U.S. patent application number 09/845805 was filed with the patent office on 2001-10-04 for compositions and administration of compositions for the treatment of blood disorders.
This patent application is currently assigned to Susan Perrine. Invention is credited to Perrine, Susan P..
Application Number | 20010027215 09/845805 |
Document ID | / |
Family ID | 26725817 |
Filed Date | 2001-10-04 |
United States Patent
Application |
20010027215 |
Kind Code |
A1 |
Perrine, Susan P. |
October 4, 2001 |
Compositions and administration of compositions for the treatment
of blood disorders
Abstract
The invention relates to novel compositions and to methods for
the pulsed administration of compositions to a patient or to cells
in vitro for the treatment of human blood disorders. Compositions
contain chemical compounds that stimulate the expression of fetal
hemoglobin and/or stimulate the proliferation of red blood cells,
white blood cells and platelets in patients and ex vivo for
reconstitution of hematopoiesis in vivo. These methods are useful
to treat or prevent the symptoms associated with anemia, sickle
cell disease, thalassemia, blood loss, and other blood disorders.
The invention also relates to methods for the pulsed administration
of compositions to patients for the treatment and prevention of
cell proliferative disorders including deficiencies such as
cytopenia and malignancies and for expansion of cells for
hematopoietic transplantation. Pulsed administration has been shown
to be more effective than continuous therapy in patients
tested.
Inventors: |
Perrine, Susan P.; (Weston,
MA) |
Correspondence
Address: |
NIXON PEABODY LLP
ATTENTION: DAVID RESNICK
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Susan Perrine
|
Family ID: |
26725817 |
Appl. No.: |
09/845805 |
Filed: |
April 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09845805 |
Apr 30, 2001 |
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09086998 |
May 29, 1998 |
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6231880 |
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60048132 |
May 30, 1997 |
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Current U.S.
Class: |
514/557 ;
514/568 |
Current CPC
Class: |
A61K 31/19 20130101;
A61K 31/192 20130101 |
Class at
Publication: |
514/557 ;
514/568 |
International
Class: |
A61K 031/19; A61K
031/192 |
Goverment Interests
[0001] This invention was made with support from the United States
government under grant numbers HL37118 and HL-15157, awarded by the
National Heart, Lung and Blood Institute of the National Institutes
of Health, and grant number 000831, awarded by the United States
Food and Drug Administration, and the United States government has
certain rights in the invention.
Claims
I claim:
1. A method for treating a human cell proliferative disorder by
stimulating cell growth, comprising administering to a patient in
need a pharmaceutically effective amount of a composition
containing an effective amount of a C.sub.1-C.sub.4 substituted
and/or phenyl substituted carboxylic acid and pharmaceutically
acceptable salts thereof, and a pharmaceutically acceptable carrier
or diluent, wherein said C.sub.1-C.sub.4 moiety and said phenyl
moiety can be substituted or unsubstituted, wherein said
substituents are selected from the group consisting of hydroxy,
halogens, phenyl, thiol, mercapto and methylthiol.
2. The method of claim 1, wherein said composition is a
C.sub.1-C.sub.4 substituted carboxylic acid.
3. The method of claim 1, wherein said composition is a
dimethyl-substituted carboxylic acid.
4. The method of claim 1 wherein the cytopenia is a red or white
blood cell anemia, a leukopenia or a thrombocytopenia.
5. The method of claim 1 wherein the disorder is a
hemoglobinopathy.
6. A method of reducing the amount of a growth stimulating compound
that must be administered to a patient having a cell proliferative
disorder comprising administering an effective amount of a
composition containing a C.sub.1-C.sub.4 substituted and/or phenyl
substituted compound, wherein said compound is selected from the
group consisting of cinnamic acid, acetic acid, butyric acid and
propionic acid, or a pharmaceutically acceptable salt thereof, in a
pharmaceutically acceptable carrier or diluert, wherein said
C.sub.1-C.sub.4 moiety and said phenyl moiety can be substituted or
unsubstituted, and said substituents are selected from the group
consisting of hydroxy, halogens, phenyl, thiol, mercapto and methyl
thiol.
7. The method of claim 6, wherein the composition is a dimethyl
substituted compound.
8. The method of claim 7, wherein the compound is selected from the
group consisting of acetic acid, phenoxyacetic acid, methoxyacetic
acid, cinnamic acid, hydrocinnamic acid, butyric acid, and
propionic acid.
9. The method of claim 1 wherein the composition is administered by
delivery of a therapeutically effective pulsed dose of said
composition over a period of time and the therapeutically effective
pulsed dose comprises less of the composition than a therapeutic
continuous dose administered over said period of time.
10. The method of claim 1 wherein the composition is administered
by injection, infusion, instillation or ingestion.
11. The method of claim 9 wherein said pulsed dose has an interval
between each pulse from about 3 to about 21 days.
12. The method of claim 1 wherein treatment stimulates the number
of circulating platelet cells or white blood cells as determined
from peripheral blood cell counts.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to composition methods for the
treatment and prevention of blood disorders such as anemia,
neutropenia, thrombocytopenia, thalassemia and sickle cell disease
using such compositions. The compositions include C.sub.1-C.sub.4
substituted and/or phenyl substituted carboylic acids such as
dimethyl substitutions onto carboxylic acids. The methods comprise
the administration of compositions that stimulate the expression of
a globin protein and, in particular, fetal hemoglobin, or the
proliferation or development of hemoglobin expressing, myeloid
cells or megakaryocytic cells.
[0004] 2. Description of the Background
[0005] The major function of red blood cells is to transport oxygen
to tissues of the body. Minor functions include the transportation
of nutrients, intercellular messages and cytoldnes, and the
absorption of cellular metabolites. Anemia, or a loss of red blood
cells or red blood cell capacity, can be grossly defined as a
reduction in the ability of blood to transport oxygen. Anemia can
be measured by determining a patient's red blood cell mass or
hematocrit. Hematocrit values are indirect, but fairly accurate
measures of the total hemoglobin concentration of a blood sample.
Anemia, as measured by a reduced hematocrit, may be chronic or
acute. Chronic anemia may be caused by extrinsic red blood cell
abnormalities, intrinsic abnormalities or impaired production of
red blood cells. Extrinsic or extra-corpuscular abnormalities
include antibody-mediated disorders such as transfusion reactions
and erythroblastosis, mechanical trauma to red cells such as
micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic
purpura and disseminated intravascular coagulation. In addition,
infections by parasites such as Plasmodium, chemical injuries from,
for example, lead poisoning, and sequestration in the mononuclear
system such as by hypersplenism can result in red blood cell
disorders and deficiencies.
[0006] Impaired red blood cell production can occur by disturbing
the proliferation and differentiation of the stem cells or
committed cells. Some of the more common diseases of red cell
production include aplastic anemia, hypoplastic anemia, pure red
cell aplasia and anemia associated with renal failure or endocrine
disorders. Disturbances of the proliferation and differentiation of
erythroblasts include defects in DNA synthesis such as impaired
utilization of vitamin B.sub.12 or folic acid and the megaloblastic
anemias, defects in heme or globin synthesis, and anemias of
unknown origins such as sideroblastic anemia, anemia associated
with chronic infections such as malaria, trypanosomiasis, HIV,
hepatitis virus or other viruses, and myelophthisic anemias caused
by marrow deficiencies.
[0007] Intrinsic abnormalities include both hereditary and acquired
disorders. Acquired disorders are those which have been induced
through, for example, a membrane defect such as paroxysmal
nocturnal hemoglobinuria. Hereditary disorders include disorders of
membrane cytoskeleton such as spherocytosis and elliptocytosis,
disorders of lipid synthesis such as an abnormally increased
lecithin content of the cellular membrane, red cell enzyme
deficiencies such as deficiencies of pyruvate kinase, hexokinase,
glutathione synthetase and glucose-6-phosphate dehydrogenase.
Although red blood cell disorders may be caused by certain drugs
and immune system disorders, the majority are caused by genetic
defects in the expression of hemoglobin. Disorders of hemoglobin
synthesis include deficiencies of globin synthesis such as
thalassemia syndromes and structural abnormalities of globin such
as sickle cell syndromes and syndromes associated with unstable
hemoglobins.
[0008] Mammalian globin gene expression is highly regulated during
development. The basic structure of the .alpha. and .beta. globin
genes are similar as are the basic steps in synthesis of .alpha.
and .beta. globin. There are at least five human .alpha. globin
genes located on chromosome 16 including two adult .alpha. globin
genes of 141 amino acids that encode identical polypeptides which
differ only in their 3'-untranslated regions, one embryonic .alpha.
gene, zeta (.zeta.), and at least two pseudo-alpha genes, psi zeta
(.psi..beta.) and omega alpha (.omega..alpha.). The human .beta.
globin gene cluster includes one embryonic gene, epsilon
(.epsilon.), two adult beta globin genes, beta (.beta.) and delta
(.delta.), two fetal beta globin genes G-gamma (G-.gamma.) and
A-gamma (A-.gamma.), which differ by only one amino acid, and at
least one pseudo-beta gene, psi beta (.psi..beta.). All are
expressed from a single 43 kilobase segment of human chromosome 11
(E. F. Fritsch et al., Nature 279:598-603, 1979).
[0009] Hemoglobin A comprises four protein chains, two alpha chains
and two beta chains (.alpha..sub.2.beta..sub.2), interwoven
together, each with its own molecule of iron and with a combined
molecular weight of about 68 kD. The hemoglobin macromolecule is
normally glycosylated and upon absorbing oxygen from the lungs
transforms into oxyhemoglobin (HbO.sub.2). There are at least six
distinct forms of hemoglobin, each expressed at various times
during development. Hemoglobin in the embryo is found in at least
three forms, Hb-Gower 1 (.zeta..sub.2.beta..sub.2), Hb-Gower 2
(.alpha..sub.2.gamma..sub.2), and Hb-Portand
(.zeta..sub.2.gamma..sub.2). Hemoglobin in the fetus comprises
nearly totally HbF (.alpha..sub.2.gamma..sub.2), whereas hemoglobin
in the adult contains about 96% HbA (.alpha..sub.2.beta..sub.2),
about 3% HbA.sub.2 (.alpha..sub.2 .delta..sub.2) and about 1% fetal
HbF (.alpha..sub.2 .gamma..sub.2). The embryonic switch of globin
expression from .zeta. to .alpha. and from .epsilon. to .gamma.
begins in the yolk sac. However, chains of embryonic .zeta. and
.epsilon. have been found in the fetal liver and complete
transition to the fetal form does not occur until late in fetal
development. The fetal switch from .gamma. to .beta. begins later
in erythropoeisis with the amount of .gamma. globin produced
increasing throughout gestation. At birth, .beta. globin accounts
for about 40% of non-.alpha. globin chain synthesis and thereafter
continues to rapidly increase. Neither the switch from embryonic to
fetal or fetal to adult appears to be controlled through cell
surface or known cytokine interactions. Control seems to reside in
a developmental clock with the switch occurring at times determined
only by the stage of fetal development.
[0010] Defects or mutations in globin chain expression are common.
Some of these genetic mutations pose no adverse or only minor
consequences to the person, however, most mutations prevent the
formation of an intact or normal hemoglobin molecule through a
functional or structural inability to effectively bind iron, an
inability of the chains or chain pairs to effectively or properly
interact, an inability of the molecule to absorb or release oxygen,
a failure to express sufficient quantities of one or more globin
chains or a combination of these malfunctions. For example,
substitutions of valine for glutamic acid at the sixth position of
the .beta. chain produces HbS and was found to occur in about 30%
of black Americans. In the HbS heterozygote, only about 40% of
total hemoglobin is HbS with the remainder being the more normal
HbA.
[0011] Upon deoxygenation, HbS molecules undergo aggregation and
polymerization ultimately leading to a morphological distortion of
the red cells which acquire a sickle or holly-leaf shape. Sickling
has two major consequences, a chronic hemolytic anemia and an
occlusion of small blood vessels that results in ischemic damage to
tissues. Further, when exposed to low oxygen tensions,
polymerization converts HbS hemoglobin from a free-flowing liquid
to a viscous gel. Consequently, the degree of pathology associated
with sickle cell anemia can be correlated with the relative amount
of HbS in the patient's system.
[0012] Individuals with severe sickle cell anemia develop no
symptoms until about five to six months after birth. In these
infants it was determined that fetal hemoglobin did not interact
with HbS and, as long as sufficient quantities were present, could
modulate the effects of HbS disease. This modulating effect of
.beta. globin is also observed with other .beta. globin disorders,
such as HbC and HbD, and other mutations of the .beta. chain. HbS
polymerization is also significantly affected by the hemoglobin
concentration of the cell. The higher the HbS concentration, the
greater the chances for contact between two or more HbS molecules.
Dehydration increases hemoglobin concentration and greatly
facilitates sickling.
[0013] To some extent, sickling is a reversible phenomenon. With
increased oxygen tensions, sickled cells depolymerize. This process
of polymerization-depolymerization is very damaging to red cell
membranes and eventually leads to irreversibly sickled cells (ISC)
which retain their abnormal shape even when fully oxygenated. The
average ISC survives for about 20 days in the body, as compared to
the normal 120 day life span. Individuals with HbS syndromes have,
frequent infections, chronic hemolysis with a striking
reticulocytosis and hyperbilirubinemia. The course of the disease
is typically punctuated with a variety of painful crises called
vaso-occlusive crises. These crises represent episodes of hypoxic
injury and infarction in the organs, abdomen, chest, extremities or
joints. Leg ulcers are an additional manifestation of the
vaso-occlusive tendency of this disease. Central nervous system
involvement is common producing seizures and even strokes. Aplastic
crises, also common, represent a temporary cessation of bone marrow
activity and may be triggered by infections, folic acid deficiency
or both. Crises are episodic and reversible, but may be fatal.
