U.S. patent application number 16/946708 was filed with the patent office on 2020-11-19 for anti-viral compositions containing pikfyve inhibitors and use thereof.
The applicant listed for this patent is AI Therapeutics, Inc. Invention is credited to Paul Beckett, Neil Beeharry, Chris Conrad, Sean Landrette, Henri Lichenstein, Jonathan M. Rothberg.
Application Number | 20200360397 16/946708 |
Document ID | / |
Family ID | 1000005004516 |
Filed Date | 2020-11-19 |
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United States Patent
Application |
20200360397 |
Kind Code |
A1 |
Lichenstein; Henri ; et
al. |
November 19, 2020 |
ANTI-VIRAL COMPOSITIONS CONTAINING PIKFYVE INHIBITORS AND USE
THEREOF
Abstract
The present invention relates to compositions containing a
PIKfyve inhibitor, preferably apilimod, APY0201 or YM-201636, most
preferably apilimod, for use in treating or preventing viral
infections, preferably Ebola or Marburg virus infections. It also
relates to a pharmaceutical pack or kit comprising apilimod and at
least one additional anti-viral agent.
Inventors: |
Lichenstein; Henri;
(Guilford, CT) ; Rothberg; Jonathan M.; (Guilford,
CT) ; Beeharry; Neil; (Guilford, CT) ;
Beckett; Paul; (Yorktown Heights, NY) ; Landrette;
Sean; (Meriden, CT) ; Conrad; Chris;
(Guilford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AI Therapeutics, Inc |
Guilford |
CT |
US |
|
|
Family ID: |
1000005004516 |
Appl. No.: |
16/946708 |
Filed: |
July 1, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15545337 |
Jul 21, 2017 |
10729694 |
|
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PCT/US2016/014254 |
Jan 21, 2016 |
|
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16946708 |
|
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62107263 |
Jan 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C07K 16/10 20130101; C12N 15/1131 20130101; A61K 31/5377 20130101;
A61K 38/21 20130101; A61K 39/39575 20130101; A61K 31/713 20130101;
C07K 2317/76 20130101; A61K 31/00 20130101; C12N 2310/14
20130101 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; A61K 31/00 20060101 A61K031/00; C12N 15/113 20060101
C12N015/113; C07K 16/10 20060101 C07K016/10; A61K 45/06 20060101
A61K045/06; A61K 31/713 20060101 A61K031/713; A61K 38/21 20060101
A61K038/21; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method for treating an Ebola or Marburg viral infection in a
mammalian subject in need thereof, the method comprising
administering to the mammalian subject a composition comprising an
effective amount of apilimod, or a pharmaceutically acceptable salt
thereof.
2. The method of claim 1, wherein the apilimod is apilimod free
base or a pharmaceutically acceptable salt thereof.
3. The method of claim 2, wherein the pharmaceutically acceptable
salt is selected from a sulfate, citrate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid, acid
phosphate, isonicotinate, lactate, salicylate, acid citrate,
tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,
succinate, maleate, besylate, gentisinate, fumarate, gluconate,
glucaronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, and pamoate.
4. The method of claim 3, wherein the pharmaceutically acceptable
salt is selected from the group consisting of a chloride,
methanesulfonate, fumarate, lactate, maleate, pamoate, phosphate,
and tartrate.
4. The method of claim 1, wherein the pharmaceutically acceptable
salt is apilimod dimesylate.
5. The method of claim 1, wherein the mammalian subject is a
human.
6. The method of claim 1, further comprising administering to the
mammalian subject at least one additional anti-viral agent, either
in the same composition as the apilimod or a pharmaceutically
acceptable salt thereof, or in a different composition.
7. The method of claim 6, wherein the anti-viral agent comprises an
antibody or a combination of antibodies.
8. The method of claim 6, wherein the anti-viral agent comprises a
small interfering RNA (siRNA) or a combination of siRNA
molecules.
9. The method of claim 8, wherein the siRNA or combination of siRNA
molecules targets one or more Ebola virus proteins.
10. The method of claim 9, wherein the siRNA or combination of
siRNA molecules targets one or more Ebola virus proteins selected
from the group consisting of the zaire Ebola L polymerase, zaire
Ebola membrane-associated protein (VP24), and zaire Ebola
polymerase complex protein (VP35).
11. The method of claim 6, wherein the anti-viral agent is an
interferon.
12. The method of claim 5, wherein the apilimod, or a
pharmaceutically acceptable salt thereof is administered via an
oral, intravenous, or subcutaneous route.
13. The method of claim 12, wherein the administration of the
apilimod, or a pharmaceutically acceptable salt thereof is once
daily, twice daily, or continuous for a period of time.
14. The method of claim 12, wherein the apilimod, or a
pharmaceutically acceptable salt thereof is administered in an
amount of 70 to 1000 mg/day.
15. The method of claim 12, wherein the amount of apilimod
dimesylate is from 0.001 mg/kg to about 1000 mg/kg.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/545,337 filed on Jul. 21, 2017, which is a
national stage entry, filed under 35 U.S.C. .sctn. 371, of
International Application No. PCT/US2016/014254, filed on Jan. 21,
2016, which claims priority to U.S. Patent Application Ser. No.
62/107,263, filed on Jan. 23, 2015, the contents of which are
hereby fully incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to anti-viral compositions and
methods of using same.
BACKGROUND OF THE INVENTION
[0003] Many viruses enter the cell via endocytosis and utilize the
cell's the endosomal network as a means to infiltrate the cell and
replicate. For example, viral entry into cells may be mediated by a
viral glycoprotein (GP), which attaches viral particles to the cell
surface, delivers them to endosomes, and catalyzes fusion between
viral and endosomal membranes. For example, Rab9 GTPase was shown
to be required for replication of HIV-1, filoviruses (such as Ebola
and Marburg), and measles virus. Murray et al. 2005 Virology
79:11742-11751. Silencing Rab9 expression dramatically inhibited
HIV replication, as did silencing the host genes encoding TIP47,
p40, and PIKfyve, which also facilitate
late-endosome-to-trans-Golgi vesicular transport. Reducing Rab9
expression also inhibited the replication of the enveloped Ebola
and Marburg filoviruses and that of measles virus, but not the
non-enveloped reovirus. US 20070087008 (Hodge et al.) describes
RAB9A, RAB11A, and modulators of those proteins as potentially
useful for decreasing viral replication, especially HIV
replication.
[0004] Additional studies indicate that the endo/lysosomal
cholestrol transporter Niemann-Pick C1 (NPC1) acts as a
post-endocytic intracellular receptor that is necessary for Ebola
and Marburg virus penetration. Carette et al., 2011 Nature 477:340.
Niemann-Pick C1 (NPC1) and the homotypic fusion and vacuole protein
sorting (HOPS) complex were identified in a genome-wide haploid
genetic screen as host factors for filovirus entry. The NPC1 locus
was the single strongest hit, with 39 independent insertions. The
HOPS complex was the next strongest hit. Additional genes whose
products are involved in the biogenesis of endosomes (PIKfyve) and
lysosomes (BLOC1S1, BLOC1S2), and in the targeting of luminal cargo
to the endocytic pathway (GNPTAB) were also identified, but only
NPC1 was validated in functional assays. For example, NPC1 function
was required for infection by Ebola and Marburg viruses in human
fibroblasts, NPC1 deficiency conferred resistance to viral
infection in HAP1 and CHO cells, and NPC1 null mice were resistant
to infection and pathogenesis of Ebola and Marburg viruses. WO
2012/103081 (Chandran et al.) describes methods for treating
filovirus infection using an agent that inhibits, inter alia, NPC1
and the HOPS.
[0005] In yeast, fusion of the phagosome membrane to the lysosome
membrane requires the HOPs complex and phosphatidylinositol
3,5-bisphosphate (PI(3,5)P.sub.2). Synthesis of PI(3,5)P.sub.2 is
mediated by phosphatidylinositol-3-phosphate 5-kinase (PIKfyve).
Phosphoinositides such as PI(3,5)P.sub.2 are important lipid
regulators of membrane trafficking and cellular signaling. Using
the inhibitor YM201636, Jefferies et al. showed that inhibiting
PIKfyve and blocking cellular production of PI(3,5)P.sub.2 disrupts
endomembrane transport and retroviral budding. Jefferies et al.
