U.S. patent application number 17/031868 was filed with the patent office on 2021-04-22 for methods of treating sickle cell disease and related disorders using fumaric acid esters.
This patent application is currently assigned to Augusta University Research Institute, Inc.. The applicant listed for this patent is Augusta University Research Institute, Inc.. Invention is credited to Vadivel Ganapathy, Pamela M. Martin.
Application Number | 20210113512 17/031868 |
Document ID | / |
Family ID | 1000005307306 |
Filed Date | 2021-04-22 |
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United States Patent
Application |
20210113512 |
Kind Code |
A1 |
Ganapathy; Vadivel ; et
al. |
April 22, 2021 |
Methods of Treating Sickle Cell Disease and Related Disorders Using
Fumaric Acid Esters
Abstract
Methods of using one or more fumaric acid esters or
pharmacologically active salts, derivatives, analogues, or prodrugs
thereof to increase expression of fetal hemoglobin (HbF) are
disclosed. The methods typically include administering to a subject
an effective amount of one or more fumaric acid esters optionally
in combination or alternation with hydroxyurea to induce HbF
expression in the subject in an effective amount to reduce one or
more symptoms of a sickle cell disorder, a hemoglobinopathy, or a
beta-thalassemia, or to compensate for a genetic mutation is the
human beta-globin gene (HBB) or an expression control sequence
thereof. Pharmaceutical dosage units and dosage regimes for use in
the disclosed methods are also provided.
Inventors: |
Ganapathy; Vadivel;
(Martinez, GA) ; Martin; Pamela M.; (Evans,
GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Augusta University Research Institute, Inc. |
Augusta |
GA |
US |
|
|
Assignee: |
Augusta University Research
Institute, Inc.
Augusta
GA
|
Family ID: |
1000005307306 |
Appl. No.: |
17/031868 |
Filed: |
September 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15666393 |
Aug 1, 2017 |
10813905 |
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17031868 |
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14105893 |
Dec 13, 2013 |
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15666393 |
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61737360 |
Dec 14, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/225 20130101;
A61K 31/17 20130101 |
International
Class: |
A61K 31/225 20060101
A61K031/225; A61K 31/17 20060101 A61K031/17 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
Agreement NEI EY018053 awarded the National Institutes of Health.
The government has certain rights in the invention.
Claims
1-15. (canceled)
16. A method for treating a hemoglobinopathy, a sickle cell-related
disorder, or a beta thalassemia in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a prodrug of monomethylfumarate.
17. The method of claim 16, which is a method for treating a
hemoglobinopathy.
18. The method of claim 17, wherein the hemoglobinopathy is a
sickle cell disorder.
19. The method of claim 18, wherein the sickle cell disorder is
sickle cell anemia.
20. The method of claim 16, which is a method for treating a beta
thalassemia.
21. The method of claim 16, which is a method for treating a sickle
cell-related disorder.
22. The method of claim 21, wherein the sickle cell-related
disorder is a retinopathy.
23. The method of claim 16, wherein the method further comprises
administering hydroxyurea to the subject.
24. The method of claim 23, wherein the subject is unresponsive to
treatment with hydroxyurea alone.
25. The method of claim 24, wherein the subject expresses lower
levels of OCTN1 than patients who respond to hydroxyurea.
26. The method of any one of claims 16-25, wherein the
administering of the prodrug of monomethylfumarate is orally.
27. The method of claim 22, wherein the administering of the
prodrug of monomethylfumarate is locally to the eye.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Application No. 61/737,360, filed Dec. 14, 2012.
FIELD OF THE INVENTION
[0003] The field of the invention is generally related to
compositions including fumaric acid esters and methods of their use
for HbF (.gamma.-globin gene) induction.
BACKGROUND OF THE INVENTION
[0004] Sickle-cell disease (SCD), also known as sickle-cell anemia
(SCA) and drepanocytosis, is an autosomal recessive genetic blood
disorder caused by a point mutation in the .beta.-globin chain of
hemoglobin. SCD is characterized by red blood cells that adopt an
abnormal, rigid, sickle shape, referred to as "sickling" under
low-oxygen conditions. Repeated episodes of sickling can damage the
blood cell's membrane and decrease its elasticity. Sickled cells
can fail to return to normal shape when normal oxygen tension is
restored. As a consequence, these rigid blood cells are unable to
deform as they pass through narrow capillaries, leading to vessel
occlusion and ischemia. The actual anemia of the illness is caused
by hemolysis, the destruction of the red cells, caused by their
misshapes.
[0005] Normally, humans have Hemoglobin A, which consists of two
alpha and two beta chains, Hemoglobin A2, which consists of two
alpha and two delta chains and Hemoglobin F, consisting of two
alpha and two gamma chains in their bodies. Of these, Hemoglobin A
makes up around 96-97% of the normal hemoglobin in humans. Fetal
hemoglobin (also hemoglobin F or HbF) is the main oxygen transport
protein in the fetus during the last seven months of development in
the uterus and in the newborn until roughly six months old.
Functionally, fetal hemoglobin differs most from adult hemoglobin
in that it is able to bind oxygen with greater affinity than the
adult form, giving the developing fetus better access to oxygen
from the mother's bloodstream.
[0006] In newborns, fetal hemoglobin is nearly completely replaced
by adult hemoglobin by approximately six months postnatally.
However, HbF can be reactivated pharmacologically, an approach that
has been investigated as a treatment for symptoms and complications
of SCD.
[0007] Several classes of pharmacological agents that reactivate
.gamma.-globin gene transcription, thereby inducing HbF production,
have been identified. However, the S-stage cytotoxic drug
hydroxyurea (HU) is the first and at present only FDA-approved drug
for treatment of SCD. While HU has been shown to reduce
vaso-occlusive episodes and associated complications such as pain
and acute chest episodes in a large number of sickle-cell patients
treated, there are a number of limitations to using HU such as bone
marrow suppression, concerns over long-term carcinogenic
complications, and a 30% non-response rate.
[0008] Therefore, it is an object of the invention to provide
compositions and methods for treating subjects with one or more
mutations in the beta-globin gene (HBB), or an expression control
sequence thereof.
[0009] It is another object of the invention to provide
compositions and methods for treating subjects with sickle cell
disease, beta thalassemia, or variants or related diseases or
conditions thereof.
[0010] It is another object of the invention to provide
compositions and methods for reducing one or more symptoms of
sickle cell disease, beta thalassemia, or variants or related
diseases or conditions thereof.
[0011] It is a further object of the invention to provide
treatments for sickle cell disease with fewer, or less severe side
effects, greater efficacy, greater response rate, or combinations
thereof compared to existing therapies such as hydroxyurea.
SUMMARY OF THE INVENTION
[0012] Monomethylfumarate induces .gamma.-globin expression and
fetal hemoglobin production in human erythroid and retinal pigment
epithelial cells. Therefore, methods of treating sickle cell
disease (SCD) or complications of SCD include administering an
effective amount of one more fumaric acid esters or
pharmacologically active salts, derivatives, analogues, or prodrugs
thereof to induce or increase expression of fetal hemoglobin (HbF)
in a subject in need thereof are disclosed. Another method for
treating SCD or complications related to SCD includes administering
one or more fumaric acid esters in combination or alternation with
hydroxyurea (HU). In one aspect, the subject treated with the
combination of fumaric acid ester and HU is typically unresponsive
or does not respond well to HU treatment alone. Preferred subjects
for treatment with the combination of fumaric acid esters and HU
have reduced expression of OCTN1 relative to subjects that respond
well to HU treatment alone.
[0013] Methods for treating retinopathy due to SCD includes
administering one or more fumaric acid esters optionally in
combination with HU in an amount effective to increase HbF in
retinal pigment epithelial cells
[0014] Examples of suitable fumaric acid esters include, but are
not limited to monoethyl fumarate (MEF), monomethyl fumarate (MMF),
diethyl fumarate (DEF), and dimethyl fumarate (DMF). In a preferred
embodiment, the fumaric acid ester is MMF, DMF, or a combination
thereof.
[0015] The one or more fumaric acid esters or pharmacologically
active salts, derivatives, analogues, or prodrugs thereof are
administered to a subject in an effective amount to increase HbF in
the subject.
[0016] The one or more fumaric acid esters or pharmacologically
active salts, derivatives, analogues, or prodrugs thereof can also
be administered in an effective amount to increase HbF expression
in a subject in need thereof to reduce one or more symptoms of a
sickle cell disorder in the subject. The sickle cell disorder can
be a sickle cell disease such as sickle cell anemia. Typically, the
subject has at least one allele of sickle cell hemoglobin (HbS). In
some embodiments, the subject has one allele of HbS and one allele
of hemoglobin C (HbC), one allele of hemoglobin E (HbE), one allele
of .beta.-0 thalassemia, or one allele of .beta.+ thalassemia. In
some embodiments, the subject has two alleles of HbS.
[0017] The fumaric acid esters or pharmacologically active salts,
derivatives, analogues, or prodrugs thereof can be used in
combination or alternation with another therapeutic agent to treat
SCD or complications of SCD. For example the fumaric acid esters
can be combined with HU. The combination of fumaric acid esters
with HU can be formulated in a unit dose form. Thus, one embodiment
is a pharmaceutical composition comprising a fumaric acid ester and
HU, optionally including an excipient. An exemplary complication of
SCD that can be treated with the disclosed compositions includes
but is not limited to retinal complications.
[0018] The one or more fumaric acid esters or pharmacologically
active salts, derivatives, analogues, or prodrugs thereof can be
administered in an effective amount to increase HbF expression in a
subject in need thereof to reduce one or more symptoms of a
beta-thalassemia in the subject. The beta-thalassemia can be, for
example, thalassemia minor, thalassemia intermedia, and thalassemia
major.
[0019] In some embodiments, the one or more fumaric acid esters or
pharmacologically active salts, derivatives, analogues, or prodrugs
thereof is administered to a subject in an effective amount to
increase HbF expression in the subject in need thereof to
compensate for a mutation in the human beta-globin gene.
Compensating for a mutation in the human beta globin gene includes
inducing expression of HbF.
[0020] Methods of increasing HbF expression in hemoglobin
synthesizing cells are also disclosed. The methods typically
include contacting cells with an effective amount of a fumaric acid
ester, or pharmacologically active salt, derivative, analogue, or
prodrug thereof to increase HbF expression in the cells. In some
embodiments the cells are erythroid precursor cells. Alternatively,
the cells are non-erythroid cells such as macrophage, retinal
pigment cells, or alveolar epithelial cells.
[0021] The one or more fumaric acid esters or pharmacologically
active salts, derivatives, analogues, or prodrugs thereof can be in
a pharmaceutical composition. The dosage can be between 1 mg/kg to
about 50 mg/kg. The dosage can be between 0.1 g and 2.0 g per day.
The fumaric acid ester, or pharmacologically active salt,
derivative, analogue, or prodrug thereof can be administered as
part of a dosage regime. The dosage regime can include dose
escalation.
[0022] The current labeled dosing of hydroxyurea for sickle cell
disease calls for the administration of an initial dose of 15
mg/kg/day in the form of a single dose, with monitoring of the
patient's blood count every 2 weeks. If the blood counts are in an
acceptable range, the dose may be increased by 5 mg/kg/day every 12
weeks until the MTD of 35 mg/kg/day is reached. Pharmaceutical
compositions can contain 1 mg/kg to 50 mg/kg of fumaric acid ester,
preferably MMF, in combination with 1 mg/kg to 35 mg/kg of HU.
[0023] For example, a dosage regime for treatment of a sickle cell
disorder can include administering to a subject with a sickle cell
disorder a low dose of a fumaric acid ester, or pharmacologically
active salt, derivative, analogue, or prodrug thereof and
administering to the subject escalating doses of the fumaric acid
ester, or pharmacologically active salt, derivative, analogue, or
prodrug thereof until the dose is effective to reduce one or more
symptoms of the sickle cell disorder.
[0024] Some of the disclosed methods include administering to the
subject a second active agent, for example, vitamin supplements,
nutritional supplements, anti-anxiety medication, anti-depression
medication, anti-coagulants, clotting factors, anti-inflammatories,
steroids such as corticosteroids, analgesic, etc. In some
embodiments, the compositions are co-administered in combination
with one or more additional active agents for treatment of sickle
cell disease, beta-thalassemia, or a related disorder. Such
additional active agents may include, but are not limited to, folic
acid, penicillin or another antibiotics, preferably a quinolone or
macrolide, antivirals, anti-malarial prophylactics, and analgesics
to control pain crises. In some embodiments, the compositions are
co-administered with one or more additional agents that increase
expression of HbF, for example, hydroxyurea.
[0025] Methods of selecting a subject with a mutation in a
beta-globin gene for treatment are also disclosed. The methods
typically include genotyping the beta-globin gene and expression
control sequence thereof in DNA isolated from a biological sample
obtained from the subject; determining if the beta-globin gene or
expression control sequence includes a mutation; selecting the
subject for treatment if the beta-globin gene or expression control
sequence includes a mutation; and treating the subject with an
effective amount of one or more fumaric acid esters, or
pharmacologically active salts, derivatives, analogues, or prodrugs
thereof.
[0026] Still another method of treatment provides administering
fumaric acid esters in combination or alternation with HU to SCD
subjects that are unresponsive to HU treatment alone. For example,
methylmonofumaric acid ester can be administered to enhance the
update of HU in subjects that are typically unresponsive to HU.
Unresponsive to HU treatment means that the subject having SCD does
not expiring a significant therapeutic effect for treating their
SCD from HU treatment. The increase in uptake of HU can also be
accompanied by an increase in HIB expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a bar graph showing the change in .gamma.-globin
mRNA expression (fold change compared to untreated control ("UT"))
in KU812 cells over time (hours) following treatment with 100 .mu.M
monomethylfumarate (MMF) treatment. Data are represented as
means.+-.standard error of the mean (SEM); *P<0.05, **P<0.01
and ***P<0.001.
