or in the presence
of a control compound.
[0111] It should be appreciated that certain compositions may
effectively inhibit viral activity in certain cell types and not
others. A composition of the invention may be useful if it is
effective in certain cell types regardless of whether it is active
in all cell types (or even in more than one cell type). For
example, compositions of the invention may be compositions that are
effective at least in one or more neural cells, for example, one or
more cells of the central nervous system (e.g., glial cells,
astrocytes, etc.). It should be appreciated that In some
embodiments, non-cytotoxic compounds (or compounds that selectively
kill infected cells) are used in compositions and methods of the
invention.
[0112] In some embodiments, the methods of treatment or prevention
comprise administering a composition comprising one or more
compounds of Table 1 and administering a vaccine against a DNA
virus. A vaccine is defined as a pharmaceutical composition that
when administered to a subject in an effective amount stimulates
the production of protective antibody or protective T-cell
response. In some embodiments, the vaccine is protein vaccine
comprising one or more polypeptide sequences encoded by a DNA virus
sequence. In some embodiments, the vaccine is a nucleic acid
vaccine comprising DNA viral nucleic acids. Administration regimes
for vaccines are known to a person of ordinary skill in the art. In
some embodiments, ranges of amounts of polypeptide vaccines for
prophylaxis of DNA viral infection are from 0.01 to 100
microgram/dose, for example 0.1 to 50 microgram/dose. Several doses
may be needed per subject in order to achieve a sufficient immune
response and subsequent protection against DNA viral infection
(e.g., "immunizing" a subject). The term "immunizing" refers to the
ability of a substance to cause a humoral and/or cellular response
in a subject, whether alone or when linked to a carrier, in the
presence or absence of an adjuvant, and also refers to an immune
response that blocks the infectivity, either partially or fully, of
an infectious agent.
[0113] In some embodiments, the methods of treatment or prevention
comprise administering one or more compounds of Table 1 and
administering an antibody against a DNA virus. In some embodiment
the methods of treatment or prevention comprise administering one
or more compounds of Table 1 and administering an antibody against
JCV. Antibodies may be used in therapy to treat subjects with PML
and/or to prevent infection by and/or suppress the activity of JCV
and other DNA viruses. Suitable antibodies or fragments thereof may
be selected for the ability to bind one or more polypeptides
encoded by a DNA virus, including JCV. The antibody or
antigen-binding fragment thereof may be an IgG1, IgG2, IgG3, IgG4,
IgM, IgA1, IgA2, IgAsec, IgD, IgE or may have an immunoglobulin
constant and/or variable domain of an IgG1, IgG2, IgG3, IgG4, IgM,
IgA1, IgA2, IgAsec, IgD or IgE. In some embodiments, the antibody
is a bispecific or multispecific antibody. In some embodiments, the
antibody is a recombinant antibody, a polyclonal antibody, a
monoclonal antibody, a humanized antibody or a chimeric antibody,
or a mixture of these. In some embodiments, the antibody is a human
antibody, e.g., a human monoclonal antibody, polyclonal antibody or
a mixture of monoclonal and polyclonal antibodies. Antigen-binding
fragments may include a Fab fragment, a F(ab').sub.2 fragment,
and/or a F.sub.v fragment CDR3. Antibodies can be raised against a
full length DNA virus protein or JCV protein or against
polypeptides variants comprising a partial sequence of a DNA virus
protein or JCV protein. Antibodies can be generated by injecting an
animal, for example a rabbit or goat or mouse, with the antigen
(e.g., a polypeptide of a DNA virus protein or JCV protein). In
order to prepare polyclonal antibodies, fusion proteins containing
a polypeptide of a DNA virus protein or JCV protein can be
synthesized in bacteria by expression of corresponding DNA
sequences in a suitable cloning vehicle. The fusion protein can
then be purified, coupled to a carrier protein and mixed with
Freund's adjuvant (to help stimulate the antigenic response by the
rabbits) and injected into rabbits or other laboratory animals.
Alternatively, the polypeptides can be isolated from cultured cells
expressing the protein. Following booster injections at bi-weekly
intervals, the rabbits or other laboratory animals are then bled
and the sera isolated. The sera can be used directly or purified
prior to use, e.g., by methods such as affinity chromatography,
Protein A-Sepharose, Antigen Sepharose, Anti-mouse-Ig-Sepharose.
The sera can then be used to probe protein extracts run on a
polyacrylamide gel to identify the DNA virus or JCV polypeptides.
Alternatively, synthetic DNA virus or JCV polypeptides can be made
and used to inoculate animals. To produce monoclonal DNA virus or
JCV antibodies, mice are injected multiple times (see above), the
mice spleens are removed and resuspended in a phosphate buffered
saline (PBS). The spleen cells serve as a source of lymphocytes,
some of which produce antibodies of the appropriate specificity.
These are then fused with a permanently growing myeloma partner
cell, and the products of the fusion are plated into a number of
tissue culture wells in the presence of a selective agent such as
HAT. The wells are then screened by ELISA to identify those
containing cells expressing useful antibody. These are then freshly
plated. After a period of growth, these wells are again screened to
identify antibody-producing cells. Several cloning procedures are
carried out until over 90% of the wells contain single clones which
are positive for antibody production. From this procedure a stable
line of clones is established to produce the antibody. A monoclonal
antibody can then be purified by affinity chromatography using
Protein A Sepharose, ion-exchange chromatography, as well as
variations and combinations of these techniques (See e.g., U.S.
Pat. No. 6,998,467). For antibodies to be used in therapy in
humans, they may be `humanized`. Humanization of antibodies
involves replacing native mouse sequences with human sequences to
lower the chance of an immune response once the therapeutic
antibody is introduced into humans. In some embodiments, human
antibodies (e.g., identified from libraries of human antibodies)
may be used.
[0114] In some embodiments, the compounds that prevent or treat DNA
viral infection or proliferation have an IC.sub.50<100 .mu.M, an
IC.sub.50<20 .mu.M, an IC.sub.50<10 .mu.M, an IC.sub.50<5
.mu.M, or an even lower IC.sub.50. The IC.sub.50 (Inhibitory
Concentration) is defined herein as the inhibitory concentration at
which 50% of JC viral infection is inhibited. In some embodiments,
the compounds that prevent or treat DNA viral infection or
proliferation have a TC.sub.50>5 .mu.M, a TC.sub.50>20 .mu.M,
a TC.sub.50>50 .mu.M, a TC.sub.50>100 .mu.M, or an even
higher TC.sub.50. The TC.sub.50 (cytoToxic Concentration) is
defined herein as the concentration of the inhibitor at which 50%
of cells are killed. In some embodiments, the compounds that
prevent or treat DNA viral infection or proliferation have an
IC.sub.50/TC.sub.50<5, an IC.sub.50/TC.sub.50<1, an
IC.sub.50/TC.sub.50<0.5, an IC.sub.50/TC.sub.50<0.2, an
IC.sub.50/TC.sub.50<0.2 or an even lower IC.sub.50/TC.sub.50. A
compound with a lower IC.sub.50/TC.sub.50 potentially has a larger
potential therapeutic window, as a lower IC.sub.50/TC.sub.50
correlates to a lower IC.sub.50 (inhibitory concentration) and a
higher TC.sub.50 (cytotoxic concentration).
[0115] In some embodiments, the compounds that prevent or treat DNA
viral infection or proliferation, upon administration have a
concentration in the target tissue of at least 0.5.times.IC.sub.50,
at least 1.times.IC.sub.50, at least 2.times.IC.sub.50, at least
3.times.IC.sub.50, at least 10.times.IC.sub.50 or higher. In some
embodiments, the compounds that prevent or treat DNA viral
infection or proliferation, upon administration have a
concentration in the target tissue of less than 1.times.TC.sub.50,
less than 0.5.times.TC.sub.50, less than 0.2.times.TC.sub.50, less
than 0.1.times.TC.sub.50, less than 0.01.times.TC.sub.50 or lower.
In some embodiments, the compounds that prevent or treat DNA viral
infection or proliferation, upon administration have an
IC.sub.50/TC.sub.50 ratio in the target tissue of less than <5,
an IC.sub.50/TC.sub.50<1, an IC.sub.50/TC.sub.50<0.5, an
IC.sub.50/TC.sub.50<0.2, an IC.sub.50/TC.sub.50<0.2 or an
even lower IC.sub.50/TC.sub.50.
[0116] Target tissue, as used herein, embraces any tissue in a
subject, including but not limited to kidney, brain, liver, spleen,
bone marrow, intestine, stomach. In some embodiments, the target
tissue is brain tissue or CNS. In some embodiments, the target
tissue is the kidney. In some embodiments, the compounds that
prevent or treat DNA viral infection or proliferation have a high
plasma level upon administration. In some embodiments, the
compounds that prevent or treat DNA viral infection or
proliferation, wherein the DNA viral infection is located in the
kidney or is expected to target the kidney, have a high plasma
level upon administration.
[0117] In some embodiments, infection with a DNA virus is
characterized by, or has a potential to result in, infection of the
brain, including the CNS and CSF. In some embodiments, one or more
of the compounds of Table 1 are administered to a subject that has
a DNA virus infection of the brain, is at risk for infection of the
brain by a DNA virus, or has a disease associated with a DNA virus
infection of the brain (e.g., PML). In some embodiments, one or
more of the compounds of Table 1 are administered in conjunction
with an agent that targets or facilitates delivery of the compounds
across the blood brain barrier.