EMBO rep. 2008 9:164-170.
[0006] Ebola is a filamentous, lipid enveloped, negative stranded
RNA virus of the family Filoviridae. There are five known strains
of Ebola, four of which infect humans. The 5 strains are
bundibugyo, sudan, tai forest, zaire, or reston; reston is not
known to infect humans. zaire or Ebola virus is responsible for the
majority of outbreaks (Burd, 2015). Initially Ebola targets
macrophages and dendritic cells (Missai and Sullivan 2014);
however, it is subsequently able to penetrate all cell types
excluding lymphocytes by binding to the plasma membrane surface
proteins. Viral entry is mediated by glycoprotein (GP), which
attaches viral particles to the cell surface, delivers viral
particles to the endosomes; and catalyzes fusion between the viral
particles and the endosomal membranes (Gray, 2014, Cook and Lee,
2013, Ansari, 2014, Carette, et al. 2012).
[0007] EVD is global public health emergency and the World Health
Organization has declared the recent epidemic to be a Public Health
Emergency of International Concern. The NIH classifies EVD as a
Category A pathogen which indicates that it poses the highest risk
to public safety and national security (Stahelin, 2014). The recent
West African outbreak was first reported in March 2014 (McCoy, et
al. 2014, Meyers et al. 20152015, Gatherer, 2014, Ansari, 2014)
with infection rates increasing by 13% in a 6 month period. The
recent epidemic is concentrated in areas of West Africa in
countries such as Guinea, Liberia, Nigeria, Senegal, and Sierra
Leone. The current outbreak has killed over 3,800 people
(documented cases) making it the most lethal outbreak of Ebola to
date. In the recent outbreak, mortality rates are approximately 50%
to 70%; while mortality rates in the 20 recognized outbreaks of
Ebola range from 25 to 90% (Meyers et al. 2015). Clinical
manifestations, duration of illness, case fatality rates and the
degree of transmission rates in this outbreak are similar to
previous outbreaks. Due to global travel and the international aid
workers who have been on the ground in West Africa, infected people
have traveled outside of West Africa. Countries such as the United
Kingdom, Canada, Spain and the United States have allowed infected
patients to re-enter and receive treatment. On Sept. 30, 2014, the
CDC confirmed the first case of travel associated EVD in the US.
Subsequently, two health care workers who were exposed to EVD
through this initial patient, tested positive for EVD; these two
cases were the first non-travel related cases of EVD in the United
States.
[0008] EVD is initially transmitted to man through contact with an
infected animal. The virus is spread in humans from direct contact
with broken skin or mucous membranes with blood and body fluids
including urine, saliva, feces, vomit, breast milk and semen;
needles and syringes that are contaminated with the virus; or
infected animals. EVD is not spread through the air, water or food
nor is it believed to be transmitted through mosquitoes or other
insect vectors. Symptoms of EVD may appear 2 to 21 days after
exposure with an average of 10 days. During the latent period of
the disease the patient may not show symptoms and is considered
noncontagious.
[0009] Initial clinical signs of Ebola consist of: Fever
(>38.6.degree. C. or 101.5.degree. F.), severe headache, muscle
pain, weakness, diarrhea, vomiting, abdominal pain, and unexplained
hemorrhage.
[0010] Over the clinical course of Ebola, patients can develop
fulminant septic shock and coagulopathy which can subsequently lead
to death. EVD is often masked upon clinical presentation because
the early signs and symptoms of EVD are similar to other common
viral infections; therefore, patient histories are critical to
understanding the potential risk of EVD. Several diagnostic tests
are available for Ebola; including RT-PCR and ELISA tests (Burd,
2015; Bishop 2014); however, none of these tests are available in a
non-hospital setting; therefore, making confirmation of the
diagnosis difficult in the field. EVD care is generally limited to
supportive care including administration of IV fluids, maintenance
of oxygen status and blood pressure and treatment other underlying
conditions or organ dysfunction or failure. Recovery from EVD is
dependent on the supportive care and immune response of the
patient.
[0011] Currently, there are no approved therapies for EVD. Two
experimental treatments for Ebola are available on an emergency use
basis in the US. They are ZMAPP (MAPP Pharmaceuticals) which is a
biologic composed of three humanized monoclonal antibodies and
TKM-Ebola, (Tekmira) a siRNA which interferes with the viral
proteins L, VP24, and VP35 (Bishop 2014).). The efficacy of both of
these experimental agents has yet to be established in controlled
clinical trials. Current supplies of ZMAPP are exhausted and the
drug product requires frozen shipment while TKM-Ebola is on partial
clinical hold due to safety issues; therefore, there exists an
urgent medical need for the development of new safe and effective
treatments.
[0012] The present invention addresses the need for antiviral
compositions and methods for the treatment of subjects infected
with viruses and the prophylaxis of subjects who are at risk for
viral infection, and particularly for human subjects infected with
or at risk of infection with Ebola virus.
SUMMARY OF THE INVENTION
[0013] The present invention provides compositions and methods
related to the use of PIKfyve inhibitors for the treatment and/or
prophylaxis of viral infections in a subject, preferably a human
subject, in need of such treatment or prevention. In one aspect,
the present invention is based upon the premise that inhibitors of
the cellular PIP complex protein, PIKfyve, represent promising
candidate anti-virals for the treatment of infections by Ebola and
Marburg filoviruses. This is based upon the unexpected finding that
it is possible to attain relatively high and sustained plasma
concentrations of a PIKfyve inhibitor, apilimod, in mammals,
concentrations that greatly exceed the amount of apilimod needed to
inhibit PIKfyve and disrupt intracellular trafficking, which is
crucial for successful infection by viruses such as Marburg and
Ebola. Moreover, the present disclosure demonstrates that apilimod
blood plasma levels as high as XX for [time] were achievable and
without observable adverse effects in mice.
[0014] In one embodiment, the invention provides a method for
treating or preventing a viral infection in a subject in need
thereof, the method comprising administering to the subject a
composition comprising a therapeutically effective amount of at
least one PIKfyve inhibitor. In one embodiment, the at least one
PIKfyve inhibitor is selected from the group consisting of
apilimod, APY0201, and YM-201636. In one embodiment, the at least
one PIKfyve inhibitor is apilimod. In one embodiment, the at least
one PIKfyve inhibitor comprises one or more apilimod compositions
selected from apilimod free base or pharmaceutically acceptable
salts, solvates, clathrates, hydrates, polymorphs, prodrugs,
analogs or derivatives thereof. In one embodiment, the PIKfyve
inhibitor comprises one or more of an apilimod composition, an
active metabolite of apilimod, and combinations thereof. In one
embodiment, the apilimod composition comprises apilimod free base
or apilimod dimesylate.
[0015] In one embodiment of the methods described herein, the
apilimod composition is combined with at least one additional
active agent in a single dosage form.
[0016] In one embodiment, the viral infection is caused by an Ebola
virus or a Marburg virus. In one embodiment, the virus is an Ebola
virus. In one embodiment, the Ebola virus belongs to a strain
selected from the group consisting of the bundibugyo, sudan, tai
forest, and zaire strains. In one embodiment, the Ebola virus is a
zaire Ebola virus.
[0017] In one embodiment, the apilimod is administered orally, for
example in the form of a tablet or capsule. In one embodiment, the
apilimod is administered injection or by addition to sterile
infusion fluids for intravenous infusion and is in the form of a
suitable sterile aqueous solution or dispersion.
[0018] In one embodiment, the therapeutically effective amount of
apilimod in humans is from about 70 to 1000 mg/day, from about 70
to 500 mg/day, from about 70 to 250 mg/day, from about 70 to 200
mg/day, from about 70 to 150 mg/day, of from about 70 to 100
mg/day.
[0019] The present invention also provides methods of treating or
preventing a viral infection, or ameliorating one or more symptoms
or complications of a viral infection, by administering to a
subject, preferably a human subject, a composition comprising an
effective amount of at least on PIKfyve inhibitor and further
comprising administering to the subject at least one additional
anti-viral agent, either in the same composition as the at least
one PIKfyve inhibitor, or in a different composition, for example
in a therapeutic regiment as part of a combination therapy for
treatment of the viral infection. In one embodiment, the at least
one additional anti-viral agent comprises an antibody or a
combination of antibodies, preferably human or humanized
antibodies, but chimeric (e.g., mouse-human chimeras) antibodies
are also acceptable. In one embodiment, the at least one additional
anti-viral agent comprises a small interfering RNA (siRNA) or a
combination of siRNA molecules. In one embodiment, the siRNA or
combination of siRNA molecules targets one or more Ebola virus
proteins. In one embodiment, the one or more Ebola virus proteins
is selected from the group consisting of the zaire Ebola L
polymerase, zaire Ebola membrane-associated protein (VP24), and
zaire Ebola polymerase complex protein (VP35). In one embodiment,
the siRNA or combination of siRNA molecules targets all three of
these proteins.