[0028] FIG. 2 is a bar graph showing the change in
.gamma./.beta.-globin mRNA expression (fold change compared to
untreated control ("UT")) in KU812 cells over time (hours)
following treatment with known fetal hemoglobin (HbF) inducers (10
mM Cysteine (Cys), 50 .mu.M Hemin, 2 mM sodium butyrate NaB, 2000
nM suberoylanilide hydroxamic acid (SAHA), 105 nM cyclic peptide
FK228 (depsipeptide), 100 .mu.M hydroxyurea (HU)). Reproduced from
Makala, et al., Anemia. 2012; 2012:428137. Epub 2012 May 14.
[0029] FIG. 3 is a bar graph showing the ratio of luciferase gene
expression (.gamma.) to .gamma.+2.times. renilla gene expression
(.gamma.+2.beta.) (fold change compared to untreated control
("UT")) in KU812 cells over time (hours) treated with increasing
doses of monomethylfumarate (MMF). The assayed KU812 cells express
a .mu.LCR.beta.prRluc.gamma.prFluc construct containing a 3.1-kb
.mu.LCR cassette linked to a 315-bp human .beta.-globin promoter
driving the renilla and a 1.4-kb A.gamma.-globin promoter driving
the firefly luciferase genes.
[0030] FIG. 4 is a bar graph showing the ratio of luciferase gene
expression (.gamma.) to .gamma.+2.times. renilla gene expression
(.gamma.+2.beta.) (fold change compared to untreated control
("UT")) in KU812 cells over time (hours) treated with increasing
doses of dimethylfumarate (DMF). The assayed KU812 cells express a
.mu.LCR.beta.prRluc.gamma.prFluc construct containing a 3.1-kb
.mu.LCR cassette linked to a 315-bp human .beta.-globin promoter
driving the renilla and a 1.4-kb A.gamma.-globin promoter driving
the firefly luciferase genes.
[0031] FIG. 5A is a photograph of a gel showing .gamma.-globin
expression in human RPE cells (ARPE-19 cell line) analyzed by
reverse transcriptase-polymerase chain reaction (RT-PCR) at various
time points after treatment with 100 .mu.M monomethylfumarate
(MMF). FIG. 5B is a bar graph showing .gamma.-globin expression in
human RPE cells (ARPE-19 cell line) analyzed by real-time
quantitative PCR (qPCR) at various time points after treatment with
100 .mu.M monomethylfumarate (MMF). Data are represented as
mean.+-.SEM; *P<0.05, **P<0.01.
[0032] FIG. 6 is a bar graph showing .gamma.-globin mRNA expression
in primary RPE cells (fold change) isolated from the eyes of
humanized mice analyzed by real-time quantitative PCR (qPCR)
following treatment with monomethylfumarate (MMF) at various
concentrations ranging from 0-1000 .mu.M for a period of 9 hours.
Data are represented as mean.+-.SEM; *P<0.01, **P<0.001.
[0033] FIGS. 7A-7E show the induction of .gamma.-globin gene
expression and HbF production by dimethylfumarate (DMF) and
monomethylfumarate (MMF) in erythroid cells. The dual luciferase
reporter KU812 stable cell line (1.times.10.sup.6 cells/assay) was
treated with varying concentrations (0-1000 .mu.M) of DMF 7A or MMF
7B for 48 h. Cells cultured in the absence of the drugs were
included as controls (UT, untreated). Firefly luciferase and
renilla luciferase activity was measured for .gamma.-globin and
.beta.-globin promoter activity, respectively. Trypan blue
exclusion was used to monitor cell viability. FIG. 7A is a bar
graph of .gamma./.gamma.+.beta. (Fold Change) versus DMF (.mu.M).
FIG. 7B is a bar graph of .gamma./.gamma.+.beta. (Fold Change)
versus DMF (.mu.M). FIG. 7C is a bar graph of .gamma./.beta. mRNA
level of primary human erythroid progenitors grown in liquid
culture treated with DMF, MMF, or HU (.mu.M). The level of
.gamma.-globin and .beta.-globin expression was normalized to GAPDH
before the .gamma./.beta. mRNA ratio was calculated. FIGS.
7D(1)-D(4) are line graphs of Relative cell counts versus log
fluorescence values for untreated cell (FIG. 7D(1), cells treated
with 100 .mu.M HU (FIG. 7D(2), cells treated with 200 .mu.M DMF
(Figure D(3), or cells treated with 1000 .mu.M MMF (FIG. 7D(4)).
FIG. 7E is a bar graph of FITC Positive Cells (%) in untreated
cells (UT, solid bar), cells treated with 200 .mu.M DMF, cells
treated with 1000 .mu.M MMF, and cells treated with 100 .mu.M HU.
Data were expressed also as the mean concentration of HbF per cell
measure.
[0034] FIGS. 8A-8F are bar graphs showing globin gene expression
and HbF production in human RPE cells. FIG. 8A shows qPCR analysis
of endogenous .alpha.-, .beta.- and .gamma.-globin gene expression
(Relative Expression) relative to that of hypoxanthine-guanine
phosphoribosyltransferase 1 (internal control) in the human RPE
cell line ARPE-19. FIG. 8B is a bar graph showing qPCR used to
evaluate .gamma.-globin mRNA expression (Fold Change) in control
(UT, untreated) and monomethylfumarate (MMF)-treated cells (1000
.mu.M; 6-24 h). FIG. 8C is a bar graph showing qPCR analysis of
endogenous .alpha.-, .beta.- and .gamma.-globin gene expression
(Relative Globin mRNA/HPRT) relative to that of
hypoxanthine-guanine phosphoribosyltransferase 1 (internal control)
in AA (solid bars) and SS (open bars) primary RPE cells. FIG. 8D is
a bar graph showing qPCR analysis of .gamma.-globin mRNA expression
((Fold Change) in control (UT, untreated), MMF-treated (1000 .mu.M)
or HU-treated (100 .mu.M; positive control) AA (solid bars) and SS
(open bars) primary RPE cells. FIG. 8E is a bar graph of induction
of HbF protein expression (FITC Positive Cells (%)) by the
indicated agents evaluated in AA (solid bars) and SS (open bars)
primary RPE cells by FACS using the FITC-conjugated
anti-.gamma.-globin antibody and, the number of FITC-positive cells
normalized to isotype controls expressed in graphical format. IMF
protein expression was confirmed by Western blot. MMF (1 mM final
concentration) or phosphate buffered saline (PBS; 0.01 M pH 7.4)
was injected intravitreally into the eyes of live AA and SS mice
(n=6); 24 h later, .gamma.-globin mRNA expression in RPE/eyecup and
HbF protein in intact retina was evaluated by OCR (FIG. 8F). FIG.
8G is a bar graph of .gamma.Globin mRNA Expression (Fold Change) in
AA and SS cells treated with 0.01 M PBS pH7.4 (solid bars) or 1 mM
final concentration of MMF (open bars).
[0035] FIG. 9 is a bar graph showing FACS analysis of HbF protein
expression. The graph shows Mean Fluorescence Intensity of primary
human erythroid progenitor cells treated with 200 .mu.M DMF, 1000
.mu.M MMF, or 100 .mu.M HU, (UT, untreated). ***p<0.001 compared
to untreated control.
[0036] FIG. 10 is a bar graph showing 13-Globin expression in AA
and SS primary RPE. The expression of human .beta.-globin mRNA
(Fold Change) relative to that of hypoxanthine guanine
phosphoribosyl transferase 1 (internal control) was analyzed by OCR
in AA (solid bar) and SS (open bar) primary RPE cells treated (24
h) with MMF (1000 .mu.M) or HU (100 .mu.M); UT, untreated control.
*p<0.05 compared to respective untreated control.
[0037] FIG. 11 is a bar graph showing the densitometeric analysis
of a Western Blot analysis of HbF protein expression in AA and SS
primary RPE. The graph is .gamma.-Globin/.beta.-Actin (Relative
Densitometry) in cells treated with MMF or HU. (UT, untreated).
[0038] FIG. 12 is a bar graph of OCTN1 mRNA Expression (Fold
Change) in KU812 cells treated with 1000 .mu.M MMF for 16 hours.
Control cells are identified as CON. Data are represented as
mean.+-.standard error of the mean; *p<0.05.
[0039] FIG. 13 is bar graph of OCTN1 mRNA Expression (Fold Change)
in ARPE-19 cells treated with 1000 .mu.M MMF, 100 .mu.m HU, or 1000
.mu.M MMF and 100 .mu.M HU.
[0040] FIG. 14 is a bar graph of mOCTN1 mRNA expression (Fold
Change) in AA or SS cells treated with 1000 .mu.M MMF.
[0041] FIG. 15 is a bar graph of OCTN1 mRNA expression (Fold
Change) in primary mouse RPE (pRPE), Muller (pMC) and ganglion
(pGC) cells.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0042] The term "expression control sequence" refers to a nucleic
acid sequence that controls and regulates the transcription and/or
translation of another nucleic acid sequence. Control sequences
that are suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and the
like. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.
[0043] The term "gene" refers to a DNA sequence that encodes
through its template or messenger RNA a sequence of amino acids
characteristic of a specific peptide, polypeptide, or protein. The
term "gene" also refers to a DNA sequence that encodes an RNA
product. The term gene as used herein with reference to genomic DNA
includes intervening, non-coding regions as well as regulatory
regions and can include 5' and 3' ends.
[0044] As generally used herein "pharmaceutically acceptable"
refers 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, organs, and/or bodily
fluids of human beings and animals without excessive toxicity,
irritation, allergic response, or other problems or complications
commensurate with a reasonable benefit/risk ratio.
[0045] The terms "subject," "individual," and "patient" refer to
any individual who is the target of treatment using the disclosed
compositions. The subject can be a vertebrate, for example, a
mammal. Thus, the subject can be a human. The subjects can be
symptomatic or asymptomatic. The term does not denote a particular
age or sex. Thus, adult and newborn subjects, whether male or
female, are intended to be covered. A subject can include a control
subject or a test subject. The test subject can be a subject
afflicted with a genetic mutation in the beta-globin gene or an
expression control sequence thereof, or a subject with a sickle
cell disorder, a globinopathy, or a beta-thalassemia.
[0046] As used herein, the term "treating" includes alleviating the
symptoms associated with a specific disorder or condition and/or
preventing or eliminating said symptoms.
[0047] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups,
alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted
alkyl groups.
[0048] In preferred embodiments, a straight chain or branched chain
alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30
for straight chains, C3-C30 for branched chains), preferably 20 or
fewer, more preferably 15 or fewer, most preferably 10 or fewer.
Likewise, preferred cycloalkyls have from 3-10 carbon atoms in
their ring structure, and more preferably have 5, 6, or 7 carbons
in the ring structure. The term "alkyl" (or "lower alkyl") as used
throughout the specification, examples, and claims is intended to
include both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl moieties having one or more
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents include, but are not
limited to, halogen, hydroxyl, carbonyl (such as a carboxyl,
alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a
thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,
phosphate, phosphonate, phosphinate, amino, amido, amidine, imine,
cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate,
sulfamoyl, ulfonamide, sulfonyl, heterocyclyl, aralkyl, or an
aromatic or heteroaromatic moiety.
[0049] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Throughout the
application, preferred alkyl groups are lower alkyls. In preferred
embodiments, a substituent designated herein as alkyl is a lower
alkyl.
[0050] It will be understood by those skilled in the art that the
moieties substituted on the hydrocarbon chain can themselves be
substituted, if appropriate. For instance, the substituents of a
substituted alkyl may include halogen, ulfonam, nitro, thiols,
amino, azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, ulfonamide, sulfamoyl
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF3, --CN and the like. Cycloalkyls can be substituted in the
same manner.
[0051] "Aryl", as used herein, refers to C5-C10-membered aromatic,
heterocyclic, fused aromatic, fused heterocyclic, biaromatic, or
bihetereocyclic ring systems. Broadly defined, "aryl", as used
herein, includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring
aromatic groups that may include from zero to four heteroatoms, for
example, benzene, pyrrole, furan, thiophene, imidazole, oxazole,
thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and
pyrimidine, and the like. Those aryl groups having heteroatoms in
the ring structure may also be referred to as "aryl heterocycles"
or "heteroaromatics". The aromatic ring can be substituted at one
or more ring positions with one or more substituents including, but
not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ulfonamide, ketone,
aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,
--CF3, --CN; and combinations thereof.
[0052] The term "aryl" also includes polycyclic ring systems having
two or more cyclic rings in which two or more carbons are common to
two adjoining rings (i.e., "fused rings") wherein at least one of
the rings is aromatic, e.g., the other cyclic ring or rings can be
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
heterocycles. Examples of heterocyclic rings include, but are not
limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,
benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,
benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,
benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl,
chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,
4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,
pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl and xanthenyl. One or more of the
rings can be substituted as defined above for "aryl".
II. Methods of Treating Sickle Cell Disease, Beta-Thalassemias, and
Related Disorders
[0053] A. Treatment of SCD with Fumaric Acid Esters
[0054] Methods of increasing expression of HbF in cells by
contacting the cells, for example erythroid and RPE cells, with an
effective amount of a fumaric acid ester, or pharmacologically
active salt, derivative, analogue or prodrug thereof are disclosed.
The methods can be used to compensate for a mutation in the human
beta-globin gene in cells that have one or more mutations in the
beta-globin gene or an expression control sequence thereof, for
example mutations that result in the expression of the Hb S form of
hemaglobin. Compensating for the mutation includes but is not
limited to increasing the amount of HbF and reducing the amount of
Hb S in the subject compared to untreated subjects. The methods can
be used for treating sickle cell disease, for example sickle cell
anemia, and other hemoglobinopathies or thalassemias as well as
complications related to SCD, for example retinopathy.
[0055] B. Treatment of Fumaric Acid Esters in Combination or
Alternation with HU.