[0118] As used herein a "blood-brain barrier targeting agent" is a
molecule or compound that is capable of crossing the blood-brain
barrier and can be used to deliver a therapeutic composition of the
invention across the blood-brain barrier and into the CNS. As used
herein a "CNS-cell targeting agent" is a molecule or compound that
delivers a composition of the invention to a region of the CNS or
to, or into, a CNS cell type once the composition is inside the
blood-brain barrier. It will be understood that in some embodiments
of the invention, a therapeutic composition may be attached to one
or more blood-brain targeting agents and may be also attached to
one or more CNS cell targeting agents. Thus there may be more than
one type of blood-brain barrier targeting agent and/or more than
one CNS cell targeting agent attached to a carrier of the
invention. In some embodiments, one or more of the compounds of
Table 1 may be packaged in a carrier (e.g., a liposome) to
facilitate or target delivery to the brain and/or CNS.
[0119] In some embodiments, one or more of the compounds of Table 1
may be administered directly to a tissue infected by, or suspected
of being infected by, the DNA virus (e.g., JCV, BCV, or other DNA
virus). In some embodiments, one or more of the compounds of Table
1 may be administered directly to the brain. In some embodiments,
one or more of the compounds of Table 1 may be administered through
intrathecal injection. An intrathecal injection is an injection
into the spinal canal which facilitates direct delivery of a
compound to the CNS and the brain, thereby circumventing the blood
brain barrier. In some embodiments, intrathecal injection is
performed as a method of administration of compounds that have a
low oral administration.
[0120] The phrase "therapeutically-effective amount" as used herein
means that amount of a compound, material, or composition
comprising one or more compounds of the present invention which are
effective for producing some desired therapeutic effect in a
subject at a reasonable benefit/risk ratio applicable to any
medical treatment. Accordingly, in some embodiments, a
therapeutically effective amount prevents, minimizes, or reverses
disease progression associated with infection with a DNA virus
(including JCV, BCV, or other DNA virus). Disease progression can
be monitored by clinical observations, laboratory and imaging
investigations apparent to a person skilled in the art. A
therapeutically effective amount can be an amount that is effective
in a single dose or an amount that is effective as part of a
multi-dose therapy, for example an amount that is administered in
two or more doses or an amount that is administered
chronically.
[0121] The effective amount of any one or more compounds may be
from about 10 ng/kg of body weight to about 1000 mg/kg of body
weight, and the frequency of administration may range from once a
day to once a month. However, other dosage amounts and frequencies
also may be used as the invention is not limited in this respect. A
subject may be administered one or more compounds described herein
in an amount effective to treat or prevent infection with a DNA
virus. As used herein, a treatment may be prophylactic and/or
therapeutic. In some embodiments, a treatment may include
preventing viral infection and/or proliferation. In certain
embodiments, a treatment may include inhibiting and or reducing
viral infection and/or proliferation. It should be appreciated that
the terms preventing and/or inhibiting may be used to refer to a
partial prevention and/or inhibition (e.g., a percentage reduction,
for example about 5%, about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, or higher or
lower or intermediate percentages of reduction). However, in some
embodiments, a prevention or inhibition may be complete (e.g., a
100% reduction or about a 100% reduction based on an assay).
[0122] In some embodiments, the effective amount is tissue
specific. In some embodiments, the effective amount is an amount of
one or more compounds of Table 1 that results in prevention or
inhibition of viral activity in a specific tissue (e.g., viral
proliferation in that tissue). In some embodiments, the effective
amount is an amount of one or more compounds of Table 1 that
results in prevention or inhibition of viral activity in the brain.
In some embodiments, the effective amount is an amount of one or
more compounds of Table 1 that results in about 5%, about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about 90%, or higher or lower or intermediate
percentages of inhibition of viral activity in the brain. In some
embodiments, the effective amount is an amount of one or more
compounds of Table 1 that results in about 5%, about 10%, about
20%, about 30%, about 40%, about 50%, about 60%, about 70%, about
80%, about 90%, or higher or lower or intermediate percentages of
reduction of viral DNA replication in a cell (e.g., as measured in
a cellular assay) when compared to the percentage reduction of
viral DNA replication in the absence of a compound (or when the
cell is exposed to a control compound that does not have antiviral
activity, e.g., saline or vehicle control). In some embodiments,
the biological sample concentration of one or more compounds of
Table 1 is 1 nM, 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100 .mu.M, 1 mM,
10 mM, 100 mM after administration of the one or more compounds of
Table 1. In some embodiments, the biological sample concentration
of one or more compounds of Table 1 is at least 10 .mu.M after
administration of the one or more compounds of Table 1. In some
embodiments, the concentration of one or more compounds of Table 1
in a specific tissue is 1 nM, 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100
.mu.M, 1 mM, 10 mM, 100 mM after administration of the one or more
compounds of Table 1. In some embodiments, the concentration of one
or more compounds of Table 1 in a specific tissue is at least 10
.mu.M after administration of the one or more compounds of Table 1.
In some embodiments, the concentration of one or more compounds of
Table 1 in the brain is 1 nM, 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100
.mu.M, 1 mM, 10 mM, 100 mM after administration of the one or more
compounds of Table 1. In some embodiments, the concentration of one
or more compounds of Table 1 in the brain is at least 10 .mu.M
after administration of the one or more compounds of Table 1.
[0123] Accordingly, methods of treatment of JCV infection and PML
may be evaluated prior to initiating treatment with an
immunosuppressive agent, during administration of an
immunosuppressive agent, or assessed after an immunosuppressive
agent has been administered or after treatment with
immunosuppressive treatment has been terminated.
[0124] As used herein, "diagnosing" and "evaluating treatment of
PML" comprises determining the presence of JCV infection.
Determining the presence of JCV infection comprises the detection
of JCV in one or more tissues or in fluids (which can include
determining the viral load in blood and/or cerebral spinal fluid)
and may include determining the sequence of JCV. Diagnostic assays
include but are not limited to histopathology,
immunohistochemistry, flow cytometry, cytology, patho-physiological
assays, including MRI and tomography, neurological assays
biochemical assays. Biochemical assays include but are not limited
to variant analysis, viral genome analysis, ELISA analysis,
including the use of antibodies against one or more proteins of
JCV, analysis of specific proteins, platelet count, etc. Those of
ordinary skill in the art will be aware of numerous diagnostic
protocols and parameters that are routinely utilized in the
art.
[0125] In some aspects of the invention, PML-specific diagnostic
tests and methods of monitoring PML symptoms may be used in
connection with monitoring and treating a subject with PML, a
subject suspected of having PML, a subject at risk for PML, a
subject being treated with immunosuppressants, and/or a subject
currently under treatment for PML. PML diagnostic criteria may be
used in the assessment of subjects being treated (e.g., to monitor
efficacy of treatment) or to assist in the decision whether to
treat a subject with a method or composition of the invention,
including, but not limited to, whether to supplement an alternative
treatment with a treatment of the invention. In some embodiments,
PML diagnostic criteria may be used to assist in the decision
regarding treatment of a subject with immunosuppressant, e.g., to
terminate, temporarily halt, or decrease the dose of
immunosuppressant treatment regimens. Symptoms of PML are
neurological and include problems with hand-eye coordination,
including difficulty writing and typing, as well as problems with
speech, hemiparesis and nonfluent aphasia. In some cases, an MRI
scan showing an increase in the extent of the high T2-weighted and
low T1-weighted signal abnormalities compared to a normal reference
is indicative of PML. MRI may be used to monitor changes in lesion
size and progression of PML. Detection of JCV DNA by PCR in CSF is
a widely used diagnostic test of PML, it has 99% specificity and
70% selectivity. Brain biopsy for detection of JCV DNA may also be
performed to diagnose or assess PML. In the absence of JCV PCR+
result for CSF, a brain biopsy may be performed and JCV DNA
detection in brain tissue is used as a positive diagnosis of PML.
Kappos et al., Natalizumab treatment for multiple sclerosis:
recommendations for patient selection and monitoring, Lancet
Neurol., 2007 May, 6(5): 431-41, describes non-limiting examples of
diagnostic and management algorithms to monitor patients (e.g.,
multiple sclerosis patients) that are treated with natalizumab.
Patients can be monitored using a combination of clinical, MRI, and
laboratory assessments. The algorithms of Kappos et al. are
incorporated herein by reference in their entirety.
[0126] As used herein, methods of the invention may be carried out
in subjects. A subject may be a human or a non-human animal,
including, but not limited to a non-human primate, cow, horse, pig,
sheep, goat, dog, cat, or rodent. In general, all embodiments
described herein may be applied to human subjects where
appropriate.
[0127] In some embodiments, the methods of treatment of the
invention comprise administration of compositions of compounds of
Table 1 in combination with candidate therapeutic compounds
identified in screens for activity against DNA viral infection or
PML. Candidate compounds that have activity against DNA viral
infection or PML can be identified through a variety of screening
methods including both in vitro screens and in in vivo screens. In
vitro screens encompass both biochemical and biological assays.
Biochemical assays encompass assays that can determine the binding
of candidate therapeutic compounds to a specific target, e.g., a
protein or other macromolecule of the DNA virus. Biological assays
encompass cellular assays, which can for instance be based on the
uptake of a virus in a cell, the release of a virus from a cell,
infection rate, viral DNA replication (e.g., measured as a viral
DNA replication rate, an amount of viral DNA in a cell, or other
measure of viral DNA replication) etc., or any combination thereof.
In general, the assays will have a readout (like fluorescence)
which allow for the determination of the potential therapeutic
efficacy of a candidate therapeutic compounds for the treatment of
infection with a DNA virus or PML. In some embodiments, an assay
mixture for testing a candidate agent comprises a candidate agent.
A candidate agent may be an antibody, a small organic compound, or
a polypeptide, and accordingly can be selected from combinatorial
antibody libraries, combinatorial protein libraries, or small
organic molecule libraries. Typically, pluralities of reaction
mixtures are run in parallel with different agent concentrations to
obtain a different response to the various concentrations.