Damage from crisis episodes tends to be cumulative and even in
those individuals with milder forms of sickle cell disorders,
life-spans can be greatly reduced. Absent alternative intervention,
patients typically die before the age of 30.
[0014] The thalassemia syndromes are a heterogeneous group of
disorders all characterized by a lack of or a decreased synthesis
of the globin chains of HbA. Deficiencies of .beta.-globin
expression are referred to as .beta.-thalassemias and deficiencies
of .alpha.-globin, .alpha.-thalassemias. The hemolytic consequences
of deficient globin chain synthesis result from decreased synthesis
of one chain and also an excess of the complementary chain. Free
chains tend to aggregate into insoluble inclusions within
erythrocytes causing premature destruction of maturing erythrocytes
and their precursors, ineffective erythropoiesis, and the hemolysis
of mature red blood cells. The underlying defects of hemoglobin
synthesis have been elucidated over the years and largely reside in
the nucleic acid sequences which express or control the expression
of .alpha. or .beta. globin protein.
[0015] Surprisingly, .alpha.-thalassemias tend to be less severe
than .beta. thalassemias. Homozygous pairs of .beta. chains are
believed to be more soluble than those derived from unpaired a
chains. Consequently, the effects associated with free or
improperly paired globin chains, which correlate with at least half
of the clinical pathology associated with thalassemia, are
minimized.
[0016] Hemoglobin H disease, a more severe form of a thalassemia,
is a deletion of three of the four .alpha. globin genes. It is
rarely found in those of African origin, but mostly in Asians. With
only a single .alpha. gene, .alpha. chain expression is markedly
depressed and there is an excess of .beta. chains forming tetramers
called HbH hemoglobin. HbH is unable to withstand oxidative stress
and precipitates with vessels or is removed by the spleen. The most
severe form of a thalassemia is hydrops fetalis and results from a
deletion of all .alpha. globin genes. In the fetus, tetramers of
.gamma. globin develop (Hb Barts) that have an extremely high
oxygen lafty and are unable to release oxygen to the tissues.
Severe tissue anoxia results and leads to intrauterine fetal
death.
[0017] Fetal .beta.-type globin, or .gamma. globin, is expressed in
the earliest stages of mammalian development and persists until
about 32 to 34 weeks of gestation. At this stage, the adult forms
of .beta. globin begin to be expressed and substitute for the fetal
proteins. Studies correlating clinical hematological results with
the locations of various mutations that correspond to switching
indicate that a region located upstream of the 5'-end of the 3-gene
may be involved in the cis suppression of .gamma.-gene expression
in adults (E. F. Fritsch et al., Nature 279:598-603, 1979). The
reason for this switch from fetal to adult protein is unknown and
does not appear to provide any significant benefit to the
adult.
[0018] Each .beta. globin gene comprises three exons which encode
about 146 amino acids, two introns and a 5'-untranslated region
containing the promoter sequences. Biosynthesis of .beta. globin
begins with transcription of the entire gene followed with RNA
processing of the message, removal of the introns by splicing, poly
A addition, capping and post-transcriptional modifications. The
mature mRNA molecule is exported from the nucleus and translated
into .beta. globin. Defects in each of these functions have been
found associated with specific thalassemias. Identified mutations
include single-nucleotide deletions, insertions and substitutions,
frame shift mutations, deletions of entire segments of coding or
controlling regions, improper termination signals, aberrant
splicing signals, and multiple mutations.
.beta..degree.-thalassemias are characterized by a complete absence
of any .beta. globin chains. .beta..sup.+-thalassemias are
characterized by a detectable presence of a reduced amount of
.beta. chains.
[0019] There are three principal categories of .beta.-thalassemia,
thalassemia major, thalassemia intermedia and thalassemia minor.
Patients with thalassemia minor may be totally asymptomatic and are
genotypically .beta..sup.+/.beta. or .beta..degree./.beta..
Although red cell abnormalities can be detected, symptoms are mild.
Thalassemia intermedia patients are most often genotypically
.beta..sup.+/.beta..sup.+ or .beta..degree./.beta. and present
severe symptoms which can be alleviated with infrequent blood
transfusions. In contrast, thalassemia major patients are
genotypically .beta..degree./.beta..degree.,
.beta..degree./.beta..sup.+ or .beta..sup.+/.beta..sup.+, and
require regular and frequent transfusions. Children suffer from
severe growth retardation and die at an early age from the profound
effects of anemia. Those that survive longer suffer from
morphological changes. The face becomes distorted due to expansion
of marrow within the bones of the skull, hepatosplenomegaly ensues,
there is a delayed development of the endocrine organs including
the sexual organs, and a progressive iron overload with secondary
hemochromatosis.
[0020] There are two direct consequences of .beta.-thalassemia.
First, there is an inadequate formation of HbA and, therefore, an
impaired ability to transport oxygen. There are also multiple
effects attributable to an imbalance between a and .beta. chain
synthesis. Surprisingly, the pathological consequences of globin
chain imbalance appears to be the more severe. Free .alpha. chains
form unstable aggregates that precipitate within red cell
precursors in the form of insoluble inclusions. These inclusions
damage cellular membranes resulting in a loss of potassium. The
cumulative effect of these inclusions on the red blood cells is an
ineffective eropoiesis. An estimated 70% to 85% of normoblasts in
the marrow are eventually destroyed. Those that do escape immediate
destruction are at increased risk of elimination by the spleen
where macrophages remove abnormal cells. Further, hemolysis
triggers an increased expression of erythropoietin which expands
populations of erythroid precursors within bone marrow and leads to
skeletal abnormalities. Another severe complication of .beta.
thalassemia is that patients tend to have an increased ability to
absorb dietary iron. As most treatments for thalassemia involve
multiple transfusions of red blood cells, patients often have a
severe state of iron overload damaging all of the organs and
particularly the liver. To reduce the amount of iron in their
systems, iron chelators are typically administered. Although
helpful, patients succumb at an average of between about 17 to 35
years of age to the cumulative effects of the disease and iron
overload.
[0021] Genotypic variation in healthy individuals have been
identified wherein adult .beta. globin is not formed, but severe
complications are avoided. These patients constituitively express
fetal or .gamma. globin protein in amounts sufficient to substitute
for the missing .beta. globin protein. This hereditary persistence
of fetal hemoglobin (HPFH) may involve one or both of the fetal
.beta.-globin genes, A-.gamma. and G-.gamma.. Apparently,
consistent production of either .gamma.-globin protein accomplishes
the necessary functions, at least in the short term, of the
abnormal or missing .beta.-globin protein (R. Bernards et al., Nuc.
Acids Res. 8:1521-34, 1980).
[0022] A variety of small molecules have been shown to effect
hemoglobin or fetal globin expression. Early experiments
demonstrated that acetate (CH.sub.3COOH), propionate
(CH.sub.3CH.sub.2COOH), butyrate (CH.sub.3CH.sub.2CH.sub.2COOH) and
isobutyrate (CH.sub.3CH(CH.sub.3)COOH) all induced hemoglobin
synthesis in cultured Friend leukemia cells (E. Takahashi et al.,
Gann 66:577-80, 1977). Additional studies showed that polar
compounds, such as acid amides, and fatty acids could stimulate the
expression of both fetal and adult globin genes in murine
erythroleukemia cells (U. Nudel et al., Proc. Natl. Acad. Sci. USA
74:1100-4, 1977). Hydroxrurea (H.sub.2NCONHOH), another relatively
small molecule, was found to stimulate globin expression (N. L.
Letvin et al., N. Engl. J. Med. 310:869-73, 1984). Stimulation,
however, did not appear to be very specific to fetal globin (S.
Charache et al., Blood 69:109-16, 1987). Hydroxyurea is also a
well-known carcinogen making its widespread and long term use as a
pharmaceutical impractical.
[0023] Expression from the .gamma.-globin genes has been
successfully manipulated in vivo and in vitro using agents such as
cytosine arabinoside (AraC), a cytotoxic agent that induces fetal
reticulocyte production (P. Constantoulakis et al., Blood
74:1963-71, 1989), and 5-azacytidine (AZA), a well-known DNA
methylase inhibitor (T. J. Ley et al., N. Engl. J. Med.
307:1469-75, 1982). Continuous intravenous administration of AZA
produced a five- to seven-fold increase in .gamma. globin mRNA of
bone marrow cells (T. J. Ley et al., Blood 62:370-380, 1983).
Additional studies have shown that there are significant
alterations in the population of stem cells in the bone marrow
after AZA treatment (A. T. Torrealba-De Ron et al., Blood
63:201-10, 1984). These experiments indicate that AZA's effects may
be more attributable to reprogramming and recruitment of erythroid
progenitor cells than to any direct effects on specific gene
expression. Many of these agents including AZA, AraC and
hydroxyurea are myelotoxdc, carcinogenic or teratogenic making
long-term use impractical.
[0024] One of the major breakthroughs in the treatment of
hemoglobinopathies was made when it was discovered that butyric
acid (butanoic acid; CH.sub.3CH.sub.2CH.sub.2COOH) accurately and
specifically stimulated transcription of the human fetal (.gamma.)
globin gene (G. A. Partington et al., EMBO J. 3:2787-92, 1984).
These findings were quickly confirmed in vivo wherein it was shown
that pharmacological doses of butyric acid greatly increased
expression of fetal globin in adult chickens rendered anemic by
injections with phenylhydrazine (G. D. Ginder et al., Proc. Natl.
Acad. Sci. USA 81:3954-58, 1984). Selective transcriptional
activation was again thought to be due to hypo-methylation of the
embryonic gene (L. J. Burns et al., Blood 72:1536-42, 1988). Others
speculated that histone acetylation, a known effect of butric acid,
may be at least partly responsible for increased fetal gene
expression (L. J. Burns et al., EMBO J. 3:2787, 1984).
[0025] Over 50 derivatives of butyric acid have since been found to
be effective in stimulating fetal globin production (S. P. Perrine
et al., Biochem. Biophys. Res. Commun. 148:694-700, 1987). Some of
these include butric acid salts such as sodium and arginine
butyrate, .alpha.-amino-n-butync acid (butyramide;
CH.sub.3CH.sub.2CH.sub.2CONH.sub- .2), and isobutyramide
(CH.sub.3CH(CH.sub.3)C0NH.sub.2). Although promising in pilot
clinical studies, treated patients were unable to maintain adequate
levels of fetal globin in their system. It was later determined
that many of these forms of butyric acid had extremely short-half
lives. Oxidation in the serum, clearance by hepatocytes and
filtration through the kidneys rapidly eliminated these agents from
the patient's system. With others, patients rapidly developed
tolerance or metabolites of compounds had the opposite desired
effect.
[0026] A number of aliphatic carboxylic acids have been tested for
their ability to specifically increase fetal globin expression in
K562 human erythroleukernia cells (S. Safaya et al., Blood
84:3929-35, 1994). Although longer chains were considered toxic to
cells, propionate (CH.sub.3CH.sub.2COOH) and valerate (pentatonic
acid; CH.sub.3CH.sub.2CH.sub.2CH.sub.2COOH) were found to be most
effective. Butyrate (CH.sub.3(CH.sub.2).sub.2COOH), caproate
(CH.sub.3(CH.sub.2).sub- .4COOH), caprylate
(CH.sub.3(CH.sub.2).sub.6COOH), nonanoate
(CH.sub.3(CH.sub.2).sub.7COOH), and caprate
(CH.sub.3(CH.sub.2).sub.8COOH- ) produced much less of an effect.
Phenyl acetate (C.sub.6H.sub.5CH.sub.2C- OOH) and its precursor,
4-phenyl butyrate (C.sub.6H.sub.5CH.sub.2CH.sub.2C- H.sub.2COOH),
were found to decrease fetal globin expressing reticulocyte
proliferation, but increase relative proportions of fetal globin
per cell in cultured erythroid progenitor cells (E. Fibach et al.,
Blood 82:2203-9, 1993). Acetate (CH.sub.3COOH), a metabolic product
of butyrate catabolism, increased both erythrocyte precursor
populations and also fetal globin synthesis. However, these studies
also demonstrated that positive effects could only be maintained
for very short periods of time (B. Pace et al., Blood 84:3198-204,
1994).
[0027] Other agents shown to affect fetal globin expression include
activin and inhibin. Inhibin, a disulfide linked hormone of two
subunits, suppresses secretion of follicle-stimulating hormone from
the pituitary gland. Activin, sometimes referred to as erythroid
differentiating factor (EDF) or follicle-stimulating hormone
releasing protein (FRP), is also a hormone and both of these
macromolecules induced hemoglobin accumulation in cultured human
erythrocytes (S. P. Perrine et al., Blood 74:114a, 1989). Recently,
studies have shown that steel factor, a product of the mouse steel
locus (D. M. Anderson et al., Cell 63:235-43, 1990), is also
capable of influencing fetal globin synthesis in erythroid
progenitors (B. A. Miller et al., Blood 79:1861-68, 1992).
[0028] Other methods to increase fetal globin expression have
focused on recruitment and reprogramming of erythroid progenitor
cells to increase total globin expression. For example, the
hematopoietic growth factor erythropoietin (EPO) was found to be a
potent, although not a fetal-specific, reticulocyte stimulator
(Al-Khatti et al., Trans. Assoc. Am. Physicians 101:54, 1988; G. P.
Rodgers et al., N. Engl. J. Med. 328:73-80, 1993). In one
experiment, animals were treated with EPO following a specific
course of therapy (U.S. Pat. No. 4,965,251). According to this
experiment, a high dose of eiythropoietin was administered in a
first time period followed by a second time period wherein
erythropoietin was withheld. Following this regimen of treatment,
typical for a cytokine, F-reticulocyte obtained from two
chronically -anemic baboons increased from 6-8% and 20%
pre-treatment to 23% and 50% post-treatment, respectively.