[0056] Methods for treating SCD or complications thereof include
administering fumaric acid esters in combination or alternation
with HU in amounts effective to induce or increase expression of
HbF and increase expression of OCTN1 in erythroid and retinal
cells. It has been discovered that MMF induce the expression of
SLC22A4 (aka OCTN1), a transporter shown recently to transport HU
(Walker A L et al. Exp. Hematol. 2011; 39(4):446-56). The
expression of OCTN1 and its induction by MMF is evident in retinal
and erythroid cells. MMF can increase the entry of HU into
erythroid progenitor cells and facilitate the action of HU on fetal
hemoglobin production. In some embodiments, MMF can reduce the
dosing of HU in SCD patients without compromising its therapeutic
efficacy, and reduce the toxic side effects associated with HU
therapy.
[0057] Subjects with SCD that are unresponsive to HU treatment can
be treated by administering a fumaric acid ester in combination or
alternation with HU. While MMF adjuvant therapy along with HU would
certainly benefit SCD patients who respond to HU, it also has
potential to work on those who do not respond to HU. In one
embodiment, the "non-responders" express lower levels of OCTN1 than
"responders." The decreased expression of the transporter would
result in decreased entry of HU into its target cells (erythroid
progenitors) and thus decrease its pharmacological effect. Since
MMF induces OCTN1, adjuvant therapy is likely to enhance the entry
of HU in `non-responders" and thereby make the patients to become
responsive to HU therapy. This is in addition to the effect of MMF
itself in increasing fetal hemoglobin production.
[0058] Fumaric acid esters have been used for greater than 50 years
in the treatment of psoriasis, and more recently multiple
sclerosis. It is believed that the beneficial effects of fumaric
acid esters in the treatment of these pathologic conditions are due
to their potent anti-inflammatory and anti-oxidant effects. It has
been discovered that in addition to the known, robust
anti-inflammatory and anti-oxidant properties, these compounds also
induce production of fetal hemoglobin (HbF) in cells. Reactivation
of HbF, which is typically absent or expressed only at low levels
in humans over six months of age, is considered a viable approach
for treating children and adults with sickle cell disease, and
other hemoglobinopathies and thalassemias. The methods disclosed
herein typically include administering a fumaric acid ester, or
pharmacologically active salt, derivative, analogue or prodrug
thereof to a subject in need thereof to increase expression of HbF
in the subject, to increase expression of OCTN1, or both.
[0059] C. Diseases to be Treated
[0060] The disclosed compositions can be used to treat subjects
with one or more mutations in the beta-globin gene (HBB gene).
Mutations in the beta globin gene can cause sickle cell disease,
beta thalassemia, or related diseases or conditions thereof. As
discussed in more detail below, mutations in the beta-globin gene
can be identified before or after manifestations of a disease's
clinical symptoms. The compositions can be administered to a
subject with one or more mutations in the beta-globin gene before
or after the onset of clinical symptoms. Therefore, in some
embodiments, the compositions are administered to a subject that
has been diagnosed with one or more mutations in the beta-globin
gene, but does not yet exhibit clinical symptoms. In some
embodiments, the compositions are administered to a subject that is
exhibiting one or more symptoms of a disease, condition, or
syndrome associated with, or caused by one or more mutations in the
beta-globin gene.
[0061] 1. Sickle Cell Disease
[0062] Sickle cell disease (SCD) typically arises from a mutation
substituting thymine for adenine in the sixth codon of the
beta-chain gene of hemoglobin (i.e., GAG to GTG of the HBB gene).
This mutation causes glutamate to valine substitution in position 6
of the Hb beta chain. The resulting Hb, referred to as HbS, has the
physical properties of forming polymers under deoxy conditions. SCD
is typically an autosomal recessive disorder. Therefore, in some
embodiments, the disclosed compositions and methods are used to
treated a subject homozygous for an autosomal recessive mutation in
beta-chain gene of hemoglobin (i.e., homozygous for sickle cell
hemoglobin (HbS)). Also referred to as HbSS disease or sickle cell
anemia (the most common form), subjects homozygote for the S globin
typically exhibit a severe or moderately severe phenotype and have
the shortest survival of the hemaglobinopathies.
[0063] Sickle cell trait or the carrier state is the heterozygous
form characterized by the presence of around 40% HbS, absence of
anemia, inability to concentrate urine (isosthenuria), and
hematuria. Under conditions leading to hypoxia, it may become a
pathologic risk factor. Accordingly, in some embodiments, the
disclosed compositions and methods are used to treat a subject
heterozygous for an autosomal recessive mutation in the beta-chain
gene of hemoglobin (i.e., heterozygous for HbS).
[0064] 2. Beta-Thalassemia Beta-thalassemias (.beta.-thalassemias)
are a group of inherited blood disorders caused by a variety of
mutational mechanisms that result in a reduction or absence of
synthesis of .beta.-globin and leading to accumulation of
aggregates of unpaired, insoluble .alpha.-chains that cause
ineffective erythropoiesis, accelerated red cell destruction, and
severe anemia. Subjects with beta-thalassemia exhibit variable
phenotypes ranging from severe anemia to clinically asymptomatic
individuals. The genetic mutations present in .beta. thalassemias
are diverse, and can be caused by a number of different mutations.
The mutations can involve a single base substitution or deletions
or inserts within, near or upstream of the .beta. globin gene. For
example, mutations occur in the promoter regions preceding the
beta-globin genes or cause production of abnormal splice
variants.
[0065] Examples of thalassemias include thalassemia minor,
thalassemia intermedia, and thalassemia major.
[0066] Thalassemia minor refers to thalassemia where only one of
beta-globin alleles bears a mutation. Individuals typically suffer
from microcytic anemia. Detection usually involves lower than
normal MCV value (<80 fL) plus an increase in fraction of
Hemoglobin A2 (>3.5%) and a decrease in fraction of Hemoglobin A
(<97.5%). Genotypes can be .beta.+/.beta. or
.beta.-0/.beta..
[0067] Thalassemia intermedia refers to a thalassemia intermediate
between the major and minor forms. Affected individuals can often
manage a normal life but may need occasional transfusions, e.g., at
times of illness or pregnancy, depending on the severity of their
anemia. Genotypes can be .beta.+/.beta.+ or .beta.-0/.beta..
[0068] Thalassemia major refers to a thalassemia where both
beta-globin alleles have thalassemia mutations. This is a severe
microcytic, hypochromic anemia. If left untreated, it causes
anemia, splenomegaly, and severe bone deformities and typically
leads to death before age 20. Treatment consists of periodic blood
transfusion; splenectomy if splenomegaly is present, and treatment
of transfusion-caused iron overload. Cure is possible by bone
marrow transplantation. Cooley's anemia is named after Thomas
Benton Cooley. Genotypes include .beta.+/.beta.-0 or
.beta.-0/.beta.-0 or .beta.+/.beta.+.
[0069] 3. Sickle Cell Related Disorders
[0070] Although carriers of sickle cell trait do not suffer from
SCD, individuals with one copy of HbS and one copy of a gene that
codes for another abnormal variant of hemoglobin, such as HbC or Hb
beta-thalassemia, have a less severe form of the disease. For
example, another specific defect in beta-globin causes another
structural variant, hemoglobin C (HbC). Hemoglobin C (abbreviated
as Hb C or HbC) is an abnormal hemoglobin in which substitution of
a glutamic acid residue with a lysine residue at the 6.sup.th
position of the .beta.-globin chain has occurred. A subject that is
a double heterozygote for HbS and HbC (HbSC disease) is typically
characterized by symptoms of moderate clinical severity.
[0071] Another common structural variant of beta-globin is
hemoglobin E or hemoglobin E (HbE). HbE is an abnormal hemoglobin
in which substitution of a glutamic acid residue with a lysine
residue at the 26.sup.th position of the .beta.-globin chain has
occurred. A subject that is a double heterozygote for HbS and HbE
has HbS/HbE syndrome, which usually causes a phenotype similar to
HbS/b+ thalassemia, discussed below.
[0072] Some mutations in the beta-globin gene can cause other
structural variations of hemoglobin or can cause a deficiency in
the amount of -globin being produced. These types of mutations are
referred to as beta-thalassemia mutations.
[0073] The absence of beta-globin is referred to as beta-zero
(.beta.-0) thalassemia. A subject that is a double heterozygote for
HbS and .beta.-0 thalassemia (i.e., HbS/.beta.-0 thalassemia) can
suffer symptoms clinically indistinguishable from sickle cell
anemia.
[0074] A reduced amount of beta-globin is referred to as
.beta.-plus (.beta.+) thalassemia. A subject that is a double
heterozygote for HbS and .beta.+ thalassemia (i.e., HbS/.beta.+
thalassemia) can have mild-to-moderate severity of clinical
symptoms with variability among different ethnicities.
[0075] Rare combinations of HbS with other abnormal hemoglobins
include HbD Los Angeles, G-Philadelphia, HbO Arab, and others.
[0076] Therefore, in some embodiments, the disclosed compositions
and methods are used to treating a subject with an HbS/.beta.-0
genotype, an HbS/.beta.+ genotype, an HBSC genotype, an HbS/HUE
genotype, an HbD Los Angeles genotype, a G-Philadelphia genotype,
or an abHbO Arab genotype.
[0077] As discussed above, retinopathy due to SCD can also be
treated by administering an effective amount of a fumaric acid
ester, for example MMF, optionally in combination or alternation
with HU in amounts effective to induce expression of HbF in retinal
cells, for example in RPE cells. Sickle retinopathy occurs when the
retinal blood vessels get occluded by sickle red blood cells and
the retina becomes ischemic, angiogenic factors are made in retina.
In sickle cell disease, this occurs mostly in the peripheral
retina, which does not obscure vision at first. Eventually, the
entire peripheral retina of the sickle cell patient becomes
occluded and many neovascular formations occur. Administration of
one or more fumaric acid esters optionally in combination with HU
can reduce or inhibit the formation of occlusions in the peripheral
retina of a sickle cell patient.
[0078] 4. Non-Erythroid Cell Related Disorders
[0079] Although red blood cells are the primary producers of
hemoglobin, reports indicate that other, non-hematopoietic cells,
including, but not limited to, macrophage, retinal pigment cells,
and alveolar epithelial cells such as alveolar type II (ATII) cells
and Clara cells which are the primary producers of pulmonary
surfactant, also synthesize hemoglobin (Newton, et al., J. Biol.
Chem., 281(9)5668-5676 (2006), Tezel, et al., Invest. Ophthalmol.
Vis. Sci., 50(4):1911-9 (2009), Liu, et al., Proc. Natl. Acad. Sci.
USA, 96(12)6643-6647 (1999)). These findings are consistent with
the conclusion that the expression of hemoglobin by non-erythroid
cells at interfaces where oxygen-carbon dioxide diffusion occurs
may be an adaptive mechanism to facilitate oxygen transport (Tezel,
et al., Invest. Ophthalmol. Vis. Sci., 50(4):1911-9 (2009).
[0080] Therefore, in some embodiments, the compositions disclosed
herein are used to increase HbF expression in non-erythroid cells
including, but not limited to, macrophage, retinal pigment cells,
and alveolar epithelial cells such as alveolar type II (ATII) cells
and Clara cells. In some embodiments, the compositions disclosed
herein are used to increase HbF expression in non-erythroid cells
at interfaces where oxygen-carbon dioxide diffusion occurs,
including, but not limited to the eyes and lungs. In some
embodiments, the compositions are used to induce, increase, or
enhance hemoglobin synthesis retinal pigment cells in an effective
amount to prevent, reduce, or alleviate one or more symptoms of
age-related macular degeneration or diabetic retinopathy.
[0081] D. Symptoms of Sickle Cell Disease, Beta-Thalassemias, and
Related Disorders
[0082] In some embodiments, the compositions disclosed herein are
administered to a subject in an effective amount to treatment one
or more symptoms of sickle cell disease, a beta-thalassemia, or a
related disorder.
[0083] Beta-thalassemia can include symptoms such as anemia,
fatigue and weakness, pale skin or jaundice (yellowing of the
skin), protruding abdomen with enlarged spleen and liver, dark
urine, abnormal facial bones and poor growth, and poor
appetite.
[0084] In subjects with sickle cell disease, or a related disorder,
physiological changes in RBCs can result in a disease with the
following signs: (1) hemolytic anemia; (2) vaso-occlusive crisis;
and (3) multiple organ damage from microinfarcts, including heart,
skeleton, spleen, and central nervous system.
[0085] Chronic Hemolytic Anemia
[0086] SCD is a form of hemolytic anemia, with red cell survival of
around 10-20 days. Approximately one third of the hemolysis occurs
intravascularly, releasing free hemoglobin (plasma free hemoglobin
[PFH]) and arginase into plasma. PFH has been associated with
endothelial injury including scavenging nitric oxide (NO),
proinflammatory stress, and coagulopathy, resulting in vasomotor
instability and proliferative vasculopathy. A hallmark of this
proliferative vasculopathy is the development of pulmonary
hypertension in adulthood.
[0087] Vaso-Occlusive Crisis
[0088] Vaso-occlusive crisis occurs when the circulation of blood
vessels is obstructed by sickled red blood cells, causing ischemic
injuries. The most common complaint is of pain, and recurrent
episodes may cause irreversible organ damage. One of the most
severe forms is the acute chest syndrome which occurs as a result
of infarction of the lung parenchyma. Vaso-occlusive crisis can be
accompanied by a pain crisis which can occur suddenly and last
several hours to several days.
[0089] The pain can affect any body part. It often involves the
abdomen, bones, joints, and soft tissue, and it may present as
dactylitis (bilateral painful and swollen hands and/or feet in
children), acute joint necrosis or avascular necrosis, or acute
abdomen. With repeated episodes in the spleen, infarctions and
autosplenectomy predisposing to life-threatening infection are
usual. The liver also may infarct and progress to failure with
time. Papillary necrosis is a common renal manifestation of
vaso-occlusion, leading to isosthenuria (i.e, inability to
concentrate urine).
[0090] Severe deep pain is present in the extremities, involving
long bones. Abdominal pain can be severe, resembling acute abdomen;
it may result from referred pain from other sites or
intra-abdominal solid organ or soft tissue infarction. Reactive
ileus leads to intestinal distention and pain.