Typically, one of these concentrations serves as a negative
control, e.g., at zero concentration of agent or at a concentration
of agent below the limits of assay detection. Any molecule or
compound can be a candidate therapeutic. Non-limiting examples of
candidate therapeutics are small molecules, RNA including siRNAs,
DNA including aptamers, and proteins including antibodies and
antibody fragments. The invention also embraces candidate
therapeutic compounds with different modes of action. Candidate
agents encompass numerous chemical classes, although typically they
are organic compounds, proteins or antibodies (and fragments
thereof that bind antigen). In some general embodiments, the
candidate agents are small organic compounds, e.g., those having a
molecular weight of more than 50 yet less than about 2500, for
example less than about 1000 and, in certain embodiments, less than
about 500. Candidate agents comprise functional chemical groups
necessary for structural interactions with polypeptides and/or
nucleic acids, and may include at least an amine, carbonyl,
hydroxyl, or carboxyl group, optionally at least two of the
functional chemical groups or at least three of the functional
chemical groups. The candidate agents can comprise cyclic carbon or
heterocyclic structure and/or aromatic or polyaromatic structures
substituted with one or more of the above-identified functional
groups. Candidate agents also can be biomolecules such as nucleic
acids, polypeptides, saccharides, fatty acids, sterols,
isoprenoids, purines, pyrimidines, derivatives or structural
analogs of the above, or combinations thereof and the like.
[0128] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides, synthetic organic
combinatorial libraries, phage display libraries of random or
non-random polypeptides, combinatorial libraries of proteins or
antibodies, and the like. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant, and animal
extracts are available or readily produced. Additionally, natural
and synthetically produced libraries and compounds can be readily
be modified through conventional chemical, physical, and
biochemical means. Further, known agents may be subjected to
directed or random chemical modifications such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs of the agents.
[0129] A variety of other reagents also can be included in the
mixture. These include reagents such as salts, buffers, neutral
proteins (e.g., albumin), detergents, etc., which may be used to
facilitate optimal protein-protein and/or protein-agent binding.
Such a reagent may also reduce non-specific or background
interactions of the reaction components. Other reagents that
improve the efficiency of the assay such as protease inhibitors,
nuclease inhibitors, antimicrobial agents, and the like may also be
used.
[0130] In some embodiments, candidate therapeutic compounds are
based on the compounds of Table 1 and comprise modified versions of
the compounds of Table 1 generated through methods of medicinal
chemistry known to the skilled artisan.
[0131] In another aspect, the present invention provides
"pharmaceutical compositions", which comprise a therapeutically
effective amount of one or more of the compounds described herein,
formulated together with one or more pharmaceutically acceptable
carriers (additives) and/or diluents. As described in detail, the
pharmaceutical compositions of the present invention may be
specially formulated for administration in solid or liquid form,
including those adapted for the following: oral administration, for
example, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g., those targeted for buccal, sublingual,
and systemic absorption, boluses, powders, granules, pastes for
application to the tongue; parenteral administration, for example,
by subcutaneous, intramuscular, intravenous or epidural injection
as, for example, a sterile solution or suspension, or
sustained-release formulation; topical application, for example, as
a cream, ointment, or a controlled-release patch or spray applied
to the skin, lungs, or oral cavity; intravaginally or
intrarectally, for example, as a pessary, cream or foam;
sublingually; ocularly; transdermally; or nasally, pulmonary and to
other mucosal surfaces.
[0132] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0133] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient, or
solvent encapsulating material, involved in carrying or
transporting the subject compound from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; pH buffered
solutions; polyesters, polycarbonates and/or polyanhydrides; and
other non-toxic compatible substances employed in pharmaceutical
formulations.
[0134] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated, and the particular mode of administration. The
amount of active ingredient that can be combined with a carrier
material to produce a single dosage form will generally be that
amount of the compound which produces a therapeutic effect.
Generally, this amount will range from about 1% to about 99% of
active ingredient, for example from about 5% to about 70%, and in
some embodiments from about 10% to about 30%.
[0135] In certain embodiments, a formulation of the present
invention comprises an excipient selected from the group consisting
of cyclodextrins, liposomes, micelle forming agents, e.g., bile
acids, and polymeric carriers, e.g., polyesters and polyanhydrides;
and a compound of the present invention. In certain embodiments, an
aforementioned formulation renders orally bioavailable a compound
of the present invention.
[0136] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0137] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0138] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol, glycerol monostearate,
and non-ionic surfactants; absorbents, such as kaolin and bentonite
clay; lubricants, such as talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, and
mixtures thereof; and coloring agents. In the case of capsules,
tablets and pills, the pharmaceutical compositions may also
comprise buffering agents. Solid compositions of a similar type may
also be employed as fillers in soft and hard-shelled gelatin
capsules using such excipients as lactose or milk sugars, as well
as high molecular weight polyethylene glycols and the like.
[0139] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made in a suitable machine in which a mixture
of the powdered compound is moistened with an inert liquid
diluent.
[0140] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be formulated for rapid release, e.g.,
freeze-dried. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions that
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0141] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0142] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0143] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0144] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0145] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0146] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0147] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0148] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0149] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Dissolving or dispersing the compound in the proper medium
can make such dosage forms. Absorption enhancers can also be used
to increase the flux of the compound across the skin. Either
providing a rate controlling membrane or dispersing the compound in
a polymer matrix or gel can control the rate of such flux.
[0150] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0151] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
sugars, alcohols, antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0152] Examples of suitable aqueous and nonaqueous carriers, which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0153] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms upon the subject
compounds may be ensured by the inclusion of various antibacterial
and antifungal agents, for example, paraben, chlorobutanol, phenol
sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into
the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
[0154] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0155] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions, which are
compatible with body tissue.
[0156] In certain embodiments, a compound or pharmaceutical
preparation is administered orally. In other embodiments, the
compound or pharmaceutical preparation is administered
intravenously. Alternative routes of administration include
sublingual, intramuscular, and transdermal administrations.
[0157] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1% to
99.5% (in certain embodiments, 0.5% to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0158] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given in forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. In some embodiments, oral
administrations are used.
[0159] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0160] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0161] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0162] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically-acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0163] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0164] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular compound being
employed, the duration of the treatment, other drugs, compounds
and/or materials used in combination with the particular compound
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0165] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required to achieve the desired therapeutic effect and then
gradually increasing the dosage until the desired effect is
achieved.
[0166] In some embodiments, a compound or pharmaceutical
composition of the invention is provided to a subject chronically.
Chronic treatments include any form of repeated administration for
an extended period of time, such as repeated administrations for
one or more months, between a month and a year, one or more years,
or longer. In many embodiments, a chronic treatment involves
administering a compound or pharmaceutical composition of the
invention repeatedly over the life of the subject. In certain
embodiments, chronic treatments involve regular administrations,
for example one or more times a day, one or more times a week, or
one or more times a month. In general, a suitable dose such as a
daily dose of a compound of the invention will be that amount of
the compound that is the lowest dose effective to produce a
therapeutic effect. Such an effective dose will generally depend
upon the factors described above. Generally doses of the compounds
of this invention for a patient, when used for the indicated
effects, will range from about 0.0001 to about 100 mg per kg of
body weight per day. In some embodiments, the daily dosage will
range from 0.001 to 50 mg of compound per kg of body weight, for
example from 0.01 to 10 mg of compound per kg of body weight.
However, lower or higher doses can be used. In some embodiments,
the dose administered to a subject may be modified as the
physiology of the subject changes due to age, disease progression,
weight, or other factors.
[0167] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0168] While it is possible for one or more compounds of the
present invention to be administered alone, in general embodiments
one or more compounds may be administered as a pharmaceutical
formulation (composition) as described herein.
[0169] The compounds according to the invention may be formulated
for administration in any convenient way for use in human or
veterinary medicine, by analogy with other pharmaceuticals.
[0170] The invention also relates to a method of making a
medicament for use in treating a subject, e.g., for treating or
preventing a DNA virus (e.g., JCV or BKV) infection, for inhiting a
DNA virus replication or proliferation. Such medicaments can be
used for prophylactic treatment of a subject at risk for or
suspected of having a DNA virus infection (e.g., for treatment of a
subject prior to, during, and/or after the subject receives an
immunomodulatory therapy). Accordingly, one or more compounds or
compositions described herein that modulate DNA virus replication
or proliferation as described herein may be used for the
preparation of a medicament for use in any of the methods of
treatment described herein. In some embodiments, the invention
provides for the use of one or more compounds or compositions of
the invention (e.g., identified as inhibiting DNA virus
replication) for the manufacture of a medicament or pharmaceutical
for treating a mammal (e.g., a human) having one or more symptoms
of, or at risk for, DNA virus infection, replication and/or
proliferation (e.g., one or more symptoms of JCV or BKV activity,
or one or more symptoms of another DNA virus activity).
Accordingly, aspects of the invention relate to the use of one or
more compounds or compositions of the invention for the preparation
of a medicament for treating or preventing PML in a subject.
[0171] Accordingly, the invention also relates to one or more
compounds or compositions of the invention for use as a medicament.
The invention also relates to one or more of these compounds or
compositions for use in methods of the invention, for example in
methods of inhibiting DNA virus (e.g., JCV or BKV) replication, or
of treating or preventing a disease associated with DNA virus
replication or proliferation (e.g., in subjects that are about to
be, are being, and/or have been treated with at least one
immunomodulatory composition).
[0172] In some aspects, the invention provides kits comprising one
or more compounds of Table 1 and instructions for administering the
one or more compounds of Table 1. In some embodiments, the kit also
comprises an immunosuppressant and instructions for administering
the one or more compounds of Table 1 and the immunosuppressants. In
some embodiments, the immunosuppressant is natalizumab. The
components of a kit can be included in a container or package
having one or more positions for each component (and each component
can be separately packaged in a dry, liquid, gel, or other
form).