[0020] In the methods described here, the at least one PIKfyve
inhibitor can be administered by any suitable route and either in
the same dosage form or in a different dosage form from the
optional additional anti-viral agent or other optional therapeutic
agent as described infra. In one embodiment, administration is via
an oral, intravenous, or subcutaneous route. In one embodiment, the
administration of the at least one PIKfyve inhibitor is once daily,
twice daily, or continuous for a period of time, for example one or
several days or one or several weeks. Continuous administration may
be performed, for example, by using slow release dosage form that
is e.g., implanted in the subject, or via continuous infusion, for
example using a pump device, which also may be implanted.
[0021] In one embodiment, the at least one PIKfyve inhibitor is an
apilimod composition and the apilimod composition is administered
in an amount of 70 to 1000 mg/day. In one embodiment,
administration is effective to achieve a plasma concentration of
the PIKfyve inhibitor in the subject in the range of from 50 to
1000 nM.
[0022] In accordance with the methods described herein, the
effective amount of the PIKfyve inhibitor administered is, for
example, an amount effective to prevent or ameliorate a cytokine
storm in the subject, inhibit or reduce the rate of viral
replication in the subject, and/or stabilize or reduce the viral
load of the subject.
[0023] The invention also provides a pharmaceutical pack or kit
comprising, in separate containers or in a single container, a unit
dose of an apilimod composition and a unit dose of at least one
additional anti-viral agent. In one embodiment, the pharmaceutical
pack or kit comprises at least one PIKfyve inhibitor that is an
apilimod composition selected from apilimod free base, apilimod
dimesylate, or a racemically pure enantiomer of an active
metabolite of apilimod, and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1: Apilimod binds with high affinity to PIKfyve (Kd=75
pM).
[0025] FIG. 2: Apilimod induces vacuolization of a representative
cancer cell line. Left: Untreated cell. Right: Live cells treated
with 500 nM apilimod 24 h.
[0026] FIG. 3: Mean Plasma Apilimod (API) or Plasma Total Apilimod
Effect Concentration (TEAC) in ng/mL versus Time. Apilimod free
base was administered in two 105 mg doses, 10 hours apart.
[0027] FIG. 4A-C: In vitro activity of apilimod dimesylate against
Ebola virus. A, Log10 total copies of virus per treatment (Rx) vs
time-for the qRT-PCR on HepG2 cells for the time-points
post-infection with ZEBOV-Kikwit: 1 hour, 48 hours and 72 hours; B,
Log10 total copies reduced per Rx as compared to EBOV mock vs time;
C, CellTiter-Glo Luminescent Cell Viability Assay of LAM-002
treatment of HepG2 cell line for 72 hours at concentration range of
10,000 nM to 19 nM at two fold dilutions.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides compositions and methods
related to the use of PIKfyve inhibitors for treating or preventing
a viral infection in a subject, preferably a human subject, in need
of such treatment or prevention.
[0029] In one embodiment, the invention provides methods for the
treatment of viral infections in a subject by administering to the
subject a therapeutically effective amount of at least one PIKfyve
inhibitor. In one embodiment, the invention provides pharmaceutical
compositions comprising a therapeutically effective amount of at
least one PIKfyve inhibitor. In one embodiment, the at least one
PIKFyve inhibitor is selected from the group consisting of
apilimod, APY0201, and YM201636, or a pharmaceutically acceptable
salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug,
analog or derivative thereof.
[0030] In one embodiment, the viral infection is caused by a virus
selected from the group consisting of measles, Ebola (EboV),
Marburg (MarV), borna disease, and human immunodeficiency virus
(HIV), severe acute respiratory system virus (SARS), and middle
east respiratory syndrome virus (MERS). In one embodiment, the
viral infection is caused by EboV or MarV.
[0031] In one embodiment, the at least one PIKfyve inhibitor is
apilimod. Apilimod is a selective inhibitor of PIKFyve (Cai et al.
2013 Chem. & Biol. 20:912-921). Based upon its ability to
inhibit IL-12/23 production, apilimod has been suggested as useful
for treating inflammatory and autoimmune diseases such as
rheumatoid arthritis, sepsis, Crohn's disease, multiple sclerosis,
psoriasis, or insulin dependent diabetes mellitus, and in cancers
where these cytokines were believed to play a pro-proliferative
role.
[0032] As used herein, the term "an apilimod composition" may refer
to a composition comprising apilimod itself (free base), or may
encompass pharmaceutically acceptable salts, solvates, clathrates,
hydrates, polymorphs, prodrugs, analogs or derivatives of apilimod,
as described below. The structure of apilimod is shown in Formula
I:
##STR00001##
[0033] The chemical name of apilimod is
2-[2-Pyridin-2-yl)-ethoxy]-4-N'-(3-methyl-benzilidene)-hydrazino]-6-(morp-
holin-4-yl)-pyrimidine (IUPAC name:
(E)-4-(6-(2-(3-methylbenzylidene)hydrazinyl)-2-(2-(pyridin-2-yl)ethoxy)py-
rimidin-4-yl)morpholine), and the CAS number is 541550-19-0.
[0034] Apilimod can be prepared, for example, according to the
methods described in U.S. Pat. Nos. 7,923,557, and 7,863,270, and
WO 2006/128129.
[0035] In one embodiment, the apilimod composition for use in the
compositions and methods of the invention is the free base or
dimesylate salt form, MW 610.7 (dimesylate salt); tPSA 83.1; pKa
5.39 (.+-.0.03), 4.54 (.+-.0.27); HBD 1. The apilimod dimesylate
salt is highly water soluble (>25 mg/mL) and shows moderate
permeability (>70% in rats). In one embodiment, the apilimod
composition for use in the compositions and methods of the
invention is an active metabolite of apilimod. Six primary
metabolites were identified in rat and human microsomal and
hepatocyte stability studies. Human, rat, rabbit and dog studies
showed a qualitatively similar metabolic profile. T.sub.max
generally occurred within 1 or 2 hours after the oral dose,
consistent with the rapid elimination of this compound from the
circulation. Reaction phenotyping studies indicated that CYP3A4 and
to a lesser extent CYP1A2 and/or CYP2D6, contribute to metabolism.
The primary metabolites are short-lived in circulation. Both
apilimod free base and the dimesylate salt are highly bound
(>99%) to rat, dog and human plasma proteins.
[0036] In one embodiment, the at least one PIKfyve inhibitor is
selected from APY0201, and YM-201636. The structures of APY0201 and
YM-201636 are shown in Formula II and Formula III respectively.
##STR00002##
[0037] The chemical name of APY0201 is
(E)-4-(5-(2-(3-methylbenzylidine)hydrazinlyl)-2-(pyridine-4-yl)pyrazolol[-
1,5-a]pyrimidin-7-yl)morpholine. APY0201 is a selective PIKfyve
inhibitor (Hayakawa et al. 2014 Bioorg. Med. Chem. 22:3021-29).
APY0201 directly interacts with the ATP-binding site of PIKfyve
kinase, which leads to suppression of PI(3,5)P.sub.2 synthesis,
which in turn suppresses the production of IL-12/23.
[0038] The chemical name for YM201636 is
6-amino-N-(3-(4-morpholinopyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phen-
yl)nicotinamide (CAS number is 371942-69-7). YM201636 is a
selective inhibitor of PIKfyve (Jefferies et al. EMBO rep. 2008
9:164-170). It reversibly impairs endosomal trafficking in NIH3T3
cells, mimicking the effect produced by depleting PIKfyve with
siRNA. YM201636 also blocks retroviral exit by budding from cells,
apparently by interfering with the endosomal sorting complex
required for transport (ESCRT) machinery. In adipocytes, YM-201636
also inhibits basal and insulin-activated 2-deoxyglucose uptake
(IC.sub.50=54 nM).
[0039] As used herein, the term "pharmaceutically acceptable salt,"
is a salt formed from, for example, an acid and a basic group of an
apilimod composition. Illustrative salts include, but are not
limited, to sulfate, citrate, acetate, oxalate, chloride, bromide,
iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
besylate, gentisinate, fumarate, gluconate, glucaronate,
saccharate, formate, benzoate, glutamate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate
(e.g., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. In a
preferred embodiment, the salt of apilimod comprises
methanesulfonate.
[0040] The term "pharmaceutically acceptable salt" also refers to a
salt prepared from an apilimod composition having an acidic
functional group, such as a carboxylic acid functional group, and a
pharmaceutically acceptable inorganic or organic base.
[0041] The term "pharmaceutically acceptable salt" also refers to a
salt prepared from an apilimod composition having a basic
functional group, such as an amino functional group, and a
pharmaceutically acceptable inorganic or organic acid.
[0042] The salts of the compounds described herein can be
synthesized from the parent compound by conventional chemical
methods such as methods described in Pharmaceutical Salts:
Properties, Selection, and Use, P. Hemrich Stalil (Editor), Camille
G. Wermuth (Editor), ISBN: 3-90639-026-8, August 2002. Generally,
such salts can be prepared by reacting the parent compound with the
appropriate acid in water or in an organic solvent, or in a mixture
of the two.
[0043] One salt form of a compound described herein can be
converted to the free base and optionally to another salt form by
methods well known to the skilled person. For example, the free
base can be formed by passing the salt solution through a column
containing an amine stationary phase (e.g. a Strata-NH2 column).
Alternatively, a solution of the salt in water can be treated with
sodium bicarbonate to decompose the salt and precipitate out the
free base. The free base may then be combined with another acid
using routine methods.
[0044] As used herein, the term "polymorph" means a solid
crystalline form of a compound of the present invention. Different
polymorphs of the same compound can exhibit different physical,
chemical and/or spectroscopic properties. Different physical
properties include, but are not limited to stability (e.g., to heat
or light), compressibility and density (important in formulation
and product manufacturing), and dissolution rates (which can affect
bioavailability). Differences in stability can result from changes
in chemical reactivity (e.g., differential oxidation, such that a
dosage form discolors more rapidly when comprised of one polymorph
than when comprised of another polymorph) or mechanical
characteristics (e.g., tablets crumble on storage as a kinetically
favored polymorph converts to thermodynamically more stable
polymorph) or both (e.g., tablets of one polymorph are more
susceptible to breakdown at high humidity). Different physical
properties of polymorphs can affect their processing. For example,
one polymorph might be more likely to form solvates or might be
more difficult to filter or wash free of impurities than another
due to, for example, the shape or size distribution of particles of
it.
[0045] As used herein, the term "hydrate" means a compound of the
present invention or a salt thereof, which further includes a
stoichiometric or non-stoichiometric amount of water bound by
non-covalent intermolecular forces.
[0046] As used herein, the term "clathrate" means a compound of the
present invention or a salt thereof in the form of a crystal
lattice that contains spaces (e.g., channels) that have a guest
molecule (e.g., a solvent or water) trapped within.
[0047] As used herein, the term "prodrug" means a derivative of a
compound described herein that can hydrolyze, oxidize, or otherwise
react under biological conditions (in vitro or in vivo) to provide
a compound of the invention. Prodrugs may only become active upon
such reaction under biological conditions, or they may have
activity in their unreacted forms. Examples of prodrugs
contemplated in this invention include, but are not limited to,
analogs or derivatives of a compound described herein that comprise
biohydrolyzable moieties such as biohydrolyzable amides,
biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable
carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate
analogues. Other examples of prodrugs include derivatives of
compounds of any one of the formulae disclosed herein that comprise
--NO, --NO.sub.2, --ONO, or --ONO.sub.2 moieties. Prodrugs can
typically be prepared using well-known methods, such as those
described by Burger's Medicinal Chemistry and Drug Discovery (1995)
172-178, 949-982 (Manfred E. Wolff ed., 5th ed).
[0048] In addition, some of the compounds suitable for use in the
methods of in this invention have one or more double bonds, or one
or more asymmetric centers. Such compounds can occur as racemates,
racemic mixtures, single enantiomers, individual diastereomers,
diastereomeric mixtures, and cis- or trans- or E- or Z- double
isomeric forms. All such isomeric forms of these compounds are
expressly included in the present invention. The compounds of this
invention can also be represented in multiple tautomeric forms, in
such instances, the invention expressly includes all tautomeric
forms of the compounds described herein (e.g., there may be a rapid
equilibrium of multiple structural forms of a compound), the
invention expressly includes all such reaction products). All such
isomeric forms of such compounds are expressly included in the
present invention. All crystal forms of the compounds described
herein are expressly included in the present invention.
[0049] As used herein, the term "solvate" or "pharmaceutically
acceptable solvate," is a solvate formed from the association of
one or more solvent molecules to one of the compounds disclosed
herein. The term solvate includes hydrates (e.g., hemi-hydrate,
mono-hydrate, dihydrate, trihydrate, tetrahydrate, and the
like).
[0050] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another but differs
slightly in composition (as in the replacement of one atom by an
atom of a different element or in the presence of a particular
functional group, or the replacement of one functional group by
another functional group). Thus, an analog is a compound that is
similar or comparable in function and appearance, but not in
structure or origin to the reference compound. As used herein, the
term "derivative" refers to compounds that have a common core
structure, and are substituted with various groups as described
herein.
Methods of Treatment
[0051] The present invention provides methods for the treatment of
viral infections in a subject in need thereof by administering to
the subject a therapeutically effective amount of at least one
PIKfyve inhibitor. In one embodiment, the at least one PIKfyve
inhibitor is selected from an apilimod composition, APY0201,
YM-201636 or a pharmaceutically acceptable salt, solvate,
clathrate, hydrate, polymorph, metabolite, prodrug, analog or
derivative thereof. The present invention further provides the use
of at least one PIKfyve inhibitor for the preparation of a
medicament useful for the treatment of viral infections.
[0052] The term "therapeutically effective amount" refers to an
amount of a PIKfyve inhibitor sufficient to treat, ameliorate a
symptom of, reduce the severity of, or reduce the duration of a
viral infection, or enhance or improve the therapeutic effect of
another therapy, e.g., another antiviral therapy, when administered
in combination with the PIKfyve inhibitor or as part of a
therapeutic regimen that includes administering a PIKfyve
inhibitor.
[0053] In one embodiment, the therapeutically effective amount is
an amount effective to achieve one or more of the following:
inhibit cellular PIKfyve activity, substantially prevent viral
entry into a subject's cells, reduce the amount of viral particles
which gain entry to a subject's cells, reduce viral replication
within the subject's cells, ameliorate one or more symptoms
associated with viral infection of the subject, and reduce the
severity of one or more symptoms associated with viral infection of
the subject.
[0054] In one embodiment, the therapeutically effective amount is
an amount sufficient to reduce the magnitude of, or prevent the
onset, of a cytokine storm in the subject.
[0055] In one embodiment, the therapeutically effective amount is
an amount sufficient to reduce viral load. In one embodiment, the
viral load is reduced by 5% or greater, 10% or greater, 20% or
greater, 30% or greater, 40% or greater, 50% or greater, or 75% or
greater. In one embodiment, the viral load is reduced by at least
0.5 log units, at least 1 log unit, at least 2 log units, at least
3 log units, at least 4 log units, at least 10 log units, at least
15 log units, or by at least 20 log units.
[0056] An effective amount can range from about 0.001 mg/kg to
about 1000 mg/kg, more preferably 0.01 mg/kg to about 100 mg/kg,
more preferably 0.1 mg/kg to about 10 mg/kg; or any range in which
the low end of the range is any amount between 0.001 mg/kg and 900
mg/kg and the upper end of the range is any amount between 0.1
mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg
and 20 mg/kg). Effective doses will also vary, as recognized by
those skilled in the art, depending on the diseases treated, route
of administration, excipient usage, and the possibility of co-usage
with other therapeutic treatments such as use of other agents. See,
e.g., U.S. Pat. No. 7,863,270, incorporated herein by
reference.