[0091] Bone pain and abdominal pain may be present. The face also
may be involved. Pain may be accompanied by fever, malaise, and
leukocytosis.
[0092] Skeletal Manifestations
[0093] Skeletal manifestations include, but are not limited to,
infarction of bone and bone marrow, compensatory bone marrow
hyperplasia, secondary osteomyelitis, secondary growth defects,
intravascular thrombosis, osteonecrosis (avascular necrosis/aseptic
necrosis), degenerative bone and joint destruction, osteolysis (in
acute infarction), Articular disintegration, myelosclerosis,
periosteal reaction (unusual in the adult), H vertebrae (steplike
endplate depression also known as the Reynold sign or codfish
vertebrae), Dystrophic medullary calcification, bone-within-bone
appearance, decreased density of the skull, decreased thickness of
outer table of skull due to widening of diploe, hair on-end
striations of the calvaria, osteoporosis sometimes leading to
biconcave vertebrae, coarsening of trabeculae in long and flat
bones, and pathologic fractures, bone shortening (premature
epiphyseal fusion), epiphyseal deformity with cupped metaphysis,
peg-in-hole defect of distal femur, and decreased height of
vertebrae (short stature and kyphoscoliosis).
[0094] Renal Manifestations
[0095] Renal manifestations include, but are not limited to,
various functional abnormalities such as hematuria, proximal tubule
dysfunction, impaired potassium excretion, and hyperkalemia; and
gross anatomic alterations, for example, hypertrophied kidneys,
with a characteristic smooth, capsular surface.
[0096] Splenic Manifestations
[0097] Splenic manifestations include, but are not limited to,
enlargement, including rapid and/or painful enlargement known as
splenic sequestration crisis, infarction, low pH and low oxygen
tension in the sinusoids and splenic cords, functional impairment,
autosplenectomy (fibrosis and shrinking of the spleen in advanced
cases), immune deficiency and increased risk of sepsis.
[0098] Other Common Symptoms
[0099] Lower serum immunoglobulin M (1 gM) levels, impaired
opsonization, and sluggish alternative complement pathway
activation, increase susceptibility to infection pneumonia,
bronchitis, cholecystitis, pyelonephritis, cystitis, osteomyelitis,
meningitis, and sepsis and other challenges from infectious agents
including, but not limited to, Mycoplasma pneumoniae, Salmonella
typhimurium, Staphylococcus aureus, and Escherichia coli; growth
delays or maturation delays during puberty in adolescents,
hand-foot syndrome, acute chest syndrome, stroke, hemiparesis,
hemosiderin deposition in the myocardium, dilation of both
ventricles and the left atrium, cholelithiasis, paraorbital facial
infarction, retinal vascular changes, proliferative retinitis, loss
of vision, leg ulcers, priapism, avascular necrosis, and pulmonary
hypertension.
III. Compositions for Use in Treating Sickle Cell Disease,
Beta-Thalassemia, or Related Disorders
[0100] A. Active Agents
[0101] 1. Fumaric Acid Esters
[0102] The methods disclosed herein typically include administering
a subject in need thereof one or more fumaric acid esters or
pharmacologically active salts, derivatives, analogues or prodrugs
thereof. In preferred embodiments, the one or more fumaric acid
esters, pharmacologically active salts, derivatives, analogues, or
prodrugs thereof are part of pharmaceutical compositions which can
include a pharmaceutically acceptable carrier. Fumaric acid esters
(FAE) are agents derived from the unsaturated dicarbonic acid,
fumaric acid. Fumaric acid is a white crystalline powder with a
characteristic acidic taste that is commonly used as a food
additive and flavoring agent in cakes and sweets. Fumaric acid is
poorly absorbed and believed to pass through the body without
causing any effects. However, esters of fumaric acid (FAEs) are
potent chemicals and recognized for their ability to treat clinical
symptoms of psoriasis and multiple sclerosis.
[0103] In one embodiment, the fumaric acid ester has the following
formula:
##STR00001##
[0104] wherein R and R' are independently selected from the group
consisting of hydrogen or substituted or unsubstituted alkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, alkenyl, heteroalkenyl,
cycloalkenyl, heterocycloalkenyl, aryl, and heteroaryl, with the
provision that R and R' are not both hydrogen.
[0105] In some embodiments, one or both of R and R' are lower alkyl
(e.g., C.sub.1-C.sub.4), such as substituted or unsubstituted
methyl or ethyl. Exemplary FAEs include, but are not limited to,
monoethyl fumarate (MEF), monomethyl fumarate (MMF), diethyl
fumarate (DEF), dimethyl fumarate (DMF), as well as
pharmacologically active salts, derivatives, analogues, or prodrugs
thereof.
[0106] Relationships between the physicochemical properties of the
fumaric acid esters, including their presystemic metabolism and
intestinal absorption, are known in the art. See, for example,
Werdenberg, et al., Biopharm. Drug Dispos., 24(6):259-73 (2003),
which reports that the intestinal permeability of the monoesters
methyl hydrogen fumarate, ethyl hydrogen fumarate, n-propylhydrogen
fumarate and n-pentyl hydrogen fumarate increase with an increase
in their lipophilicity, however, their presystemic metabolism rates
likewise increase with increasing ester chain length. Therefore, it
is believed that for fumarates, an increase in intestinal
permeability of the more lipophilic derivatives is counterbalanced
by an increase in first-pass extraction.
[0107] Additional studies on characterizing the intestinal
absorption of fumaric acid esters indicate that uncharged diester
dimethylfumarate displays a high presystemic metabolic liability
and high permeability in an in vitro small intestinal cell model
(Werdenberg, et al., Biopharm. Drug Dispos., 24(6):259-73 (2003)).
Results also show complete metabolism of DFM in the intestinal
tissue.
[0108] DMF is rapidly hydrolysed by esterases to the metabolite
MMF. Accordingly, in a preferred embodiment the fumaric acid ester
is DMF, MMF, or a combination thereof. In some embodiments, the
compositions also includes Monoethyl fumarate.
[0109] Formulations including dimethyl fumarate and ethyl hydrogen
fumarate have been used in the treatment of psoriasis for many
years. One family of such formulations are marketed under the
tradename FUMADERM. FUMADERM is in the form of tablets intended for
oral use and it is available in two different dosage strengths
(FUMADERM Initial and FUMADERM):
TABLE-US-00001 TABLE 1 Components and Quantitative Composition of
FUMADERM (U.S. patent application 2008/0004344) Fumaderm .RTM.
Initial Fumaderm .RTM. Dimethylfumarate 30 mg 120 mg
Ethylhydrogenfumarate, 67 mg 87 mg calcium salt
Ethylhydrogenfumarate, 5 mg 5 mg Magnesium salt
Etylhydrogenfumarate, 3 mg 3 mg Zinc salt
[0110] For the treatment of psoriasis, the two strengths are
typically applied in an individually based dose regimen starting
with FUMADERM Initial in an escalating dose, and then after e.g.,
three weeks of treatment switching to FUMADERM. Both FUMADERM
Initial and FUMADERM are enteric coated tablets. In some
embodiments, the composition used in the methods disclosed herein
includes FUMADERM Initial, FUMADERM, or a combination thereof.
[0111] Another marketed composition is FUMARAAT 120 which contains
120 mg of dimethylfumarate and 95 mg of calcium monoethylfumarate
(TioFarma, Oud-Beijerland, Netherlands). In the publication
(Litjens et al. Br. J. Clin. Pharmacol. 2004, vol. 58:4, pp.
429-432), the pharmacokinetic profile of FUMARAAT 120 was reported
in healthy subjects. The results show that a single oral dose of
FUMARAAT 120 is followed by a rise in serum monomethylfumarate
concentration and only negligible concentrations of
dimethylfumarate and fumaric acid is observed. The results indicate
that dimethylfumarate is rapidly hydrolyzed to monomethylfumarate
in an alkaline environment, but according to the authors not in an
acid environment. As the composition is enteric coated, it is
believed that the uptake of fumarate takes place mainly in the
small intestine. It is believed that dimethylfumarate is either
hydrolysed to the monoester before uptake due to an alkaline
environment or it is rapidly converted to monoester by esterases in
the circulation. In some embodiments, the composition used in the
methods disclosed herein includes FUMARAAT 120.
[0112] The study also shows that time to peak concentration
(T.sub.max) and peak concentration (C.sub.max) are subject to food
effect, i.e., T.sub.max is prolonged (mean for fasted conditions is
182 min, whereas for fed conditions mean is 361 min) [lag time is
90 min for fasted and 300 min for fed] and C.sub.max is decreased
(fasted: 0.84 mg/l, fed: 0.48 mg/l) by concomitant food-intake.
[0113] Another study, in healthy subjects with two tablets of
FUMADERM, revealed C.sub.max values (determined as monoethyl- or
monomethylfumarate) in a range from 1.0 to 2.4 .mu.g/ml and a
T.sub.max in a range of from 4.8 to 6.0 hours (Reddingius W. G.
Bioanalysis and Pharmacokinetics of Fumarates in Humans.
Dissertation ETH Zurich No. 12199 (1997)).
[0114] U.S. Published Application 2012/0165404, which is
specifically incorporated by reference herein in its entirety,
describes compositions referred to as BG00012, an orally available
formulation of dimethyl fumarate (DMF) which is in clinical
development for treatment of relapsing-remitting multiple sclerosis
(RRMS). U.S. Published Application 2012/0165404, describes that
some embodiments in which dimethyl fumarate is administered to a
patient the DMF is formulated in capsules containing enteric coated
microtablets referred to "BG-12" or "BG00012." The coating of the
tablets is composed of different layers. The first layer is a
methacrylic acid-methyl methacrylate copolymer/isopropyl alcohol
solution which isolates the tablet cores from potential hydrolysis
from the next applied water suspensions. Enteric coating of the
tablet is then conferred by an aqueous methacrylic acid-ethyl
acrylate copolymer suspension. In some embodiments, the composition
used in the methods disclosed herein includes BG00012. The complete
components and quantitative composition of the capsules are given
in Table 2.
TABLE-US-00002 TABLE 2 Components and Quantitative Composition of
BG00012, (U.S. patent application No. 2012/0165404) Amount/
Ingredients capsule Function Core Microtablets Active ingredients:
Dimethyl Fumarate* 120.00 mg active ingredient Excipients:
Croscarmellose sodium 15.00 mg disintegrant Microcrystalline
Cellulose 131.60 mg filler Magnesium stearate 5.00 mg lubricant
Talcum 19.80 mg glidant Silica colloidal anhydrous 2.60 mg glidant
Mass core microtablets 294.00 mg Coating Microtablets Excipients:
Triethyl Citrate** 7.60 mg plasticizer Methacrylic Acid-Methyl 5.50
mg film coating agent Methacrylate Copolymer (1:1) as Methacrylic
Acid-Methyl (44.00 mg) Methacrylate Copolymer (1:1) solution
12.5%** Simeticone (corresponding to 0.17 mg anti-foam agent
Simeticone Ph Eur) as Simeticone Emulsion USP** (0.53 mg) Talcum
micronised** 13.74 mg lubricant Methacrylic acid - Ethyl Acrylate
33.00 mg film coating agent Copolymer (1:1) as Methacrylic acid -
Ethyl (110.00 mg) Acrylate Copolymer (1:1) dispersion 30%** Mass
enteric coated microtablets 354.01 mg Mass of gelatin capsule 96.00
mg Mass of filled capsule 450.01 mg
[0115] In some embodiments, the fumaric acid ester is a prodrug of
a fumaric acid ester. Preliminary results from a Phase 1 clinical
trial in healthy adults designed to assess the pharmacokinetics
(PK), safety and tolerability of single doses of four different
oral formulations of a fumaric acid ester compound that is a
prodrug of monomethyl fumarate (MMF) referred to as XP23829 were
favorable (XenoPort Press Release, "XenoPort Reports Favorable
Metabolism and Pharmacokinetics of XP23829, a Novel Fumaric Acid
Ester, in Phase 1 Trial" (2012)). XP23829 is being developed for
the potential treatment of relapsing-remitting multiple sclerosis
(RRMS) and/or psoriasis. The trial showed that administration of
XP23829 resulted in the expected levels of MMF in the blood. The
four formulations produced different PK profiles of MMF, including
one formulation that could potentially be dosed two or three times
a day and at least one formulation that may be suitable for
once-.alpha.-day dosing. XP23829 was generally well-tolerated in
the trial. U.S. Patent Application Nos. 2012/0157523, 2012/0095003,
and 2010/00048651, each of which is incorporated by reference in
its entirety, discuss prodrugs of methyl hydrogen fumarate,
pharmaceutical compositions thereof and methods of use. In some
embodiments, the fumaric acid ester is a fumaric acid ester prodrug
such as XP23829, or pharmacologically active salt, an analogue or
derivative thereof.
[0116] Other suitable fumaric acid ester compounds, pharmaceutical
compositions, and formulations suitable for use in the disclosed
methods are known in the art. See for example, U.S. Pat. Nos.
6,509,376, 6,436,992, 6,277,882, 6,355,676, 6,509,376, 4,959,389,
and U.S. Patent Application No. 2008/0004344, each of which is
incorporated by reference in its entirety. U.S. Pat. Nos. 6,509,376
and 6,436,992 discuss formulations containing DMF and/or MMF. U.S.
Pat. Nos. 6,277,882 and 6,355,676 describe the use of alkyl
hydrogen fumarates and certain fumaric acid mono alkyl ester salts,
respectively, for preparing micro tablets for treating psoriasis,
psoriatic arthritis, neurodermatitis and enteritis regionalis. U.S.
Pat. No. 6,509,376 describes the use of certain dialkyl fumarates
pharmaceutical preparations for use in transplantation medicine or
the therapy of autoimmune diseases in the form of micro tablets or
pellets, U.S. Pat. No. 4,959,389, which describes compositions
containing different salts of fumaric acid monoalkyl ester alone or
in combination with dialkyl fumarate, and U.S. Patent Application
No. 2008/0004344, which describes salts of fumaric acid
monoalkylesters and their pharmaceutical use. The Case report
"Treatment of disseminated granuloma annulare with fumaric acid
esters" from BMC Dermatology, vol. 2, no. 5, 2002, relates to
treatment with fumaric acid esters.