[0173] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting.
EXAMPLES
Example 1
Detection of JCV and JCV Variants by PCR
[0174] Nucleic acids are isolated from a biological sample using
established protocols (e.g., cell lysis). Because the viral DNA may
have integrated in the genomic DNA or may still be present as a
smaller entity, both genomic DNA and shorter DNA sequences may be
isolated and subjected to PCR analysis. Upon isolation the nucleic
acids are resuspended in a buffer that will facilitate PCR
analysis. Buffers that facilitate PCR analysis are known to the
skilled artisan and are also commercially available from
manufacturers of PCR enzymes (e.g., New England Biolabs, Beverly,
Mass.). Nucleotide primers are designed to result in the
amplification of a JCV gene. PCR amplification is an established
laboratory technique and comprises the addition of nucleotide
primers, a polymerase and single nucleotides, and polymerase buffer
and subjection this mixture to cycles of annealing, amplification
and dissociation resulting in the amplification of a desired DNA
sequence. Upon amplification, the JCV gene is separated from the
residual DNA and excess single nucleotides. The amplified JCV DNA
is sequenced and the resulting nucleotide sequence is translated
into a peptide sequence to determine if JCV polypeptides and
polypeptide variants are present in the biological sample.
Example 2
Detection of JCV and JCV Variants Using ELISA
[0175] Proteins and peptides are isolated from a biological sample
using standard laboratory techniques. Both the cellular proteins
and proteins of non-cellular components can be subjected to the
analysis. In one assay the sample is interrogated for the presence
of JCV polypeptides. The polypeptides are detected using sandwich
ELISA comprising antibodies specific for JCV polypeptides. The
antibodies are generated by inoculating animals (e.g., rabbits)
with the JCV polypeptides of the invention resulting in polyclonal
antibodies. If so desired, cells can be harvested from the
inoculated animal to generate monoclonal antibodies. Methods for
the generation of both polyclonal and monoclonal antibodies are
routine in the art. The antibodies against JCV polypeptide and JCV
polypeptide variants are immobilized on a solid surface (e.g., a
96-well plate), with one antibody type per well or surface area.
The biological samples comprising the polypeptides are added to the
wells and incubated with the immobilized antibodies. JCV
polypeptides and JCV polypeptide variants present in the sample
will bind to an antibody specific for the polypeptide. After
incubation, the sample is removed and the solid surfaces are washed
to remove any unbound material. As a next step, a solution
containing additional antibodies specific for JCV peptides is added
to the wells. This second aliquot of antibodies will create the
"sandwich" (e.g., immobilized antibody: JCV polypeptide: second
antibody). This second antibody can be detected using, for
instance, a labeled tertiary antibody, allowing for the detection
of JCV variant polypeptides. Alternatively, the secondary antibody
itself may be labeled.
[0176] In a second ELISA assay, biological samples are assayed for
the presence of antibodies against one or more CV polypeptides or
JCV variant polypeptides. This assay can be use to determine
whether a subject is currently infected with, or has previously
been exposed to, JCV or a JCV variant. Even if a specific JCV
variant is no longer present, antibodies against the variant may
still be present in the biological sample and can be detected. In
this ELISA assay JCV polypeptides are attached to a solid surface
and the biological samples are incubated with these polypeptides.
If antibodies specific for these polypeptides are present in the
biological samples they will bind to the polypeptides. Any unbound
material is again removed. The presence of bound antibody is
detected using a labeled secondary antibody.
Example 3
Treatment of JCV Infection
[0177] SV40 transformed glial cells were seeded in 10% FBS media.
The cells were seeded at 2000 cells per well in 75 .mu.l of media
per well. On day 2, the media was removed and replaced by 35 .mu.l
of a 1.times. concentrated drug aliquot combined with a 100.times.
diluted JCV Turbo aliquot in 35 .mu.l of 2% FBS media (JCV Turbo is
a hybrid Mad-1/SVE.DELTA. virus constructed by insertion of the
regulatory region of SV40 into the regulatory region of the Mad-1
strain of JCV (Mad-1/SVE), Vacante et al., Virology 1989, 170:
353-361). The cells were incubated for an hour, after which another
65 uL aliquot of 1.times. concentrated drug in 2% FBS was added. On
day 5 the cells were stained with DAPI (for total cell number) and
with a mouse monoclonal antibody against SV40 VP1, which cross
reacts with the JCV VP1 protein. The JCV VP1 protein is displayed
on the cell surface when a cell is infected by JVC1. The ability of
the drug to suppress or inhibit JCV infection is shown in Table 2.
The information is tabulated as % inhibition (as measured by the
number and percentage of JCV positive cells). The cytotoxocity of
the drug is displayed in the third column.
[0178] In a separate experiment the IC.sub.50 was determined. FIG.
1 shows the inhibition curve for different cell seedings. The
y-axis shows the percentage inhibition depending on the
concentration of neutralizing antibody added (Neutralizing antibody
is a rabbit polyclonal from anti-JCV neutralizing serum).
[0179] In a further experiment the correlation between
concentration of JCV and % infection was determined. FIG. 2 shows
that a JCV dilution of 1:50 results in 6% of infected cells, while
a JCV dilution of 1:500 results in a 1.5% infection.
TABLE-US-00002 TABLE 2 Inhibition of JCV infection % Inhibition %
Inhibition (total % Inhibition total Cell Molecular #JCV+) (% JCV+)
Number MOLENAME Weight @10 uM sd @10 uM sd @10 uM sd
CHLOROACETOXYQUINOLINE 221.64276 38 0 20 2 20 2 DEMETHYLNOBILETIN
388.37384 38 0 20 2 20 2 PROPANIL 218.08202 12 14 21 0 -12 17
AMINOETHOXYDIPHENYLBORANE 225.09822 25 5 21 12 3 8 5-NITRO-2-
300.31409 14 9 21 1 -9 9 PHENYLPROPYLAMINOBENZOIC ACID [NPPB]
3beta- 414.62848 25 5 21 12 3 8 HYDROXYISOALLOSPIROST-9(11)- ENE
LEOIDIN 413.21021 14 9 21 1 -9 9 PICROPODOPHYLLOTOXIN 414.41168 19
4 22 1 -1 5 THIABENDAZOLE 201.25182 19 4 22 1 -1 5 HARMANE
182.22488 24 0 22 8 4 8 6,4'-DIHYDROXYFLAVONE 254.242 18 0 22 6 -5
8 GENTIOPICROSIDE 356.32929 32 5 22 3 11 2 (R)-ANGOLENSIN 272.30063
18 0 22 6 -5 8 PTAEROXYLIN 258.27374 32 5 22 3 11 2 DIPYRIDAMOLE
504.63297 24 8 22 3 3 6 NABUMETONE 228.29083 20 5 23 4 -2 1
ROSIGLITAZONE 357.43323 22 3 23 3 -1 8 DILTIAZEM HYDROCHLORIDE
450.98602 20 1 24 0 -4 0 BETAMETHASONE 392.4675 34 0 24 4 14 4
ICHTHYNONE 408.40741 34 0 24 4 14 4 AMCINONIDE 502.5798 16 14 25 7
-11 9 RILUZOLE 234.2018 16 14 26 9 -12 5 FLUFENAMIC ACID 281.23413
37 8 26 8 14 1 CHRYSIN 254.24199 18 5 26 8 -12 5 DICTAMNINE
199.20902 35 1 26 3 12 5 PIPLARTINE 317.34152 21 11 27 2 -7 11
PEUCENIN 260.28964 21 11 27 2 -7 11 METHOXYVONE 266.29636 38 1 27 2
15 4 ISOTRETINOIN 300.44104 39 10 28 1 15 13 CHLOROXYLENOL
156.61157 36 2 29 10 10 9 TOMATINE 994.13745 40 8 29 15 12 7
PRIMULETIN 238.24258 40 8 29 15 12 7 MEFENAMIC ACID 241.28966 29 4
29 2 0 9 DIETHYLSTILBESTROL 268.35556 42 10 29 13 18 1
CHLORAMPHENICOL PALMITATE 505.43799 20 5 29 9 -13 6
METHYLXANTHOXYLIN 210.22975 33 3 30 1 5 2 L-ALANINOL 75.110596 13 2
31 3 -23 9 DICLOFENAC SODIUM 318.13409 44 2 31 1 18 4 FLUNIXIN
MEGLUMINE 491.46429 30 11 33 12 -4 2 DEHYDROABIETAMIDE 299.45636 40
4 33 7 11 3 PACHYRRHIZIN 336.30057 45 0 34 1 17 2 DICUMAROL
336.3006 36 13 35 11 3 4 DIFFRACTIC ACID 374.39035 44 0 36 2 13 2
ACEMETACIN 415.82962 32 7 38 3 -8 6 GINKGOLIC ACID 346.51007 33 11
39 6 -8 8 XANTHONE 196.20534 39 0 39 3 1 4 FUSIDIC ACID 516.7182 39
6 40 4 -2 16 POLYMYXIN B SULFATE 1301.5724 36 3 41 8 -8 10 PYRANTEL
PAMOATE 594.68781 48 2 42 0 12 3 4-(3-BUTOXY-4- 278.35132 48 2 42 0
12 3 METHOXYBENZYL)IMIDAZOLIDIN- 2-ONE MICONAZOLE NITRATE 479.14554
46 1 42 1 7 1 CANDESARTAN CILEXTIL 610.66943 54 5 44 4 19 3
ENDOSULFAN 406.92694 54 3 44 2 19 2 DIOXYBENZONE 244.24689 55 8 49
8 11 1 TOLFENAMIC ACID 261.70752 61 2 53 0 18 5 MEFLOQUINE 378.3172
66 10 64 7 5 10
Example 4
Identification and Characterization of Mefloquine Efficacy Against
JC Virus
[0180] In order to identify the drugs with anti-JCV activity, a
commercially available collection of approved drugs and bioactive
compounds were screened in in vitro JC viral infection assay. As a
primary screen, inhibition of the viral infection rate was
monitored in human glial cell line SVG-A (Major, Miller et al.