[0057] In one embodiment, the therapeutically effective amount of
apilimod in humans is from about 70 to 1000 mg/day, from about 70
to 500 mg/day, from about 70 to 250 mg/day, from about 70 to 200
mg/day, from about 70 to 150 mg/day, of from about 70 to 100
mg/day.
[0058] In one embodiment, the method comprises administering to a
subject a therapeutically effective amount of a PIKfyve inhibitor
as monotherapy for the treatment of a viral infection. In another
embodiment, described more fully below, the PIKfyve inhibitor, or
more than one PIKfyve inhibitor, is administered as part of a
combination therapy or therapeutic regimen, for example with one or
more anti-viral agents or an anti-viral regimen.
[0059] The methods may comprise administering the PIKfyve inhibitor
according to a specified dosing schedule or therapeutic regimen.
For example, the PIKfyve inhibitor can be administered once daily
or from two to five times daily. In one embodiment, apilimod,
APY0201, or YM-201636 is administered thrice daily, twice daily,
once daily, fourteen days on (four times daily, thrice daily or
twice daily, or once daily) and 7 days off in a 3-week cycle, up to
five or seven days on (four times daily, thrice daily or twice
daily, or once daily) and 14-16 days off in 3 week cycle, or once
every two days, or once a week, or once every 2 weeks, or once
every 3 weeks.
[0060] In the context of the methods described herein, the amount
of a PIKfyve inhibitor, preferably an apilimod composition,
administered to the subject is a therapeutically effective amount.
The term "therapeutically effective amount" refers to an amount
sufficient to treat, ameliorate a symptom of, reduce the severity
of, or reduce the duration of the disease or disorder being
treated, or enhance or improve the therapeutic effect of another
therapy, or sufficient to exhibit a detectable therapeutic effect
in the subject. In one embodiment, the therapeutically effective
amount of a PIKfyve inhibitor, preferably an apilimod composition,
is the amount effective to inhibit PIKfyve kinase activity.
[0061] An effective amount of a PIKfyve inhibitor, preferably an
apilimod composition, can range from about 0.001 mg/kg to about
1000 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 10 mg/kg to
about 250 mg/kg, about 0.1 mg/kg to about 15 mg/kg; or any range in
which the low end of the range is any amount between 0.001 mg/kg
and 900 mg/kg and the upper end of the range is any amount between
0.1 mg/kg and 1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5
mg/kg and 20 mg/kg). Effective doses will also vary, as recognized
by those skilled in the art, depending on the diseases treated,
route of administration, excipient usage, and the possibility of
co-usage with other therapeutic treatments such as use of other
agents. See e.g., U.S. Pat. No. 7,863,270, incorporated herein by
reference.
[0062] In more specific aspects, a PIKfyve inhibitor, preferably an
apilimod composition, is administered at a dosage regimen of
70-1000 mg/day (e.g., 70, 75, 80, 85, 90, 95, 100, 125, 125, 150,
175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, or 1000 mg/day) for at least 1 week,
in some embodiments for 1 to 4 weeks, from 2 to 6 weeks, from 2 to
8 weeks, from 2 to 10 weeks, or from 2 to 12 weeks, 2 to 16 weeks,
or longer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 36, 48, or
more weeks). In one embodiment, a PIKfyve inhibitor, preferably an
apilimod composition, is administered at a dosage regimen of
70-1000 mg/day for 2, 4, 12, or 16 weeks. Alternatively or
subsequently, a PIKfyve inhibitor, preferably an apilimod
composition, is administered at a dosage regimen of 35 mg-500 mg
twice a day for 4 weeks, 8 weeks, 12 weeks, 16 weeks, or
longer.
[0063] An effective amount of the PIKfyve inhibitor can be
administered once daily, twice daily, from two to five times daily,
up to two times or up to three times daily, or up to eight times
daily. In one embodiment, the inhibitor is administered thrice
daily, twice daily, once daily, fourteen days on (four times daily,
thrice daily or twice daily, or once daily) and 7 days off in a
3-week cycle, up to five or seven days on (four times daily, thrice
daily or twice daily, or once daily) and 14-16 days off in 3 week
cycle, or once every two days, or once a week, or once every 2
weeks, or once every 3 weeks.
[0064] In accordance with the methods described herein, a "subject
in need of" is a subject having a viral disease, or a subject
having an increased risk of developing a viral disease, relative to
the population at large. The subject in need thereof can be one
that is "non-responsive" or "refractory" to a currently available
therapy for the viral disease. In this context, the terms
"non-responsive" and "refractory" refer to the subject's response
to therapy as not clinically adequate to relieve one or more
symptoms associated with the viral infection. In one aspect of the
methods described here, the subject in need thereof is a subject
having a viral disease caused by an Ebola or Marburg virus who is
refractory to standard therapy.
[0065] A "subject" includes a mammal. The mammal can be e.g., any
mammal, e.g., a human, primate, vertebrate, bird, mouse, rat, fowl,
dog, cat, cow, horse, goat, camel, sheep or a pig. Preferably, the
mammal is a human. The term "patient" refers to a human
subject.
[0066] The present invention also provides a monotherapy for the
treatment of a viral disease, as described herein. As used herein,
"monotherapy" refers to the administration of a single active or
therapeutic compound to a subject in need thereof.
[0067] As used herein, "treatment", "treating" or "treat" describes
the management and care of a patient for the purpose of combating a
viral disease and includes the administration of a PIKfyve
inhibitor, preferably an apilimod composition, to alleviate the
symptoms or complications of the viral disease.
[0068] As used herein, "prevention", "preventing" or "prevent"
describes reducing or eliminating the onset of the symptoms or
complications of the viral disease, includes the administration of
a PIKfyve inhibitor, preferably an apilimod composition, to reduce
the onset, development or recurrence of symptoms of the viral
disease.
Combination Therapies
[0069] The present invention also provides methods comprising
combination therapy. As used herein, "combination therapy" or
"co-therapy" includes the administration of a therapeutically
effective amount of a PIKfyve inhibitor, preferably an apilimod
composition, with at least one additional active agent, as part of
a specific treatment regimen intended to provide a beneficial
effect from the co-action of the active agents in the regimen.
"Combination therapy" is not intended to encompass the
administration of two or more therapeutic compounds as part of
separate monotherapy regimens that incidentally and arbitrarily
result in a beneficial effect that was not intended or
predicted.
[0070] Thus, the invention provides methods of treating a subject
for a viral disease or viral infection (the terms "viral disease"
and "viral infection" are used interchangeably herein) using a
combination therapy comprising a PIKfyve inhibitor, preferably an
apilimod composition, and at least one additional active agent in
an anti-viral regimen for the treatment of the viral disease.
[0071] The at least one additional active agent may be a
therapeutic agent, for example an anti-viral agent, or a
non-therapeutic agent, and combinations thereof. With respect to
therapeutic agents, the beneficial effect of the combination
includes, but is not limited to, pharmacokinetic or pharmacodynamic
co-action resulting from the combination of therapeutically active
compounds. With respect to non-therapeutic agents, the beneficial
effect of the combination may relate to the mitigation of a
toxicity, side effect, or adverse event associated with a
therapeutically active agent in the combination.
[0072] In one embodiment, the at least one additional agent is a
non-therapeutic agent which mitigates one or more side effects of
an apilimod composition, the one or more side effects selected from
any of nausea, vomiting, headache, dizziness, lightheadedness,
drowsiness and stress. In one aspect of this embodiment, the
non-therapeutic agent is an antagonist of a serotonin receptor,
also known as 5-hydroxytryptamine receptors or 5-HT receptors. In
one aspect, the non-therapeutic agent is an antagonist of a 5-HT3
or 5-HT1a receptor. In one aspect, the non-therapeutic agent is
selected from the group consisting of ondansetron, granisetron,
dolasetron and palonosetron. In another aspect, the non-therapeutic
agent is selected from the group consisting of pindolol and
risperidone.
[0073] In one embodiment, the at least one additional agent is a
therapeutic agent. In one embodiment, the therapeutic agent is
selected from an anti-viral agent, an anti-viral vaccine, a
nucleotide analogue, a cytokine (e.g., an interferon), and an
immunoglobulin, and combinations thereof. In one embodiment, the at
least one additional agent is selected from an inhibitor of one or
more of NPCI, VPSII, VPSI6, VPSI8, VPS33A, VPS39, VPS41, BLOCISI,
BLOCIS2, GNPT-AB, PIKFYVE, ARGHGAP23, COPI, COPII, TIP47, P40, Rab
GTP-binding proteins (e.g., Rab9), clathrin, AP1, AP3, t-/v-SNARE
complex, ARFs, Ras GTP-ases, and a combinations thereof. In one
embodiment, the at least one additional therapeutic agent is
selected from an antibody, a PIKfyve inhibitor, and an inhibitor of
phosphotransferase activity.
[0074] Non-limiting examples of anti-viral agents that may be used
in combination with a PIKfyve inhibitor as described herein include
Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine;
Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone;
Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine
Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;
Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine
Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet
Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium;
Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine
Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;
Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;
Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;
Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;
Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate;
Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.
[0075] In certain embodiments the at least one PIKfyve inhibitor is
provided in a single dosage form in combination with one or more
additional therapeutic agents. In one embodiment, the at least one
PIKfyve inhibitor is an apilimod composition and the therapeutic
agent is an antiviral agent. In another embodiment, the apilimod is
provided in combination with one or more additional PIKfyve
inhibitors, for example APY0201 and YM201636. Where more than one
therapeutic agent is present in a single dosage form, the
therapeutically effective amount is based upon the total amount of
therapeutic agents in the dosage form.
[0076] In one embodiment the at least one PIKfyve inhibitor is
provided in a separate dosage form from the one or more additional
therapeutic agents. Separate dosage forms are desirable, for
example, in the context of a combination therapy in which the
therapeutic regimen calls for administration of different
therapeutic agents at different frequencies or under different
conditions, or via different routes.
[0077] In one embodiment, administration of the at least one
PIKfyve inhibitor as described herein is accomplished via an oral
dosage form suitable for oral administration. In another embodiment
administration is by an indwelling catheter, a pump, such as an
osmotic mini-pump, or a sustained release composition that is, for
example, implanted in the subject.
Pharmaceutical Compositions and Formulations
[0078] The present invention provides pharmaceutical compositions
comprising an effective amount of at least one PIKfyve inhibitor
and at least one pharmaceutically acceptable excipient or carrier,
wherein the effective amount is as described above in connection
with the methods of the invention.
[0079] In one embodiment, the PIKfyve inhibitor is selected from
one or more of an apilimod composition as described above, APY0201,
YM-201636, and pharmaceutically acceptable salts, solvates,
clathrates, hydrates, polymorphs, metabolites, prodrugs, analogs
and derivatives thereof. In one embodiment, the PIKfyve inhibitor
is an apilimod composition.
[0080] In one embodiment, the at least one PIKfyve inhibitor is
combined with at least one additional therapeutic agent in a single
dosage form. In one embodiment, the at least one additional
therapeutic agent is selected from an anti-viral agent (as
described above), an anti-viral vaccine, a nucleotide analogue, a
cytokine (e.g., an interferon), and an immunoglobulin, and
combinations thereof. In one embodiment, the at least one
additional therapeutic agent is selected from an inhibitor of one
or more of NPCI, VPSII, VPSI6, VPSI8, VPS33A, VPS39, VPS41,
BLOCISI, BLOCIS2, GNPT-AB, PIKFYVE, ARGHGAP23, COPI, COPII, TIP47,
P40, Rab GTP-binding proteins (e.g., Rab9), clathrin, AP1, AP3,
t-/v-SNARE complex, ARFs, Ras GTP-ases, and a combinations thereof.
In one embodiment, the additional therapeutic agent is selected
from an antibody, a PIKfyve inhibitor, and an inhibitor of
phosphotransferase activity.
[0081] The term "pharmaceutically acceptable" refers to those
compounds, materials, compositions, carriers, 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.
[0082] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes excipient that is acceptable for
veterinary use as well as human pharmaceutical use. Examples of
pharmaceutically acceptable excipients include, without limitation,
sterile liquids, water, buffered saline, ethanol, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like), oils, detergents, suspending agents, carbohydrates
(e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g.,
ascorbic acid or glutathione), chelating agents, low molecular
weight proteins, or suitable mixtures thereof.
[0083] A pharmaceutical composition can be provided in bulk or in
dosage unit form. It is especially advantageous to formulate
pharmaceutical compositions in dosage unit form for ease of
administration and uniformity of dosage. The term "dosage unit
form" as used herein refers to physically discrete units suited as
unitary dosages for the subject to be treated; each unit containing
a predetermined quantity of active compound calculated to produce
the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved. A dosage unit form can be an
ampoule, a vial, a suppository, a dragee, a tablet, a capsule, an
IV bag, or a single pump on an aerosol inhaler.
[0084] In therapeutic applications, the dosages vary depending on
the agent, the age, weight, and clinical condition of the recipient
patient, and the experience and judgment of the clinician or
practitioner administering the therapy, among other factors
affecting the selected dosage. Generally, the dose should be a
therapeutically effective amount. Dosages can be provided in
mg/kg/day units of measurement (which dose may be adjusted for the
patient's weight in kg, body surface area in m.sup.2, and age in
years). Exemplary doses and dosages regimens for the compositions
in methods of treating viral infections are described above.
[0085] A dose may be provided in unit dosage form. For example, the
unit dosage form can comprise 1 nanogram to 2 milligrams, or 0.1
milligrams to 2 grams; or from 10 milligrams to 1 gram, or from 50
milligrams to 500 milligrams or from 1 microgram to 20 milligrams;
or from 1 microgram to 10 milligrams; or from 0.1 milligrams to 2
milligrams.
[0086] The pharmaceutical compositions can take any suitable form
(e.g, liquids, aerosols, solutions, inhalants, mists, sprays; or
solids, powders, ointments, pastes, creams, lotions, gels, patches
and the like) for administration by any desired route (e.g,
pulmonary, inhalation, intranasal, oral, buccal, sublingual,
parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, intrapleural, intrathecal, transdermal,
transmucosal, rectal, and the like). For example, a pharmaceutical
composition of the invention may be in the form of an aqueous
solution or powder for aerosol administration by inhalation or
insufflation (either through the mouth or the nose), in the form of
a tablet or capsule for oral administration; in the form of a
sterile aqueous solution or dispersion suitable for administration
by either direct injection or by addition to sterile infusion
fluids for intravenous infusion; or in the form of a lotion, cream,
foam, patch, suspension, solution, or suppository for transdermal
or transmucosal administration.
[0087] A pharmaceutical composition can be in the form of an orally
acceptable dosage form including, but not limited to, capsules,
tablets, buccal forms, troches, lozenges, and oral liquids in the
form of emulsions, aqueous suspensions, dispersions or solutions.
Capsules may contain mixtures of a compound of the present
invention with inert fillers and/or diluents such as the
pharmaceutically acceptable starches (e.g., corn, potato or tapioca
starch), sugars, artificial sweetening agents, powdered celluloses,
such as crystalline and microcrystalline celluloses, flours,
gelatins, gums, etc. In the case of tablets for oral use, carriers
which are commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, can also be added.
For oral administration in a capsule form, useful diluents include
lactose and dried corn starch. When aqueous suspensions and/or
emulsions are administered orally, the compound of the present
invention may be suspended or dissolved in an oily phase is
combined with emulsifying and/or suspending agents. If desired,
certain sweetening and/or flavoring and/or coloring agents may be
added.
[0088] A pharmaceutical composition can be in the form of a tablet.
The tablet can comprise a unit dosage of a compound of the present
invention together with an inert diluent or carrier such as a sugar
or sugar alcohol, for example lactose, sucrose, sorbitol or
mannitol. The tablet can further comprise a non-sugar derived
diluent such as sodium carbonate, calcium phosphate, calcium
carbonate, or a cellulose or derivative thereof such as methyl
cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and
starches such as corn starch. The tablet can further comprise
binding and granulating agents such as polyvinylpyrrolidone,
disintegrants (e.g. swellable crosslinked polymers such as
crosslinked carboxymethylcellulose), lubricating agents (e.g.
stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT),
buffering agents (for example phosphate or citrate buffers), and
effervescent agents such as citrate/bicarbonate mixtures.
[0089] The tablet can be a coated tablet. The coating can be a
protective film coating (e.g. a wax or varnish) or a coating
designed to control the release of the active agent, for example a
delayed release (release of the active after a predetermined lag
time following ingestion) or release at a particular location in
the gastrointestinal tract. The latter can be achieved, for
example, using enteric film coatings such as those sold under the
brand name Eudragit.RTM..
[0090] Tablet formulations may be made by conventional compression,
wet granulation or dry granulation methods and utilize
pharmaceutically acceptable diluents, binding agents, lubricants,
disintegrants, surface modifying agents (including surfactants),
suspending or stabilizing agents, including, but not limited to,
magnesium stearate, stearic acid, talc, sodium lauryl sulfate,
microcrystalline cellulose, carboxymethylcellulose calcium,
polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan
gum, sodium citrate, complex silicates, calcium carbonate, glycine,
dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate,
lactose, kaolin, mannitol, sodium chloride, talc, dry starches and
powdered sugar. Preferred surface modifying agents include nonionic
and anionic surface modifying agents. Representative examples of
surface modifying agents include, but are not limited to, poloxamer
188, benzalkonium chloride, calcium stearate, cetostearyl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon
dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum
silicate, and triethanolamine.
[0091] A pharmaceutical composition can be in the form of a hard or
soft gelatin capsule. In accordance with this formulation, the
compound of the present invention may be in a solid, semi-solid, or
liquid form.
[0092] A pharmaceutical composition can be in the form of a sterile
aqueous solution or dispersion suitable for parenteral
administration. The term parenteral as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intra-arterial, intrasynovial, intrasternal,
intrathecal, intralesional and intracranial injection or infusion
techniques.
[0093] A pharmaceutical composition can be in the form of a sterile
aqueous solution or dispersion suitable for administration by
either direct injection or by addition to sterile infusion fluids
for intravenous infusion, and comprises a solvent or dispersion
medium containing, water, ethanol, a polyol (e.g., glycerol,
propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof, or one or more vegetable oils. Solutions or suspensions of
the compound of the present invention as a free base or
pharmacologically acceptable salt can be prepared in water suitably
mixed with a surfactant. Examples of suitable surfactants are given
below. Dispersions can also be prepared, for example, in glycerol,
liquid polyethylene glycols and mixtures of the same in oils.
[0094] The pharmaceutical compositions for use in the methods of
the present invention can further comprise one or more additives in
addition to any carrier or diluent (such as lactose or mannitol)
that is present in the formulation. The one or more additives can
comprise or consist of one or more surfactants. Surfactants
typically have one or more long aliphatic chains such as fatty
acids which enables them to insert directly into the lipid
structures of cells to enhance drug penetration and absorption. An
empirical parameter commonly used to characterize the relative
hydrophilicity and hydrophobicity of surfactants is the
hydrophilic-lipophilic balance ("HLB" value). Surfactants with
lower HLB values are more hydrophobic, and have greater solubility
in oils, while surfactants with higher HLB values are more
hydrophilic, and have greater solubility in aqueous solutions.
Thus, hydrophilic surfactants are generally considered to be those
compounds having an HLB value greater than about 10, and
hydrophobic surfactants are generally those having an HLB value
less than about 10. However, these HLB values are merely a guide
since for many surfactants, the HLB values can differ by as much as
about 8 HLB units, depending upon the empirical method chosen to
determine the HLB value.
[0095] Among the surfactants for use in the compositions of the
invention are polyethylene glycol (PEG)-fatty acids and PEG-fatty
acid mono and diesters, PEG glycerol esters, alcohol-oil
transesterification products, polyglyceryl fatty acids, propylene
glycol fatty acid esters, sterol and sterol derivatives,
polyethylene glycol sorbitan fatty acid esters, polyethylene glycol
alkyl ethers, sugar and its derivatives, polyethylene glycol alkyl
phenols, polyoxyethylene-polyoxypropylene (POE-POP) block
copolymers, sorbitan fatty acid esters, ionic surfactants,
fat-soluble vitamins and their salts, water-soluble vitamins and
their amphiphilic derivatives, amino acids and their salts, and
organic acids and their esters and anhydrides.
[0096] The present invention also provides packaging and kits
comprising pharmaceutical compositions for use in the methods of
the present invention. The kit can comprise one or more containers
selected from the group consisting of a bottle, a vial, an ampoule,
a blister pack, and a syringe. The kit can further include one or
more of instructions for use in treating and/or preventing a
disease, condition or disorder of the present invention, one or
more syringes, one or more applicators, or a sterile solution
suitable for reconstituting a pharmaceutical composition of the
present invention.
[0097] All percentages and ratios used herein, unless otherwise
indicated, are by weight. Other features and advantages of the
present invention are apparent from the different examples. The
provided examples illustrate different components and methodology
useful in practicing the present invention. The examples do not
limit the claimed invention. Based on the present disclosure the
skilled artisan can identify and employ other components and
methodology useful for practicing the present invention.
EXAMPLES
Example 1: Apilimod is a Highly Selective Binder of PIKfyve
Kinase
[0098] Protein kinase profiling of apilimod was conducted to
identify kinase targets (DiscoveRx, Fremont, Calif.). A
dissociation constant (Kd) study was performed using apilimod at
increasing concentrations (0.05-3000 nM) against PIKfyve, a known
target of apilimod. The experiment was performed in duplicate and
the Kd was determined to be 0.075 nM (range 0.069-0.081 nM) (see
FIG. 1).
[0099] Apilimod was also screened against a comprehensive panel of
kinases (PIKfyve not included). In total, 456 kinases, including
disease-relevant kinases, were assayed for their ability to bind
with apilimod. The screening concentration of apilimod was 1 mM, a
concentration that is >10,000 times greater than the Kd for
apilimod against PIKfyve. The results from the screen showed that
apilimod did not bind to any of the 456 kinases tested.
[0100] Together, these results demonstrate that apilimod binds with
high selectivity to a single cellular kinase, PIKfyve.
Example 2: Apilimod Induces Vacuolization and Disrupts
Intracellular Traficking in Cells
[0101] Apilimod has been demonstrated to be a potent and specific
inhibitor of the phosphoinositide kinase PIKfyve, an enzyme that
binds to PI(3)P and catalyzes the formation of the lipid second
messengers PI(3,5)P2 and PI(5)P. PIKfyve is associated with the
cytosolic leaflet of early endosomes and its activity is required
for endomembrane homeostasis, endolysosomal function and proper
retrograde transport from the endosome to the trans-Golgi network.
Introduction of a kinase dead mutant into cells induces a swollen
vacuole phenotype that can be rescued by the injection of
PI(3,5)P2. Inhibition of PIKfyve by pharmacological methods as well
as RNAi also produces swollen vacuoles and disruption of
endomembrane dynamics. It has been discovered that pharmacological
disruption of PIKfyve with apilimod induces selective lethality of
specific cancer cell lines through disruption of intracellular
trafficking (see FIG. 2).
Example 3: Prediction of in vivo Anti-Ebola Activity in Humans
[0102] Inhibition of cancer cell proliferation and inhibition of
Ebola virus infection share a common mechanism i.e. inhibition of
PIKfyve leading to vacuole formation and loss of intracellular
trafficking. In the above clinical study, the trough TAEC values
were greater than 25 mg/mL (60 nM). Since vacuole formation in
cells at apilimod concentrations as low as 20 nM have been
observed, one could conclude that oral administration of apilimod
free base at doses ranging from 70 to 1000 mg/day should provide
continuous PIKfyve inhibition to maintain vacuolization of cells
and block Ebola infection in clinical therapy in patients.
[0103] Furthermore, it was shown in female Balb/c mice, a strain
often used for in vivo Ebola infection studies, that constant
infusion of apilimod as the bis-mesylate salt, using subcutaneously
implanted osmotic mini-pumps (e.g. Alzet models 1007D, 15
mg/kg/day; model 2001, 30 mg/kg/day; vehicle: 25% DMSO, 25%
Cremaphor, 50% sterile water) can provide sustained blood
concentrations of apilimod in excess of 0.5 .mu.M and 1 .mu.M,
respectively, as measured at 24 h (Table 1). Apilimod bis-mesylate
was well tolerated under these conditions, with no visible adverse
effects. In contrast, when apilimod was administered by
intraperitoneal injection (30 mg/kg in 0.5%, methylcellulose in
water) the blood concentration was below the level of quantitation
at the 24 h time point.
[0104] Therefore, intravenous infusion of apilimod bis-mesylate to
humans in an appropriate formulation (highly water soluble) at an
appropriate rate, is expected provide potent Ebola virucidal
activity and may be useful in the acute, critical care setting.
TABLE-US-00001 TABLE 1 Comparison of Apilimod plasma concentrations
following I.P. injection, continuous S.C. Infusion, or both
simultaneously in Balb/C mice Dose (Bis-mesylate Route Vehicle
salt) I.P. I.P. I.P. Apilimod Pump (injec- Pump (injec- Pump
(injec- Ave. Plasma Group (S.C.) tion) (S.C.) tion) (S.C.) tion)
Conc. (.mu.M) 1 DRW MC -- -- -- 2 -- -- MC -- 30 mg/kg BQL 3 DRW MC
~15 mg/kg/d 30 mg/kg 0.682 4 DRW MC ~30 mg/kg/d 30 mg/kg 1.01 5 DRW
DRD -- 30 mg/kg -- 6 -- -- DRD -- 30 mg/kg BQL 7 DRW DRD ~15
mg/kg/d 30 mg/kg 0.613 8 DRW DRD ~30 mg/kg/d 30 mg/kg 1.76 9 -- DRW
-- ~15 mg/kg/d -- 0.755 10 -- DRW -- ~30 mg/kg/d -- 1.87
[0105] The following study protocol was carried out to obtain the
results discussed above:
Study Duration
[0106] Dosing: Days 1 to 6, PK determination: Day 7
Formulations
[0107] DRW: 25% DMSO, 25% Cremophor RH40, 50% Sterile water.
[0108] MC: 0.5% methylcellulose in water.
[0109] DRD: 10% DMSO, 13.5% Cremophor RH40, 76.5% 5% dextrose in
water.
Alzet Mini-Pumps
[0110] 1007D Alzet pump used to dose .about.15 mg/kg/d (Reservoir
volume=100 .mu.L)
[0111] 2001 Alzet pump used to dose .about.30 mg/kg/d (Reservoir
volume=200 .mu.L)
[0112] Drug concentration is 25 mg/mL in both pumps
Dosing Volume
[0113] 10 mL/kg for I.P. injection
[0114] 0.5 .mu.L/h for 1007D pump
[0115] 1.0 .mu.L/h for 2001 pump
Dose Groups
[0116] MC formulation with I.P. injection was used in mice in
Groups 1 to 4.
[0117] DRD formulation with I.P. injection was used in mice in
Groups 5 to 8. DRW formulation was administered by Alzet mini-pump
in all groups except 2 and 6. Group 1 and 5 are DRW formulation
controls (i.e. no drug).
[0118] All groups received I.P. drug or I.P. control except groups
9 and 10--the latter are the mini-pump infusion ONLY groups.
Conclusions
[0119] 1.) S.C. continuous mini-pump infusion of apilimod
bis-mesylate, with (Groups 3 and 4, Groups 7 and 8) or without
(Groups 9 and 10) concomitant I.P. injection, delivers
therapeutically relevant steady state plasma concentrations of
apilimod in Balb/c mice (based on Day 7 data).
[0120] 2.)Steady state plasma concentration is roughly proportional
to infusion rate. Since the drug concentration is the same (25
mg/mL) for both infusion rates, steady state plasma concentration
is roughly proportional to dose (Group 3 vs 4, Group 7 vs 8, Group
9 vs 10).
[0121] 3.) I.P. injection alone fails to provide measurable plasma
concentration of apilimod 24 h after the final dose, irrespective
of formulation (Groups 2 and 6).
[0122] 4.) S.C. continuous mini-pump infusion alone is sufficient
to provide therapeutically relevant steady state plasma
concentrations of apilimod.
Example 4: In vitro Efficacy Against Ebola Proliferation
[0123] There are five species in the genus Ebolavirus: zaire
Ebolavirus (ZEBOV), sudan Ebolavirus (SUDV), reston Ebolavirus
(RESTV), tai forest Ebolavirus (TAFV) and bundibugyo Ebolavirus
(BDBV) (Olszanecki R, 2014). These EBOV species have been sequenced
including the ZEBOV strain Kikwit, revealing RNA genome encodes
genes for viral proteins: nucleoprotein (NP), glycoprotein, RNA
dependent RNA polymerase (L) and matrix proteins (VP24, 68 VP30,
VP35 and VP40). A number of the ZEBOV genes are used as surrogate
markers of viral load and propagation. For the following in vitro
study, the VP30 gene was used.
Methods
[0124] HepG2 cells were infected at a multiplicity of infection
(MOI) 0.1 with the virus ZEBOV-Kikwit, rocking every 15 minutes at
37 degrees Celsius for one hour. The inoculum was removed and cells
washed 4 times with media and replaced with 10% FBS EMEM alone or
10% FBS EMEM with 6.times.5-fold dilutions of LAM-002 starting at
500 nanomolar (nM), e.g. 500, 100, 20, 4, 0.8, and 0.16. Samples
for quantitative reverse transcription polymerase chain reaction
(qRT-PCR) and plaque assay were collected at 48 and 72 hours
post-infection.
[0125] RNA was isolated from supernatants utilizing the Viral RNA
mini-kit (Qiagen) using 100 .mu.L of supernatant into 600 .mu.L of
buffer AVL. Primers/probe targeting the VP30 gene of EBOV were used
for quantitative real-time PCR (qRT-PCR) with the probe used here
being 6-carboxyfluorescein (6FAM)-5'CCG TCA ATC AAG GAG CGC CTC
3'-6 carboxytetramethylrhodamine (TAMRA) for the Kikwit in vitro
studies (Life Technologies). EBOV RNA was detected using the CFX96
detection system (BioRad Laboratories) in One-step probe qRT-PCR
kits (Qiagen) with the following cycle conditions: 50.degree. C.
for 10 minutes, 95.degree. C. for 10 seconds, and 40 cycles of
95.degree. C. for 10 seconds and 59.degree. C. for 30 seconds.
Threshold cycle (CT) values representing EBOV genomes were analyzed
with CFX Manager Software, and data are shown as genome equivalents
(GEq). To create the GEq standard, RNA from EBOV stocks was
extracted and the number of EBOV genomes was calculated using
Avogadro's number and the molecular weight of the EBOV genome.
[0126] Virus titration was performed by plaque assay with Vero E6
cells from all supernatants. Briefly, increasing 10-fold dilutions
of the samples were adsorbed to Vero E6 monolayers in duplicate
wells (200 .mu.L); the limit of detection was 25 pfu/mL.
Results
[0127] Results of a representative experiment are shown in two
formats in FIG. 1A and FIG. 1B. FIG. 4A shows a bar graph of the
results of Log10 total copies of virus per treatment (Rx) vs
time-for the qRT-PCR on HepG2 cells for the time-points
post-infection with ZEBOV-Kikwit: 1 hour, 48 hours and 72 hours.
FIG. 4B shows bar graph of the FIG. 1A results Log10 total copies
reduced per Rx as compared to EBOV mock vs time. After one hour of
treatment no change in ZEBOV concentration level. After 48 hours
treatment with LAM-002 (apilimod dimesylate) at 20 nM, 100 nm, and
500 nM; the ZEBOV virus propagation was lowered by 0.5, 1.0 and 1.0
log10 number of virus copies, respectively. After 72 hours of
treatment with LAM-002 at 20 nM, 100 nm, and 500 nM; the ZEBOV
virus propagation was again lowered by the values of 0.5, 1.0 and
2.0 log10 number of virus copies, respectively.
[0128] FIG. 4C shows the CellTiter-Glo Luminescent Cell Viability
Assay of LAM-002 treatment of HepG2 cell line for 72 hours at
concentration range of 10,000 nM to 19 nM at two fold dilutions,
performed according to manufacturer's instructions (Promega). The
viability of the HepG2 cells is greater than 73% at 625 nM, 87% at
325 nm, 100% at 156 nM and lower concentrations of LAM-002. The
data of viability over range of LAM-002 verifies that the
observations of lower virus levels were not due to decreased cell
number or cell toxicity.
[0129] The experimental data show a LAM-002 dose dependent decrease
of ZEBOV levels and provide evidence of the in vitro efficacy of
LAM-002 as a small molecule therapy to reduce Ebola virus
propagation and viral load.
* * * * *