[0117] 2. Co-Administration
[0118] The compositions disclosed herein can optionally include, or
be co-administered with one or more additional active agents.
Co-administration can include the simultaneous and/or sequential
administration of the one or more additional active agents and one
or more fumaric acid ester, or pharmacologically active salt,
derivative, analogue, or prodrug thereof. The one or more
additional active agents and the fumaric acid ester, or
pharmacologically active salt, derivative, analogue, or prodrug
thereof can be included in the same or different pharmaceutical
formulation. The one or more additional active agents and the
fumaric acid ester, or pharmacologically active salt, derivative,
analogue, or prodrug thereof can achieve the same or different
clinical benefit. An appropriate time course for sequential
administration may be chosen by the physician, according to such
factors as the nature of a patient's illness, and the patient's
condition. In certain embodiments, sequential administration
includes the co-administration of one or more additional active
agents and the nanoparticle gene carriers within a period of one
week, 72 hours, 48 hours, 24 hours, or 12 hours.
[0119] The additional active agent can be chosen by the user based
on the condition or disease to be treated. Example of additional
active agents include, but are not limited to, vitamin supplements,
nutritional supplements, anti-anxiety medication, anti-depression
medication, anti-coagulants, clotting factors, anti-inflammatories,
steroids such as corticosteroids, analgesic, etc.
[0120] In some embodiments, the compositions disclosed herein are
co-administered in combination with one or more additional active
agents for treatment of sickle cell disease, beta-thalassemia, or a
related disorder. Such additional active agents may include, but
are not limited to, folic acid, penicillin or another antibiotics,
preferably a quinolone or macrolide, antivirals, anti-malarial
prophylactics, and analgesics to control pain crises.
[0121] In some embodiments, the compositions are co-administered
with one or more additional agents that increase expression of HbF,
for example, hydroxyurea.
[0122] In some embodiments, the compositions are co-administered
with one or more additional treatment protocols, for example,
transfusion therapy, stem cell therapy, gene therapy, bone marrow
transplants, dialysis or kidney transplant for kidney disease,
gallbladder removal in people with gallstone disease, hip
replacement for avascular necrosis of the hip, surgery for eye
problems, and wound care for leg ulcers.
[0123] B. Effective Amounts
[0124] In some embodiments, the compositions are administered in an
amount effective to induce a pharmacological, physiological, or
molecular effect compared to a control that is not administered the
composition. In some embodiments, a fumaric acid ester, or
pharmacologically active salt, derivative, analogue, or prodrug
thereof is administered to a subject in need thereof to increase
expression of HbF in the subject. For example, HbF expression can
be increased in an amount effective to compensate for, or reduce
the effects of a mutation in the HBB gene. In some embodiments, the
fumaric acid ester, or pharmacologically active salt, derivative,
analogue, or prodrug thereof is administered in an effective amount
to reduce the sickling of red blood cells in a patient relative to
a control.
[0125] In some embodiments, the fumaric acid ester, or
pharmacologically active salt, derivative, analogue, or prodrug
thereof is provided in an effective amount to prevent, reduce or
alleviate one or more symptoms of a disease or disorder to be
treated. For example, the compositions disclosed herein can be
administered to a subject in need thereof in an effective amount to
reduce or alleviate one or more symptoms of sickle cell disease, a
beta-thalassemia, or a sickle cell related disorder, including, but
not limited to, the symptoms discussed above.
[0126] Suitable controls are known in the art and can be determined
based on the disease to be treated. Suitable controls include, but
are not limited to a subject, or subjects without sickle cell
disease, a beta-thalassemia, or a sickle cell related disorder; or
a condition or status of a subject with the disease or disorder
prior to initiation of the treatment. For example, in some
embodiments, treatment of a subject with a fumaric acid ester, or
pharmacologically active salt, derivative, analogue, or prodrug
thereof improves one or more pharmacological, physiological, or
molecular effects; reduces or alleviates one or more symptoms of
the disease or disorder to be treated; or a combination thereof
compared to a subject or subjects without the disease or disorder
to be treated. In some embodiments, treatment of a subject with a
fumaric acid ester, or pharmacologically active salt, derivative,
analogue, or prodrug thereof improves one or more pharmacological,
physiological, or molecular effects; reduces or alleviates one or
more symptoms of the disease or disorder to be treated; or a
combination thereof in the subject compared to the same
pharmacological, physiological, or molecular effects; or symptoms
of the disease or disorder in the subject prior to administration
of the fumaric acid ester, or pharmacologically active salt,
derivative, analogue, or prodrug thereof to the subject.
[0127] In some embodiments, the fumaric acid ester, or
pharmacologically active salt, derivative, analogue, or prodrug
thereof is administered to a subject in need thereof in an
effective amount to improve one or more pharmacological,
physiological, or molecular effects, or to reduce or alleviate one
or more symptoms of the disease or disorder with higher efficacy,
lower toxicity, or a combination thereof compared to a subject
treated with an different therapeutic agent such as hydroxyurea
(HU).
[0128] C. Dosages and Dosage Regimes
[0129] For all of the disclosed compounds, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age, and general health of the recipient, will be able to ascertain
proper dosing. The selected dosage depends upon the desired
therapeutic effect, on the route of administration, and on the
duration of the treatment desired. Generally dosage levels of 0.001
to 100 mg/kg of body weight daily are administered to mammals.
Generally, for intravenous injection or infusion, dosage may be
lower.
[0130] As discussed above some fumaric acid esters have been
administered to treat patients for psoriasis and multiple
sclerosis.
[0131] For example, an exploratory, prospective, open-label study
of fumaric acid esters (FAE, FUMADERM) was conducted in patients
with relapsing-remitting multiple sclerosis. The study consisted of
the following four phases: 6-week baseline, 18-week treatment
(target dose of 720 mg/day), 4-week washout, and a second 48-week
treatment phase (target dose of 360 mg/day) (Schimrigk, et al.,
Eur. J. Neurol., 13(6):604-10 (2006)). Following this dosage
regime, patients were stable or slightly improved for clinical
outcomes including Expanded Disability Status Scale (EDSS) score,
ambulation index (AI), and nine-hole peg test (9-HPT). The most
common adverse effects were gastrointestinal symptoms and flushing,
and all adverse effects were reported as mild and reversible.
[0132] In an exemplary treatment regimen of fumaric acid esters for
treatment of psoriasis includes a gradual increase in dosage
according to the schedule depicted in Table 3.
TABLE-US-00003 TABLE 3 Dosage schedule of fumaric acid esters used
for patients with psoriasis (Reproduced from Roll, et al., Indian
J. Dermatol. Venereol. Leprol., 73: 133-7 (2007)). Week Fumaderm
.RTM. initial Fumaderm .RTM. Dosage of DMF 1 1-0-0 30 mg 2 1-0-1 60
mg 3 1-1-1 90 mg 4 1-0-0 120 mg 5 1-0-1 240 mg 6 1-1-1 360 mg 7
2-1-1 480 mg 8 2-1-2 600 mg 9 2-2-2 720 mg
[0133] This schedule was shown to improve gastrointestinal
tolerance (Nast A, et al., J. German Soc. Dermatol., 4:51-5
(2006)). Most patients treated with fumaric acids require two to
four tablets of FUMADERM, for treatment of psoriasis.
[0134] Therefore, daily dosages for fumaric acid esters can range
from about 1 mg to about 5,000 mg, preferably about 10 mg to about
2,500 grams, more preferably about 50 mg to about 2,000 grams of a
fumaric acid ester, or a pharmacologically active salt, derivative,
analogue or prodrug thereof.
[0135] In some embodiments the compositions include DMF, MMF, or a
combination thereof. For DMF or MMF, the therapeutically effective
amount can range from about 1 mg/kg to about 50 mg/kg (e.g., from
about 2.5 mg/kg to about 20 mg/kg or from about 2.5 mg/kg to about
15 mg/kg). Effective doses will also vary, as recognized by those
skilled in the art, dependent on route of administration, excipient
usage, and the possibility of co-usage with other therapeutic
treatments including use of other therapeutic agents. For example,
an effective dose of DMF or MMF to be administered to a subject,
for example orally, can be from about 0.1 g to about 1 g or more
than 1 g per day; from about 200 mg to about 800 mg per day; from
about 240 mg to about 720 mg per day; from about 480 mg to about
720 mg per day; or about 720 mg per day. The daily dose can be
administered in separate administrations of 2, 3, 4, or 6 equal
doses.
[0136] In some embodiments of the one or more fumaric acid esters,
or pharmacologically active salts, derivatives, analogues or
prodrugs thereof are present in a pharmaceutical preparation. In
some embodiments the composition is administered to the patient
three times per day (TID). In some embodiments the pharmaceutical
preparation is administered to the patient two times per day (BID).
In some embodiments, the composition is administered at least one
hour before or after food is consumed by the patient.
[0137] In some embodiments, the composition is administered as part
of a dosing regimen. For example, the patient can be administered a
first dose of the composition for a first dosing period; and a
second dose of the composition for a second dosing period,
optionally followed by one or more additional doses for one or more
additional dosing periods. The first dosing period can be less than
one week, one week, or more than one week.
[0138] In some embodiments the dosage regime is a dose escalating
dosage regime. The first dose can be a low dose. For example, in
some embodiments, the composition includes DMF, and a low dose of
DMF, for example about 30 mg, can be the starting dose for a
dose-escalation protocol. Dose escalation can be continued until a
satisfactory biochemical or clinical response is reached. Next, the
dosages can be maintained or steadily reduced to a maintenance
dose. In some embodiments, the final dosage can be about 1-2 grams
per day (i.e., 6 tablets of FUMADERM).
[0139] Studies on the use of fumaric acid esters to treat psoriasis
show that dosage may not be related to body weight or to the
activity of the disease (Nast A, et al., J. German Soc. Dermatol.,
4:51-5 (2006)). Accordingly, the dosage and dosage regime for each
patient can be adjusted according to the individual's response and
the onset or severity of adverse effects.
[0140] The most common side effects are gastrointestinal symptoms
such as abdominal pain, diarrhea, nausea and malaise. These signs
and symptoms occur primarily within the first few weeks after
initiation of treatment and within 90 minutes to six hours after
oral intake of the drug. They last for several minutes up to half
an hour and can be alleviated by intake of tablets with milk.
[0141] Flushing of the skin is another common complaint, ranging
from rapid sensation of heat to long-lasting facial redness.
Improvement of the latter side effect has been seen on treatment
with acetylsalicylic acid but this has not yet been confirmed
scientifically. Typically, the adverse effects discussed above are
dose-dependent and they decrease in frequency during the course of
the treatment.
[0142] Less commonly observed side effects are lymphocytopenia,
leukocytopenia and elevated eosinophil counts. A decrease of
lymphocytes below 500/mm.sup.3 should lead to dosage reduction or
withdrawal of treatment. The eosinophilia is transient and usually
observed between the fourth and tenth week of treatment.
[0143] Rarely, moderate elevations of liver enzymes and bilirubin
have been observed. Proteinuria has been noted too, but it proved
to be transient. An increased risk for infections has not been
documented.
[0144] Relapse or rebound phenomena do not typically occur using
fumaric acid esters such as FUMADERM. Therefore, treatment may be
discontinued abruptly if needed.
[0145] The current labeled dosing of hydroxyurea for sickle cell
disease calls for the administration of an initial dose of 15
mg/kg/day in the form of a single dose, with monitoring of the
patient's blood count every 2 weeks. If the blood counts are in an
acceptable range, the dose may be increased by 5 mg/kg/day every 12
weeks until the MTD of 35 mg/kg/day is reached. Pharmaceutical
compositions can contain 1 mg/kg to 50 mg/kg of fumaric acid ester,
preferably MMF, in combination with 1 mg/kg to 35 mg/kg of HU. The
combination formulation can contain 5, 10, 15, 20, 25, 30, 35, 40,
45 or 50 mg/kg of HU.
[0146] D. Formulations
[0147] Pharmaceutical compositions including a fumaric acid ester,
or pharmacologically active salt, derivative, analogue, or prodrug
thereof are disclosed. The pharmaceutical compositions may be for
administration by oral, parenteral (intramuscular, intraperitoneal,
intravenous (IV) or subcutaneous injection), transdermal (either
passively or using iontophoresis or electroporation), or
transmucosal (nasal, vaginal, rectal, or sublingual) routes of
administration or using bioerodible inserts and can be formulated
in unit dosage forms appropriate for each route of
administration.
[0148] Red blood cells, which are cells of erythroid lineage, are
the primary producers of hemoglobin. Therefore, in a preferred
embodiment the fumaric acid esters are administered to a subject in
an effective amount to induce HbF in hematopoietic stems cells. In
the early fetus, erythropoiesis takes place in the mesodermal cells
of the yolk sac. By the third or fourth month, erythropoiesis moves
to the spleen and liver. After seven months, erythropoiesis occurs
primarily in the bone marrow, however, in certain disease states
erythropoiesis can also occurs outside the bone marrow, within the
spleen or liver, in adults. Therefore, in some embodiments, the
compositions are administered in an effective amount to induce HbF
expression in cells of erythroid lineage in the bone marrow (i.e.,
the red bone marrow), the liver, the spleen, or combinations
thereof.
[0149] Preferably the composition induces HbF in cells synthesizing
or committed to synthesize hemoglobin. For example, in preferred
embodiments, the fumaric acid ester, or pharmacologically active
salt, derivative, analogue, or prodrug thereof induces HbF in
basophilic normoblast/early normoblast also commonly called
erythroblast, polychromatophilic normoblast/intermediate
normoblast, orthochromatic normoblast/late normoblast, or a
combination thereof.
[0150] In a preferred embodiment, the composition is an oral
formulation. Oral formulations of DMF or MMF such as FUMADERM can
be absorbed by the small intestine where MMF can enter systemic
circulation.