1985) infected with JCV strain Mad1/SVE.DELTA. (Vacante, Traub et
al. 1989). The infection rate was measured as a percent of cells
expressing viral envelop protein VP1 using Cellomics ArrayScan
(Pittsburgh, Pa.). Out of 2000 compounds in the SPECTRUM collection
screened, 14 were identified that had inhibited the number of
infected cells by >50% at the concentrations <20 .mu.M
(IC.sub.50<20 .mu.M). Since PML is a result of uncontrolled
viral replication in the CNS, the compounds were evaluated to
determine their ability to cross the blood brain barrier in
sufficient concentration to be therapeutically effective. Based on
the published literature, mefloquine shows CNS penetration that
could be expected to achieve in vitro derived efficacious
concentrations in humans (Jones, Kunsman et al. 1994; Pham, Nosten
et al. 1999).
[0181] Using qPCR to quantify the number of viral copies in the
culture, it also was shown that mefloquine inhibits viral DNA
replication. Further experiments with mefloquine demonstrated its
ability to inhibit infection by another JCV strain, Mad4 and in a
different cell type, primary human astrocytes. Mefloquine is
similarly effective in the inhibition of JCV infection rate even
when added to the culture system 24 hrs after infection of cells
with the virus, suggesting that it inhibits virus in previously
infected cells. Both (+) and (-) enantiomers of mefloquine racemate
were similarly potent at inhibiting JCV infection in the JCV
inhibition assay.
[0182] Mefloquine hydrochloride is an antimalarial agent indicated
for the treatment and prophylaxis of mild to moderate acute malaria
caused by mefloquine-susceptible strains of P. falciparum and P.
vivax and has a significant history of use in human population,
with 11 million patients treated since 1984, when it was first
registered. Although no animal model of PML or JCV infection is
available to test mefloquine in vivo, in vitro results and
published literature show that mefloquine is an effective anti-JCV
therapy.
Materials and Methods
[0183] Compounds: The 2,000 compounds Spectrum Collection
(MicroSource Discovery Inc., Groton, Conn.) consists of .about.1000
Drugs defined according to the name designations as set forth in
the USP Dictionary of USAN and International Drug Names (2005, US
Pharmacopeia), including Food and Drug Administration (FDA)
approved drugs, and other bioactive compounds and natural products.
An alphabetical list of the compounds is available at "The Spectrum
Collection" internet site. The compounds are supplied as 10 mM
solutions in dimethyl sulfoxide (DMSO). Mefloquine was purchased
from Sigma (Sigma-Aldrich, St. Louis, Mo.). Two mefloquine
enantiomers were separated from a commercial mefloquine sample by
Chiral Technologies (Chiral Technologies, West Chester, Pa.) using
chiral HPLC on a CHIRALPAK IA column.
[0184] Cell Lines: The human glial cell line SVG-A (a gift from
Walter Atwood), established by transformation of human fetal glial
cells by an origin-defective SV40 mutant (Major, Miller et al.
1985), was cultured in 1.times. Eagle Minimal Essential Media (MEM)
supplemented with 10% heat-inactivated fetal bovine serum, 4 mM
L-glutamine (Mediatech, Holy Hill, Fla.). Infection was performed
in 1.times. Eagle Minimal Essential Media (MEM) supplemented with
2% heat-inactivated fetal bovine serum, 4 mM L-glutamine
(Mediatech, Inc.). Human astrocytes (ScienCell Research
Laboratories, San Diego, Calif.) were isolated from fetal cerebral
cortex and cultured in proprietary basal medium, supplemented with
2% fetal bovine serum, 1% astrocyte growth, and
penicillin/streptomycin (ScienCell Research Laboratories).
Infection of astrocytes was performed in this media.
[0185] JC Polyoma Viruses: Hybrid Mad-1/SVEDelta virus
(Mad-1/SVE.DELTA. virus) (a gift from Walter Atwood) was
constructed by insertion of the regulatory region of SV40 into the
regulatory region of the Mad-1 strain of JCV (Mad-1/SVE) (Vacante,
Traub et al. 1989). Virus was propagated on SVG-A cells and
purified as previously described (Liu, Hope et al. 1998). Briefly,
SVGA cells plated at 50% confluence are infected with a 1:50
dilution of the MAD1-SVE delta strain of JC virus for 1 hour at
37.degree. C. That concentration of virus has been shown to give a
maximal infection rate of SVGA cells. Cells are cultured for 3
weeks with weekly changes of media. After 3 weeks of culture, cells
are scraped from the flasks, pooled, including loose cells from
prior media changes, and pelleted. The cell pellet is then
resuspended in 20 ml of supernatant and disrupted in a
microfluidizer (Microfluidics inc., Newton, Mass.). Deoxycholate is
added to a final concentration of 0.25% and incubated at 37.degree.
C. for 30 minutes. The virus-containing cellular supernatant is
then centrifuged at 10,000 RPM for 30 minutes in a SA600 rotor. The
supernatant is then aliquoted and stored at -80.degree. C. The
nonarchetypal strain of JC, Mad4 (Major, Vacante et al. 1987; Frye,
Trebst et al. 1997) was obtained from ATCC (Manassas, Va.).
[0186] Detection antibodies: PAB597 (a gift from Walter Atwood), a
mouse monoclonal to SV40 V antigen, cross-reacts to JCV VP1
(Atwood, Wang et al. 1995) and was used to visualize JC infection
with secondary detection using an Alexa-Fluor 488-labeled goat
anti-mouse secondary antibody (Molecular Probes). Cell nuclei were
stained with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen,
Carlsbad, Calif.). Neutralizing anti-JCV rabbit antisera was a gift
from Walter Atwood (Atwood 2001).
[0187] JCV Infectivity Assay: SVG-A cells were seeded at 2,000
cells/well/0.075 ml of culture media in 96 flat-bottom well plate
(Corning, N.Y.). The next day compounds were prepared in assay
medium (2% heat-inactivated fetal bovine serum, 4 mM L-glutamine,
1.times.MEM). A master viral plate was prepared by mixing equal
volumes of [2.times.] compound and [2.times.] diluted virus for the
final 1.times. concentration of the compound and the virus. The
plate containing cells was gently inverted and shaken to remove
media. From the master plate, 0.035 ml compound/virus was added to
designated wells. The cells were incubated with the compound/virus
mixture for 60 minutes in a humidified 37.degree. C. CO.sub.2
incubator. At that time final concentration of the drug in media
was added to designated wells to bring the final volume up to 0.1
ml/well. The plates were incubated for an additional three days,
the cells were then washed once with 1.times.PBS, and fixed in 2%
paraformaldehyde/1.times.PBS for 30 minutes at room temperature.
The fixative was removed and the cells were solubilized with 0.5%
Triton X100 in PBS for an additional 30 minutes. Infection by
Mad-1/SVEA virus was visualized by staining with PAB597, a
monoclonal antibody against the major capsid protein VP1, the cells
were incubated with 0.05 ml of PAB597 (2 .mu.g/ml in 1.times.PBS)
for 60 minutes at 37.degree. C. Following a wash step with
1.times.PBS, primary antibody was detected with an Alexa-Fluor
488-labeled goat anti-mouse secondary antibody at 1:100 dilution in
1.times.PBS, cells were counterstained with DAPI at 1 .mu.g/ml
(0.05 ml/well) for 30 minutes at 37.degree. C. Cells were washed
with 1.times.PBS and 0.1 ml 1.times.PBS was added to cells. Field
images of each well were acquired and analyzed using the Cellomics
ArrayScan (Thermo Scientific Inc, Waltham, Mass.) using the Target
Activation software. Human astrocytes follow the same basic assay
protocol with some notably exceptions. The cells are seeded at
4,000 cells/well/0.075 ml of culture media in 96 flat-bottom well
plate (Corning). The culture media is used for the infection. The
length of the infection is six to ten days (instead of the three
days for SVG-A cells).
[0188] Real time PCR: Taqman forward and reverse primers and MGB
probes were designed for JC virus TAg using Primer Express v1.0
(Applied Biosystems, Foster City, Calif.) according to
manufacture's recommendation. To create a copy number standard
curve for absolute quantification, pUC19 plasmid containing JC
virus genome was linearized using SmaI. Linearization was confirmed
by capillary electrophoresis using an Agilent 12000 kit according
to manufacturer's recommendation. Concentrations were determined by
A.sub.260 nm measurement on a nanodrop spectrophotometer.
Linearized plasmid was diluted 1/10 in TE starting at
5.times.108/uL. Quadruplicate PCR reactions were run in a 384 well
optical plate (Applied Biosystems, Foster City, Calif.). Real time
reactions were cycled in a 7900HT (Applied Biosystems, Foster City,
Calif.) thermal cycler under the following conditions: 50.degree.
C. for 2 minutes (uracil N-deglycosylase digest), 95.degree. C. 10
minutes (activation of Taq thermostable polymerase), and 40 cycles
of 95.degree. C. for 15 seconds and 60.degree. C. for 60 seconds
with 900 nM forward and reverse primers, 200 nM Taqman probe, and
1.times. Universal master mix (Applied Biosystems, Foster City,
Calif.). The fluorescence emission was collected every seven
seconds for the length of the run for each reaction well. Copy
number was determined for each experimental sample by comparison to
the absolute JC plasmid standard curve using Sequence Detection
Software (Applied Biosystems, Foster City, Calif.). The adjusted
copy number was calculated by first subtracting the experimentally
determined mass of JC virus DNA from the total DNA mass that was
added to each 20 ul rxn well. P-values were calculated using a
Student t-test. JCV copy number was quantified by comparison to a
standard and normalized by the total DNA extracted from a sample.