[0029] These methods were somewhat advantageous to artificially
phlebotomized baboons, but could be counter-productive to patients
with a hemoglobinopathy. Thalassemic patients express high levels
of EPO, supplemental treatments with EPO and do not improve the
globin chain imbalance, but result in more thalassemic cells.
Sickle cell patients and other patients with unstimulated levels
would also not benefit from supplemental EPO treatments because
absolute amounts of both .alpha.-globin and non .alpha.-globin
would increase. Treatments with EPO can increase the frequency and
number of sickle cell crises due to increasing the blood viscosity
with more Hbs, both of which are to be avoided in such
patients.
[0030] Other hematopoietic growth factors, such as
granulocyte/macrophage-- colony stimulating factor (GM-CSF) and
interleukin 3 (IL-3), were also tested in vivo or in vitro for the
ability to stimulate F-reticulocytes (M. Giabbianell et al., Blood
74:2657, 1989; A. R. Migliaccio et al., Blood 76:1150, 1990). Both
of these factors were found to non-specifically increase
fetal-globin synthesis in tissue culture cells.
SUMMARY OF THE INVENTION
[0031] The invention provides novel compositions and methods for
the treatment and prevention of blood disorders.
[0032] We have found that certain compositions provide improved
advantages such as prolonged induction of growth related genes,
e.g., C-myb and C-myc gene, unexpectedly better cell proliferation,
enhanced stability, and have a sparing or abrogating effect for
growth factor requirements such as IL-3 or EPO.
[0033] The compositions include C.sub.1-C.sub.4 alkyl and/or phenyl
substitution on carboxylic acids such as
.alpha.-methylhydrocinnamic acid, 3,4 dimethoxycinnamic acid,
2-methylhydrocinnamic acid, 2- and 3-methoxycinnamic acid, 3,4
dimethoxyphenylacetic acid, 3-3,4 dimethoxyphenylpropionic acid,
2,5 dimethoxyphenylacetic acid, 2,2 dimethylbutyric acid, 2,2
dimethylpropionic acid, 2,2 dimethylphenoxyacetic acid, 2,2
dimethymethoxyacetic acid, and 2,2 dimethylphenylpropionic acid.
The alkyl group can be substituted or non-substituted. Substituents
include hydroxy, halogens phenyl, thiol, mercapto, and methylthiol,
Dimethyl substitutions onto the carboxylic acids are preferred.
Pharmaceutically acceptable salts of these compositions are also
included herein.
[0034] The compositions can be administered by any of a range of
methods. Preferred methods include as oral compositions or by pulse
administration.
[0035] One embodiment of the invention is directed to methods for
the treatment of blood disorders and other maladies such as
neoplasia by administering compositions to a patient in pulses.
Pulse therapy according to the methods of the invention is much
more effective than continuous therapy. The effective dose as well
as the total amount of composition needed by the patient to be
therapeutically effective is decreased as compared to amounts
required for similar effect with continuous therapy. Further, as
most chemical compositions are non-toxic at all effective doses,
pulsed administration can be continued for very long periods with
no adverse effects to the patient.
[0036] Another embodiment of the invention is directed to methods
for the stimulation of cell proliferation by the administration of
erythropoietin or other cell stimulatory agent to a patient and the
administration of a chemical composition of the invention in
pulses. Such a treatment regimen prepares bone marrow cells for
stimulation and increases overall hemoglobin expression and
production in the body.
[0037] These compositions can be used, either with or without
pulsing, for the treatment of not only blood disorders, but for
other disorders such as neoplasia.
[0038] Other objects and advantages of the invention are set forth
in part in the description which follows, and in part, will be
obvious from this description, or may be learned from the practice
of the invention.
DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows primer extension analysis of globin mRNA
demonstrates a 2.4-26 fold increase in .gamma.-globin mRNA was
induced over constitutive levels in untreated control K562 cells by
Arg (arginine butyrate), PAA (ST 1; phenoxyacetic acid), ST 7
(AMHCA; .alpha.-methylhydrocinnamic acid), ST 20 (DMB or DMBA; 2,2
dimethylbutyric acid), ST 32 (2-methoxycinnamic acid), ST 33 (2
methyl hydrocinnamic acid), ST 34 (cis-2-methoxycinnamic acid), ST
37 (3,4 dimethoxy phenyl acetic acid), ST 38 (3-3,4-dimethoxy
phenyl propionic acid), ST 40 (2,5 {dimethoxy phenyl} acetic acid),
ST 44 (3,5 dimethoxy 4-hydroxy cinnamic acid), ST 47 (transcinnamic
acid). Butyric acid produced a 2-fold increase in .gamma.-globin
expression compared to untreated control cells. Fold increase over
control levels is shown.
[0040] FIGS. 2A and 2B show comparisons on cell proliferation. FIG.
2A shows comparison of the proliferation of 32D cells in the
presence of optimal IL-3 (25 U/ml), low IL-3 (0.5 U/ml; 50 fold
depletion) and in the absence of IL-3, which results in uniform
cell death by apoptosis.
[0041] FIG. 2B shows comparison of proliferative rates of
multi-lineage IL-3 dependent cells in the presence of a low
concentration of IL3 alone and with the addition of erythropoietin
(EPO) at 3 U/ml. G-CSF (granulocyte-colony stimulating factor) at
100 U/ml, and 1.0 mM concentrations of PAA, AMHCA, DMB PMBA),
butyric acid, DMHAA (dimethylhydroxyacetic acid). Withdrawal of
IL-3 completely and addition of butyrate to the low concentration
(0.5 U/ml) of IL-3 resulted in decreased cell proliferation and
cell death. Addition of test compounds resulted in continued cell
proliferation at rates similar to those induced by EPO and
G-CSF.
[0042] FIG. 3 shows induction of reticulocytes in C57 mice treated
with AMHCA (ST 7 or ST 007) for 7 days. Increases of 2.5 and 6 fold
over baseline reticulocytes was observed (shows a 3 and 6 fold
increase in RBC production). The treatment period is shown by the
horizontal bar above the graph. A similar increase was not observed
in controls which were similarly handled and treated with saline
and bled (phlebotomized) the same amount for 21 days. Controls had
no significant increase in redculocyte counts.
[0043] FIG. 4 shows pharmacokinetics after oral administration of
single doses of PAA, DMBA (in humans) and AMHCA (in monkeys) in
primates. Plasma levels persisted in the millimolar range far above
concentrations which are necessary for hematologic effects in vitro
(shown by arrow) for greater than 6 hours following oral doses of
40-500 mg/kg body weight. This demonstrates that these compounds
are useful in vivo and are resistant to rapid metabolism.
[0044] FIG. 5 shows the rate of increase in c-myb and c-myc
expression in 32D cells compared to control cells cultured with low
IL-3 treated with G-CSF (positive control), EPO (positive control),
ST 7 or 7 (AMHCA), ST 14 or 14 (DMHCA; 2, 2 dimethylhydrocinnamic
acid), ST 20 or 20 (DMBA), PAA, ST 30 or 30 (BMHCA or
.beta.-aminohydrocinnamic acid), DL-.beta.ABA (DL-.beta. amino
butyric acid), ST 24 or 24 (DMPA; 2,2 dimethyl propionic acid), and
ST 27 or 27 (DMMAA or 2, 2 dimethyl methoxy acetic acid). White
bars represent fold increases at day 1 and black bars fold
increases at day 7. Baseline (or the 0 level) represents 0.5 U/ml
IL-3. The myb gene has been shown to be an important regulator of
hematopoietic cell proliferation, differentiation and
apoptosis.
[0045] FIG. 6 shows the rate of increase or decrease in histone and
actin expression (as negative control) in 32D cells treated with
G-CSF, EPO, ST 7, 14, 20, PAA, 30, DL-.beta.ABA, 24 and 27. No
significant change in the expression of these genes was observed
with exposure to the test compounds. This demonstrates that the
increase in c-myb and c-myc expression is specific.
[0046] FIG. 7 shows the rate of proliferation of 32D cells with low
IL-3 (0.5 U/ml) after treatment with AMHCA (ST 007) (to increase
c-myc and c-myb expression) as compared to treatment with butyrate.
Cells die and are do not proliferate in the presence of butyrate
whereas proliferation increases 5 fold over 4 days in the presence
of ST 007 (i. e. increased c-myc and c-myb expression translates
into increased cellular proliferation).
[0047] FIG. 8 shows hematologic effects of ST 007 in a Baboon (RBC
proliferation translates from in vitro data to in vivo data).
[0048] FIG. 9 shows effects of ST 7 on hemoglobin and platelets (i.
e. ST 7 acts on multiple cell lineages).
[0049] FIG. 10 shows how compounds act on very primitive and
multipotential stem cells as shown in this chart.
[0050] FIG. 11 shows Northern Blots for the growth related
genes.
[0051] FIG. 12 shows the increase in reticulocytes vs. days of
treatment in mice with phenylacetic acid.
[0052] FIG. 13 shows white blood cell stimulation in a baboon by a
methylhydrocinnamic acid (AMHCA).
DESCRIPTION OF THE INVENTION
[0053] As embodied and broadly described herein, the present
invention is directed to compositions and methods for the
administration of pharmaceutical compositions useful for the
treatment and prevention of disorders including cell proliferative
disorders such as malignancies and cytopenias, and blood disorders
such as an anemia, sickle cell syndrome and thalassemia.
[0054] We have found that a number of compositions provide
excellent results in treating many of these disorders. The
compounds include .alpha.-methylhydrocinnamic acid (trans and cis);
2-methylhydrocinnamic acid (trans and cis); 2- and
3-methoxycinnamic acid (trans and cis); 4-chlorophenoxy
-2-propionic acid; 3,4 dimethoxycinnamic acid; 3,4 dimethoxyphenyl
acetic acid; 3-3,4 dimethoxy phenyl propionic acid;
2-(4'-methoxyphenoxy)propionic acid; 2,5 dimethoxyphenyl acetic
acid; hydrocinnamic acid; 3-phenylpropionic acid; 2,2
dihydrocinnamic acid; 2,methylbutyric acid, 2,2 dimethylbutyric
acid; 2,2 dimethylpropionic acid; 2,2 dimethylphenoxy acetic acid;
2,2 dimethylmethoxy acetic acid; 2,2 dimethylphenyl propionic acid;
.alpha.-methyl lactate methyl ether; benzoyl formic acid; D,L
.alpha.-amino butyric acid; D,L 0-amino butyric acid;
.beta.-aminohydrocinnamic acid; .alpha.-methyl lactic acid; and
dimethyl hydroxyacetic acid.
[0055] A preferred group of compositions include C.sub.1-C.sub.4
substituted and/or phenyl-substitutions on carboxylic acids.
Preferably it is a C.sub.1-C.sub.4 alkyl substitution. The alkyl or
phenyl moiety can be substituted or non-substituted. Preferred
substituents include hydroxy, halogens, phenyl, thiol, mercapto and
methyl thiol.
[0056] Preferred carboxylic acids include cinnamic acids (such as
hydrocinnamic acid), acetic acids and propionic acids.
[0057] The C.sub.1-C.sub.4 alkyl is preferably methyl. Preferably,
it is a dimethyl substitution.
[0058] Preferred compounds include C.sub.1-C.sub.4 substituted
phenoxyacetic acid, C.sub.1-C.sub.4 substituted cinnamic acid,
C.sub.1-C.sub.4 phenoxy acetic acid, C.sub.1-C.sub.4 substituted
propionic acid and C.sub.1-C.sub.4 substituted butyric add. More
preferred compounds include C.sub.1-C.sub.4 alkyl and/or phenyl
substitution on carboxylic adds such as .alpha.-methylhydrocinnamic
acid, 3,4 dimethoxycinnamic acid, 2-methylhydrocinnamic acid, 2-
and 3-methoxycinnamic acid, 3,4 dimethoxyphenylacetic acid, 3-3,4
dimethoxyphenylpropionic acid, 2,5 dimethoxyphenylacetic acid, 2,2
dimethylbutyric acid, 2,2 dimethylpropionic acid, 2,2
dimethylphenoxyacetic acid, 2,2 dimethymethoxyacetic acid, and 2,2
dimethylphenylpropionic acid. The alkyl group can be substituted or
non-substituted. Substituents include hydroxy, halogens phenyl,
thiol, mercapto, and methylthiol, Dimethyl substitutions onto the
carboxylic acids are preferred. Pharmaceutically acceptable salts
of these compositions are also included herein.
[0059] These compounds can be administered by known techniques such
as orally, intraperitoneally, etc.
[0060] Preferably, the compounds are manufactured in such means
that they can be administered orally.
[0061] In another embodiment, the compounds are administered
intravenously.
[0062] In a preferred method they are delivered by pulse
therapy.
[0063] It has been discovered that a variety of chemicals useful
for the treatment of blood and other disorders are more effective
when administered to a patient in pulses. Pulse therapy is not a
form of discontinuous administration of the same amount of a
composition over time, but comprises administration of the same
dose of the composition at a reduced frequency or administration of
reduced doses.
[0064] One embodiment of the invention is directed to compositions
with a mechanism of action involving regulation of histone
deacetylase by a chemical compound such as glycerol, acetic acid,
butric acid, and an amino-n-butric acid (such as d- or
1-amino-n-butyric acid, .alpha.- or .beta.-amino-n-butyric acid).
Some butyric acid compounds, such as arginine butyrate or
isobutamide may also be useful. See also, U.S. Pat. Nos. 4,822,821
and 5,025,029. Thus, one can regulate histone deacetylase to
enhance globin production by administering an effective amount of a
compound selected from the group consisting of glycerol, acetic
acid, butyric acid, and amino-n-butyric acid, in a pharmaceutically
acceptable carrier or diluent. Preferably, the compound is an
amino-n-butric acid.
[0065] According to these methods, blood and other disorders can be
effectively treated and without unnecessary adverse side effects to
the patient. Although most compositions are generally safe and
non-toxic at therapeutic doses, pulsed administration further
reduces risks associated with, for example, toxicity, allergic
reactions, the build-up of toxic metabolites and inconveniences
associated with conventional treatment. In addition, chemical
compositions, being useful at a reduced dose and frequency, have a
substantially reduced risk of induced tolerance. Drugs are not
inactivated by cellular enzymes or cleared from cells and organs
prior to having the desired effect. Further, long-term therapy,
typically required for the amelioration of many blood disorders,
becomes possible. Consequently, doses necessary for maintaining a
constant effect for the patient are steady and material costs and
inconveniences associated with administration are substantially
reduced.
[0066] One embodiment of the invention is directed to the pulsed
administration of pharmaceutical compositions for the treatment or
prevention of a blood disorder. Pulsed administration is
surprisingly more effective than continuous treatment as pulsed
doses are often lower than would be expected from continuous
administration of the same composition. Each pulse dose can be
reduced and the total amount of drug administered over the course
of treatment to the patient is minimized.
[0067] In traditional forms of therapy, repeated administration is
designed to maintain a desired level of an active ingredient in the
body. Very often, complications that develop can be attributed to
dosage levels that, to be effective, are near toxic or otherwise
harmful to normal cells. In contrast, with pulse therapy, in vivo
levels of drug drop below that level required for effective
continuous treatment. Therefore, pulsing is not simply the
administration of a sufficiently large bolus such that there will
be therapeutically sufficient drug available for a long period of
time. Pulsed administration can substantially reduce the amount of
the composition administered to the patient per dose or per total
treatment regimen with an increased effectiveness. This represents
a significant saving in time, effort and expense and, more
importantly, a lower effective dose substantially lessens the
number and severity of complications that may be experienced by the
patients. As such, pulsing is surprisingly more effective than
continuous administration of the same composition.
[0068] Preferably, compositions contain chemicals that are
substantially non-toxic. Substantially non-toxic means that the
composition, although possibly possessing some degree of toxicity,
is not harmful to the long-term health of the patient. Although the
active component of the composition may not be toxic at required
levels, there may also be problems associated with administering
the necessary volume or amount of the final form of the composition
to the patient. For example, if the composition contains a salt,
although the active ingredient may be at a concentration that is
safe and effective, there can be a harmful build-up of sodium,
potassium or another ion. With a reduced requirement for the
composition or at least the active component of that composition,
the likelihood of such problems can be reduced or even eliminated.
Consequently, although patients may have minor or short term
detrimental side-effects, the advantages of taking the composition
outweigh the negative consequences.
[0069] Methods for the pulsed administration of compositions of the
invention are preferably used for the treatment of blood disorders
such as hemoglobinopathies (e.g. sickle cell anemia, thalassemia),
neoplastic diseases including tumors, leukemias,
lymphoproliferative disorders and metastases, and cell
proliferative disorders such as viral-induced malignancies (e.g.
latent virus infections) and cytopenia including red and white
blood cell anemia, leukopenia, neutropenia and thrombocytopenia.
Compositions most effective at pulsed administration are typically
non-toxic or non-cytotoxic chemicals without any substantial
proteinaceous active component at the therapeutically effective
pulsed dose. Preferably, treatment does not stimulate apoptosis in
the cells being directly treated or in the otherwise normal cells
of the body which will also be exposed to the composition.
[0070] Individual pulses can be delivered to the patient
continuously over a period of several hours, such as about 2, 4, 6,
8, 10, 12, 14 or 16 hours, or several days, such as 2, 3, 4, 5, 6,
or 7 days, preferably from about 1 hour to about 24 hours and more
preferably from about 3 hours to about 9 hours. Alternatively,
periodic doses can be administered in a single bolus or a small
number of injections of the composition over a short period of
time, typically less than 1 or 2 hours. For example, arginine
butyrate has been administered over a period of 4 days with
infusions for about 8 hours per day or overnight, followed by a
period of 7 days of no treatment. This has been shown to be an
effective regimen for many thalassemic disorders. Fetal hemoglobin
levels rise substantially and there is a significant rise in the
number of both adult and fetal hemoglobin expressing cells.
Substantially means that there are positive consequences that raise
the patient's standard of living such as, for example, increased
activity or mobility, fewer side-effects, fewer hospital stays or
visits to the physician, or fewer transfusions.
[0071] The interval between pulses or the interval of no delivery
is greater than 24 hours and preferably greater than 48 hours, and
can be for even longer such as for 3, 4, 5, 6, 7, 8, 9 or 10 days,
two, three or four weeks or even longer. As the results achieved
may be surprising, the interval between pulses, when necessary, can
be determined by one of ordinary skill in the art. Often, the
interval between pulses can be calculated by administering another
dose of the composition when the composition or the active
component of the composition is no longer detectable in the patient
prior to delivery of the next pulse. Intervals can also be
calculated from the in vivo half-life of the composition. Intervals
may be calculated as greater than the in vivo half-life, or 2, 3,
4, 5 and even 10 times greater the composition half-life. For
compositions with fairly rapid half lives such as arginine butyrate
with a half-life of 15 minutes, intervals may be 25, 50, 100, 150,
200, 250 300 and even 500 times the half life of the chemical
composition.
[0072] The number of pulses in a single therapeutic regimen may be
as little as two, but is typically from about 5 to 10, 10 to 20, 15
to 30 or more. In fact, patients can receive drugs for life
according to the methods of this invention without the problems and
inconveniences associated with current therapies. Compositions can
be administered by most any means, but are preferable delivered to
the patient orally or as an injection (e.g. intravenous,
subcutaneous, intraarterial, infusion or instillation, and more
preferably by oral ingestion. Various methods and apparatus for
pulsing compositions by infusion or other forms of delivery to the
patient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958;
4,948,592; 4,965,251 and 5,403,590.
[0073] Compositions administered in pulses have the surprising
benefit of reducing the overall load of drug on the patient as the
total amount of drug administered can be substantially less than
that amount that has been therapeutically administered by
conventional continuous therapy. For example, arginine butyrate has
been shown to be effective at continuous administration at about
2000 mg/kg patient weight. Doses of between about 400 to 1500
mg/kg, preferably from about 600 to 1000 mg/kg and more preferably
from 700 to 800 mg/kg, when administered in pulses, are
surprisingly more beneficial as measured by a rise in fetal
hemoglobin levels in thalassemic patients. Typical pulsed amounts
of arginine butyrate are from about 2 to about 20 g/kg/month, and
preferably from about 3 to about 10 g/kg/month wherein the patient
receives a total of less than about 20 kg per month, preferably
less than about 15 kg per month and more preferably less than about
10 kg per month. The amounts administered per pulse as well as the
total amount of the composition received by the patient over the
regimen is substantially reduced. Preferably, the therapeutically
effective pulsed dose is less than the continuous dose, or less
than one half, one third, one quarter, one fifth, one tenth or even
one twentieth of the therapeutic continuous dose of the same
composition or even less.
[0074] A treatment regimen can be considered effective if it
stimulates globin chain expression or the proliferation of
erythroblasts or other erythroid progenitor cells, for example with
hemoglobinopathy patients, the proliferation of cells such as white
blood cells or platelet forming cells, or reduces the number of
proliferating cells in, for example, a tumor or other malignancy.
Cell numbers are usually most easily determined from peripheral
blood sampling or from calculations of tumor size.
[0075] Another embodiment of the invention is directed to methods
for the pulsed administration of compositions to a patient along
with the pulsed or non-pulsed administration of other compositions
or therapies for the treatment or amelioration of a disorder.
Pulsing of either or both of the compositions can, in part,
synchronize cell development, as there is an increased
proliferation of erythrocytes and an increased expression of
hemoglobin, specifically, fetal hemoglobin. Compositions and
therapies which can be pulsed include most of the known or
conventional or already well-known treatment regimens. One
preferable treatment involves the pulsed or continuous
administration of erythropoietin, or another bone marrow cell
stimulant, followed by the pulsed administration of a chemical
composition of the invention. This regimen has the beneficial
effect of stimulating the process of E/Mega cell to erythrocyte
development and proliferation which can be followed by stimulation
of fetal globin gene expression from the newly proliferated cells.
Following such treatments, fetal globin levels in the body rise
substantially and much higher than would have been expected from
conventional continuous therapy.
[0076] A blood disorder is any disease or malady which could be
characterized as a direct or indirect consequence of a defect or
disease of hemoglobin producing cells or the production of
hemoglobin. The blood disorder may be associated with an anemia
such as sickle cell anemia, hemolytic anemia, infectious anemia,
aplastic anemias, hypoproliferative or hypoplastic anemias,
sideroblastic anemias, myelophthisic anemias, antibody-mediated
anemias, anemias due to enzyme-deficiencies or chronic diseases,
anemias due to blood loss, radiation therapy or chemotherapy,
thalassemias including .alpha.-like and .beta.-like thalassemias.
Treatable blood disorders also include syndromes such as hemoglobin
C, D and E disease, hemoglobin lepore disease, and HbH and HbS
diseases. Treatment ameliorates one or more symptoms associated
with the disorder. Symptoms typically associated with blood
disorders include, for example, anemia, tissue hypoxia, organ
dysfunction, abnormal hematocrit values, ineffective
erythropoiesis, abnormal reticulocyte (erythrocyte) count, abnormal
iron load, the presence of ring sideroblasts, splenomegaly,
hepatomegaly, impaired peripheral blood flow, dyspnea, increased
hemolysis, jaundice, anemic crises and pain such as angina
pectoris.
[0077] Compositions to be administered according to the methods of
the invention are preferably physiologically stable and safe, and
contain one or more chemical compounds that increase the extent or
magnitude of hematopoiesis, increase the proliferation of
hemoglobin expressing and other cells, increase or balance the
expression of globin proteins or increase or stimulate the specific
expression of functional globin protein such as .gamma.-globin.
Stimulation of specific gene expression involves activation of
transcription or translation promoters or enhancers, or alteration
of the methylation pattern or histone distribution along the gene
to promote expression. Expression may also be stimulated by
inhibition of specific transcription or translation repressors,
activation of specific transcription or translation activation
factors, or activation of receptors on the surface of particular
populations of cells. Stimulation may recruit additional cells to
marrow, reprogram differentiated cells to express hemoglobin or
switch to the expression of an embryonic, fetal or other
globin-like peptide. Stimulation may also activate a previously
dormant or relatively inactive genes which substitutes for the
defective or damaged gene products such as, for example, the
post-natally suppressed genes which encode .epsilon., .delta. or
.gamma. globin, which can substitute for adult .beta. globin, or
.zeta. globin which can substitute for a defective or deficient
.alpha. globin.
[0078] Alternatively, compositions may be used to turn down the
expression of those genes whose products are being over expressed
and thereby disrupting the balanced production of normal globin
proteins. Genes whose expression or whose balanced expression can
be effected by the compositions include the globin genes such as
the various forms of the .zeta.-type genes, the .epsilon.-type
genes, the .alpha.-type genes, the .beta.-type genes, the
.delta.-type genes, the .gamma.-type genes and at least partially
functional pseudo-globin genes.
[0079] The mechanism of action of many of the chemical compounds or
active ingredients of compositions for the treatment of blood
disorders involves effecting one or more of the processes of cell
proliferation, cell recruitment, specific hemoglobin expression,
heme synthesis or globin chain synthesis. Cell proliferation may be
increased, for example, by stimulating stem cells, CFUs, BFUs,
megakaryocytes, myeloid cells, platelets, white blood cells or
pro-erythrocyte colony growth, or decreased, for example, by
effecting a cell's period in or ability to transverse a stage (S,
G.sub.0, G.sub.1, M) of the cell cycle. Cell recruitment may be
promoted through the expression of specific cytokines such as cell
surface receptors or secreted factors. Hemoglobin expression can be
increased or decreased by affecting heme expression, globin peptide
expression, heme/globin peptide assembly, globin peptide
glycosylation or globin transport through the golgi apparatus.
Globin expression can be increased or decreased by altering
chromatin and/or nucleosome structure to render .alpha. genetic
element more or less susceptible to transcription, by altering DNA
structure, for example, by methylation of G residues, by affecting
the activity of cell-specific transcription or translation factors
such as activators or repressors, or by increasing the rate of
transcription or translation. For example, useful chemical
compounds include C.sub.1-C.sub.4 alkyl substituted or phenyl
substituted carboxylic acid compounds such as phenoxyacetic acid,
methoxyacetic acid, substituted-cinnamic acid such as dimethyl
hydrocinnamic acid, .alpha.-methyl cinnamic acid and
.alpha.-methylhydrocinnamic acid (AMHCA) stimulate alterations in
binding or removal of transcription factors from the proximal
promoter region of certain genes of the .gamma.- and .beta.-globin
gene clusters and thereby increase post-natally suppressed gene
expression.
[0080] Chemical compounds preferably increase the expression of
hemoglobin, increase the expression of one or more embryonic or
fetal globin genes or increase the number of hemoglobin expressing
or fetal globin expressing reticulocytes. Preferably, compositions
increase embryonic or fetal globin gene expression or embryonic or
fetal reticulocyte counts greater than about 2%, more preferably
greater than about 5%, and even more preferably greater than about
9%. For comparative purposes, a 4% increase in fetal globin gene
expression equates to about 20% to 25% rise or increase in fetal
globin in peripheral blood samples. Consequently, an increase of
greater than about 1% fetal globin expression, preferably greater
than about 3%, or about 1% fetal globin expressing cells,
preferably greater than about 3%, can alleviate symptoms associated
with beta globin disorders.
[0081] Hemoglobin expression, globin expression and cell
proliferation can be assayed by measuring fold increases in
expressed amounts of specific protein or numbers of specific cells
in treated samples as compared to untreated controls. Using this
criteria, compositions preferably increase the amount of hemoglobin
expression, the amount of globin expression, the number of
hemoglobin expressing cells or the number of globin expressing
cells by greater than or equal to about two-fold, preferably about
four-fold and more preferably about eight-fold.
[0082] Chemical compounds are preferably optically pure with a
specific conformation (plus {+} or minus {-}), absolute
configuration (R or S), or relative configuration (D or L).
Particular salts such as sodium, potassium, magnesium, calcium,
choline, amino acid, ammonium or lithium, or combinations of salts
may also be preferred, however, certain salts may be more
advantageous than others. For example, chemical compounds that
require high doses may introduce too much of a single salt to the
patient. Sodium is generally an undesirable salt because at high
doses, sodium can increase fluid retention resulting in tissue
destruction. In such instances, combinations of different salts or
alternative salts can be used.
[0083] In addition to the above chemical compounds, other compounds
include derivatives of these chemicals. Derivatives are chemical or
biological modifications of the parent compound and include
analogs, homologs, next adjacent homologs and compounds based on
any of the foregoing. Analogs include both structural and
functional analogs. Functional analogs are those compounds which
are functionally related to the activity of the parent compound.
Structural analogs are those compounds related to the parent
compound in the arrangement or number of carbon atoms. For example,
such compounds may have double or triple covalent bonds wherein the
parent has a single covalent bond. Homologs are those compounds
which have the same number of carbon atoms as the parent compound,
but further comprise additional moieties such as one or more
phosphate groups (PO.sub.4), sulfate groups (SO.sub.3), amines and
amides (NH.sub.3), nitrate groups (NO.sub.2), acidified or
esterified carbon atoms or combinations thereof. Next adjacent
homologs are those compounds with one more or less carbon atom.
Related compounds include those compounds which have been modified
such as by substitutions and/or additions. For example, compounds
of the invention may be substituted with one or more halogens such
as chlorine (Cl), fluorine (F), iodine (I), bromine (Br) or
combinations of these halogens. As known to those of ordinary skill
in the art, halogenation can increase the polarity, hydrophilicity
or lipophilicity or a chemical compound which can be a desirable
feature, for example, to transform a chemical compound into a
composition which is more easily tolerated by the patient or more
readily absorbed by the epithelial lining of the gastrointestinal
tract. Such compositions could be orally administered to
patients.
[0084] Therapeutically effective chemical compounds may be created
by modifying any of the above chemical compounds so that after
introduction into the patient, these compounds metabolize into
active forms, such as the forms above, which have the desired
effect on the patient. Compounds may also be created which are
metabolized in a timed-release fashion allowing for a minimal
number of introductions which are efficacious for longer periods of
time. Combinations of chemical compounds can also produce useful
new compounds from the interaction of the combination. Such
compounds may also produce a synergistic effect when used in
combination with other known or other compounds.
[0085] Compositions may also comprise proteinaceous agents such as
cytokines that will increase the extent or magnitude of
hematopoiesis, increase the proliferation of hemoglobin expressing
cells, increase or balance the expression of hemoglobin
macromolecules or increase or stimulate the specific expression of
alternate globin genes such as .gamma.-globin. Such proteinaceous
agents include steel factor, insulin, erythropoietin (EPO),
interferon (IFN), insulin growth factor (IGF), stem cell factor
(SCF), macrophage-colony stimulating factor (M-CSF),
granulocyte-colony stimulating factor (G-CSF), GM-CSF, growth
factors such as fibroblast-derived growth factor (FGF), epidermal
growth factor (EGF) and platelet-derived growth factor (PDGF),
nerve growth factor (NGF), vascular endothelial growth factor
(VEGF), bone morphogenic proteins (BMPs), the interleukins (IL)
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, etc., activin also
referred to as erythroid differentiation factor (EDF) or
follicle-stimulating hormone releasing protein (FRP), inhibin, stem
cell proliferation factor (SCPF) and active fragments, subunits,
derivatives and combinations of these proteins. Erythropoietin,
activin and SCF all stimulate the proliferation of stem cells,
committed cells and erythroid progenitor cells, and can also
stimulate the expression of embryonic globin, fetal globin or
partly functional pseudo-globin expression. The hematopoietic
factor, steel factor, also referred to as kit ligand, mast cell
growth factor and stem cell factor, recruits and stimulates the
proliferation of hemoglobin expressing cells and the specific
expression of embryonic or fetal globin. Proteinaceous agents of
the invention may also be animated, glycosylated, acylated,
neutralized, phosphorylated or otherwise derivatized to form
compositions which are more suitable for the method of
administration to the patient or for increased stability during
shipping or storage.
[0086] Compositions may be physiologically stable at
therapeutically effective concentrations. Physiological stable
compounds are compounds that do not break down or otherwise become
ineffective upon introduction to a patient prior to having a
desired effect. Compounds are structurally resistant to catabolism,
and thus, physiologically stable, or coupled by electrostatic or
covalent bonds to specific reagents to increase physiological
stability. Such reagents include amino acids such as arginine,
glycine, alanine, asparagine, glutamine, histidine or lysine,
nucleic acids including nucleosides or nucleotides, or substituents
such as carbohydrates, saccharides and polysaccharides, lipids,
fatty acids, proteins, or protein fragments. Useful coupling
partners include, for example, glycol such as polyethylene glycol,
glucose, glycerol, glycerin and other related substances.
[0087] Physiological stability can be measured from a number of
parameters such as the half-life of the compound or the half-life
of active metabolic products derived from the compound. Certain
compounds of the invention have in vivo half lives of greater than
about fifteen minutes, preferably greater than about one hour, more
preferably greater than about two hours, and even more preferably
greater than about four hours, eight hours, twelve hours or longer.
Although a compound is stable using this criteria, physiological
stability can also be measured by observing the duration of
biological effects on the patient. These effects include
amelioration or elimination of patient symptoms, an increase in
number or appearance of hemoglobin producing cells, or an
alteration, activation or suppression of specific gene expression,
such as, for example, the persistence of fetal globin chain
expression in blood cells.
[0088] Symptoms may be clinically observed or biologically
quantified. For example, observed symptoms are those which can be
clinically perceived and include pathological alterations in
cellular morphology such as red cell sickling, anemic crises,
jaundice, splenomegaly, hepatomegaly, hemorrhaging, tissue damage
due to hypoxia, organ dysfunction, pain such as angina pectoris,
fatigue including shortness of breath, weakness and poor exercise
ability, and pallor. Clinical symptoms which are important from the
patient's perspective include a reduced frequency or duration, or
elimination of the need for transfusions or chelation therapy.
Quantifiable biological symptoms are those which can be more
accurately measured such as anemia, enzyme activity, hematocrit and
hemoglobin levels, decreased cell viability, ineffective
erythropoiesis, abnormal reticulocyte count, abnormal iron loads,
inadequate peripheral blood flow, anuria, dyspnea, hemolysis and
specific gene expression. Other quantifiable biological activities
include, for example, the ability to recruit and stimulate the
proliferation of hemoglobin expressing cells, the ability to
increase hemoglobin expression, the ability to balance .alpha.-type
and .beta.-type globin gene expression or the ability to increase
expression of embryonic, fetal or at least partially functional
pseudo-globin genes. Preferably, a stable compound of the invention
has an in vivo half-life of greater than about 15 minutes, a serum
half-life of greater than about 15 minutes, or a biological effect
which continues for greater than 15 minutes after treatment has
been terminated or the serum level of the compound has decreased by
more than half.
[0089] Compositions are not significantly biotransformed, degraded
or excreted by catabolic processes associated with metabolism.
Although there may be some biotransformation, degradation or
excretion, these function are not significant if the composition is
able to exert its desired effect. Catabolic processes include
deamination of aminases, hydrolysis of esters and amides,
conjugation reactions with, for example, glycine or sulfate,
oxidation by the cytochrome p450 oxidation/reduction enzyme system
and degradation in the fatty acid pathway. Hydrolysis reactions
occur mainly in the liver and plasma by a variety of non-specific
hydrolases and esterases. Both deaminases and amidases, also
localized in the liver and serum, carry out a large part of the
catabolic process. Reduction reactions occur mainly intracellularly
in the endoplasmic reticulum and transferases perform conjugation
reactions mainly in the kidneys and liver.
[0090] Compositions are also preferably safe at effective dosages.
Safe compositions are compositions that are not substantially toxic
(e.g. cytotoxic or myelotoxic), or mutagenic at required dosages,
do not cause adverse reactions or side effects, and are well
tolerated. Although side effects may occur, compositions are
substantially safe if the benefits achieved from their use outweigh
disadvantages that may be attributable to side effects. Unwanted
side effects include nausea, vomiting, hepatic or renal damage or
failure, hypersensitivity, allergic reactions, cardiovascular
problems, gastrointestinal disturbances, seizures and other central
nervous system difficulties, fever, bleeding or hemorrhaging, serum
abnormalities and respiratory difficulties.
[0091] Compositions useful for treating blood disorders preferably
do not substantially affect the viability of a cell such as a
normal mammalian cell, the cell being treated or effected by the
chemical compound. Normal cell viability, the viability of an
untransformed or uninfected cell, can be determined from analyzing
i Ad the effects of the composition on one or more biological
processes of the cell. Detrimental interference with one or more of
these cellular processes becomes significant when the process
becomes abnormal. Examples of quantitatable and qualifiable
biological processes include the processes of cell division,
protein synthesis, nucleic acid (DNA or RNA) synthesis, nucleic
acid (principally DNA) fragmentation and apoptosis. Others
processes include specific enzyme activities, the activities of the
cellular transportation systems such as the transportation of amino
acids by system A (neutral), system B (acidic) or system C (basic),
and the expression of a cell surface protein. Each of these
parameters is easily determined as significantly detrimental, for
example, in tissue culture experiments, in animal experiments or in
clinical studies using techniques known to those of ordinary skill
in the art. Abnormal cell division, for example, can be mitosis
which occurs too rapidly, as in a malignancy, or unstably,
resulting in programmed cell death or apoptosis, detected by
increased DNA degradation. The determination of abnormal cell
viability can be made on comparison with untreated control cells.
Compositions preferably increase normal cell viability. Increased
cell viability can be determined by those of ordinary skill in the
art using, for example, DNA fragmentation analysis. A decreased
amount of fragmentation indicates that cellular viability is
boosted. Determinations of increased or decreased viability can
also be concluded from an analysis of the results of multiple
different assays. Where multiple tests provide conflicting results,
accurate conclusions can still be drawn by those of ordinary skill
based upon the cell type, the correctness or correlation of the
tests with actual conditions and the type of composition.
[0092] Compositions can be prepared in solution as a dispersion,
mixture, liquid, spray, capsule or as a dry solid such as a powder
or pill, as appropriate or desired. Solid forms may be processed
into tablets or capsules or mixed or dissolved with a liquid such
as water, alcohol, saline or other salt solutions, glycerol,
saccharides or polysaccharide, oil or a relatively inert solid or
liquid. Liquids administered orally may include flavoring agents
such as mint, cherry, guava, citrus, cinnamon, orange, mango, or
mixed fruit flavors to increase palatability. Pills, capsules or
tablets administered orally may also include flavoring agents.
Additionally, all compositions may further comprise agents to
increase shelf-life, such as preservatives, anti-oxidants and other
components necessary and suitable for manufacture and distribution
of the composition. Compositions further comprise a
pharmaceutically acceptable carrier. Carriers are chemical or
multi-chemical compounds that do not significantly alter or effect
the active ingredients of the compositions. Examples include water,
alcohols such as glycerol and polyethylene glycol, glycerin, oils,
salts such as sodium, potassium, magnesium and ammonium, fatty
acids, saccharides or polysaccharides. Carriers may be single
substances or chemical or physical combinations of these
substances.
[0093] Another embodiment of the invention is directed to
combinations of compositions comprising a chemical compound in
combination with an agent know to positively affect hemoglobin
expression or hemoglobin expressing cells. The agent may be a
chemical compound such as acetic acid, butric acid, D- or
L-amino-n-butyric acid, .alpha.- or .beta.-amino-n-butric acid,
arginine butyrate or isobutyramide, all disclosed in U.S. Patent
Nos. 4,822,821 and 5,025,029. Others include butyrin, 4-phenyl
butyrate (C.sub.6H.sub.5CH.sub.2CH.sub.2CH.sub.2COOH),
phenylacetate (C.sub.6H.sub.5CH.sub.2COOH), phenoxy acetic acid,
all of which and more are disclosed in U.S. Pat. No. 4,704,402, and
U.S. patent application Ser. No. 08/398,588 (entitled "Compositions
for the Treatment of Blood Disorders" filed Mar. 3, 1995), and
derivatives, salts and combination of these agents. Alternatively,
the agent may be a hematopoietic protein such as erythropoietin,
steel factor, insulin, an interleukin, a growth factor, hormones
such as activin or inhibin, disclosed in U.S. Pat. Nos. 5,032,507
and 4,997,815, and active fragments and combinations of these
proteins either with each other or with other chemical compounds.
Such composition may have additive or synergistic effects.
[0094] Another embodiment of the invention is directed to methods
for the treatment of patients with blood disorder comprising the
pulsed administration of one or more compositions. Compositions to
be administered contain a therapeutically effective pulsed amount
of a chemical compound or proteinaceous agent. A therapeutical
effective pulsed amount is that amount which has a beneficial
effect to the patient by alleviating one or more symptoms of the
disorder or simply reduce premature mortality. For example, a
beneficial effect may be a decrease in pain, a decrease in
duration, frequency or intensity of crises, an increased
hematocrit, an improved erythropoiesis, a reduced or eliminated
necessity for chelation therapy, an increased reticulocyte count,
an increased peripheral blood flow, a decreased hemolysis,
decreased fatigue or an increased strength. Preferably, a
therapeutic amount is that amount of chemical compound or agent
that stimulates or enhances the expression of non-adult globin such
as embryonic or fetal globin, or the proliferation of embryonic,
fetal or adult globin expressing cells. A therapeutically effective
amount for continuous therapy is typically greater than a
therapeutically amount that is effective in pulsed therapy.
Consequently, pulsed therapy exposes the patient to lower levels of
the composition and/or the active ingredient than would be needed
with non-pulse therapy.
[0095] Compositions provided to the patient may include any
combination of the proteins or chemical compounds described herein
or known to those of ordinary skill in the art. The patient may be
a domesticated animal such as a dog, cat, horse, cow, steer, pig,
sheep, goat or chicken, or a wild animal, but is preferably a human
or another primate. Administration may be to an adult, an
adolescent, a child, a toddler, a neonate or an infant, or
administered in utero. Administration of the composition may be
short term, continuous or sporadic as necessary. Patients with a
suspected or diagnosed with a blood disorder may only require
composition treatment for short periods of time or until symptoms
have abated or have been effectively eliminated.
[0096] Compositions can be directly or indirectly administered to
the patient. Indirect administration is performed, for example, by
administering the composition to cells ex vivo and subsequently
introducing the treated cells to the patient. The cells may be
obtained from the patient to be treated or from a genetically
related or unrelated patient. Related patients offer some advantage
by lowering the immunogenic response to the cells to be introduced.
For example, using techniques of antigen matching, immunologically
compatible donors can be identified and utilized.
[0097] Direct administration of a composition may be by oral,
parenteral, sublingual, rectal such as suppository or enteral
administration, or by pulmonary absorption or topical application.
Parenteral administration may be by intravenous injection,
subcutaneous injection, intramuscular injection, intra-arterial
injection, intrathecal injection, intra peritoneal injection or
direct injection or other administration to one or more specific
sites. Injectable forms of administration are sometimes preferred
for maximal effect in, for example, bone marrow. When long term
administration by injection is necessary, venous access devices
such as medi-ports, in-dwelling catheters, or automatic pumping
mechanisms are also preferred wherein direct and immediate access
is provided to the arteries in and around the heart and other major
organs and organ systems.
[0098] Another effective method of administering the composition is
by transdermal transfusion such as with a dermal or cutaneous
patch, by direct contact with, for example, bone marrow through an
incision or some other artificial opening into the body.
Compositions may also be administered to the nasal passages as a
spray. Arteries of the nasal area provide a rapid and efficient
access to the bloodstream and immediate access to the pulmonary
system. Access to the gastrointestinal tract, which can also
rapidly introduce substances to the blood stream, can be gained
using oral, enema, suppository, or injectable forms of
administration. Compositions may be administered as a bolus
injection or spray. Compositions that may or may not be pulsed may
be given sequentially over time (episodically) such as every two,
four, six or eight hours, every day (QD) or every other day (QOD),
or over longer periods of time such as weeks to months.
Compositions may also be administered in a timed-release fashion
such as by using slow-release resins and other timed or delayed
release materials and devices.
[0099] Orally active compositions are more preferred as oral
administration is usually the safest, most convenient and
economical mode of drug delivery. Oral administration is usually
disadvantageous because compositions are poorly absorbed through
the gastrointestinal lining. Compounds which are poorly absorbed
tend to be highly polar. Consequently, compounds which are
effective, as described herein, may be made orally bioavailable by
reducing or eliminating their polarity. This can often be
accomplished by formulating a composition with a complimentary
reagent which neutralizes its polarity, or by modifying the
compound with a neutralizing chemical group. Oral bioavailability
is also a problem because drugs are exposed to the extremes of
gastric pH and gastric enzymes. These problems can be overcome in a
similar manner by modifying the molecular structure to withstand
very low pH conditions and resist the enzymes of the gastric mucosa
such as by neutraing an ionic group, by covalently bonding an ionic
interaction, or by stabilizing or removing a disulfide bond or
other relatively labile bond.
[0100] Treatments to the patient may be therapeutic or
prophylactic. Therapeutic treatment involves administration of one
or more compositions of the invention to a patient suffering from
one or more symptoms of the disorder. Symptoms typically associated
with blood disorders include, for example, anemia, tissue hypoxia,
organ dysfunction, abnormal hematocrit values, ineffective
erythropoiesis, abnormal reticulocyte count, abnormal iron load,
splenomegaly, hepatomegaly, impaired peripheral blood flow,
dyspnea, increased hemolysis, jaundice, anemic crises and pain such
as angina pectoris. Relief and even partial relief from one or more
of these symptoms corresponds to an increased life span or simply
an increased quality of life. Further, treatments that alleviate a
pathological symptom can allow for other treatments to be
administered.
[0101] Prophylactic treatments involve pulsed administration of a
composition to a patient having a confirmed or suspected blood
disorder without having any overt symptoms. For example, otherwise
healthy patients who have been genetically screened and determined
to be at high risk for the future development of a blood disorder
may be administered compositions of the invention prophylactically.
Administration can begin at birth and continue, if necessary, for
life. Both prophylactic and therapeutic uses are readily acceptable
because these compounds are generally safe and non-toxic.
[0102] Another embodiment of the invention is directed to a method
for regulating the expression of .alpha. globin gene in a mammalian
cell. Briefly, the cell is exposed to an effective amount of a
composition. A poorly expressed or quiescent globin gene of the
cell is stimulated to increase the expression of its protein
product. An effective amount of the composition is that amount
which increases the extent or magnitude of hematopoiesis, increases
the proliferation of hemoglobin expressing cells, increases,
decreases or balances expression from one or more globin genes, or
increases or stimulates the specific expression of one or more
globin genes such as an alpha (.alpha.) globin gene, a zeta
(.zeta.) globin gene, an epsilon (.epsilon.) globin gene, a beta
(.beta.) globin gene, a delta (.delta.) globin gene, .alpha. gamma
(G-.gamma. or A-.gamma.) globin gene, or an, at least, partly
functional pseudo-globin gene. Cells can be treated in culture or
in vivo. Cultures of treated cells will produce increased amounts
of hemoglobin and preferably embryonic or fetal globin. This
hemoglobin can be harvested for introduction to a patient or the
stimulated cells themselves can be administered to the patient.
Alternatively, recombinant cells containing .alpha. globin gene
which can be stimulated by compositions of the invention can be
utilized. These recombinant cells may be heterologous or homologous
natural cells, or synthetically created cells such as a lipid
vesicles.
[0103] Another embodiment of the invention is directed to a method
for regulating the proliferation of red blood cells and,
preferably, specifically regulating the expression of fetal
hemoglobin. As above, an effective amount of a composition is
administered in pulses to, for example, a cell population obtained
from stem cells, bone marrow, cord blood, yolk sac cells, or fetal
cells such as fetal liver cells, or combinations thereof, ex vivo.
The pulse-treated cells, or purified products harvested from these
cells, are then administered to a patient in vivo. This method can
be utilized to treat blood disorders in patients by increasing the
amount of one or more different types of globin or hemoglobin
expressing cells can alleviate symptoms associated with a blood
disorder. Cells can be obtained from volunteers or the patients to
be treated. Alternatively, treated cells or products derived from
treated cells can be harvested, purified by, for example, column
chromatography, and utilized for other medical applications such as
diagnostic or other treatment monitoring screening kits.
[0104] Another embodiment of the invention is directed to a method
for ameliorating a blood disorder by administering a
therapeutically effective amount of a pharmaceutical composition
containing an agent that stimulates the expression of .alpha.
globin gene or stimulates the proliferation of hemoglobin
expressing cells wherein the composition does not significantly
decrease viability of the cell being treated or a normal cell. The
therapeutically effective amount is that amount which ameliorates
one or more symptoms of the blood disorder or reduces premature
mortality. A normal cell is a relatively healthy mammalian cell
that is not otherwise infected or transformed. Viability can be
assayed by determining the effect of the composition on cell
division, protein or nucleic acid synthesis, biochemical salvage
pathways, amino acid or nucleotide transport processes, nucleic
acid fragmentation or apoptosis and comparing the effects observed
to control cells. Pulsing, according to the described treatment
regimens, can also be used to administer these and other
compositions of the invention and their effects tested in tissue
culture, in vivo or by cell counting.
[0105] Patients with blood disorders are typically quite infirm
with, for example, iron damaged organs and systems. Most treatments
further tax the patient's already frail health in an effort to
combat the disorder. This is true for both arginine butyrate and
isobutyramide which decrease cell viability as determined in DNA
fragmentation assays. To decrease cell viability is not desired for
the treatment of blood disorders and may even be harmful.
Surprisingly, many of the pulsed compositions maintain or,
preferably, increase cell viability. This is a great benefit in the
treatment of blood disorders and can significantly increase thee
chances for a successful outcome for the patient. For example, the
pulsed administration of phenoxyacetic acid or butyric acid ethyl
ester both reduce DNA fragmentation in fragmentation assays, and
phenoxyacetic acid and .alpha.-methyl hydrocinnamic acid do not
significantly alter system A transport of amino acids.
[0106] As such, pulsed composition can be used to treat or prevent
iron overloaded or iron deficient systems such as occurs in
transfused patients and anemic patients with thalassemia or sickle
cell anemia. As chemicals of the compositions of the invention
regulate systems that exploit iron, the amount of free and the
amount of available iron in a patient's system can be regulated and
carefully controlled. Chelation therapy, often the only
conventional treatment available for iron over-loaded transfusion
patients, may be lessened or avoided entirely. As chelation therapy
is often uncertain and with some risk of its own, the long-term
prognosis for these patients is greatly improved.
[0107] Another embodiment of the invention is directed to a method
for increasing fetal hemoglobin comprising the pulsed
administration of a composition to a patient. For example,
hemoglobin F content of blood so treated is increased greater than
about 2%, preferably greater than about 5% and more preferably
greater than about 10%. Patients which can be treated include any
mammal such as a human. Chemical compounds which could be utilized
include C.sub.1-C.sub.4 substituted and phenyl substituted phenoxy
acetic acid, C.sub.1-C.sub.4 substituted and phenyl substituted
cinnamic acid, C.sub.1-C.sub.4 substituted and/or phenyl
substituted hydrocinnamic acid, .alpha.-methyl hydrocinnamic acid,
C.sub.1-C.sub.4 substituted and phenyl substituted acetic acid,
C.sub.1-C.sub.4 substituted and phenyl substituted propionic acid,
and C.sub.1-C.sub.4 substituted and/or phenyl substituted butyric
acid, or a derivative or modification thereof. Such methods are
useful to treat or prevent blood disorders in the same or a
different patient. For example, to treat the same patient, the
compound can be pulse administered for a therapeutically effective
period of time to allow the hemoglobin content of just the globin
protein content to rise. Alternatively, the patient can be treated
and the patient's blood collected at peak times of hemoglobin or
globin production, collected and stored, and administered to
another patient or re-administered to the same patient. Such
treatments would be useful therapies for those being treated with
radiator therapy, chemotherapy, bone marrow transplants, blood
diseases, such as sickle cell disease and thalassemia, and other
disorders which would be alleviated with an increased blood
hemoglobin content.
[0108] Another embodiment of the invention is directed to methods
for the treatment of a patient with an infection or a neoplastic
disorder comprising the pulsed administration of a therapeutically
effective composition. Treatable infectious diseases include
bacterial infections such as sepsis and pneumonia, infections
caused by bacterial pathogens such as, for example, Pneumococci,
Streptococci, Staphylococci, Neisseria, Chlamydia, Mycobacteria,
Actinomycetes and the enteric microorganisms such as enteric
Bacilli; viral infections caused by, for example, a hepatitis
virus, a retrovirus such as HIV, an influenza virus, a papilloma
virus, a herpes virus (HSV I, HSV II, EBV), a polyoma virus, a slow
virus, paramyxovirus and corona virus; parasitic diseases such as,
for example, malaria, trypanosomiasis, leishmania, amebiasis,
toxoplasmosis, sarcocystis, pneumocystis, schistosomiasis and
elephantitis; and fungal infections such as candidiasis,
phaeohyphomycosis, aspergillosis, mucormycosis, cryptococcosis,
blastomycosis, paracoccidiodomycosis, coccidioidomycosis,
histomycosis, actinomycosis, nocardiosis and the Dematiaceous
fungal infections.
[0109] Anti-neoplastic activity includes, for example, the ability
to induce the differentiation of transformed cells including cells
which comprise leukemias, lymphomas, sarcomas, neural cell tumors,
carcinomas including the squamous cell carcinomas, seminomas,
melanomas, neuroblastomas, mixed cell tumors, germ cell tumors,
undifferentiated tumors, neoplasm due to infection (e.g. viral
infections such as a human papilloma virus, herpes viruses
including Herpes Simplex virus type I or II or Epstein-Barr virus,
a hepatitis virus, a human T cell leukemia virus (HTLV) or another
retrovirus) and other malignancies. Upon differentiation, these
cells lose their aggressive nature, no longer metastasize, are no
longer proliferating and eventually die and/or are removed by the T
cells, natural killer cells and macrophages of the patient's immune
system. The process of cellular differentiation is stimulated or
turned on by, for example, the stimulation and/or inhibition of
gene specific transcription. Certain gene products are directly
involved in cellular differentiation and can transform an actively
dividing cell into a cell which has lost or has a decreased ability
to proliferate. An associated change of the pattern of cellular
gene expression can be observed. To control this process includes
the ability to reverse a malignancy. Genes whose transcriptional
regulation are altered in the presence of compositions of the
invention include the oncogenes myc, ras, myb, jun, fos, abi and
srcr The activities of these gene products as well as the
activities of other oncogenes are described in J. D. Slamon et al.
(Science 224:256-62, 1984).
[0110] Another example of anti-neoplastic activity includes the
ability to regulate the life cycle of the cell, the ability to
repress angiogenesis or tissue regeneration through the blockade or
suppression of factor activity, production or release, the ability
to regulate transcription or translation, or the ability to
modulate transcription of genes under angiogenesis, growth factor
or hormonal control. These activities are an effective therapy
particularly against prostatic-neoplasia and breast carcinomas.
Additional anti-neoplastic activities include the ability to
regulate the cell cycle for example by effecting time in and
passage through S phase, M phase, G.sub.1 phase or G.sub.0 phase,
the ability to increase intracellular cAMP levels, the ability to
inhibit or stimulate histone acetylation, the ability to methylate
nucleic acids and the ability to maintain or increase intracellular
concentrations of anti-neoplastic agents.
[0111] The neoplastic disorder may be any disease or malady which
could be characterized as a neoplasm, a tumor, a malignancy, a
cancer or a disease which results in a relatively autonomous growth
of cells. Neoplastic disorders prophylactically or therapeutically
treatable with compositions of the invention include small cell
lung cancers and other lung cancers, rhabdomyosarcomas, chorio
carcinomas, glioblastoma multiformas (brain tumors), bowel and
gastric carcinomas, leukemias, ovarian cancers, prostate cancers,
osteosarcomas or cancers which have metastasized. Diseases of the
immune system which are treatable by these compositions include the
non-Hodgkin's lymphomas including the follicular lymphomas,
Burlitt's lymphoma, adult T-cell leukemias and lymphomas,
hairy-cell leukemia, acute myelogenous, lymphoblastic or other
leukemias, chronic myelogenous leukemia, and myelodysplastic
syndromes. Additional diseases treatable by the compositions
include virally -induced cancers wherein the viral agent is EBV,
HPV, HIV, CMV, HTLV-1 or HBV, breast cell carcinomas, melanomas and
hematologic melanomas, ovarian cancers, pancreatic cancers, liver
cancers, stomach cancers, colon cancers, bone cancers, squamous
cell carcinomas, neurofibromas, testicular cell carcinomas and
adenocarcinomas.
[0112] In another embodiment of the invention, compositions may be
pulse administered in combination with other anti-neoplastic agents
or therapies to maximize the effect of the compositions in an
additive or synergistic manner. Cytokines which may be effective in
combination with the compositions include growth factors such as B
cell growth factor (BCGF), fibroblast-derived growth factor (FDGF),
granulocyte/macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), macrophage colony
stimulating factor (M-CSF), epidermal growth factor (EGF), platelet
derived growth factor (PDGF) nerve growth factor (NGF), stem cell
factor (SCF), and transforming growth factor (TGF). These growth
factors plus a composition may further stimulate cellular
differentiation and/or the expression of certain MHC antigens or
tumor specific antigens. For example, BCGF plus a composition may
be effective in treating certain B cell leukemias. NGF plus a
composition may be useful in treating certain neuroblastomas and/or
nerve cell tumors. In a similar fashion, other agents such as
differentiating agents may be useful in combination with a
composition to prevent or treat a neoplastic disorder. Other
differentiating agents include B cell differentiating factor
(BCDF), erythropoietin (EPO), steel factor, activin, inhibin, the
bone morphogenic proteins (BMPs), retinoic acid or retinoic acid
derivatives such as retinol, the prostaglandins, and TPA.
[0113] Alternatively, other cytokines and related antigens in
combination with a composition may also be useful to treat or
prevent neoplasia. Potentially useful cytokines include tumor
necrosis factor (TNF), the interleukins (IL-1, IL-2, IL-3, etc.),
the interferon proteins (IFN) IFN-.alpha., IFN-.gamma., and
IFN-.gamma., cyclic AMP including dibutyryl cyclic AMP, hemin,
hydroxyurea, hypoxanthine, glucocorticoid hormones, dimethyl
sulfoxide (DMSO), and cytosine arabinoside, and anti-virals such as
acyclovir and gemciclovirs. Therapies using combinations of these
agents would be safe and effective against malignancies and other
forms of cancer. Combinations of therapies may also be effective in
inducing regression or elimination of a tumor or some other form of
cancer such as pulsed compositions plus radiation therapy, toxin or
drug conjugated antibody therapy using monoclonal or polyclonal
antibodies directed against the transformed cells, gene therapy or
specific anti-sense therapy. Effects may be additive, logarithmic,
or synergistic, and methods involving combinations of therapies may
be simultaneous protocols, intermittent protocols or protocols
which are empirically determined.
[0114] Another embodiment of the invention comprises methods for
the pulse administration of compositions for the treatment of
neoplastic disorders by augmenting conventional chemotherapy,
radiation therapy, antibody therapy, and other forms of therapy.
Compositions containing chemical compounds in combination with
chemotherapeutic agents, enhance the effect of the chemotherapeutic
agent alone. Compositions decrease the expression or activity of
proteins responsible for lowering the intra -cellular concentration
of chemotherapeutic agents. Proteins responsible for resistance to
drugs and other agents, the multi-drug resistance (MDR) proteins,
include the .beta.-glycoprotein (Pgp) encoded by the mdr-1 gene.
Consequently, conventional drugs for the treatment of neoplastic
disorders accumulate at higher concentrations for longer periods of
time and are more effective when used in combination with the
compositions herein. Some conventional chemotherapeutic agents
which would be useful in combination therapy with compositions of
the invention include the cyclophosphamide such as alkylating
agents, the purine and pyrimidine analogs such as mercapto-purine,
the vinca and vinca -like alkaloids, the etoposides or etoposide
like drugs, the antibiotics such as deoxyrubocin and bleomycin, the
corticosteroids, the mutagens such as the nitrosoureas,
antimetabolites including methotrexate, the platinum based
cytotoxic drugs, the hormonal antagonists such as antiinsulin and
antiandrogen, the antiestrogens such as tamoxifen an other agents
such as doxorubicin, L-asparaginase, dacarbazine (DTIC), amsacrine
(mAMSA), procarbazine, hexamethylmelamine, and mitoxantrone. The
chemotherapeutic agent could be given simultaneously with the
compounds of the invention or alternately as defined by a protocol
designed to maximize drug effectiveness, but minimize toxicity to
the patient's body.
[0115] Another embodiment of the invention is directed to aids for
the treatment of human disorders such as infections, neoplastic
disorders and blood disorders. Aids contain compositions of the
invention in predetermined amounts which can be individualized in
concentration or dose for a particular patient. Compositions, which
may be liquids or solids, are placed into reservoirs or temporary
storage areas within the aid. At predetermined intervals, a set
amount of one or more compositions are administered to the patient.
Compositions to be injected may be administered through, for
example, mediports or in-dwelling catheters. Aids may further
comprise mechanical controls or electrical controls devices, such
as a programmable computer or computer chip, to regulate the
quantity or frequency of administration to patients. Examples
include both single and dual rate infusers and programmable
rinsers. Delivery of the composition may also be continuous for a
set period of time. Aids may be fixed or portable, allowing the
patient as much freedom as possible.
[0116] The following examples are offered to illustrate embodiments
of the present invention, but should not be viewed as limiting the
scope of the invention.
EXAMPLES
[0117] Treatment of K562 Cells and Analysis of Globin mRNA
[0118] K562 cells kindly provided by Dr. George Atweh were cultured
with 10% fetal bovine serum (Sigma, St. Louis, Mo.) and RPMI media
(Grand Island Biological Company, Grand Island, New York) in a
humidified atmosphere with 5% CO2/95% air. Compounds were tested at
a final concentration of 1 mM at neutral pH and included butyric
acid, phenoxyacetic acid, dimethylbutric acid, alpha
methylhydrocionamic acid, 2,3, and 4-methoxyhydrocinnamic acid,
dihydrocinnamic acid, methoxycinnamic acid, methoxyacetic acid,
phenylpropionic acid, amino hydrocinnamic acid, DL .beta.- and
DL-.beta.amino-n-butyric acid, cinnamic acid, and 2
methylhydrocinnamic acid (Aldrich Chemical Company, St. Louis,
Mo.). Additional compounds studied included dimethylhydroxy acetic
acid, dimethylpropionic acid, dimethylphenoxyacetic acid, and
dimethylmethoxyacetic acid. After three days of culture with these
agents, mRNA was purified and .alpha., .beta., and .gamma. globin
mRNA was analyzed by primer extension using oligonucleotide primers
and quantitation on a PhosphoImager as previously described. A
representative autoradiogram and a summary of the globin expression
induced by the effective compounds is shown in FIG. I and Table
I.
[0119] Proliferation Studies Using 32D Cells
[0120] 32D cells were cultured in RPMI media with 10% fetal bovine
serum (Sigma, St. Louis, Mo.), 100 mM glutamine (GIBCO), and murine
IL3 (20 U/ml) (Biosource International). Growth factor controls
used included the standard concentration of IL-3 required for
proliferation of these cells (25 U/ml) and a 50-fold lower
concentration (0.5 U/ml), and erythropoietin (3 U/ml) or G-CSF (
U/ml), (Amgen, Thousand Oaks, Calif.). The test compounds were
added at final concentrations of 1 mM. As a cell density of
2.5-10.times.10.sup.5 is necessary for growth of this cell line,
this density was maintained by passing the cells at three day
intervals or by concentrating the cells when apoptosis occurred.
Proportions of cells which were viable or apoptotic, and the
fraction of cells in each part of the cell cycle was assessed by
incubating the cells with Trypan blue and enumeration, and with
propidium iodide incubation and FACScan analysis as previously
described.
[0121] In Vivo Administration in Mice
[0122] To determine if a prototype test compound has in vivo
activity in stimulating erythropoeisis, methylhydrocinnamic acid
was administered to C.sub.57 black mice. Mice were cared for and
experiments were performed according to regulations of the
Committee on Animal Research at the University of Southern Alabama.
The test compound was administered by intraperitoneal injection
three times per date for seven days at a total daily dose of 300
mg/kg. Blood (50 .mu.l) was sampled from the retro-orbital space
and reticulocytes were quantitated by staining with 1% brilliant
cresyl blue and counting the percentage of reticulum positive cells
in 1000 cells. Reticulocytes were computed to control mice which
were injected with the same volume of normal saline and which
received a 50 .mu.l daily phlebotomy for twenty-one days without a
significant change in hematocrit or a significant increase in
reticulocyte counts (B. Pace, unpublished observations).
[0123] Phamrucokinetic Studies
[0124] Baboons were cared for according to regulations of the
Committee on Animal Care at the University of Oklahoma Health
Sciences Center. Chronic indwelling venous and arterial catheters
which were maintained using sterile technique for blood sampling.
Compounds were administered by nasogastric tube and blood was
collected to determine drug plasma levels at regular intervals
following single oral doses. Three doses of one compound were also
studied in two human volunteers. The test compounds were analyzed
after ether extraction of the plasma, separation by HPLC, and
quantitated by comparison to a spilled internal standard of
heptanoic acid according to previously described methods.
[0125] The effects of the representative compounds which have been
synthesized or selected for resistance to beta oxidative metabolism
and glucuronidation in stimulating .gamma. globin gene expression
in a human erythroid-like cell line and for their effects on cell
growth utilizing a multi-lineage murine hematopoietic cell line,
32D. This cell line is dependent on high concentrations of IL-3 for
growth. 32D cells undergo apoptotic cell death if IL-3 is
completely withdrawn and do not proliferate when IL-3
concentrations are reduced by 50-fold over the levels required for
proliferation. No condition or growth factor has been found to
abrogate the IL-3 dependency of this cell line for cell
proliferation (Patel, Oncogene 13:1197 (1996)). In the presence of
IL3 depletion, these cells also terminally differentiate along the
erythroid lineage in the presence of erythropoietin or terminally
differentiate into mature granulocytes in the presence of G-CSF.
Some test compounds which stimulated .gamma. globin expression also
supported proliferation of this multi-lineage cell line and
prevented apoptotic cell death when IL-3 was withdrawn. In vivo
activity was also found with a prototype test compound administered
mice. Finally. half-lives for three prototype compounds were found
to be several hours following oral administration to baboons,
demonstrating potential therapeutic utility.
RESULTS
[0126] Effects of the test compounds on globin gene expression were
assessed by comparing the ratios of I globin:a globin mRNA and the
ratio of .gamma. globin mRNA in treated cells were compared to
.gamma. globin MRNA in control cells, adjusted for an internal
control. .gamma. globin mRNA increased by 2.4 to 26-fold over
untreated (control) K562 cells in the presence of several of the
test compounds, as shown in Table I. The most active compounds in
stimulating .gamma. globin compared to control cells were
phenoxyacetic acid, 2-methylhydrocinnamic acid and
.alpha.-methylhydrocinnamic acid, 2-methoxycinnamic acid,
dimethoxyphenyl acetic acid, butyrate, and 2,2-dimethylbutyrate.
These results are consistent with previous finding that these and
similar compounds stimulate .gamma. globin expression in erythroid
progenitors cultured from human subjects and from CD34+ cells
isolated from fetal liver.