[0151] In some embodiments, the composition is administered
locally, to the site in need of therapy. Although red blood cells
are the primary producers of hemoglobin, reports indicate that
other, non-hematopoietic cells, including macrophage, retinal
pigment cells, and alveolar epithelial cells such as alveolar type
H (ATII) cells and Clara cells which are the primary producers of
pulmonary surfactant, also synthesize hemoglobin (Newton, et al.,
J. Biol. Chem., 281(9)5668-5676 (2006), Tezel, et al., Invest.
Ophthalmol. Vis. Sci., 50(4):1911-9 (2009), Liu, et al., Proc.
Natl. Acad. Sci. USA, 96(12)6643-6647 (1999)). These findings are
consistent with the conclusion that the expression of hemoglobin by
non-erythroid cells at interfaces where oxygen-carbon dioxide
diffusion occurs may be an adaptive mechanism to facilitate oxygen
transport.
[0152] Therefore, in some embodiments, the composition is
administered locally to interfaces where oxygen-carbon dioxide
diffusion occurs, including but not limited, to the eye or
lungs.
[0153] In some embodiments, the composition is administered locally
to the eye to treat a retinopathy, or another ocular manifestation
associated with sickle cell disease, or a related disorder.
[0154] 1. Formulations for Enteral Administration
[0155] In a preferred embodiment the compositions are formulated
for oral delivery. Oral solid dosage forms are described generally
in Remington's Pharmaceutical Sciences, 18.sup.th Ed. 1990 (Mack
Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms
include tablets, capsules, pills, troches or lozenges, cachets,
pellets, powders, or granules or incorporation of the material into
particulate preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, etc., or into liposomes. Such compositions
may influence the physical state, stability, rate of in vivo
release, and rate of in vivo clearance of the disclosed. See, e.g.,
Remington's Pharmaceutical Sciences, 18.sup.th Ed. (1990, Mack
Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein
incorporated by reference. The compositions may be prepared in
liquid form, or may be in dried powder (e.g., lyophilized) form.
Liposomal or proteinoid encapsulation may be used to formulate the
compositions. Liposomal encapsulation may be used and the liposomes
may be derivatized with various polymers (e.g., U.S. Pat. No.
5,013,556). Sec also, Marshall, K. In: Modern Pharmaceutics Edited
by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the
formulation will include the peptide (or chemically modified forms
thereof) and inert ingredients which protect peptide in the stomach
environment, and release of the biologically active material in the
intestine.
[0156] The fumaric acid ester, or pharmacologically active salt,
derivative, analogue, or prodrug thereof may be chemically modified
so that oral delivery of the compound is efficacious. Generally,
the chemical modification contemplated is the attachment of at
least one moiety to the component molecule itself, where the moiety
permits uptake into the blood stream from the stomach or intestine,
or uptake directly into the intestinal mucosa. Also desired is the
increase in overall stability of the component or components and
increase in circulation time in the body. PEGylation is a preferred
chemical modification for pharmaceutical usage. Other moieties that
may be used include: propylene glycol, copolymers of ethylene
glycol and propylene glycol, carboxymethyl cellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, polyproline,
poly-1,3-dioxolane and poly-1,3,6-tioxocane [see, e.g., Abuchowski
and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as
Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York,
N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.
4:185-189].
[0157] Another embodiment provides liquid dosage forms for oral
administration, including pharmaceutically acceptable emulsions,
solutions, suspensions, and syrups, which may contain other
components including inert diluents; adjuvants such as wetting
agents, emulsifying and suspending agents; and sweetening,
flavoring, and perfuming agents.
[0158] Controlled release oral formulations may be desirable.
Fumaric acid esters, or pharmacologically active salt, derivatives,
analogues, or prodrugs thereof can be incorporated into an inert
matrix which permits release by either diffusion or leaching
mechanisms, e.g., gums. Slowly degenerating matrices may also be
incorporated into the formulation. Another form of a controlled
release is based on the Oros therapeutic system (Alza Corp.), i.e.,
the drug is enclosed in a semipermeable membrane which allows water
to enter and push drug out through a single small opening due to
osmotic effects.
[0159] For oral formulations, the location of release may be the
stomach, the small intestine (the duodenum, the jejunem, or the
ileum), or the large intestine. Preferably, the release will avoid
the deleterious effects of the stomach environment, either by
protection of the agent (or derivative) or by release of the agent
(or derivative) beyond the stomach environment, such as in the
intestine. To ensure full gastric resistance a coating impermeable
to at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D.TM., Aquateric.TM., cellulose acetate phthalate (CAP),
Eudragit L.TM., Eudragit S.TM., and Shellac.TM.. These coatings may
be used as mixed films.
[0160] 2. Topical or Mucosal Delivery Formulations
[0161] Compositions can be applied topically. The compositions can
be delivered to the lungs while inhaling and traverses across the
lung epithelial lining to the blood stream when delivered either as
an aerosol or spray dried particles having an aerodynamic diameter
of less than about 5 microns.
[0162] A wide range of mechanical devices designed for pulmonary
delivery of therapeutic products can be used, including but not
limited to, nebulizers, metered dose inhalers, and powder inhalers,
all of which are familiar to those skilled in the art. Some
specific examples of commercially available devices are the
Ultravent.TM. nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the
Acorn II.TM. nebulizer (Marquest Medical Products, Englewood,
Colo.); the Ventolin.TM. metered dose inhaler (Glaxo Inc., Research
Triangle Park, N.C.); and the Spinhaler.TM. powder inhaler (Fisons
Corp., Bedford, Mass.).
[0163] Formulations for administration to the mucosa will typically
be spray dried drug particles, which may be incorporated into a
tablet, gel, capsule, suspension or emulsion. Standard
pharmaceutical excipients are available from any formulator. Oral
formulations may be in the form of chewing gum, gel strips, tablets
or lozenges.
[0164] Transdermal formulations may also be prepared. These will
typically be ointments, lotions, sprays, or patches, all of which
can be prepared using standard technology. Transdermal formulations
will require the inclusion of penetration enhancers.
[0165] 3. Controlled Delivery Polymeric Matrices
[0166] Controlled release polymeric devices can be made for long
term release systemically following implantation of a polymeric
device (rod, cylinder, film, disk) or injection (microparticles).
The matrix can be in the form of microparticles such as
microspheres, where peptides are dispersed within a solid polymeric
matrix or microcapsules, where the core is of a different material
than the polymeric shell, and the peptide is dispersed or suspended
in the core, which may be liquid or solid in nature. Unless
specifically defined herein, microparticles, microspheres, and
microcapsules are used interchangeably. Alternatively, the polymer
may be cast as a thin slab or film, ranging from nanometers to four
centimeters, a powder produced by grinding or other standard
techniques, or even a gel such as a hydrogel.
[0167] Either non-biodegradable or biodegradable matrices can be
used for delivery of disclosed compounds, although biodegradable
matrices are preferred. These may be natural or synthetic polymers,
although synthetic polymers are preferred due to the better
characterization of degradation and release profiles. The polymer
is selected based on the period over which release is desired. In
some cases linear release may be most useful, although in others a
pulse release or "bulk release" may provide more effective results.
The polymer may be in the form of a hydrogel (typically in
absorbing up to about 90% by weight of water), and can optionally
be crosslinked with multivalent ions or polymers.
[0168] The matrices can be formed by solvent evaporation, spray
drying, solvent extraction and other methods known to those skilled
in the art. Bioerodible microspheres can be prepared using any of
the methods developed for making microspheres for drug delivery,
for example, as described by Mathiowitz and Langer, J. Controlled
Release 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers
6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci.
35:755-774 (1988).
[0169] The devices can be formulated for local release to treat the
area of implantation or injection--which will typically deliver a
dosage that is much less than the dosage for treatment of an entire
body--or systemic delivery. These can be implanted or injected
subcutaneously, into the muscle, fat, or swallowed.
IV. Methods of Diagnosis
[0170] The methods of treatment disclosed herein can include a
first step of selecting a subject for treatment. In some
embodiments, the subject is selected for treatment when the subject
exhibits one or more of the clinical symptoms of sickle cell
disease, beta-thalassemia, or a related disorder such as those
discussed above. In some embodiments, the subject is selected for
treatment when the subject exhibits a genetic or biochemical
indicator of sickle cell disease, beta-thalassemia, or a related
disorder. For example, the subject can be selected for treatment
based on identification of a genetic alteration, defect, or
mutation in the beta-globin gene or an expression control sequence
thereof, by biochemical or morphological alterations in hemoglobin
or hemoglobin synthesizing cells, or combinations thereof.
[0171] In some embodiments, the subject is selected when a
combination of clinical symptoms and genetic or biochemical
alterations are identified. In some embodiments, the subject is
selected based on one or more clinical symptoms, or one or more
genetic or biochemical alterations. For example, subjects can be
selected for treatment based on the identification of a genetic
alteration, a biochemical or morphological alteration, or a
combination thereof, before the subject exhibits clinical symptoms
of sickle cell disease, beta-thalassemia, or a related
disorder.
[0172] A. Identification of Genetic Alterations
[0173] In some embodiments, the subject is selected for treatment
based on identification of one or more genetic alterations in one
or more alleles of the human beta-globin gene or expression control
sequence thereof. Genetic alterations indicative of sickle cell
disease, beta-thalassemia, or related disorders include the
exemplary mutations discussed above, or other mutations that lead
to a reduction in the synthesis, structure, or function of human
beta-globin protein.
[0174] Methods of selecting a subject having one or more genetic
alterations in one or more alleles of the beta-globin gene or
expression control sequences thereof include the steps of obtaining
a biological sample containing nucleic acid from the subject and
detecting the presence or absence one or more genetic alterations
in one or more alleles of the beta-globin gene or expression
control sequences thereof in the biological sample. Any biological
sample that contains the DNA of the subject to be diagnosed can be
employed, including tissue samples and blood samples, with
nucleated blood cells being a particularly convenient source. The
DNA may be isolated from the biological sample prior to testing the
DNA for the presence or absence of the genetic alterations.
[0175] The detecting step can include determining whether the
subject is heterozygous or homozygous for a genetic alteration. The
step of detecting the presence or absence of the genetic alteration
can include the step of detecting the presence or absence of the
alteration in both chromosomes of the subject (i.e., detecting the
presence or absence of one or two alleles containing the marker or
functional polymorphism). More than one copy of a genetic
alterations (i.e., subjects homozygous for the genetic marker) can
indicate a greater risk of developing sickle cell disease,
beta-thalassemia, or related disorder. In some embodiments, the
subject is heterozygous for two or more genetic alterations in the
beta-globin gene (also referred to herein as double heterozygotes,
triple heterozygotes, etc.). One copy of two or more genetic
alterations in the beta-globin gene can indicate a greater risk of
developing sickle cell disease, beta-thalassemia, or related
disorder.
[0176] The process of determining the genetic sequence of human
beta-globin gene is referred to as genotyping. In some embodiments,
the human beta-globin gene is sequenced. Methods for amplifying DNA
fragments and sequencing them are well known in the art. For
example, automated sequencing procedures that can be utilized to
sequence the beta-globin gene, include, but not limited to,
sequencing by mass spectrometry single-molecule real-time
sequencing (Pacific Bio), ion semiconductor (ion torrent
sequencing), pyrosequencing (454), sequencing by synthesis
(Illumina), sequencing by ligation (SOLiD sequencing), chain
termination (Sanger sequencing).
[0177] In some embodiments, the genotype of the subject is
determined by identifying the presence of one or more single
nucleotide polymorphisms (SNP) associated with sickle cell disease,
beta-thalassemia, or a related disorder. Methods for SNP genotyping
are generally known in the art (Chen et al., Pharmacogenomics J.,
3(2):77-96 (2003); Kwok, et al., Curr. Issues Mol. Biol.,
5(2):43-60 (2003); Shi, Am. J. Pharmacogenomics, 2(3):197-205
(2002); and Kwok, Anna. Rev. Genomics Hum. Genet., 2:235-58
(2001)).
[0178] SNP genotyping can include the steps of collecting a
biological sample from a subject (e.g., sample of tissues, cells,
fluids, secretions, etc.), isolating gnomic DNA from the cells of
the sample, contacting the nucleic acids with one or more primers
which specifically hybridize to a region of the isolated nucleic
acid containing a target SNP under conditions such that
hybridization and amplification of the target nucleic acid region
occurs, and determining the nucleotide present at the SNP position
of interest, or, in some assays, detecting the presence or absence
of an amplification product (assays can be designed so that
hybridization and/or amplification will only occur if a particular
SNP allele is present or absent). In some assays, the size of the
amplification product is detected and compared to the length of a
control sample; for example, deletions and insertions can be
detected by a change in size of the amplified product compared to a
normal genotype.
[0179] The neighboring sequence can be used to design SNP detection
reagents such as oligonucleotide probes and primers. Common SNP
genotyping methods include, but are not limited to, TaqMan assays,
molecular beacon assays, nucleic acid arrays, allele-specific
primer extension, allele-specific PCR, arrayed primer extension,
homogeneous primer extension assays, primer extension with
detection by mass spectrometry, pyrosequencing, multiplex primer
extension sorted on genetic arrays, ligation with rolling circle
amplification, homogeneous ligation, multiplex ligation reaction
sorted on genetic arrays, restriction-fragment length polymorphism,
single base extension-tag assays, and the Invader assay. Such
methods may be used in combination with detection mechanisms such
as, for example, luminescence or chemiluminescence detection,
fluorescence detection, time-resolved fluorescence detection,
fluorescence resonance energy transfer, fluorescence polarization,
mass spectrometry, and electrical detection.
[0180] Other suitable methods for detecting polymorphisms include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science, 230:1242 (1985); Cotton, et al., PNAS, 85:4397 (1988); and
Saleeba, et al., Meth. Enzymol., 217:286-295 (1992)), comparison of
the electrophoretic mobility of variant and wild type nucleic acid
molecules (Orita et al., PNAS, 86:2766 (1989); Cotton, et al,
Mutat. Res., 285:125-144 (1993); and Hayashi, et al., Genet. Anal.