Zero copies of JC virus were detected in a non-infected negative
control.
[0189] DNA extraction and sample prep: DNA was extracted using to
QIAamp 96 blood kit (cat#51161; Qiagen Inc., Valencia, Calif.) with
optional RNase A treatment. DNA was quantitated using Quant-iT
dsDNA high sensitivity assay according to manufacturer's
recommendations (cat # Q33120 Molecular Probes Inc., Eugene,
Oreg.). Purified DNA was stored at -20.degree. C. until use.
[0190] Calculations: The rate of JCV infection was calculated by
normalizing the number of JCV infected cells by the total number of
nucleated cells as % JCV.sup.+ cells=((total # VP1.sup.+
cells)/(total # DAPI.sup.+ cells))*100%. The % of viral inhibition
by a compound was calculated using a JCV infection rate instead of
using total number of JCV infected cells (e.g., VP1.sup.+ cells).
Percent JCV Inhibition=100%*(1-(% JCV.sup.+ cells with a compound-%
JCV.sup.+ cells in positive control)/(% JCV.sup.+ cells in positive
control-% JCV.sup.+ cells in negative control). The positive
control is the cells infected with the virus in the absence of any
compound, negative control are the cells not infected with any
virus. The number of JCV.sup.+ cells in the negative control
samples quantitated by Cellomics ArrayScan were always <1% of
the number of JCV.sup.+ in the positive control. The percent of JCV
DNA inhibition was calculated as 100%*(1-(JCV copy# with a
compound-JCV copy# in positive control)/JCV copy# in positive
control. Zero copies of JCV genome were detected in no-infected
negative control samples. For high throughput screening of
compounds, the
Z-factor=1-3*((.sigma.p+.sigma.n)/.parallel..mu.p-.mu.n|; the mean
(.mu.) and standard deviation (.sigma.) of both the positive (p)
and negative (n) controls (Zhang, Chung et al. 1999) were
calculated. Intraplate intragroup CV was always below 20% and
Z'>0.5. IC.sub.50 values were calculated using Prism software
(GraphPad Software Inc., USA).
Primary Screen: JC Viral Infection Assay
[0191] In order to identify the drugs with anti-JCV activity, a
commercially available collection of approximately 2000 approved
drugs and bioactive compounds called the SPECTRUM collection were
screened for anti-JCV activity in an in vitro viral infectivity
assay (Pho, Ashok et al. 2000). As a primary screen, the inhibition
of the viral infection rate was monitored in a human fetal
astroglial cell line (SVG-A) infected with the JCV strain
Mad1/SVE.DELTA.. The SVG-A cell line (Major, Miller et al. 1985)
was chosen for the primary screening assay, as it is one of the few
available cell lines permissive for JC viral replication.
Mad1/SVE.DELTA. JCV strain is a wild type Mad-1 virus originally
isolated from the PML patient that had its regulatory non-coding
region replaced with SV40 regulatory region (Vacante, Traub et al.
1989). That insertion had been demonstrated to extend species and
cell-type host range of the virus. Infection of SVG-A cells with
Mad1/SVE.DELTA.JCV strain allows for very fast viral replication
and detection of virally infected cells within 3 days of infection
unlike 6-15 days need with other cell types and viral strains.
[0192] Uniformity of a cell line, consistent infection rate and
relatively short assay time are all important factors in creating a
robust assay for screening of a large number of compounds. To
facilitate screening of the large compound collection, this JCV
infectivity assay (Pho, Ashok et al. 2000) was used in a 96-well
format and a Cellomics ArrayScan was used to measure JCV
replication. FIG. 3 shows the detection and measurement of cellular
infection with JCV. In FIG. 3A SVG-A cells infected with JCV strain
Mad1/SVE.DELTA. 3 days earlier were fixed and stained with murine
monoclonal antibodies specific for VP1 protein (green staining).
The total cells present in the culture were visualized with DAPI
DNA nuclear staining (blue) and picture was taken with Cellomics
ARRAYSCAN.RTM. camera at .times.20 magnification. FIG. 3B
illustrates the number of infected cells (e.g., VP1.sup.+ cells)
per group plotted against the dilution factor of the viral stock
used to infect the cells (mean.+-.SD, n=2). The total number of
cells (bars) is similar for all groups. FIGS. 3C and 3D illustrate
results for cells that were infected in the presence of various
dilutions of JCV neutralizing antiserum or cidofovir. Three days
later cells were fixed; stained and total number of VP1.sup.+ cells
and DAPI.sup.+ events per treatment group was enumerated using
Cellomics ARRAYSCAN.RTM..
[0193] In this system infected cells can be identified by
immunofluorescent staining with antibodies specific for JCV capsid
protein VP1. Total number of cells in culture was visualized via
staining with DNA stain DAPI (FIG. 3A). ARRAYSCAN.RTM. was used to
identify and count every single event in the assay well, routinely
counting 300-800 VP1.sup.+ cells and 8,000-16,000 DAPI.sup.+ events
per well in a 96-well plate, thus minimizing variability due to
non-uniform cell growth pattern and/or intrawell viral spread and
producing a very consistent result. As can be seen from FIG. 3B,
the number of infected cells (e.g., VP1.sup.+ cells) at the end of
culture period is proportional to the number of infectious viral
particles used to infect the cell culture. In these experiments,
the highest dilution of the viral stock that was convenient for
application was used. Using neutralizing rabbit anti-JCV serum as a
positive control for viral inhibition it also was shows that the
assay responds to viral inhibition in a predictive fashion (FIG.
3C).
[0194] During screening of the library for antiviral activity at
single dose (10 .mu.M), it was discovered that some of the
compounds that most dramatically inhibited the number of virally
infected cells (e.g., VP1.sup.+ cells) also dramatically reduced
the total number of cells (e.g., DAPI.sup.+ events) likely due to
their cytotoxic/cytostatic effects. To determine whether a
particular compound had decreased the number of virally infected
cells not just due its cytotoxic effect but due to its antiviral
effect, the percent of viral inhibition by a compound was
calculated using the JCV infection rate (e.g., % JCV.sup.+
cells=((total # VP1.sup.+ cells)/(total # DAPI.sup.+ cells))*100%)
rather than total number of JCV infected cells (e.g., the total
number of VP1.sup.+ cells). In this system JCV infection leads to
4-7% of all cells being infected in 72 hours. As can be seen from
the example of treatment with cidofovir, a drug tested for efficacy
against PML (FIG. 3D), while it inhibited the number of infected
cells in culture they also inhibited the total number of cells in
culture to the same degree so the percent inhibition of infection
rate (e.g., % JCV.sup.+ cells) was not significant. Similar effect
was observed with other drugs with well reported cytotoxic effects,
e.g., Mytomicin C and cytarabine (data not shown).
Drug Screening and Selection
[0195] Following screening the library at a single compound
concentration of 10 .mu.M, a number of drugs and compounds that
effectively inhibited JCV infection rate by >20% without
significant cell toxicity (<20% total cell number inhibition)
were identified (FIG. 4). A level of 20% was chosen as a cut off
for the first pass screening because the CV of the assay was
consistently <20%. 67 compounds matched those criteria (Table 1)
and were subsequently tested in the same assay across a full dose
response curve to further evaluate their therapeutic potential.
Based on the results from full dose curves 14 drugs proved to be
effective, demonstrating >90% inhibition of the virally infected
cells (IC.sub.50<20 .mu.M) without statistically significant
cell toxicity (<20% total cell number inhibition) at those
concentrations (Table 3). Only the drugs that did not reduce total
cell numbers were selected in order to diminish the chance of
confounding an anti-viral effect of the drug with its
cytotoxic/cytostatic effect. The compounds that had reduced total
cell number by >80% were retested at the lower concentrations,
but none were identified that demonstrated clear anti-JCV effect
without concomitant cytotoxic/cytostatic effect (FIG. 4). FIG. 4
shows the flow chart for SPECTRUM collection screening. The primary
assay employed an SVG-A cell line and JCV Mad1/SVE.DELTA. viral
strain. The assay was performed as described for FIG. 3
(*IC.sub.50-inhibition of % JCV infected cells by 50%;
TC.sub.50-inhibition of total cell numbers by 50%).
[0196] The published literature was reviewed for information on
pharmacokinetics and brain distribution of these drugs in humans
and in animal models. Since JC virus uncontrollably replicates in
the oligodendrocytes and astrocytes of the effected individuals
during PML but not all drugs are capable of crossing blood brain
barrier it would be advantageous for any potential PML therapy if
the drug candidate is capable of achieving efficacious
concentration in the brain. Based on the published literature
(Table 3), mefloquine had been demonstrated to accumulate in the
brains of treated patients at the level of its in vitro efficacious
concentration (FIG. 3A; IC.sub.50=3.9.+-.2.1 .mu.M). Brain
concentration of mefloquine based on postmortem brain analysis of
people taking the drug prior to their deaths was 35-50 nmol per
gram of brain tissue, which could be approximated as 35-50 .mu.M
(Jones, Kunsman et al. 1994; Pham, Nosten et al. 1999). This
indicates that potentially efficacious doses of mefloquine can be
achieved in the brain patients who would be receiving approved
doses of the drug.