[0127] Under culture conditions containing recombinant murine IL-3
at 50 U/ml, the optimal concentrations for cell proliferation,
apoptosis was detected in less than 10% of the cell population and
32D cells doubled after 3 days. Apoptosis in 32D cells increased to
80% when IL-3 levels were decreased by 50-fold, from 25 U/ml to 0.5
U/ml. The cells underwent 100% apoptosis in the complete absence of
IL-3 (FIG. 2). In contrast, when IL-3 was decreased to 0.5 U/ml,
the minimum required to prevent apoptosis, cell numbers did not
significantly, increase and plateaued after 2 days. In the presence
of 0.5 U/ml IL-3 and addition of erythropoietin or G-CSF, cell
proliferation occurred along the erythroid and myeloid pathways
respectively as has been previously reported, and cell numbers
increased by 2-3 fold over 5 days, shown in FIG. 2. In the presence
of phenoxyacetic acid, alpha methylhydrocinnamic acid,
dimethylbutyric acid, DL-.beta.amino-n-butyric acid and
dimethylhydroxyacetic acid, however, cell proliferation increased 2
to 3-fold despite the low concentration of IL-3 (FIG. 2). In
contrast, addition of 1 mM butyrate with the low concentration of
IL-3 resulted in cell death. Addition of 1 mM test compounds with
the same low concentration of IL-3 resulted in a 2.5-3-fold
increase in cell proliferation with several compounds above that
observed with the marginal IL-3 concentration alone and resulted in
a degree of proliferation similar to that induced by erythropoietin
and G-CSF.
[0128] Bioavailability and Pharmacokinetic studies of certain test
compounds were performed in juvenile baboons using oral delivery of
the test compounds via gavage. Millimolar plasma levels were
detected following single oral doses of phenoxyacetic acid,
dimethylbutyrc acid, and methylhydrocinnamic acid and these levels
persisted for 6 hours or longer. Calculated half-lives were 6.5,
6.8, and 7.6 hours respectively, following doses of 100-500 mg/kg.
These peak plasma levels are higher than the concentration of
compound which was required for .gamma. globin stimulation in
primary hematopoietic cells in vitro.
[0129] To determine how general the effects of these compounds may
be, one lead compound, alpha methylhydrocinnamic acid was also
administered to mice. Administration of the compound resulted in a
200-600% (2-6 fold) increase in reticulocytes over baseline.
Reticulocytosis was observed in a step-wise manner and in a
time-frame consistent with the time required for development and
maturation of late and early murine erythroid progenitors (3 and 6
days, respectively). Reticulocytes increased by only 6-8% after 21
days of saline-injections in control mice phlebotomized to the same
(50 .mu.l/day) degree. Hematocrits did not change in controls over
this time (B. Pace, unpublished observations).
[0130] Cell proliferation stimulation is transgenic mice, baboons,
human cell culture, and a murine multi-lineage cell line by the
active compounds, genes whose expression is increased early in cell
proliferation induced by hematopoietic growth factors such as IL-3
and erythropoietin were examined. RNA was extracted from 32D cells
treated with the compounds for one day and for 11 days. Northern
blots were prepared with probes for the early growth related genes
c-myb and c-myc and beta actin and histone H.sub.3 were used as
controls. Increased expression of c-myb occurs transiently, and
early, when growth is induced by erythropoietin and IL-3. See FIG.
5.
[0131] Of multiple compounds tested, c-myb was induced by 3-4 fold
by the compounds methylhydrocinnamic acid, dimethylbutyric acid,
phenoxyacetic acid, DL-beta and D-alpha-amino butyric acid,
2,2-dimethyl methoxyacetic acid, and dimethyl propionic acid (alpha
dimethyl hydrocinnamic acid). C-myb was induced 2-fold with beta
amino hydrocinnamic acid. The growth-related gene c-myc was induced
2-fold by the same active compounds. Actin and histone H.sub.3
mRNAs were not affected by the compounds. See FIGS. 5 and 6.
[0132] FIG. 11 shows the relative steady-state accumulation of
c-myb, c-myc, histone-3, and beta-actin mRNA in IL3-dependent 32D
cells at different time points after exposure of cells to different
test compounds. The first lane is from cells cultured in no IL-3,
lane 2 in 25 U/ml murine IL3 and lanes 3-18 have low IL-3
concentration (0.5 U/ml) plus test compounds. In addition, cells in
lane 4 were treated with 100 U/ml G-CSF, lane 5
2,2-dimethyl-methoxy acetic acid, lane 6 alpha methylhydrocinnamic
acid, lane 7 phenoacetic acid, lane 8 arginine butyrate day 1 and
5, lane 9 .alpha.-dimethyl hydroacetic acid, lane 10
2,2-dimethylbutyric acid, lane 11 beta aminohydrocinnamic acid,
lane 12 2-2-dimethylpropionic acid, lane 13 dimethylhydroxy acetic
acid/a-methyl lactic acid, lane 14 2-2-dimethylphenoxy acetic acid,
lane 15 2,2 dimethyl-l-phenoxyacetic acid, lane 16 cis-2 methoxy
cinnamic acid, lane 17 thioctic acid days 1 and 5, and lane 18
4-chlorophenoxy-2-propionic-ac- id days 1 and 5. All compounds were
tested here at 1 mM. Each set of treated cells is denoted by one
numbered and one unnumbered lane consisting of mRNA from the same
cells treated for days 1 and 11 respectively, except where cells
did not survive to day 11 and only day 1 of treatment is shown. 20
ug of total RNA from each sample were subjected to Northern blot
analysis using specific probes for c-myb, c-myc, actin, and histone
H.sub.3. One day and 11-day samples from the same treated cells
were quantitated by PhosphoImager.
[0133] In Vivo Experiments in Mice Transgenicfor the Human Beta
Globin Gene Locus.
[0134] Three prototype compounds, methylhydrocinnamic acid (MHCA),
phenoxyacetic acid (PAA), and dimethylbutyric acid PMB), were
administered at doses from 100 to 250 mg/kg in two daily doses by
intraperitoneal injection to mice transgenic for a human beta
globin locus YAC containing a silenced gamma globin gene.
Reticulocytes, newly synthesized red blood cells, were counted
daily and non-alpha globin in MRNA was analyzed by Rnase
protection. Only 50 microliters of blood were removed daily for
testing. A 5 to 10-fold increase in reticulocytes and a 1.7-2.4
fold increase in gamma globin mRNA was observed within one week of
therapy with the three protype compounds. In contrast, control mice
to which normal saline was administered, with the same degree of
phlebotomy for testing, had no significant changes in reticulocytes
or globin in mRNA.
[0135] Mice have more rapid metabolic rates than do larger animals,
such as humans and these compounds are still active in mice.
Furthermore, gamma 9fetal) globin has not been readily inducible by
compounds such as alpha amino-n-butyric acid in these same mice.
Accordingly, the results are significant. See the following
table:
1 Reticulocytes .gamma./.gamma. + .beta. mRNA (fold (fold Animal
Day 0 Peak increase) Day 0 Peak increase) DMB-1 2.4 17.7 (7.3) 0.20
0.36 (1.8) DMB-2 4.2 21.3 (5.1) 0.17 0.31 (1.7) MHCA-1 2.9 17.9
(5.6) 0.33 0.80 (2.4) MHCA-2 2.3 23.3 (10.1) 0.14 0.18 (1.5)
[0136] Control mice, to which normal saline was similarly
administered, had no changes in reticulocytes or globin mRNA. [Mice
have a higher metabolic rate than do larger animals, and .gamma.
globin has not always been inducible by rapidly metabolized
butyrates in these mice.]
[0137] FIG. 12 shows increase in young, newly proliferating red
blood cells after treatment with phenoxy acetic acid in four
transgenic mice. Each curette represents one animal. Reticulocytes
increased from 2.5 to 7-fold with the highest increase resulting
from the higher dose.
[0138] Hematopoietic stimulation in a baboon by the compounds AMHCA
is shown in FIG. 13. An increase in multiple blood cell lineages
resulted when a prototype hemoldne compound (a methylhydrocinnamic
acid) was administered for five days to an anemic baboon, which was
being phlebotomized 5% of its blood volume daily. An increase in
both white blood cells and total hemoglobin was observed.
[0139] Mononuclear cells from patients with sickle cell disease or
thalassemia trait were isolated on Ficoll Hypague, washed, and
cultured in methylcellulose media with optimal concentrations of
hematopoietic growth factors IL-3, GM-CSF, Stem Cell Factor, IL6, 3
U/ml Eiythropoietin, insulin, bovine serum albumin, and 0.2-0.5 mM
concentrations of test compounds of derivatives of cinnamic acid
and hydrocinnamic acid. An increase in numbers of erythroid
colonies over and was observed compared to control cultures
containing optimal concentrations of growth factors alone. The
following illustrates some representative cultures:
2 TABLE 1 Mean BFU-E/culture 4 cultures averaged % Increase (per
0.2 million cells) over control Control 192 2 methylbutyric acid
297 55% 3,5 dimethoxy4-hydroxycinnamic 215 11% acid Control 272
Transcinnamic acid 322 18% Control 176 Alpha methylhydrocinnamic
acid 223 32.4% 2 Methylhydrocinnamic acid 212 20.5% 4
Methoxycinnamic acid 191 8.5%
[0140]
3TABLE 2 Effect of Compounds on Fetal and Alpha Globin mRNAs in
K562 Cells Radioactivity .alpha. Fetal Globin Alpha Globin Compound
b (.gamma.) (.alpha.) .gamma./.alpha. Control 915479 118789 7.7
Arginine butyrate 2176523 296132 7.3 Phenoxyacetic acid 2755891
507148 5.4 .alpha.-Methylhydrocinnamic acid 1648056 92979 17.7
2,2-Dimethylbutyric acid 1697936 178751 9.5 trans-2-Methoxycinnamic
acid 957146 36751 26.0 2-Methylhydrocinnamic acid 1388899 89473
15.5 cis-2-Methoxycinnamic acid 2255627 105452 21.4
(3,4-Dimethoxyphenyl)acetic acid 1206529 106875 11.3
3-(3,4-Dimethoxyphenyl)propionic 1858358 191985 9.7 acid
(2,5-Dimethoxyphenyl)acetic acid 1240100 85941 14.4 .alpha.
Radioactivity was determined by phosphorimager. b Compounds were
tested at a final concentration of 1 mM.
DISCUSSION
[0141] Suppression or inhibition of erythropoiesis and general
hematopoiesis in a dose-dependent fashion can be limitations of
butrates and hydroxyurea, respectively, in the treatment of the
p-hemoglobinopathies. Further disadvantages of the butyrates as
optimal therapeutics include their extremely rapid metabolism in
uivo. The current studies arose from a search to identify novel
orally -bioavailable compounds with long in vivo half-lives, which
induce y globin gene expression without simultaneously inducing
cell growth arrest. Extensive investigation of agents which affect
hematopoiesis during the past decade has focused on multipotential
hematopoietic growth factors which stimulate proliferation of
multiple lineages such as IL-3 and GM-CSF, the lineage-specific
growth factors erythropoietin and G-CSF, the differentiating agents
DMSO, butyric acid, retinoic acid, and HMBA and inhibitory factors,
such as TGF-.beta. and IFN-.gamma.. Previous comparison of the
effects of butyric acid, which inhibits erythroid proliferation and
.alpha. amino-n-butrric acid, which slightly stimulates erythroid
progenitor growth, suggested that compounds with slight
modifications may also modulate erythroid cell growth. The findings
herein demonstrate that several classes of simple compounds, with
specific modifications in structure, stimulate the proliferation of
hematopoietic cells and can decrease the requirements for the
multipotential growth factor IL-3. Abrogation of IL-3 requirements
has not been previously found. As these compounds diffuse into
cells freely without requiring receptors and diffuse into
mitochondria, the compounds likely exert their growth stimulating
activities through metabolic pathways as well as through
traditional signaling pathways, and through transcriptional
regulation of growth-related genes.
[0142] The pattern of globin gene stimulation induces in K562 cells
by some of these compounds is complex, in that certain compounds
(butrric and phenoxyacetic acid) stimulated expression of both
.alpha. and .gamma. globin MRNA. This may represent an effect of
inducing differentiation of these cells or of inducing expression
of different globin genes. Other compounds (cis
2-methoxyhydrocinnamic acid) curiously decreased expression of
.alpha. globin, which accentuated the K562 .alpha. thalassemic
phenotype. Such an effect would not be deleterious in human
.beta.-thalassemia, and would be expected to improve overall globin
chain balance. Phenoxyacetic acid, derivatives of hydro-cinnamic
and cinnamic acid, and dimethylbutyric acid induced I globin MRNA
and cellular proliferation. Such compounds particularly merit
further investigation for future consideration as therapeutics of
the beta thalassemias, as the accelerated erythroid apoptosis
characteristic of these diseases severely limits the time-frame
during which any Hemoglobin F stimulant can act to improve globin
chain balance before cell death occurs.
[0143] Several of the compounds studied here do not undergo rapid
metabolism in vivo, as do the simple fatty acids. The phenoxyacetic
and phenylalkylacids and the dimethylated carboxylic acid
derivatives were selected for their structural resistance to usual
routes of metabolism in vivo. A prototype of these compounds, a
methylhydrocinnamic acid, did indeed have activity in mice, and
three prototype compounds had prolonged half-lives in the baboon.
This result is significant because mice have higher rates of
metabolism than do humans and because similar doses of butyrate
were previously not effective in mice transgenic for the human
.gamma. globin gene without previous treatment with 5-azacytidine
or when given at much higher doses. These and similar compounds
particularly with modifications at the fourth position of a phenyl
ring and the 2,2 dimethyl substituted carboxylic acids, appear
attractive as hematopoietic stimulants for all lineages and as
fetal hemoglobin-inducing agents.
* * * * *