Tech. Appl., 9:73-79 (1992)), and assaying the movement of
polymorphic or wild-type fragments in polyacrylamide gels
containing a gradient of denaturant using denaturing gradient gel
electrophoresis (DGGE) (Myers et al., Nature, 313:495 (1985)).
Sequence variations at specific locations can also be assessed by
nuclease protection assays such as Rnasc and S1 protection or
chemical cleavage methods.
[0181] Another method for genotyping SNPs is the use of two
oligonucleotide probes in an oligonucleotide ligation assay (OLA)
(U.S. Pat. No. 4,988,617). In this method, one probe hybridizes to
a segment of a target nucleic acid with its 3'-most end aligned
with the SNP site. A second probe hybridizes to an adjacent segment
of the target nucleic acid molecule directly 3' to the first probe.
The two juxtaposed probes hybridize to the target nucleic acid
molecule, and are ligated in the presence of a linking agent such
as a ligase if there is perfect complementarity between the 3'-most
nucleotide of the first probe with the SNP site. If there is a
mismatch, ligation would not occur. After the reaction, the ligated
probes are separated from the target nucleic acid molecule, and
detected as indicators of the presence of a SNP.
[0182] Other methods that can be used to genotype the SNPs include
single-strand conformational polymorphism (SSCP), and denaturing
gradient gel electrophoresis (DGGE). SSCP identifies base
differences by alteration in electrophoretic migration of single
stranded PCR products. Single-stranded PCR products can be
generated by heating or otherwise denaturing double stranded PCR
products. Single-stranded nucleic acids may refold or form
secondary structures that are partially dependent on the base
sequence. The different electrophoretic mobilities of
single-stranded amplification products are related to base-sequence
differences at SNP positions. DGGE differentiates SNP alleles based
on the different sequence-dependent stabilities and melting
properties inherent in polymorphic DNA and the corresponding
differences in electrophoretic migration patterns in a denaturing
gradient gel.
[0183] Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can
also be used to score SNPs based on the development or loss of a
ribozyme cleavage site. Perfectly matched sequences can be
distinguished from mismatched sequences by nuclease cleavage
digestion assays or by differences in melting temperature. If the
SNP affects a restriction enzyme cleavage site, the SNP can be
identified by alterations in restriction enzyme digestion patterns,
and the corresponding changes in nucleic acid fragment lengths
determined by gel electrophoresis.
[0184] B. Identification of Biochemical and Morphological
Alterations
[0185] In some embodiments, subjects are selected for treatment
based on identification of biochemical or morphological alterations
or abnormalities in hemoglobin, or hemoglobin synthesizing cells
such as hematopoietic stem cells, erythrocyte progenitor cells,
erythrocytes, macrophage, retinal pigment epithelial cells,
alveolar type II (ATII) cells, and others. The methods typically
include identifying one or more biochemical or morphological
alterations that is associated with a genetic alteration in the
human beta-globin gene, or otherwise diagnostic of sickle cell
disease, a beta-thalassemia, or a related disorder. Methods of
diagnosing sickle cell disease, beta-thalassemia, or a related
disorder according to biochemical or morphological alterations in
the hemoglobin or hemoglobin synthesizing cells are known in the
art, and include but are not limited to, analysis of erythrocyte
morphology, osmotic fragility, hemoglobin composition, globin
synthesis rates, and red blood cell indices (Rowley, American
Journal of Hematology, 1(0:129-137, (1976)).
[0186] In some embodiments, the method includes testing a subject's
blood for HbS, and selecting the subject for treatment if HbS is
present. Methods for testing a subject's blood for the presence of
HbS include solubility tests (e.g., SICKLEDEX) and sickling test.
The SICKLEDEX test operates on the principle that Hb-S tends to
form tactoids or liquid crystals within the erythrocytes under
conditions of low oxygen tension resulting in the characteristic
"sickle shape" distortion of the red cell. A reducing agent (i.e.,
dithionite) is mixed with whole blood and buffer. If Hb-S is
present, it becomes insoluble and forms a cloudy suspension. Other
hemoglobins are more soluble and will form a transparent solution.
A sickling test can be used to determine if a red blood cell
changes into a sickle shape after a blood sample is mixed with a
reducing agent and identifying morphological changes to shape of
red blood cells (i.e., "sickling") by microscopy.
[0187] Other suitable tests include, hemoglobin electrophoresis,
which employs gel electrophoretic techniques to separate out the
various types of hemoglobin from a blood sample obtained from the
subject. The test can detect abnormal levels of HbS, as well as
other abnormal hemoglobins, such as hemoglobin C. It can also be
used to determine whether there is a deficiency of any normal form
of hemoglobin, as in various thalassemias. Alternatives to
electrophoretic techniques include isoelectric focusing and
chromatographic techniques.
[0188] Other tests that can be used to select a subject for
treatment with the compositions and methods disclosed herein
include tests typically employed as part of a hemoglobinopathy
screen, for example, a complete blood count (CBC) or iron study
(ferritin). For example, a blood count can be used to detect
anemia, and a blood smear and be used to identify sickled
cells.
EXAMPLES
Example 1: Monomethylfumarate (MMF) Induces .gamma.-Globin (Hbf)
Gene and Protein Expression in Cells of Erythroid Lineage
Materials and Methods
[0189] Pharmaceutical Agents
[0190] The fumaric acid esters dimethylfumarate (DMF) and
monomethylfumarate (MMF) are the primary constituents of Fumaderm
and BG00012, drugs currently marketed for treatment of psoriasis.
BG00012 is also completing phase III clinical testing for treatment
of multiple sclerosis. MMF is the major bioactive component of
each.
[0191] Cell Culture
[0192] KU812, a human leukemic cell line that expresses the fetal
.gamma.-globin and adult .beta.-globin genes, is a commonly used
system for screening and discovery of novel HbF inducers; this is
because of comparable globin gene response patterns in KU812 and
primary erythroid cells after treatments with drug inducers.
Results
[0193] KU812 cells were cultured in the presence or absence of MMF
for time periods ranging from 0-24 hours and evaluated for changes
in .gamma.-globin gene expression relative to 18S ribosomal RNA
expression (internal experimental control) by qPCR (FIG. 1). In
comparison to control, untreated (UT) cells, significant increases
in .gamma.-globin gene expression were observed as early as 3 hours
post-incubation with low-dose (100 .mu.M) MMF treatment. This
MMF-induced increase in .gamma.-globin expression persisted up to
24 h post-exposure to the compound. Data in FIG. 1 are represented
as means.+-.standard error of the mean (SEM); *P<0.05,
**P<0.01 and ***P<0.001.
[0194] FIG. 1 shows that 100 .mu.M MMF increases the expression of
.gamma.-globin gene expression in KU812 cells .about.2-4-fold
depending upon the incubation time. FIG. 2 shows the induction of
.gamma.-globin transcription by known HbF inducers in KU812 cells
cultured under conditions similar to those described above. FIG. 2
is reproduced from MaKala et al., Anemia, Volume 2012, Article ID
428137) (2012). The last bar of FIG. 2 shows data for hydroxyurea
(HU). At 100 .mu.M, the same concentration that we used for MMF, HU
induces .gamma.-globin gene expression .about.2-fold. This data
supports the conclusion that MMF is at least equivalent, or even
better than HU, in terms of its ability to induce .gamma.-globin
gene expression in this cell system.
Example 2: Monomethylfumarate (MMF) Drives Expression by Activation
the .gamma.-Globin (Hbf) Gene Promoter
Materials and Methods
From MaKala et al., Anemia, Volume 2012, Article ID 428137)
(2012)
[0195] KU812 Stable Lines
[0196] KU812 stable cell lines were created by co-transfecting
wild-type KU812 cells with pEGFP-NI (G418 selectable marker) and
the .mu.LCR.beta.prRluc A.gamma.prFluc dualreporter a kind gifts
from Dr. George Stamatoyannopoulos (University of Washington).
Briefly, the 315-bp human .beta.-globin gene promoter sequence was
inserted upstream of the Renilla along with a polyadenylation
signal downstream to create P.beta.prRluc. Likewise, 1.4 kb of
human A.gamma.-globin promoter was inserted upstream of firefly
luciferase to create A.gamma.prFluc. The .mu.LCR (locus control
region), P.beta.prRluc, and A.gamma.prFluc fragments were
subsequently cloned into the mammalian vector, pRL-null. The
dual-luciferase reporter lines were produced using 10 .mu.g each of
linearized .mu.LCR.beta.prRluc A.gamma.prFluc and pEGFP-NI plasmids
co-transfected into KU812 cells by electroporation at 260 V, 975
.mu.F (Bio-Rad, Hercules Calif.). After 72 hr, G418 was added at a
concentration of 900 .mu.g/.mu.l for 3 days then maintained under
selection pressure indefinitely at a concentration of 400
.mu.g/.mu.l. KU812 stable lines were treated with the various drugs
at the same concentrations described above. FK228 and analogues
were screened at concentrations between 1-1000 nM for 48 hr and
cell toxicity was monitored by 2% Trypan blue exclusion. The effect
of drug treatments on .gamma.-globin and 3-globin promoter activity
was monitored by luciferase assay.
[0197] Dual Luciferase Assay
[0198] Luciferase activity was monitored under the different
experimental conditions using the Dual Luciferase Assay Reporter
System (Promega, Madison, Wis.). The activity of firefly luciferase
represents .gamma.-globin promoter activity (.gamma.F), while the
renilla luciferase is the read-out for .beta.-globin promoter
activity (.beta.R). The .beta.-globin promoter was strategically
cloned between the LCR and .gamma.-globin promoter to increase
.beta. expression, while simultaneously increasing the sensitivity
of detection of .gamma.-globin gene inducers. After drug
treatments, KU812 stable cells were washed with 1.times. phosphate
buffered saline and lysed in 1.times. Passive Lysis Buffer for 15
min, then protein extracts were added to the Luciferase Assay
Reagent II and firefly luciferase activity quantified in a Turner
Designs TD-20/20 luminometer (Sunnyvale, Calif.). To measure
.beta.R activity, Stop & Glo Reagents was added to measure the
renilla luciferase activity. Total protein was determined by
Bradford assay on a Beckman DU 640 spectrophotometer (Chaska,
Minn.) and luciferase activity was corrected for total protein.
Results
[0199] The fumaric acid ester was also in the KU812 dual-luciferase
reporter system, an assay system using a stable KU812 cell line
created with the .mu.LCR.beta.prRluc.gamma.prFluc construct
containing a 3.1-kb .mu.LCR cassette linked to a 315-bp human
.beta.-globin promoter driving the renilla and a 1.4-kb
A.gamma.-globin promoter driving the firefly luciferase genes.
Since, the firefly luciferase gene (.gamma.F) has approximately 50%
greater luminescence than the renilla gene (.beta.R), renilla
activity was multiplied by two to adjust for the difference in
luminescence yielding the .gamma./.gamma.+2.beta. final
measurement. This assay system was previously reported as an
efficacious screening tool for the identification of .gamma.-globin
gene activators (MaKala et al., Anemia, Volume 2012, Article ID
428137) (2012)).
[0200] Luciferase activity, an indicator of .gamma.-globin promoter
induction, was monitored KU812
.mu.LCR.beta.prRluc.gamma.prFluc-stable cells cultured in the
presence or absence (untreated control, UT) of MMF at varying
dosages for 48 hour according to the method discussed above and in
MaKala et al., Anemia, Volume 2012, Article ID 428137) (2012). MMF
induced .gamma.-globin promoter activity in a dose-dependent manner
(FIG. 3). Cell viability by Trypan blue exclusion remained at
90-95% for the concentrations shown, indicating that even at higher
concentrations, MMF exhibits little or no cellular toxicity.
Example 3: Dimethylfumarate (DMF) Drives Expression by Activation
the .gamma.-Globin (Hbf) Gene Promoter
[0201] MMF is the primary bioactive metabolite derived from
metabolism of FUMADERM and BG00012, the fumaric acid ester drugs
presently used clinically, and DMF is the primary ingredient.
Therefore, the effectiveness of DMF was tested as an inducer of
.gamma.-globin gene expression under experimental conditions
identical to those described above in Example 2. As with MMF,
treatment of cells with DMF also induced .gamma.-globin promoter
activity significantly (FIG. 4). At concentrations in the 100-200
.mu.M range, induction of .gamma.-globin promoter activity
increased .about.2-10 fold in comparison to control, untreated (UT)
cells. However, at higher concentrations the effectiveness of DMF
as an inducer of .gamma.-globin promoter activity declined
substantially. Trypan blue exclusion revealed that this decrease
was likely due to an increase in cellular toxicity as indicated by
a decrease in cell viability.
Example 4: Monomethylfumarate (MMF) Induces .gamma.-Globin (Hbf)
Gene and Protein Expression in a Retinal Pigment Epithelial (RPE)
Cell Line
[0202] Red blood cells, cells of erythroid lineage, are the primary
producers of hemoglobin. However, recent reports suggest that
other, non-hematopoietic cells are capable of the same. This
includes retinal pigment epithelial (RPE) cells, a cell type
critical to normal visual function. Therefore, an assay was
designed to test the induction of .gamma.-globin gene expression by
MMF in ARPE-19, a transformed human RPE cell line commonly used as
an in vitro model of human RPE ARPE-19 cells were cultured in the
presence or absence or MMF (100 .mu.M) for varying periods of time
(0-24 h) and the expression of .gamma.-globin analyzed by reverse
transcriptase-polymerase chain reaction (RT-PCR) (FIG. 5A). A
time-dependent increase in .gamma.-globin gene expression was
observed in these cells when cultured in the presence of MMF.
[0203] The results presented in FIG. 5A were data were corroborated
using real-time quantitative PCR (qPCR) (FIG. 5B). Data are
represented as mean.+-.SEM; *P<0.05, **P<0.01.