Characterization of Mefloquine Activity in Primary Cell Culture
[0197] In order to further characterize the effect of mefloquine in
JC virus infectivity, experiments were performed to evaluate
whether the JCV inhibitory effect of mefloquine was dependent on
the cell line used in the primary screen. Since SVG-A cells is a
cell line propagated in vitro for many generations, and is
transformed with SV40 large T antigen that can enhance JCV
replication, experiments were performed to evaluate whether
mefloquine is capable of inhibiting viral replication in cells that
more closely resemble JCV target in human brain and do not express
SV40 TAg. Since in vitro infection culture of primary
oligodendrocytes, a primary JCV target during PML, is not
established, the JCV infection of human fetal astrocytes was used
to test the ability of mefloquine to inhibit the viral infection in
primary cell culture. FIG. 5 shows the efficacy of mefloquine
against two different viral strains (Mad1 and Mad4) in two
different cell types (SVG-A and primary astrocytes). FIG. 5A
illustrates SVG-A cells with Mad1/SVE.DELTA. JCV (n=12) virus. FIG.
5B illustrates primary human fetal astrocytes with Mad1/SVE.DELTA.
JCV. FIG. 5C illustrates SVG-A cells with JCV strain Mad-4 (n=5). %
JCV Inhibition was calculated as 100%*(1-(% JCV.sup.+ cells with a
compound-% JCV.sup.+ cells in negative control)/(% JCV.sup.+ cells
in positive control-% JCV cells in negative control). Inhibition of
total cell numbers (e.g., DAPI events) was less than 20% for all
drug concentrations plotted. Unless otherwise noted, one
representative graph out of the number presented on the graph is
shown. IC.sub.50 are calculated as an average of all those
experiments. As can be seen from FIG. 5A, mefloquine inhibits JCV
infection of primary human fetal astrocytes with essentially the
same efficacy as it inhibits viral infection in SVG-A cells. This
result shows that anti-JCV effect of mefloquine is not dependent on
cell type being infected by the virus, and that mefloquine is
effective at inhibiting the virus in its "natural" setting of glial
cell infection.
Characterization of Mefloquine Activity on Different JC Viral
Strains
[0198] Experiments were also performed to determine whether the
inhibitory effect of mefloquine is observed with other JC viral
strains, and is not limited to Mad1/SVE.DELTA. JCV strain used in
the primary screening assay. The primary screen was conducted with
the Mad1/SVE.DELTA. JCV strain because of its fast replication in
tissue culture. However, this viral strain contains a transcription
regulatory region (non-coding) from its polyomavirus family member
SV40. To ensure that mefloquine's anti-viral activity is not
limited only to the virus with this modification, mefloquine's
ability to inhibit an unmodified JCV strain, wild type virus Mad4
that was originally isolated from a PML patient (Major, Vacante et
al. 1987; Frye, Trebst et al. 1997), was tested. As can be seen
from FIG. 5C, based on the results from 5 independent experiments,
mefloquine inhibits Mad4 infection of SVG-A cells with the same
efficacy as Mad1/SVE.DELTA. infection. This result demonstrates
that anti-JCV effect of mefloquine does not depend on the viral
strain.
Effect on JCV Viral DNA Replication
[0199] In order to better understand the mechanism of action of
mefloquine and address whether this drug inhibits rate of cell
infection by JC virus via inhibition of viral DNA replication,
viral DNA was quantified using qPCR and a probe set specific for
JCV T Antigen. As can be seen from FIG. 6, the percent of JCV DNA
inhibition by mefloquine almost overlaps the percent of infection
rate inhibition. A linear correlation between the viral DNA copy
number and a number of virally infected cells was observed (data
not shown). This result demonstrates that mefloquine inhibits
infection rate via its inhibition of viral DNA replication, and not
VP1 protein expression. FIG. 6 shows the effect of mefloquine on
JCV DNA replication. SVG-A cells were infected with Mad1/SVE.DELTA.
JCV 3 days earlier in the presence of various drug concentrations.
% JCV DNA inhibition (.diamond.) was calculated using JCV copy
numbers determined by qPCR with a probe set specific for TAg.
Inhibition of infection rate (x) was measured in a replicate plate
as described herein.
Effect of Mefloquine on Established JCV Infection
[0200] Experiments were performed to demonstrate that mefloquine is
effective against established JCV infection. While it is apparent
from the above experiments that mefloquine is effective at
inhibiting JCV infection when added at the same time as the virus,
it was not clear from those experiments whether mefloquine
inhibited viral entry into the cells, or the viral life cycle in
the cell. Since during PML many cells are already infected with JC
virus, in some embodiments a drug candidate for treatment of PML
may be selected as one that can inhibit viral life cycle in already
infected cells. As seen from FIG. 7, when added in 3 or 24 hours
post infection, mefloquine was just as effective at inhibiting cell
infection as it was when added to the cells together with the
virus. Since most of the virus enters the cells within 1 hour, and
all of the virus enters the cells within first 24 hours after
infection (unpublished data), this result shows that mefloquine is
effective in inhibiting viral replication in already infected
cells. In FIG. 7 various concentration of the drug were added to
the culture of primary human fetal astrocytes infected with JC
virus at the same time as virus (circles; T=0), 3 hours after virus
addition (squires; T=3 h), or 24 hours after (triangles; T=24 h).
Ten days after infection with the virus cells were fixed and number
of virally infected cells was enumerated. Results of a
representative experiment out of 4 different experiments with
primary astrocytes or SVG-A cells are shown.
Characterization of Mefloquine Enantiomers
[0201] Mefloquine is a racemic mix of (+) and (-) enantiomers of
(R*, S*)-.alpha.-2-piperidinyl-2,8-bis
(trifluoromethyl)-4-quinolinemethanol hydrochloride (FIG. 8). FIG.
8 shows the efficacy of different isomers of mefloquine. FIG. 8A
shows IC.sub.50s and structure of (R,S) and (S,R) enantiomers
constituting drug racemate commercial mefloquine. (+) and (-)
enantiomers were separated from a racemate via chiral
chromatography and added to SVG-A cells simultaneously with JCV
strain Mad1/SVE.DELTA. 3 days earlier. After fixation and staining
total number of VP1.sup.+ cells and DAPI.sup.+ events per treatment
group was enumerated using Cellomics ARRAYSCAN.RTM.. A
representative experiment out of six performed is shown. While
there is a minimal difference between the activities of the
enantiomers against malaria (Basco, Gillotin et al. 1992; Brocks
and Mehvar 2003), the (-) enantiomer is much more potent in
antagonistic activity against A2a receptor (Weiss, Benwell et al.
2003). The two enantiomers also display different pharmacokinetics
and brain penetration properties (Bourahla, Martin et al. 1996;
Baudry, Pham et al. 1997). In order to better understand the
anti-JCV effects of these two components of the marketed mefloquine
racemate, each enantiomer from a racemate was separated using
chiral chromatography and compared them in a JCV inhibition assay.
As can be seen from FIG. 8, both enantiomers have very similar
efficacy in inhibiting JC virus. FIG. 9 shows the efficacy of
different isomers of mefloquine and
2,8-Bis-trifluoromethyl-quinolin. The compounds were added to SVG-A
cells simultaneously with JCV strain Mad1/SVE.DELTA. 3 days
earlier. After fixation and staining total number of VP1.sup.+
cells and DAPI.sup.+ events per treatment group was enumerated
using Cellomics ARRAYSCAN.RTM.. A representative experiment out of
six performed is shown. The illustrated compounds all inhibited JC
virus replication. S,S-mefloquine and R,R-mefloquine have similar
IC.sub.50 similar to R,S-mefloquine and S,R-mefloquine, while a
structurally related compound that has a aromatized ring has a
slightly higher IC.sub.50. FIG. 10. shows that mefloquine anti-JCV
activity is not inhibited by cerebrospinal fluid (CSF).
[0202] Based on this result and on the reported brain concentration
for mefloquine enantiomers (Baudry, Pham et al. 1997) it can be
concluded that mefloquine can be administered at concentrations
efficacious in JCV inhibition in the brain of patients.
Furthermore, lack of significant differences between anti-JCV
activities of the enantiomers implies that this effect is not
mediated by A2a receptor.
[0203] Mefloquine has been identified as a potential treatment for
PML based on the results of screening a commercially available
collection of approved drugs and bioactive compounds in in vitro JC
viral infection assay. Follow-up experiments demonstrated that the
effect of mefloquine is not limited to one cell type or a single
viral strain. Furthermore, mefloquine is capable of inhibiting
viral replication in cells already infected with the virus. Both
(+) and (-) enantiomers of mefloquine racemate were almost
equipotent at inhibiting JCV infection, indicating that isolating
individual enantiomers would not improved the benefit of the
existing drug. In addition, the literature shows that mefloquine
crosses the blood brain barrier and can accumulate in the brain at
the in vitro defined efficacious concentration for inhibition of JC
viral infectivity. Although no animal model is available for PML,
the in vitro results and literature data show that mefloquine is an
effective anti-JCV therapy.
TABLE-US-00003 TABLE 3 Selected inhibitors (anti-JCV IC50
.ltoreq.20 .mu.M, Therapeutic index (IC50/TC50) <0.5) TC.sub.50,
IC.sub.50, Brain, plasma References for MOLENAME THERAPY STATUS
.mu.M .mu.M .mu.M Cmax, .mu.M PK data ISOTRETINON antiacne, USP,
INN, ND 4.4 1100 ng/ml (Clamon, antineoplastic BAN Chabot et al.
1985; Colburn and Gibson 1985) MEFLOQUINE antimalarial USAN, INN,
16.1 4.0 30-50 6.0 (Jones, Kunsman BAN et al. 1994; Pham, Nosten et
al. 1999) DICLOFENAC antiinflammatory USP, JAN 30.5 8.3 SODIUM
DILTIAZEM Ca USP, INN, >40 8.5 HYDROCHLORIDE channel BAN, JAN
blocker FUSIDIC antibacterial USAN, INN, ND 8.6 1 .mu.g/ml 10-90
.mu.g/ml (Mindermann, ACID BAN Zimmerli et al. 1993; Turnidge 1999)
MICONAZOLE antifungal USP, JAN 22.9 8.6 nd 10 ng/ml (Stevens,
Konsil NITRATE (topical) et al. 2002) MEFENAMIC antiinflammatory,
USP, INN, ND 10.9 0.8 .mu.g/ml# 10-20 .mu.g/ml (Glazko 1966; ACID
analgesic BAN, JAN Fukuda, Kitaichi et al. 2005) FLUNIXIN
analgesic, USP, 47.7 16.6 MEGLUMINE antiinflammatory veterinarian
PROPANIL herbicide >40 7.8 DEHYDROABIETAMIDE NA >40 13.0
DIFFRACTIC NA >40 14.4 ACID HARMANE NA >40 14.4 XANTHONE NA
ND 16.8 METHOXYVONE NA >40 17.2 #animal data USP, United States
Pharmacopeia INN, International Nonproprietary Name BAN, British
Approved Name JAN, Japanese Approved Name Clamon, G., G. G. Chabot,
et al. (1985). "Phase I study and pharmacokinetics of weekly
high-dose 13-cis-retinoic acid." Cancer Res 45(4): 1874-8. Colburn,
W. A. and D. M. Gibson (1985). "Isotretinoin kinetics after 80 to
320 mg oral doses." Clin Pharmacol Ther 37(4): 411-4. Fukuda, M.,
K. Kitaichi, et al. (2005). "Altered brain penetration of
diclofenac and mefenamic acid, but not acetaminophen, in Shiga-like
toxin II-treated mice." J Pharmacol Sci 97(4): 525-32. Glazko, A.
J. (1966). "Experimental observations on flufenamic, mefenamic and
meclofenamic acids. 3. Metabolic disposition." Ann Phys Med Suppl:
23-36. Jones, R., G. Kunsman, et al. (1994). "Mefloquine
distribution in postmortem cases." Forensic Sci Int 68(1): 29-32.
Mindermann, T., W. Zimmerli, et al. (1993). "Penetration of fusidic
acid into human brain tissue and cerebrospinal fluid." Acta
Neurochir (Wien) 121(1-2): 12-4. Pham, Y. T., F. Nosten, et al.
(1999). "Cerebral uptake of mefloquine enantiomers in fatal
cerebral malaria." Int J Clin Pharmacol Ther 37(1): 58-61. Stevens,
R. E., J. Konsil, et al. (2002). "Bioavailability study of a 1200
mg miconazole nitrate vaginal ovule in healthy female adults." J
Clin Pharmacol 42(1): 52-60. Turnidge, J. (1999). "Fusidic acid
pharmacology, pharmacokinetics and pharmacodynamics." Int J
Antimicrob Agents 12 Suppl 2: S23-34.
Example 5
Identification and Characterization of Compounds that are Active
Against JC Virus
[0204] FIG. 11 shows the dose response of several compounds that
have anti-JCV activity. FIGS. 12 and 13 show selected arylalkanoic
acid NSAIDs and their anti-JCV activity. FIG. 14 shows the result
of modeling studies with mefloquine and related compounds and the
IC.sub.50s of a number of compounds structurally related to
mefloquine. A pharmacophore-based alignment method Catalyst and
shape-based alignment method ROCS were used to study the similarity
between the compounds. The overlays below (or above) were derived
in ROCS using scoring scheme that combines shape and weighted
chemical force-field overlap between molecules. Similar
relationships between molecules were detected through pharmacophore
matching.
[0205] Table 4 shows the anti-JCV activity of a number of
structural analogs to mefloquine (See also FIG. 15). Table 5 shows
the anti-JCV activity of a number of structural analogs to fusidic
acid (See also FIG. 16).
[0206] It should be appreciated that any one or more of the
structural analogs described herein and/or any one or more of the
compounds listed in Tables 1-5 can be used according to methods of
the invention if they have suitable properties as described
herein.
TABLE-US-00004 TABLE 4 Selected compounds from mefloquine
functional fingerprint in silico screen anti- JCV activ- Structure
MOLREGNO MDLNUMBER MOLNAME CAS# ity
Nc1ncnc2n(c(nc12)Cl)C1OC(CO)C(O)C1O 129461 MFCD01076457
8-CHLOROADENOSINE 34408-14-5 Yes Nc1nccc2n(cnc12)C1OC(CO)C(O)C1O
64582 MFCD00153951 3-DEAZAADENOSINE 6736-58-9 Yes
Nc1nc2ncnc2c(n1)OCC1CCCCC1 289926 MFCD05664734
O6-Cyclohexylmethylguanine Yes
CCC(CO)Nc1nc(c2ncn(c2n1)C(C)C)NCc1ccccc1 182843 MFCD02266402
ROSCOVITINE, (S)-ISOMER Yes Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O 5424
MFCD00005752 ADENOSINE 58-61-7 No
NC1.dbd.Nc2n(cnc2C(.dbd.O)N1)C1OC(CO)C(O)C1O 9593 MFCD00010182
GUANOSINE 118-00-3 No CC(C)C(.dbd.O)c1c2n(nc1C(C)C)C.dbd.CC.dbd.C2
123831 MFCD00864808 IBUDILAST 50847-11-5 No
OCC1OC(C(O)C1O)n1cnc2c(ncnc12)NCc1ccccc1 5414 MFCD00005740
6-BENZYLAMINOPURINE 4294-16-0 NA RIBOSIDE
Nc1c2ncn(c2nc[n+]1[O--])C1OC(CO)C(O)C1O 21536 MFCD00037993
ADENOSINE-N'-OXIDE NA NC1.dbd.Nc2n(cnc2C(.dbd.O)N1)C1OC(CO)C(O)C1O
38045 MFCD00064103 BETA-L-GUANOSINE NA
OCC1OC(C(O)C1O)n1cnc2c(ncnc12)NC1CC2CCC1C2 40907 MFCD00069271
(2S)-N6-[2-ENDO- NA NORBORNYL]ADENOSINE Cn1cnc2c(ncnc12)NC1CCCC1
64492 MFCD00153844 N-0840 NA Cc1cc(c2nccc(c2c1)C)C 74766
MFCD00191505 4,6,8- NA TRIMETHYLQUINOLINE
Nc1nc2ncnc2c(n1)OCc1ccccc1 91708 MFCD00269931 O6-BENZYLGUANINE
19916-73-5 NA Cc1cc(c2cccc(c2n1)C)C 93735 MFCD00272331 2,4,8-
18441-61-7 NA TRIMETHYLQUINOLINE
CN1C(.dbd.O)N(C)c2ncn(c2C1.dbd.O)CC1OCCO1 123874 MFCD00865218
DOXOFYLLINE 69975-86-6 NA CC(C)Cn1cnc2c(nc3ccccc3c12)N 124036
MFCD00866946 IMIQUIMOD 99011-02-6 NA
CCCCN1C(.dbd.O)N(CCCC)c2ncn(c2C1.dbd.O)CC(C).dbd.O 124063
MFC00867152 DENBUFYLLINE 57076-71-8 NA
CN1N.dbd.C(N)c2cn(c3ncnc1c23)C1OC(CO)C(O)C1O 124573 MFCD00932413
6-AMINO-4-METHYL-8- 35943-35-2 NA (BETA-D-RIBOFURANOSYL)-
4H,8H-PYRROLO[4,3,2- DE]PYRIMIDO[4,5- C]PYRIDAZINE
N(c1ccccc1)C1.dbd.CC(.dbd.Nc2ncnn21)c1ccccc1 124680 MFCD00951199
BUTTPARK 57\40-37 NA CC(C)n1cnc2c(nc(nc12)NCCCO)NCc1ccccc1 280770
MFCD04974145 6-BENZYLAMINO-2-(3- NA HYDROXYPROPYLAMINO)-
9-ISOPROPYLPURINE
OC(.dbd.O)c1cc2cc(c(cc2c(n1)C(.dbd.O)c1ccc(c(c1)O)O)O)O 280811
MFCD04974186 1-(3',4'- NA DIHYDROXYBENZOYL)-6,7-
DIHYDROXYISOQUINOLINE- 3-CARBOXYLIC ACID
TABLE-US-00005 TABLE 5 Selected fusidic acid analogs anti- JCV
activ- Structure MOLREGNO MDLNUMBER MOLNAME ity
CC(.dbd.O)OC1CCC2C3CCC4.dbd.CC(O)CCC4C3CCC12C 93067 MFCD00271609
4-ESTREN-3- Yes BETA, 17- BETA-DIOL 17-ACETATE
CC(OC(C).dbd.O)C1CCC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C 93343
MFCD00271896 5-BETA- Yes PREGNAN- 3-ALPHA, 6-ALPHA, 20-BETA- TRIOL
20- ACETATE CC(OC(C).dbd.O)C1CCC2C3CCC4.dbd.CC(O)CCC4(C)C3CCC12C
93383 MFCD00271937 4-PREGNEN- Yes 3-BETA, 20- BETA-DIOL 20-ACETATE
CC(OC(C).dbd.O)C1CCC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C 93342
MFCD00271895 5-BETA- No PREGNAN- 3-ALPHA, 6-ALPHA, 20-ALPHA- TRIOL
20-ACETATE
CC(CC(O).dbd.O)C1CCC2C3C(CC4CC(O)CCC4(C)C3CC(OC(C).dbd.O)C12C)OC(C).dbd.O
234323 MFCD03695615 23-NOR-5- No BETA- CHOLANIC ACID-3- ALPHA, 7-
ALPHA, 12- ALPHA- TRIOL 7, 12-DIACE- TATE
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[0235] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the
invention.
[0236] All publications, patents and sequence database entries
mentioned herein, including those items listed below, are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.
* * * * *