[0204] Immunofluorescence localization techniques utilizing a
FITC-labeled anti-HbF antibody confirmed that the MMF-induced
increase in .gamma.-globin mRNA was associated with a corresponding
increase in HbF protein expression.
Example 5: Monomethylfumarate (MMF) Induces .gamma.-Globin (HUI)
Gene and Protein Expression in Primary Retinal Pigment Epithelial
(RPE) Cells
[0205] The effect of MMF on primary RPE cells was also
investigated. RPE cells were isolated from the eyes of humanized
mice and used to establish primary RPE cell cultures. These animals
have been genetically engineered such that they express human
rather than mouse beta and gamma globin genes and hence synthesize
human hemoglobin. Primary RPE cells were cultured in the presence
or absence of MMF at concentrations ranging from 0-1000 .mu.M for a
period of 9 hours. Total RNA was prepared and .gamma.-globin gene
expression analyzed by qPCR. A dose-dependent increase in
.gamma.-globin gene expression was observed also in these cells
when cultured in the presence of MMF (0-1000 .mu.M) (FIG. 6). Data
are represented as mean.+-.SEM; *P<0.01, **P<0.001.
Example 6: Evaluation of the Induction of .gamma.-Globin by DMF and
MMF
[0206] Methods and Materials
[0207] Induction of .gamma.-globin by DMF and MMF (Sigma, St.
Louis, Mo.) in the dual-luciferase KU812 stable line using a dual
luciferase assay was investigated. HU (100 .mu.M; Sigma) was
included as a positive control and cell viability was monitored by
trypan blue exclusion. Findings in KU812 cells were confirmed in
human primary erythroid progenitor cells grown in liquid culture
using a published protocol. Globin expression was measured by qPCR
also as previously published. HbF protein was measured relative to
that of isotype control using FITC conjugated anti-human HbF
antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, Calif.) and
fluorescence activated cell sorting (sec supplemental methods for
details). HbF protein expression was confirmed by Western blot
analysis using anti-human HbF antibody (1:1000; Bethyl
Laboratories, Inc., Montgomery, Tex.) and horseradish
peroxidase-conjugated sheep IgG (1:1000; Santa Cruz).
[0208] Identical experiments were performed using the human RPE
cell line ARPE-19, an established model for the study of RPE19, and
primary RPE cell cultures established from the eyes of HbAA- and
HbSS-expressing Townes humanized knock-in SCD mice (Jackson
Laboratories, Bar Harbor, Me.) per our published method.
Additionally, MMF (1 mM final concentration) or PBS (control) was
injected intravitreally into the eyes of HbAA and HbSS mice and
retinal .gamma.-globin and HbF expression analyzed by qPCR and
immunofluorescence 24 h post-injection. Animal studies were
approved by the Georgia Regents University Institutional Committee
for Animal Use in Research and Education.
[0209] Primary Erythroid Culture
Erythroid progenitors were generated in vitro from adult CD34' stem
cells (STEMCELL Technologies, Inc. Vancouver, Canada) using a
2-stage culture system that achieves terminal erythroid
differentiation.sub.18. CD34' stem cells (500,000) were grown in
First medium consisting of Iscove Modified Dulbecco Media
containing human AB serum, interleukin-3 (10 ng/mL), stem cell
factor (10 ng/mL) and erythropoietin (2 IU/mL). On day 7, the
erythroblasts were placed in Second medium with 2 IU/mL
erythropoietin for the duration. On day 8, erythroid cells were
treated with monomethylfumarate (MMF, 1000 .mu.M), dimethylfumarate
(DMF, 200 .mu.M) or hydroxyurea (HU, 100 .mu.M). Then cells were
harvested for total RNA and protein for qPCR, FACS and western blot
analyses.
[0210] Fluorescence Activated Cell Sorting (PACS)
[0211] After drug treatments, 500,000 cells were washed twice with
phosphate buffered saline and then fixed in 4% paraformaldehyde and
permeated with ice-cold acetone/methanol (4:1). Cells were
incubated with anti-.gamma.-globin-FITC antibody (Santa Cruz
Biotechnology, Santa Cruz, Calif.) in PBT (PBS/01% BSA/0.1% triton
X100) solution for 20 min to stain intracellular HbF antigens. The
labeled cells were analyzed by Bectin Dickerson LSR-II flow
cytometer (BD Bioscience). All experiments were performed in
triplicate.
[0212] Intravitreal Injection
[0213] HbAA- and HbSS-expressing Townes humanized knock-in sickle
cell disease mice (6 weeks old; n=6) were used for intravitreal
injection of MMF following our published protocol.sub.9. Briefly,
animals were weighed and anesthetized using 174 (1 .mu.L/g body
weight) of a solution of ketamine (80 mg/mL) and xylazine (12
mg/mL). Then 5 .mu.L of proparacaine solution (5% w/v) was
administered topically to the eyes. MMF (1 .mu.L; 10 mM solution
prepared in PBS) was then injected into the vitreous body of the
right eye of each animal at the limbus; the left eye served as a
contralateral control and received an equal volume of phosphate
buffered saline (PBS, 0.01 M pH 7.4). Taking into account a total
estimated vitreous volume of 10 .mu.L per mouse eye, the final
concentration of MMF achieved in our experimental system was 1 mM.
At 24 h postinjection, mice were sacrificed via CO.sub.2
inhalation, and eyes were harvested. Some eyes (n=3 per treatment
group) were flash frozen in liquid nitrogen and cryosectioned for
use in immunofluorescence analyses while the remaining were
dissected to isolate RPE/eyecup from neural retina and total RNA
prepared.
[0214] Results
[0215] Pharmacologic induction of HbF remains the best treatment
approach to ameliorate the clinical complications of SCD. The
pleiotropic actions of FAE in a broad spectrum of tissues, high
tolerability and oral bioavailability, and recent FDA approval of
Tecfidera (BG-12; Biogen Idec, Weston, Mass.) for use in multiple
sclerosis make these agents attractive for rapid extrapolation to
clinical trials in SCD. Therefore, the ability of DMF and MMF to
induce .gamma.-globin expression and HbF production in erythroid
cells was investigated. The induction of .gamma.-globin promoter
activity by DMF and MMF was observed in KU812 cells by dual
luciferase assay with maximal induction at 200 .mu.M for DMF and
1000 .mu.M MMF (FIGS. 7A and 7B); findings were confirmed in
primary human erythroid cells (FIGS. 7C-7E). Levels of
.gamma./.beta.-globin mRNA were induced significantly by both DMF
and MMF (4- and 8-fold, respectively; FIG. 7C). FACS demonstrated a
28- and 32-fold increase in HbF positive cells in the presence of
these compounds (FIGS. 7D(1)-7D(4) and 7E), which is significantly
higher than levels produced by HU (15-fold); see also FIG. 9. HbF
protein expression was confirmed by Western blot (data not
shown).
[0216] HbF protein production in ARPE-19 cells exposed to MMF (1000
.mu.M) for 24 h was evaluated using a FITC-conjugated HbF antibody
and fluorescence microscopy (data not shown). Cell nuclei were
counterstained with DAPI. Treatments identical to those detailed
above were performed using primary RPE cells isolated from the eyes
of HbAA- or HbSS-expressing Townes humanized knock-in sickle cell
disease mice and the expression of .gamma.-globin mRNA evaluated by
qPCR using primer pairs specific to the human .gamma.-globin
gene.
[0217] Data (mean.+-.SEM) are from at least five data points
generated from at least three independent drug treatments. For in
vivo studies, six animals were included per group and samples were
run in duplicate. Paired student t-test was performed and a
P<0.05 was considered significant.
[0218] These data demonstrate the ability of DMF and MMF to induce
HbF synthesis in human erythroid progenitors and support further
testing in a pre-clinical sickle cell mouse model.
[0219] Following oral intake, DMF is not detectable in plasma as it
is rapidly hydrolyzed and converted into MMF. Based on this
information, only MMF for studies with RPE cells were used (FIGS.
8A-8G). Findings in ARPE-19 and primary RPE cells mirrored closely
those obtained in primary erythroid progenitors. This work confirms
a single prior report of .beta.-globin gene expression in RPE14
(FIGS. 8A and C) and demonstrates for the first time the induction
of .gamma.-globin expression and HbF production in these cells by
MMF and HU (FIGS. 8B, 8D, 8E and FIG. 11). These data are further
supported by in vivo studies demonstrating the significant
elevation of .gamma.-globin mRNA and HbF protein in RPE/eyecup and
neural retina isolated from the eyes of HbAA and HbSS mice injected
intravitreally with MMF (FIGS. 8F and 8G).
[0220] Though SR is thought to be largely a vascular disease, there
is clinical evidence of early, non-vascular cell involvement,
specifically of photoreceptor cell (PRC) dysfunction. PRCs, first
order neurons in the visual pathway, have a high oxygen demand.
Given their isolation from a vascular supply, they depend solely
upon RPE cells for metabolic support. The synthesis of Hb by
non-erythroid cells has been reported at other interfaces where
O.sub.2/CO.sub.2 diffusion occurs; this may also be the case in RPE
cells. The physiological importance of Hb production in RPE is yet
to be determined; it is possible that defects in RPE Hb expression
may contribute to retinal dysfunction and degeneration in SCD3.
[0221] Little is known regarding the impact of HbF-inducing
therapies in the retina. A recent study by Estepp et al.
demonstrated an inverse correlation between SR and plasma HbF
concentration. Follow-up studies on a larger scale are required to
substantiate HbF-inducing therapies to treat SR. Such therapy may
confer benefit in patients of HbSS and HbSC genotypes, where the
incidence of SR is highest. The present findings that FAE induce
.gamma.-globin expression and HbF production are new and support
the possible re-purposing of BG-12 for treatment of SCD.
Additionally, a new cellular target was identified for the
therapeutic management of SR, a factor of high clinical relevance
given the 10% incidence of vision loss and blindness among SCD
patients and the lack of effective strategies for prevention and
treatment.
Example 7: MMF Induces Expression of SLC22A4 (Aka OCTN1)
[0222] Immunofluorescence analysis of human OCTN1 expression
revealed the robust expression of the transporter in human primary
erythroid cells generated in liquid culture from adult CD34' stem
cells.
[0223] KU812, a human leukemic cell line that expresses the fetal
.gamma.-globin and adult .beta.-globin genes, is a commonly used
system for screening and discovery of novel HbF inducers (see
above). FIG. 7 shows the robust induction of .gamma.-globin mRNA
and HbF production in these cells by MMF. FIG. 11 shows OCTN1
expression is also induced in these cells by MMF treatment (MMF,
1000 .mu.M, 16 h). Data are represented as mean.+-.standard error
of the mean; *p<0.05.
[0224] Red blood cells, cells of erythroid lineage, are the primary
producers of hemoglobin. However, recent reports suggest other,
nonhematopoietic cells to be capable of doing the same. This
includes retinal pigment epithelial (RPE) cells, a cell type
critical to normal visual function. FIG. 8 shows that induction of
.gamma.-globin gene expression and HbF protein production by MMF
occurs in RPE. FIG. 12 shows that MMF also induces OCTN1 expression
in these cells; HU alone had little to no effect on OCTN1
expression. Data are represented as mean.+-.SEM; *p<0.05.
Example 8: MMF Induces OCTN1 mRNA and Protein Expression in Primary
RPE Cells Isolated from HbAA- and HbSS-Expressing Mouse Retinas
[0225] Given that ARPE-19 is a transformed human RPE cell line, the
findings in RPE cells isolated freshly from the living animals as
such cells was investigated to provide a more accurate
representation of RPE cells in their native environment.
Additionally, the humanized knock-in SCD mouse model (the Townes
mouse), a rodent model engineered such that animals express human
.alpha., .beta., and .gamma. globin rather than the rodent globin
genes, allows for the pre-clinical study of parameters highly
reflective of the human condition. The effects of MMF on OCTN1
expression in HbAA and HbSS-expressing primary RPE cells from this
model was investigated. Treatment with 1000 .mu.M MMF induced
expression of OCTN1 robustly both at the RNA and protein level.
(FIG. 13, *p<0.001).
Example 9: MMF Induces OCTN1 Protein Expression In Vivo in HbAA-
and HbSS-Expressing Mouse Retinas
[0226] To determine whether the findings obtained in isolated
retinal cells can be extrapolated to the in vivo condition, MMF (1
mM final concentration) was delivered intravitreally into the eyes
of HbAA- and HUSS-expressing mice. 24 h post-injection, animals
were sacrificed and OCTN1 expression evaluated by
immunofluorescence. OCTN1 protein expression was upregulated
throughout the entire retina (FIG. 14). Given that these animals
are of a pigmented background and RPE is loaded with melanin
pigment, a property that may interfere or mask fluorescent signal
intensity, the expression of OCTN1 in the RPE cell layer
specifically is not as apparent using this method. However, based
upon data in primary RPE cells isolated from these animals (FIG.
13), it is upregulated in the RPE cell layer. It is important to
note also, the expression of OCTN1 in other retinal regions namely,
the retinal ganglion cell (rgc) layer and, the filamentous labeling
from the rgcs to outer nuclear layer (onl), a pattern of
localization consistent with the labeling of Muller cells. These
data are congruent with our previous analysis of HbF protein
expression, evaluated using immunofluorescence in similar
cryosections, which revealed the MMF-induced upregulation of HbF in
the RPE cell layer and throughout the neural retina (see FIG.
8).
[0227] qPCR analysis of OCTN1 mRNA expression in primary RPE,
Muller and ganglion cells isolated from normal mouse retinas
revealed expression of OCTN1 all three retinal cell types (FIG.
15). Interestingly, OCTN1 expression appeared to be highest in
pGC's. The axons of the GC's communicate directly with the brain
for higher visual processing/enabling of sight as they actually
bundle as they exit retina to form the optic nerve. In keeping with
this, an increase in OCTN1 expression and likely also HbF protein
induced by MMF in these cells would be highly beneficial in
protecting these neurons from the damaging effects of hypoxia,
oxidative stress and inflammation produced characteristically in
sickle cell disease.
[0228] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0229] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *