HK1100262B - Methods and compositions for the treatment and management of hemoglobinopathy and anemia - Google Patents

Abstract

The present invention is directed to the use of immunomodulatory compounds, particularly members of the class of compounds known as IMiDs<TM>, and more specifically the compounds 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione, to induce the expression of fetal hemoglobin genes, genes essential for erythropoiesis, and genes encoding alpha hemoglobin stabilizing protein, within a population of CD34<+> cells. These compounds are used to treat hemoglobinopathies such as sickle cell anemia or beta-thalassemia, or anemias caused by disease, surgery, accident, or the introduction or ingestion of toxins, poisons or drugs.

Description

Methods and compositions for the treatment and management of hemoglobinopathies and anemia

This application claims priority to U.S. provisional application 60/526,910 filed on 2.12.2003, which is incorporated herein by reference in its entirety.

1. Field of the invention

The present invention relates to a method for the treatment, prevention and/or management of hemoglobinopathies (e.g., sickle cell disease) and other leukemias (e.g., disease or drug-induced anemia) comprising the administration of a compound called IMiD^TMOf thalidomide analogs, in particular IMiD^TM4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (also known as α - (3-aminophthalimido) glutarimide; Celgene Corporation) and 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione (also known as 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione; Celgene Corporation), and pharmaceutical compositions comprising such compounds.

2. Background of the invention

2.1. Sickle cell disease and other hemoglobinopathies

Sickle cell disease ("SCA") is a hereditary hemolytic anemia associated with abnormal hemoglobin (called hemoglobin S). It is reported that this disease is caused by a decrease in the charge in hemoglobin S due to the substitution of amino acids, thereby decreasing the solubility of the substituted hemoglobin S. The Merck Manual of Diagnosis and Therapy, 17 th edition, Merck research Laboratories, Whitehouse Station, NJ, page 878 (1999). The hemoglobin S with reduced solubility makes the rod-like aggregates form semisolid gel, and makes the red blood cells in crescent sickle shape. These deformed and hardened red blood cells adhere to the vascular endothelium, blocking small arterioles and capillaries, causing occlusion and infarction. Because sickle-shaped red blood cells are too fragmented to withstand the mechanical pressure of the blood circulation, hemolysis occurs when they enter the circulation.

SCA is usually associated with a particular race, i.e. descendants of sub-saharan populations in african-americans and other tropical africans. The patient suffers from acute pain caused by sickle-shaped red blood cell occlusion. Sickle-shaped red blood cells have a survival time of about two weeks, while normal red blood cells have a survival time of about four months. Thus, a shortened survival time leads to chronic anemia.

Symptoms of SCA include damage to generation and development; an increased likelihood of infection; a tower-shaped skull; bone changes such as cortical thinning, irregular bone density and new bone formation within the medullary canal; the spleen became smaller due to an automated splenectomy; an increased chance of rheumatic or congenital heart disease; progressive decline in lung and kidney function; and acute chest syndrome. Acute chest syndrome is the leading cause of death and is characterized by a sudden cold, chest pain, leukocytosis and lung hepatocyte infiltration in chest x-rays.

Existing techniques for treating SCA include induction of fetal hemoglobin, relaxation of blood vessels, reduction of red blood cell adhesion, and use of Gardos channel antagonists. Iyamu and Asakura, Expert opin. ther patents, 13 (6): 807-813(2003). Gardos channels are calcium activated potassium channels described by Gardos (Curr. Top. Membr. Transp. 10: 217-277(1978) and Nature London 279: 248-250 (1979)).

Most SCA treatments studied and used are oral administration of Hydroxyurea (HU). It is believed that HU exerts its effects by inducing the production of fetal hemoglobin (HbF). However, HU is not effective in all patients; some patients do not respond at all to HU, while others develop myelosuppression. Iyamu and Asakura, supra. Has used what is called HEMOXIN^TM(formerly known as NIPRISAN)^TM) The extract exerts its anti-sickle formation effect by binding to HbS. See U.S. patentAnd 5,800,819. Iyamu and Asakura, supra. HEMOXIN^TMHas not been approved by the FDA for the treatment of SCA. One group is currently investigating the use of clotrimazole and other Gardos channel blockers to reduce the dehydration properties of sickle-shaped red blood cells. Iyamu and Asakura, supra. However, the efficacy of this compound has not been demonstrated. Other SCA treatments include intravenous solutions of glucose and electrolytes, narcotic analgesics, and infusions for cases of extreme severe anemia. Given the nascent state of most SCA treatments, safe and effective therapies for treating and controlling SCA are needed.

Treatments that increase fetal hemoglobin production are of great interest because they can increase the total hemoglobin content of individuals with hemoglobinopathies or anemia. In adults, two hemoglobins (hemoglobin α and hemoglobin β) predominate, with few other hemoglobin types. In contrast, in the fetus, there are two other hemoglobins, hemoglobin epsilon and hemoglobin gamma. Hemoglobin epsilon is the major form of early development, but disappears in the fetus by about eight weeks. In contrast, hemoglobin γ is present in early development, peaking at about 6-30 weeks of gestation, accounting for 45% of total hemoglobin. Then, it disappears from total hemoglobin from about 6 weeks before birth to about 40 weeks after birth. Although it is still present in the individual after 40 weeks, it thereafter represents less than 2% of the total hemoglobin in the bloodstream.

2.2.IMID^TM

A class of compounds has been identified, called IMiD^TM(Celgene Corporation) or immunomodulatory drugs that exhibit strong inhibition of TNF- α and significant inhibition of LPS-induced monocyte IL1 β and IL12 production. LPS-induced IL-6 is also inhibited, albeit partially, by immunomodulatory compounds. These compounds are potent stimulators of LPS-induced IL-10. IMiD has been demonstrated^TMCD34 regulated along the bone marrow and red blood cell pathway⁺Differentiation of the cells. See U.S. patent application 2003/0235909, published 2003, 12/25, the entire contents of which are incorporated herein by referenceFor reference. IMiD^TMSpecific examples of (D) include, but are not limited to, substituted 2- (2, 6-dioxopiperidin-3-yl) phthalimides and substituted 2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindoles as described in U.S. Pat. Nos. 6,281,230 and 6,316,471 to G.W. Muller et al. Heretofore IMiD^TMHave not been used as candidates for the treatment of hemoglobinopathies or anemia, or as modulators of genes involved in erythropoiesis.

3. Summary of the invention

The present invention relates to methods of treating a subject suffering from anemia or hemoglobinopathy comprising administering an effective amount of a compound of the present invention. Accordingly, in one embodiment, the invention provides a method of treating a subject having anemia or hemoglobinopathy, comprising administering to the subject an immunomodulatory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. In a specific embodiment, the anemia is anemia induced by or associated with administration of a drug or chemotherapy. In another specific embodiment, the immunomodulatory compound is an amino-substituted thalidomide. In a more specific embodiment, the immunomodulatory compound is IMiD^TM. In a more specific embodiment, the IMiD^TMIs α - (3-aminophthalimido) glutarimide (also known as 4- (amino) -2- (2, 6-dioxo- (3-piperidyl)) -isoindoline-1, 3-dione); an analog or prodrug of α - (3-aminophthalimido) glutarimide; 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione; an analog or prodrug of 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione, or a compound of the formula:

in another more specific embodiment, the IMiD is 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -4-aminoisoindoline, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -6-aminoisoindoline, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline, 1, 3 dioxo-2- (2, 6-dioxopiperidin-3-yl) -4-aminoisoindoline, or 1, 3 dioxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline.

In another embodiment, the method of treatment further comprises treating the subject with a second compound, wherein the second compound is a compound that induces fetal hemoglobin, a compound that relaxes blood vessels, a compound that reduces hemoglobin S self-aggregation when covalently bound to hemoglobin S, a compound that acts as a Gardos channel antagonist, or a compound that reduces red blood cell adhesion. In a more specific embodiment, the second compound is hydroxyurea, a guanidino derivative, nitrous oxide, a butyrate or butyrate derivative, an aldehyde or aldehyde derivative, a plant extract having anti-sickle cell forming activity (e.g., NIPRISAN)^TM(HEMOXIN^TM) Clotrimazole, derivatives of triarylmethane, monoclonal antibodies or polyethylene glycol derivatives.

In another specific embodiment, the method of treatment further comprises treating the subject with at least one cytokine. In a more specific embodiment, the at least one cytokine is erythropoietin (Epo), SCF, GM-CSF, Flt-3L, TNF α, IL-3, or any combination thereof. In another specific embodiment of the method, the subject is a mammal. In a more specific embodiment, the individual is a human.

In another embodiment, the invention provides a method of modulating CD34⁺A method of differentiating a stem cell or a precursor cell into a red blood cell, the method comprising differentiating the cell under suitable conditions in the presence of an immunomodulatory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. In a more specific embodimentIn one embodiment, the immunomodulatory compound is an amino-substituted thalidomide. In another more specific embodiment, the immunomodulatory compound is IMiD. In a more specific embodiment, the IMiD is α - (3-aminophthalimido) glutarimide (also known as 4- (amino) -2- (2, 6-dioxo- (3-piperidyl)) -isoindoline-1, 3-dione); an analog or prodrug of α - (3-aminophthalimido) glutarimide; 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione; an analog or prodrug of 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione, or a compound of the formula:

in another more specific embodiment, the IMiD is 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -4-aminoisoindoline, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -6-aminoisoindoline, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline, 1, 3 dioxo-2- (2, 6-dioxopiperidin-3-yl) -4-aminoisoindoline, and 1, 3 dioxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline. In another specific embodiment, said CD34⁺The stem cells or precursor cells are in vitro cells. In another specific embodiment, said CD34⁺The stem cell or precursor cell is an in vivo cell.

In another specific embodiment, the method further comprises contacting the cell with at least one cytokine. In a more specific embodiment, the at least one cytokine is erythropoietin, SCF, GM-CSF, Flt-3L, TNF α, IL-3, or any combination thereof.

The invention also provides pharmaceutical compositions comprising a compound of the invention and another compound or cytokine. Accordingly, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrierA carrier, wherein the second compound is a fetal hemoglobin inducing compound, a vascular relaxing compound, a compound that reduces hemoglobin S self-aggregation when covalently bound to hemoglobin S, a compound that is a Gardos channel antagonist, or a compound that reduces red blood cell adhesion. In a more specific embodiment, the second compound is hydroxyurea, a guanidino derivative, nitrous oxide, a butyrate or butyrate derivative, an aldehyde or aldehyde derivative, a plant extract having anti-sickle cell forming activity (e.g., hemaxin)^TM) Clotrimazole, triarylmethane derivatives, monoclonal antibodies or polyethylene glycol derivatives.

The invention also provides a pharmaceutical composition comprising IMiD and at least one cytokine in a pharmaceutically acceptable carrier. In particular embodiments, the cytokine is erythropoietin (Epo), SCF, GM-CSF, Flt-3L, TNF α, IL-3, or any combination thereof.

The present invention also provides a method of treating a subject having a hemoglobinopathy or anemia, the method comprising administering to the subject a compound in an amount and for a time such that a detectable increase in the level of Alpha Hemoglobin Stabilizing Protein (AHSP) is produced. In one embodiment of this method, the compound is IMiD^TM. In a specific embodiment, the compound is α - (3-aminophthalimido) glutarimide (also known as 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione) or 3- (4 'aminoisoindoline-1' -one) -1-piperidine-2, 6-dione.

As used herein, the term "hemoglobinopathy" refers to any defect in the structure or function of any hemoglobin in an individual, including defects in the primary, secondary tertiary or quaternary structure of hemoglobin caused by any mutation, such as a deletion or substitution mutation in the coding region of any hemoglobin gene, or a mutation in the promoter or enhancer of a gene or deletion of the promoter or enhancer that reduces the amount of hemoglobin produced relative to normal or standard conditions. The term also includes any decrease in the amount or potency of hemoglobin, whether normal or abnormal, caused by an external agent (e.g., disease, chemotherapy, toxin, poison, etc.).

As used herein, "anemia" refers to any decrease in the amount of hemoglobin in the blood stream as compared to normal. This decrease may be due to blood cell loss, iron deficiency, toxins, poisons, diseases, or any other physiological cause.

As used herein, the terms "symptoms of hemoglobinopathy" and "symptoms of anemia" refer to any physiological or biological symptom associated with any hemoglobinopathy or anemia, including, but not limited to, dizziness, shortness of breath, loss of consciousness, fatigue, weakness, hemolysis, pain associated with abnormal hemoglobin, decreased red blood cell count (i.e., decreased hematocrit), decreased oxygen carrying capacity of a given volume of water as compared to normal blood volume, microscopic examination of red blood cell deformation, and the like. The term also includes negative psychological symptoms such as depression, low self-esteem, affection, physical limitation, and the like.

As used herein, the term "IMiD" refers to the class of compounds disclosed in section 5.2 below, including the compounds 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (also known as α - (3-aminophthalimido) glutarimide) and 3- (4 'aminoisoindoline-1' -one) -1-piperidine-2, 6-dione.

As used herein, the terms "CC-5013" and "Revimid" are used^TM"refers to the compound 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione (also known as 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione).

As used herein, the terms "CC-4047" and "Actimid" are used^TM"refers to the compound 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (also known as α - (3-aminophthalimido) glutarimide).

As used herein, the term "CD 34⁺Cell "means CD34⁺Stem cells, progenitor cells, or precursor cells.

As used herein, the term HEMOXIN is used^TMAnd NIPRISAN^TMRefers to a plant extract as disclosed in U.S. patent 5,800,819, characterized by a mixture of about 12 to about 17 parts by weight of Piperguineense seeds, about 15 to about 19 parts by weight of Pterocarpus osun stems, about 12 to about 18 parts by weight of Eugenia caryophylata fruits, and about 25 to about 32 parts by weight of Sorghum bicolor leaves, and optionally 15 to 22 parts by weight of potassium carbonate, wherein the mixture is extracted with cold water. The plant extract has anti-sickle cell formation activity.

4. Description of the drawings

FIG. 1 shows CD34 in the presence of SCF, Flt3-L, GM-CSF and TNF α in the presence of DMSO (control) or 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione⁺Time line for cell differentiation.

FIG. 2 shows the fetal hemoglobin gene hemoglobin epsilon₁Hemoglobin gamma_AAnd hemoglobin gamma_BExpresses a response to DMSO (control) or 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione. Also shown is the 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (CC-4047) pair with haemoglobin epsilon₁Influence of induction of EST concerned.

FIG. 3 shows CD34 after 6 days of culture in the presence of 0, 0.01, 0.1, 1.0, 10 or 100. mu.M 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione⁺The level of glycophorin A marker in the cell.

FIG. 4 shows the results at 0, 0.01, 0.1, 1.0, 10 or 100. mu.M 4 after 6 days of culture(amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione in the presence of CD34⁺Levels of fetal hemoglobin in the cells.

FIG. 5 shows a portion of a microarray showing the relative expression levels of erythrocyte specific genes at days 0,3 and 6 of culture in media containing SCF, Flt3-L, GM-CSF and TNF α. The expression level was determined by hybridizing RNA-derived biotin-labeled cRNA to AffymetrixU133A microarray.

FIG. 6 shows CD34 in the presence of SCF, Flt3-L and IL-3 in the presence of DMSO (control) or 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione⁺Time line for cell expansion followed by differentiation in the presence of SCF and erythropoietin.

FIG. 7 is the results of FACS analysis showing a slight decrease in glycophorin A expression after differentiation in the presence of Epo and SCF in the presence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or DMSO (control). The values in each quadrant indicate the percentage of cells expressing glycophorin a and/or CD 71.

FIG. 8 shows CD34 after 6 days of differentiation in the presence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione, compared to the presence of DMSO control and SCF (50ng/ml) + Epo (4 units/ml)⁺Expression of fetal hemoglobin in the cell is elevated. The concentration of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione was varied from 0.001. mu.M to 10. mu.M. The data points show the percentage of cells expressing fetal hemoglobin as measured by flow cytometry.

FIG. 9 is a FACS analysis showing that increased fetal hemoglobin expression is associated with decreased adult hemoglobin expression (Y-axis). The values in each quadrant indicate the percentage of cells expressing fetal hemoglobin and/or adult hemoglobin. Cells were differentiated for 6 days in the presence of Epo, SCF, and 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or DMSO.

FIG. 10 shows that 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione increases the expression of fetal hemoglobin compared to hydroxyurea or 5-azacytidine. Cells were cultured for 6 days in the presence of SCF (50ng/ml) and Epo (2U/ml), and DMSO (control), 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (0.1, 1, 10. mu.M), 5-azacytidine (0.1, 1. mu.M; toxic 10. mu.M) or hydroxyurea (0.1, 1, 10M). Each bar shows the percentage of cells expressing fetal hemoglobin.

FIG. 11 is a flow cytometric analysis demonstrating the synergistic effect of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione and hydroxyurea in increasing fetal hemoglobin expression. CD34⁺Cells were differentiated for 6 days in the presence of SCF and Epo, and 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione or 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (see section 5.2). The values in each graph show the percentage of cells expressing fetal hemoglobin.

FIG. 12 shows STAT5 gels from UT-7 in the presence or absence of Epo, using 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or DMSO (control). The following figures: absolute levels of STAT5 protein. The upper diagram: levels of phosphorylated STAT5 protein.

5. Detailed description of the invention

5.1.CD34⁺Differentiation of cells into the erythrocyte lineage

The present invention provides for modulating CD34⁺A method for the differentiation of stem cells, precursor cells or progenitor cells into a primary erythroid lineage. The inventors have found that IMiD, when referred to as^TMWhen contacted with such cells under suitable conditions, the class of immunomodulatory compounds that differentiate into red bloodIs a ball system. This differential diversion can be evidenced by characteristic changes in gene expression, including but not limited to increased expression of genes encoding glycophorin A and fetal hemoglobin (e.g., hemoglobin γ and hemoglobin ε). Thus, the methods of the present invention are very useful in providing a method that can increase the yield of a hemoglobin-producing cell population that can replace a naturally-occurring hemoglobin-producing cell population in an individual.

IMiD^TMCan also make differentiated CD34⁺Increased expression of alpha hemoglobin stabilizing protein in a cell, alpha hemoglobin stabilizing protein being one that preferentially binds alpha hemoglobin and not beta hemoglobin or hemoglobin a (Hb alpha hemoglobin₂β₂) The protein of (1). This is advantageous because when the amount of alpha hemoglobin exceeds that of beta hemoglobin, a precipitate is easily formed which damages red blood cells. Thus, AHSP and IMiD-mediated increases in AHSP expression are predicted to be able to modulate pathological states of alpha hemoglobin excess, including beta thalassaemia trait. This effect on AHSP expression, and the increase in fetal hemoglobin expression, is an advantage of IMiD treatment over other drugs that increase fetal hemoglobin expression.

Thus, the present invention provides, in a first aspect, a method of modulating CD34⁺A method of differentiating a cell into a erythroid lineage, comprising differentiating the cell under suitable conditions and in the presence of an immunomodulatory compound, such as IMiD, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. IMiD useful in the present invention^TMExamples of which are disclosed in detail in section 5.2 below. However, IMiD is particularly preferred^TMAre 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione and 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione.

CD34⁺The cells may be any stem cells, progenitor cells, or committed cells capable of differentiating into erythrocytes. These cells may be totipotent or pluripotent, or may be committed to a hematopoietic lineage. CD34⁺The cells may be derived from any source; particularly preferred are "embryoid" derived from placenta"Stem cells. For a description of such embryonic-like stem cells and methods for obtaining them, see U.S. application publication No. US 2003/0180269A 1, published 25/9/2003, which is incorporated herein by reference in its entirety. Other CDs 34 for use in the methods of the invention⁺Cells include stem cells (e.g., hematopoietic stem cells or embryonic stem cells) obtained from any tissue and amorphous progenitor cells obtained from any tissue. When these cells whose differentiation is regulated according to the method of the invention are used for the treatment of anemia or hemoglobinopathies, these CD34 are present in relation to the target receptor⁺The cells may be xenogeneic or autologous.

CD34⁺Differentiation of cells typically occurs within 3-6 days. Culturing CD34 in the presence of IMiD⁺In vitro analysis of the cells (described in the examples) showed that changes in gene expression along the red blood cell pathway were observed on day 3 of culture. The expression of the erythrocyte-specific gene is remarkably increased, and the phenotypic characteristics of the erythrocyte appear in CD34 cultured on the 6 th day⁺In the cell.

Thus, according to the present invention, CD34 may be cultured in vitro in the presence of a compound of the invention (e.g., an immunomodulatory compound, particularly IMiD)⁺The cells are cultured for a period of time sufficient for erythrocyte-specific gene expression, particularly fetal hemoglobin gene expression, and/or cellular characteristics to occur. In various embodiments, CD34⁺The cells may be cultured for 3,6, 9, or 12 days, or longer. The compounds of the invention may be added at once at the beginning of the culture and the culture continued until differentiation is substantially complete, or for 3,6, 9, 12 days or more. Alternatively, the compound of the present invention may be added to the cultured CD34 multiple times during the culture process⁺In the cell. CD34 may be cultured in the presence of one compound of the invention or in the presence of a plurality of different compounds of the invention⁺A cell.

The compounds of the present invention can be used at any concentration of 0.01. mu.M to 10 mM. Preferably, the concentration of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione is 0.01-10. mu.M, and the concentration of 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione is preferably 0.01-100. mu.M.

In addition to in vitro differentiation of CD34⁺Outside the cells, these cells can also be differentiated in the individual. Such an individual is preferably a mammal, more preferably a human. Like in vitro differentiation of CD34⁺As with the cells, CD34 in an individual can be differentiated by administering one or more immunomodulatory compounds of the invention⁺A cell. Such administration may be in a single dose form. Alternatively, one or more compounds of the invention may be administered to an individual multiple times. Such administration can be, for example, over 3,6, 9, 12 or more days, and can be according to the dosage regimen and form described in section 5.4 below.

If CD34 is differentiated in vivo⁺Cells, differentiation may then be accomplished using an immunomodulatory compound alone or in combination with one or more cytokines. For example, for individuals with hemoglobinopathies such as sickle cell anemia or thalassemia, in vivo differentiation can be accomplished by administering one or more immunomodulatory compounds (e.g., 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione) if their SCF and/or erythropoietin levels are higher than normal. Conversely, if the individual has a anemia arising from or characterized by a lower than normal level of erythropoietic cytokines (e.g., SCF or erythropoietin), the cytokines may be administered alone, or concurrently with or prior to administration of the immunomodulatory compound. For example, one or more cytokines (e.g., a combination of SCF, Flt-3L and IL-3) may be administered, e.g., for 3-6 days, followed by, e.g., 3-6 days, of one or more immunomodulatory compounds of the invention, particularly concurrently with SCF and erythropoietin, in an amount sufficient to cause CD34 in the individual to be⁺The fetal hemoglobin expression in the cell produces a detectable increase. Alternatively, such individuals may be given CD34⁺Cells contacted with one or more in vitro cytokines (e.g., SCF, Flt-3L and I)L-3), for example, for 3-6 days, and then administering the cells to the subject with SCF and erythropoietin in an amount sufficient to result in CD34⁺The fetal hemoglobin expression in the cell produces a detectable increase. Such administration may be performed one or more times, and any one or more of such administrations may be effected by administering a compound of the invention (see section 5.3), a second compound (see below), or a combination of all three.

Any of the compounds of the invention (e.g., 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione) can be reacted with CD34⁺The stem cells, progenitor cells or precursor cells are contacted to induce one or more genes associated with or necessary for erythropoiesis and/or hematopoiesis in the cells, particularly one or more genes encoding fetal hemoglobin. In one embodiment, the invention provides a method of inducing one or more genes associated with or necessary for erythropoiesis or hematopoiesis, the method comprising contacting a hematopoietic stem cell, progenitor cell, or precursor cell with an immunomodulator, in the presence of erythropoietin and a stem cell factor, wherein the immunomodulator is present in an amount sufficient for the hematopoietic stem cell, progenitor cell, or precursor cell to express one or more genes encoding fetal hemoglobin. In a specific embodiment, the hematopoietic stem, progenitor, or precursor cells are CD34⁺A cell. In another specific embodiment, the one or more genes associated with or necessary for erythropoiesis or hematopoiesis are genes encoding: kruppel-like factor 1 erythrocytes; a rhesus monkey blood group-related glycoprotein; glycophorin B; integrin α 2 b; a factor associated with red blood cells; glycophorin A; kell blood group precursor; hemoglobin α 2; solute carrier 4, anion exchanger; carbonic anhydrase 1; hemoglobin γ a; hemoglobin γ G; hemoglobin epsilon 1; or any combination of the above. In another specific embodiment, the immunomodulator is IMiD^TM. In a more specific embodiment, the IMiD^TMIs alpha- (3-aminophthalimido) glutarimide; alpha is alphaAn analog or prodrug of (3-aminophthalimido) glutarimide; 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione; an analog or prodrug of 3- (4 'aminoisoindolin-1' -one) -1-piperidine-2, 6-dione; or a compound of the formula:

in addition to one or more compounds of the invention, CD34⁺Cells may also undergo additional differentiation in vivo or in vitro in the presence of one or more cytokines. For guiding CD34⁺Cytokines that differentiate cells along the erythrocytic differentiation pathway include, but are not limited to, erythropoietin (Epo), TNF α, Stem Cell Factor (SCF), Flt-3L, and granulocyte macrophage-colony stimulating factor (GM-CSF). Epo and SCF are well known erythropoietic cytokines. Thus, in one embodiment, CD34 is differentiated in the presence of Epo or SCF⁺A cell. In another preferred embodiment, CD34 is differentiated in the presence of Epo and SCF⁺A cell. In another embodiment, CD34 is differentiated in the presence of a combination of TNF α, SCF, Flt-3L and GM-CSF⁺A cell. In another embodiment, the differentiated cells are one or more cells in a cell culture medium. In another embodiment, the differentiated cell is a cell in an individual. In one embodiment of in vitro differentiation, one or more of Epo, TNF α, SCF, Flt-3L and GM-CSF are combined with one or more IMiDs^TMAnd (4) contacting. In one embodiment of in vivo differentiation, one or more IMiDs are administered^TMIn the same treatment regimen, one or more of Epo, TNF α, SCF, Flt-3L, and GM-CSF are administered to the individual.

The cytokine used in the method of the invention may be a naturally occurring cytokine or an artificial derivative or analogue of the cytokine. For example, analogs or derivatives of erythropoietin that may be used with the compounds of the invention include, but are not limited to, Aranesp^TMAnd Darbopoetin^TM。

The cytokines used may be purified from natural sources or produced recombinantly. Examples of recombinant cytokines useful in the methods of the invention include filgrastim; or recombinant granulocyte-colony stimulating factor (G-CSF), which is known in the United states under the trade name Neupogen(Amgen, Thousand Oaks, Calif.); sargrastim; or recombinant GM-CSF, which is available under the trade name Leukine in the United states(Immunex, Seattle, WA); recombinant Epo, tradename Epogen in the United states(Amgen, Thousand Oaks, Calif.); and methylthio Stem Cell Factor (SCF), which is sold in the united states under the trade name of ansetim. Recombinant and mutant forms of GM-CSF can be prepared as described in U.S. Pat. Nos. 5,391,485, 5,393,870, and 5,229,496, which are incorporated herein by reference. Recombinant and mutant forms of G-CSF can be prepared as described in U.S. Pat. Nos. 4,810,643, 4,999,291, 5,528,823, and 5,580,755, which are incorporated herein by reference.

Other cytokines that promote survival and/or proliferation of hematopoietic precursor cells and immunocompetent hematopoietic cells under in vitro or in vivo conditions, or stimulate division and differentiation of committed erythrocyte progenitor cells in the cells under in vitro or in vivo conditions, may be used. Such cytokines include, but are not limited to: interleukins, such as IL-2 (including recombinant IL-II ("rIL 2") and canarypox (canarypox) IL-2), IL-10, IL-12 and IL-18; interferons such as interferon alpha-2 a, interferon alpha-2 b, interferon alpha-n 1, interferon alpha-n 3, interferon beta-Ia and interferon gamma-Ib; and G-CSF.

When administered to a patient with hemoglobinopathy, the compounds of the present invention, particularly in the presence of Epo, particularly in the presence of a combination of TNF α, SCF, Flt-3L, and GM-CSF, and more particularly in the presence of Epo and SCF, induce the production of red blood cells, fetal hemoglobin, and AHSP. As noted above, the cytokines used may include purified or recombinant forms, or analogs or derivatives of the particular cytokine.

The compounds of the present invention may also be used in combination with one or more second compounds known to have or likely to have beneficial effects on hemoglobinopathies. As used herein, "beneficial effect" refers to any reduction in the symptoms of any hemoglobinopathy or anemia.

For example, for a particular hemoglobinopathy sickle cell disease, the second compound may be a compound known or likely to induce fetal hemoglobin production in addition to the compound of the present invention. Such compounds include hydroxyurea, butyrate or butyrate derivatives. The second compound may also be a vasorelaxing compound such as nitrous oxide, for example, administered topically or with an external source of nitrous oxide. The second compound may also be a compound that binds directly to hemoglobin S, preventing the induction of sickle structure formation. For example, it is called HEMOXIN^TM(NIPRISAN^TM(ii) a See U.S. patent 5,800,819) which is an extract of a mixture of about 12 to about 17 parts by weight of Piperguineense seeds, about 15 to about 19 parts by weight of Pterocarpus osun stems, about 12 to about 18 parts by weight of Eugenia caryophylata fruits, and about 25 to about 32 parts by weight of Sorghum bicolor leaves, and optionally 15 to 22 parts by weight of potassium carbonate, wherein the mixture is extracted with cold water and has activity against sickle cell formation. The second compound may also be a Gardos channel antagonist. Examples of Gardos channel antagonists include clotrimazole and triarylmethane derivatives. The second compound may also be a compound that reduces the adhesion of red blood cells, thereby reducing the amount of blood clots in sickle cell anemia.

Other hemoglobinopathies may be treated with a second compound known or likely to be effective for the particular condition. For example, thalassemia may be treated with a second compound, deferoxamine mesylate, an iron chelator that prevents iron accumulation in the blood or folic acid (vitamin B9. thalassemia or sickle cell anemia may also be treated with protein C as a second compound (U.S. Pat. No. 6,372,213. there is some evidence that herbal therapy may ameliorate the symptoms of hemoglobinopathies, such therapy and any particular active compound contained therein may be used as the second compound in the methods of the invention, see, for example, Wu Zhikui et al, "the Effect of Bushen Shengxue Fang on β -thalassemia at the Gene Level", Journal of Traditional Chinese Medicine 18 (4): 300-303 (1998); treatment of autoimmune hemolytic anemia, U.S. patent 6,538,023, "Therapeutic Uses of Green Tea Polyphenols for Sickle cell disease" may include the use of corticosteroids as the second compound.

The second compound may be a protein and may be a derivative or analogue of another protein. Such derivatives may include, but are not limited to, proteins that lack the carbohydrate moiety present in their native form (e.g., non-glycosylated forms), pegylated derivatives, and fusion proteins, such as fusion proteins formed by fusing IgG1 or IgG3 to a protein or an active portion of a protein of interest. See, e.g., penichent, m.l. and Morrison, s.l., j.immunological Methods 248: 91-101(2001).

Effective cytokines and/or other compounds for treating anemia or hemoglobinopathy may be administered concurrently with the immunomodulatory compounds of the invention. In this regard, the cytokine or other compound may be administered in a different dosage form than the immunomodulatory compound, or, if possible, mixed with the immunomodulatory compound and administered as a single pharmaceutical composition. Alternatively, the cytokine, other compound, or both, may be administered separately from the immunomodulatory compounds used in the methods of the invention, and may be administered according to the same or different dosage regimen. In preferred embodiments, immunomodulatory compounds such as IMiD^TMThe cytokine, and any other compound used to treat anemia or hemoglobinopathy are administered simultaneously, butAre administered in separate pharmaceutical dosage forms to ensure flexibility of administration.

In addition to the above therapeutic combinations, the treated individuals may also be transfused. The infusion may be a blood transfusion, preferably a matching blood, or a blood substitute, such as Hemospan^TMOr Hemospan^TMPS(Sangart)。

In any of the above therapeutic combinations, the individual treated is eukaryotic. Preferably, the individual treated is a mammal, more preferably a human.

The methods of the invention can be used to treat any anemia, including those caused by hemoglobinopathies. The origin of hemoglobinopathies and anaemias which can be treated by the method of the invention may be hereditary, such as sickle cell anaemia or thalassemia. Hemoglobinopathies may be caused by diseases such as cancer, including but not limited to hematopoietic or lymphatic system cancers. Other diseases that may be treated by the methods of the present invention include hyperfunction, splenectomy indications, intestinal resection, and bone marrow infiltration. The methods of the invention may also be used to treat anemia arising from the deliberate or accidental introduction of a poison, toxin or drug. For example, anemia arising from cancer chemotherapy may be treated with the methods and compounds of the present invention. Likewise, the methods of the invention may be used when anemia or hemoglobinopathy is the primary disease to be treated, or is a secondary disease caused by a previous disease or treatment regimen.

5.2. Compounds of the invention

The compounds of the present invention are commercially available or may be prepared according to the methods described in the patents or patent publications disclosed herein. In addition, optically pure compounds can be asymmetrically synthesized or resolved using known resolving agents or chiral columns, as well as other standard organic chemical synthesis techniques. The compounds used in the present invention include immunomodulatory compounds that are racemic, stereomerically enriched or stereomerically pure, as well as pharmaceutically acceptable salts, solvates, stereoisomers and prodrugs thereof.

Preferred compounds for use in the present invention are small organic molecules having a molecular weight of less than about 1,000g/mol, and are not proteins, peptides, oligonucleotides, oligosaccharides or other macromolecules.

As used herein, unless otherwise indicated, the terms "immunomodulatory compound" and "IMiD" are used^TM"(Celgene Corporation) included small organic molecules that significantly inhibited TNF- α, LPS-induced monocyte IL1 β and IL12, and partially inhibited IL6 production. Specific immunomodulatory compounds are discussed below.

TNF- α is an inflammatory cytokine produced by macrophages and monocytes during acute inflammation. TNF- α triggers different signaling events (signaling events) within the cell. Without being limited by theory, one of the biological effects produced by the immunomodulatory compounds of the invention is the reduction of TNF- α synthesis. Immunomodulatory compounds of the invention enhance the degradation of TNF- α mRNA.

Furthermore, without being limited by theory, immunomodulatory compounds used in the invention may also be potent T cell co-stimulators and can significantly increase cell proliferation in a dose-dependent manner. The immunomodulatory compounds of the invention have greater co-stimulatory effects on the CD8+ T cell subset than on the CD4+ T cell subset. In addition, the compounds of the present invention preferably have anti-inflammatory properties and are effective in co-stimulating T cells. Furthermore, without being bound by a particular theory, immunomodulatory compounds used in the invention may act indirectly on or directly on natural killer ("NK") cells via cytokine activation and increase the ability of NK cells to produce beneficial cytokines, such as, but not limited to, IFN- γ.

Specific examples of immunomodulatory compounds include, but are not limited to: cyano and carboxyl derivatives of substituted styrenes such as those disclosed in U.S. Pat. No. 5,929,117; 1-oxo-2- (2, 6-dioxo-3-fluoropiperidin-3-yl) isoindoline and 1, 3-dioxo-2- (2, 6-dioxo-3-fluoropiperidin-3-yl) isoindoline, such as those described in U.S. Pat. nos. 5,874,448 and 5,955,476; tetrasubstituted 2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolines, which are described in U.S. Pat. No. 5,798,368; 1-oxo and 1, 3-dioxo-2- (2, 6-dioxopiperidin-3-yl) isoindolines (e.g., 4-methyl derivatives of thalidomide), including, but not limited to, those disclosed in U.S. Pat. Nos. 5,635,517, 6,476,052, 6,555,554, and 6,403,613; 1-oxo and 1, 3-dioxoisoindolines substituted at the 4-or 5-position of the indole ring (e.g., 4- (4-amino-1, 3-dioxoisoindolin-2-yl) -4-carbamoylbutyric acid), which is described in U.S. Pat. No. 6,380,239; isoindolin-1-ones and isoindoline-1, 3-diones substituted at the 2-position with a 2, 6-dioxo-3-hydroxypiperidin-5-yl group (e.g., 2- (2, 6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl) -4-aminoisoindolin-1-one), which are described in U.S. Pat. No. 6,458,810; a class of non-polypeptide cyclic amides disclosed in U.S. Pat. nos. 5,698,579 and 5,877,200; aminothalidomide and analogs, hydrolysates, metabolites, derivatives and precursors of Aminothalidomide, as well as substituted 2- (2, 6-dioxopiperidin-3-yl) phthalimides and substituted 2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolines, such as those described in U.S. Pat. Nos. 6,281,230 and 6,316,471; and isoindole-imide compounds such as those described in U.S. patent application 09/972,487 (filed 5/10/2001), U.S. patent application 10/032,286 (filed 21/11/2001), and international application PCT/US01/50401 (international publication No. WO 02/059106). Each of the patents and patent applications listed herein are incorporated by reference in their entirety. Immunomodulatory compounds do not include thalidomide.

Other specific immunomodulatory compounds of the invention include, but are not limited to: 1-oxo and 1, 3 dioxo-2- (2, 6-dioxopiperidin-3-yl) isoindolines substituted with amino in the phenyl ring are described in U.S. Pat. No. 5,635,517, which is incorporated herein by reference. These compounds have structure I:

wherein one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂，R²Is hydrogen or lower alkyl, especially methyl. Specific immunomodulatory compounds include, but are not limited to:

1-oxo-2- (2, 6-dioxopiperidin-3-yl) -4-aminoisoindoline;

1-oxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline;

1-oxo-2- (2, 6-dioxopiperidin-3-yl) -6-aminoisoindoline;

1-oxo-2- (2, 6-dioxopiperidin-3-yl) -7-aminoisoindoline;

1, 3-dioxo-2- (2, 6-dioxopiperidin-3-yl) -4-aminoisoindoline; and

1, 3-dioxo-2- (2, 6-dioxopiperidin-3-yl) -5-aminoisoindoline.

Other specific immunomodulatory compounds of the invention belong to a class of substituted 2- (2, 6-dioxopiperidin-3-yl) phthalimides and substituted 2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindoles such as those described in U.S. Pat. Nos. 6,281,230, 6,316,471, 6,335,349, and 6,476,052, and International patent application PCT/US97/13375 (International publication No. WO 98/03502), each of which is incorporated herein by reference. Representative compounds have the formula:

wherein:

one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；

(i)R¹、R²、R³、R⁴Each independently of the others is halogen, alkyl of 1 to 4 carbon atoms or alkoxy of 1 to 4 carbon atomsOr (ii) R¹、R²、R³、R⁴One is-NHR⁵，R¹、R²、R³、R⁴The remainder of (1) is hydrogen;

R⁵is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶is hydrogen, alkyl of 1-8 carbon atoms, benzyl or halogen;

if X and Y are C ═ O and (i) R¹、R²、R³、R⁴Each of (i) is fluorine or (ii) R¹、R²、R³、R⁴One is amino, then R⁶Is not hydrogen.

Representative compounds of this class have the formula:

wherein R is¹Is hydrogen or methyl. In a separate embodiment, the present invention encompasses the use of these compounds in enantiomerically pure form (e.g., the optically pure (R) or (S) enantiomer).

Other specific immunomodulatory compounds of the invention belong to the class of isoindole-imides, which are disclosed in U.S. patent application publication Nos. 2003/0096841 and 2003/0045552 and International patent application No. PCT/US01/50401 (International publication No. WO 02/059106), each of which is incorporated herein by reference. Representative compounds are of formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates, enantiomers, diastereomers, racemates, and mixtures of stereoisomers thereof, wherein:

one of X and Y is C ═ O, and the other is CH₂Or C ═ O;

R¹is H, (C)₁-C₈) Alkyl radical (C)₃-C₇) Cycloalkyl group, (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, (C)₀-C₄) Alkyl radical- (C)₁-C₆) Heterocycloalkyl group, (C)₀-C₄) Alkyl radical- (C)₂-C₅) Heteroaryl, C (O) R³，C(S)R³，C(O)OR⁴，(C₁-C₈) alkyl-N (R)⁶)₂，(C₁-C₈) alkyl-OR⁵，(C₁-C₃) alkyl-C (O) OR⁵，C(O)NHR³，C(S)NHR³，C(O)NR³R³’，C(S)NR³R³' or (C)₁-C₈) alkyl-O (CO) R⁵；

R²Is H, F, benzyl, (C)₁-C₈) Alkyl radical (C)₂-C₈) Alkenyl, or (C)₂-C₈) An alkynyl group;

R³and R³' independently is (C)₁-C₈) Alkyl radical (C)₃-C₇) Cycloalkyl group, (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, (C)₀-C₄) Alkyl radical- (C)₁-C₆) Heterocycloalkyl group, (C)₀-C₄) Alkyl radical- (C)₂-C₅) Heteroaryl group, (C)₀-C₈) alkyl-N (R)⁶)₂，(C₁-C₈) alkyl-OR⁵，(C₁-C₈) alkyl-C (O) OR⁵，(C₁-C₈) alkyl-O (CO) R⁵Or C(O)OR⁵；

R⁴Is (C)₁-C₈) Alkyl radical (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl (C)₁-C₄) alkyl-OR⁵Benzyl, aryl, (C)₀-C₄) Alkyl radical- (C)₁-C₆) Heterocycloalkyl, or (C)₀-C₄) Alkyl radical- (C)₂-C₅) A heteroaryl group;

R⁵is (C)₁-C₈) Alkyl radical (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, or (C)₂-C₅) A heteroaryl group;

R⁶each occurrence is independently H, (C)₁-C₈) Alkyl radical (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, (C)₂-C₅) Heteroaryl, or (C)₀-C₈) alkyl-C (O) O-R⁵Or R is⁶Groups may be linked to form a heterocycloalkyl group;

n is 0 or 1; and

^*represents a chiral carbon center.

In specific compounds of formula II, when n is 0, R¹Is (C)₃-C₇) Cycloalkyl group, (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, (C)₀-C₄) Alkyl radical- (C)₁-C₆) Heterocycloalkyl group, (C)₀-C₄) Alkyl radical- (C)₂-C₅) Heteroaryl, C (O) R³，C(O)OR⁴，(C₁-C₈) alkyl-N (R)⁶)₂，(C₁-C₈) alkyl-OR⁵，(C₁-C₈) alkyl-C (O) OR⁵，C(S)NHR³Or (C)₁-C₈) alkyl-O (CO) R⁵；

R²Is H or (C)₁-C₈) An alkyl group; and

R³is (C)₁-C₈) Alkyl radical (C)₃-C₇) Cycloalkyl group, (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, (C)₀-C₄) Alkyl radical- (C)₁-C₆) Heterocycloalkyl group, (C)₀-C₄) Alkyl radical- (C)₂-C₅) Heteroaryl group, (C)₅-C₈) alkyl-N (R)⁶)₂；(C₀-C₈) alkyl-NH-C (O) O-R⁵；(C₁-C₈) alkyl-OR⁵，(C₁-C₈) alkyl-C (O) OR⁵，(C₁-C₈) alkyl-O (CO) R⁵OR C (O) OR⁵(ii) a And the other variables have the same definitions.

In other specific compounds of formula II, R²Is H or (C)₁-C₄) An alkyl group.

In other specific compounds of formula II, R¹Is (C)₁-C₈) Alkyl or benzyl.

In other specific compounds of formula II, R¹Is H, (C)₁-C₈) Alkyl, benzyl, CH₂OCH₃，CH₂CH₂OCH₃Or is or

In another embodiment of the compounds of formula II, R¹Is that

Or

Wherein Q is O or S, and R⁷Each occurrence is independently H, (C)₁-C₈) Alkyl radical (C)₃-C₇) Cycloalkyl group, (C)₂-C₈) Alkenyl, (C)₂-C₈) Alkynyl, benzyl, aryl, halogen, (C)₀-C₄) Alkyl radical- (C)₁-C₆) Heterocycloalkyl group, (C)₀-C₄) Alkyl radical- (C)₂-C₅) Heteroaryl group, (C)₀-C₈) alkyl-N (R)⁶)₂，(C₁-C₈) alkyl-OR⁵，(C₁-C₈) alkyl-C (O) OR⁵，(C₁-C₈) alkyl-O (CO) R⁵OR C (O) OR⁵Or adjacent occurrence of R⁷Together form a bicycloalkyl or aromatic ring.

In other specific compounds of formula II, R¹Is C (O) R³。

In other specific compounds of formula II, R³Is (C)₀-C₄) Alkyl radical- (C)₂-C₅) Heteroaryl group, (C)₁-C₈) Alkyl, aryl, or (C)₀-C₄) alkyl-OR⁵。

In other specific compounds of formula II, the heteroaryl group is pyridyl, furyl, or thienyl.

In other specific compounds of formula II, R¹Is C (O) OR⁴。

In other embodiments of formula II, H of C (O) NHC (O) can be replaced by (C)₁-C₄) Alkyl, aryl, or benzyl.

Other examples of such compounds include, but are not limited to: [2- (2, 6-dioxo-piperidin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-ylmethyl) -amide; (2- (2, 6-dioxo-piperidin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-ylmethyl) -carbamic acid tert-butyl ester; 4- (aminomethyl) -2- (2, 6-dioxo (3-piperidyl) -isoindoline-1, 3-dione, N- (2- (2, 6-dioxo-piperidin-3-yl) -1, 3-dioxo-2, 3-dihydro-1H-isoindol-4-ylmethyl) -acetamide, N- {2- (2, 6-dioxo (3-piperidyl) -1, 3-dioxo-isoindolin-4-yl) methyl } cyclopropyl-carboxamide, 2-chloro-N- { (2- (2, 6-dioxo (3-piperidyl) -1, 3-dioxo-isoindolin-4-yl) methyl } acetamide, N- (2- (2, 6-dioxo (3-piperidinyl) -1, 3-dioxoisoindolin-4-yl) -3-pyridylcarboxamide; 3- { 1-oxo-4- (benzylamino) isoindolin-2-yl } piperidine-2, 6-dione; 2- (2, 6-dioxo (3-piperidyl)) -4- (benzylamino) isoindoline-1, 3-dione; n- { (2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl) methyl } propionamide; n- { (2- (2, 6-dioxo (3-piperidyl) -1, 3-dioxoisoindolin-4-yl) methyl } -3-pyridylcarboxamide,; N- { (2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl) methyl } heptanamide,; N- { (2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl) methyl } -2-furancarboxamide; { N- (2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl) carbamoyl } methyl acetate,; N- (2- (2, 6-dioxo (3-piperidinyl) -1, 3-dioxoisoindolin-4-yl) pentanamide; n- (2- (2, 6-dioxo (3-piperidinyl) -1, 3-dioxoisoindolin-4-yl) -2-thienylcarboxamide;

n- { [2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl ] methyl } (butylamino) carboxamide; n- { [2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl ] methyl } (octylamino) carboxamide; and N- { [2- (2, 6-dioxo (3-piperidyl)) -1, 3-dioxoisoindolin-4-yl ] methyl } (benzylamino) carboxamide.

Other specific immunomodulatory compounds of the invention belong to the class of isoindole-imides, which are disclosed in U.S. patent application 2002/0045643, international publication No. WO 98/54170, and U.S. patent No. 6,395,754, each of which is incorporated herein by reference. Representative compounds have formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates, enantiomers, diastereomers, racemates, and mixtures of stereoisomers thereof, wherein:

one of X and Y is C ═ O, and the other is C ═ O or CH₂；

R is H or CH₂OCOR’；

(i)R¹、R²、R³、R⁴Each independently of the other is halogen, alkyl of 1 to 4 carbon atoms or alkoxy of 1 to 4 carbon atoms, or (ii) R¹、R²、R³、R⁴One of which is nitro or-NHR⁵，R¹、R²、R³、R⁴The remainder of (1) is hydrogen;

R⁵is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro or fluoro;

r' is R⁷-CHR¹⁰-N(R⁸R⁹)；

R⁷Is m-phenylene or p-phenylene or- (C)_nH_2n) -, where n is 0 to 4;

R⁸and R⁹Each independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R⁸And R⁹Linked together being tetramethylene, pentamethylene, hexamethylene, or-CH₂CH₂X¹CH₂CH₂-, wherein X¹is-O-, -S-, or-NH-;

R¹⁰is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl; and

^*represents a chiral carbon center.

Other representative compounds have the formula:

wherein:

one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；

(i)R¹、R²、R³、R⁴Each independently is halogen, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms, or (ii) R¹、R²、R³、R⁴One is-NHR⁵，R¹、R²、R³、R⁴The remainder of (1) is hydrogen;

R⁵is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro or fluoro;

R⁷is m-phenylene or p-phenylene or- (C)_nH_2n) -, where n is 0 to 4;

R⁸and R⁹Each independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R⁸And R⁹Linked together being tetramethylene, pentamethylene, hexamethylene, or-CH₂CH₂X¹CH₂CH₂-, wherein X¹is-O-, -S-, or-NH-; and

R¹⁰is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl.

Other representative compounds have the formula:

wherein:

one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；

R¹、R²、R³、R⁴Each independently is halogen, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms, or (ii) R¹、R²、R³、R⁴One being nitro or protected amino, R¹、R²、R³、R⁴The remainder of (1) is hydrogen; and

R⁶is hydrogen, alkyl of 1-8 carbon atoms, benzo, chloro or fluoro.

Other representative compounds have the formula:

wherein:

one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；

(i)R¹、R²、R³、R⁴Each independently is halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or (ii) R¹、R²、R³、R⁴One is-NHR⁵，R¹、R²、R³、R⁴The remainder of (1) is hydrogen;

R⁵is hydrogen, alkyl of 1 to 8 carbon atoms, or CO-R⁷-CH(R¹⁰)NR⁸R⁹Wherein R is⁷、R⁸、R⁹And R¹⁰Each as defined above; and

R⁶is alkyl of 1 to 8 carbon atoms, benzo, chloro or fluoro.

Specific examples of compounds have the formula:

wherein:

one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；

R⁶Is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro or fluoro;

R⁷is m-phenylene or p-phenylene or- (C)_nH_2n) -, where n is 0 to 4;

R⁸and R⁹Each independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R⁸And R⁹Linked together being tetramethylene, pentamethylene, hexamethylene, or-CH₂CH₂X¹CH₂CH₂-, wherein X¹is-O-, -S-, or-NH-; and

R¹⁰is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl.

Preferred immunomodulatory compounds of the invention are 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione and 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione. Such compounds can be obtained by standard synthetic methods (see, e.g., U.S. Pat. No. 5,635,517, which is incorporated herein by reference). The compound is available from Celgene Corporation, Warren, NJ.. 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione has the following chemical structure:

the compound 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione has the following chemical structure:

in another embodiment, specific immunomodulatory compounds of the invention include polymorphic forms of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione, such as forms A, B, C, D, E, F, G and H disclosed in U.S. provisional application 60/499,723, filed on 9/4/2003 and in the corresponding U.S. non-provisional application filed on 9/3/2004, which are incorporated herein by reference. For example, form a of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a non-solvated crystalline, which can be obtained from a non-aqueous solvent system. The X-ray powder diffraction pattern of form a comprises distinct peaks at about 8, 14.5, 16, 17.5, 20.5, 24, and 26 degrees 2 Θ, with a differential scanning calorimetry maximum melting temperature of about 270 ℃. Form a is weakly or non-hygroscopic and is the thermodynamically most stable anhydrous polymorph of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione to date.

Form B of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a hemihydrate crystalline, which can be obtained from a variety of solvent systems, including but not limited to hexane, toluene, and water. The X-ray powder diffraction pattern for form B contained distinct peaks at about 16, 18, 22, and 27 degrees 2 theta, with the DSC curve showing endotherms at about 146 and 268 ℃, and dehydration and melting as confirmed by hot-stage microscopy experiments. Interconversion studies have shown that form B converts to form E in aqueous solvent systems and to other forms in acetone and other anhydrous systems.

Form C of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a semi-solvated crystalline, which can be obtained from a solvent such as, but not limited to, acetone. Form C has an X-ray powder diffraction pattern comprising distinct peaks at about 15.5 and 25 degrees 2-theta with a differential scanning calorimetry maximum melting temperature of about 269 ℃. Form C is not hygroscopic below about 85% RH, but can be converted to form B at higher relative humidity.

Form D of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a crystalline solvated polymorph prepared from a mixture of acetonitrile and water. Form D has an X-ray powder diffraction pattern comprising distinct peaks at about 27 and 28 degrees 2 Θ and a differential scanning calorimetry maximum melting temperature of about 270 ℃. Form D is weakly or non-hygroscopic, but generally converts to form B when under pressure at higher relative humidity.

Form E of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a dihydrate crystal, which can be obtained by slurrying 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione in water, and slowly evaporating 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione in a solvent system of acetone: water of about 9: 1. Form E has an X-ray powder diffraction pattern comprising distinct peaks at about 20, 24.5, and 29 degrees 2 Θ and a differential scanning calorimetry maximum melting temperature of about 269 ℃. Form E can be converted to form C in an acetone solvent system and form G in a THF solvent system. Form E is the most stable form in aqueous solvent systems. The anti-dissolution experiment for form E showed that form E was converted to form B when heated at about 125 ℃ for about 5 minutes. Form B converts to form F upon heating at 175 ℃ for about 5 minutes.

Form F of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a non-solvated crystalline material, which can be obtained by dehydrating form E. The X-ray powder diffraction pattern for form F contains significant peaks at about 19, 19.5, and 25 degrees 2 Θ, with a differential scanning calorimetry maximum melting temperature of about 269 ℃.

Form G of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a non-solvated crystalline material that can be obtained from a slurry of forms B and E in a solvent such as, but not limited to, Tetrahydrofuran (THF). The X-ray powder diffraction pattern of form G contains significant peaks at about 21, 23, and 24.5 degrees 2 Θ, with a differential scanning calorimetry maximum melting temperature of about 267 ℃.

Form H of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione is a partially hydrated crystal (about 0.25 mole) that can be obtained by exposing form E to 0% relative humidity. The X-ray powder diffraction pattern of form H contains significant peaks at about 15, 26, and 31 degrees 2 Θ, with a differential scanning calorimetry maximum melting temperature of about 269 ℃.

Other specific immunomodulatory compounds of the invention include, but are not limited to: 1-oxo-2- (2, 6-dioxo-3-fluoropiperidin-3-yl) isoindoline and 1, 3-dioxo-2- (2, 6-dioxo-3-fluoropiperidin-3-yl) isoindoline, such as those described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each of which is incorporated herein by reference. Representative compounds have the formula:

wherein Y is oxygen or H₂And are and

R¹、R²、R³、R⁴each independently hydrogen, halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino.

Other specific immunomodulatory compounds of the invention include, but are not limited to: tetrasubstituted 2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolines, which are described in U.S. Pat. No. 5,798,368, which is incorporated herein by reference. Representative compounds have the formula:

wherein R is¹、R²、R³、R⁴Each independently is a halogen, an alkyl group having 1 to 4 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms.

Other specific immunomodulatory compounds of the invention include, but are not limited to, 1-oxo and 1, 3 dioxo-2- (2, 6 dioxopiperidin-3-yl) isoindolines, which are disclosed in U.S. Pat. No. 6,403,613, which is incorporated herein by reference. Representative compounds have the formula:

wherein:

y is oxygen or H₂，

R¹And R²The first of (A) is halogen, alkyl, alkoxy, alkylamino, dialkylamino, cyano or carbamoyl, R is¹And R²The second of (a) is, independently of the first, hydrogen, halogen, alkyl, alkoxy, alkylamino, dialkylamino, cyano or carbamoyl, and

R³is hydrogen, alkyl or benzyl.

Specific examples of compounds of the invention have the formula:

wherein R is¹And R²The first of which is halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, dialkylamino (wherein each alkyl group has 1 to 4 carbon atoms), cyano or carbamoyl,

R¹and R²Is independent of the firstIs hydrogen, halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylamino (wherein the alkyl group has 1 to 4 carbon atoms), dialkylamino (wherein each alkyl group has 1 to 4 carbon atoms), cyano or carbamoyl, and

R³is hydrogen, alkyl of 1 to 4 carbon atoms or benzyl. Specific examples include, but are not limited to, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -4-methylisoindoline.

Other representative compounds have the formula:

wherein R is¹And R²The first of which is halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, dialkylamino (wherein each alkyl group has 1 to 4 carbon atoms), cyano, or carbamoyl,

R¹and R²The second of (a) is, independently of the first, hydrogen, halogen, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylamino (wherein the alkyl group has 1 to 4 carbon atoms), dialkylamino (wherein each alkyl group has 1 to 4 carbon atoms), cyano, or carbamoyl, and

R³is hydrogen, alkyl having 1 to 4 carbon atoms, or benzyl.

Specific examples include, but are not limited to, 1-oxo-2- (2, 6-dioxopiperidin-3-yl) -4-methylisoindoline.

Other specific immunomodulatory compounds of the invention include, but are not limited to, 1-oxo and 1, 3-dioxoisoindolines substituted at the 4-or 5-position of the indoline ring, described in U.S. patent 6,380,329 and co-pending U.S. application 10/900,270 filed 7/28/2004, which are incorporated herein by reference. Representative compounds have the formula:

wherein is represented by C^*The carbon atom(s) constituting the chiral centre (when n is other than 0 and R¹And R²When not identical); x¹And X²One of which is amino, nitro, alkyl of 1 to 6 carbon atoms or NH-Z, X¹Or X²Is hydrogen; r¹And R²Each independently is hydroxy or NH-Z; r³Is hydrogen, alkyl of 1 to 6 carbon atoms, halogen or haloalkyl; z is hydrogen, aryl, alkyl with 1-6 carbon atoms, formyl or acyl with 1-6 carbon atoms; and n has a value of 0, 1 or 2; provided that if X is¹Is amino and n is 1 or 2, then R¹And R²Are not hydroxyl; and salts thereof.

Other representative compounds have the formula:

wherein when n is not 0 and R¹Is different from R²When is represented as C^*The carbon atom of (a) constitutes a chiral center; x¹And X²One of which is amino, nitro, alkyl of 1 to 6 carbon atoms or NH-Z, X¹Or X²Is hydrogen; r¹And R²Each independently is hydroxy or NH-Z; r³Is alkyl of 1 to 6 carbon atoms, halogen or hydrogen; z is hydrogen, aryl, or alkyl or acyl of 1 to 6 carbon atoms; and n has a value of 0, 1 or 2.

Specific examples include, but are not limited to, 2- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -4-carbamoyl-butyric acid and 4- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -4-carbamoyl-butyric acid, which have the following structures, and pharmaceutically acceptable salts, solvates, prodrugs, and stereoisomers thereof, respectively:

and

other representative compounds have the formula:

wherein when n is not 0 and R¹And R²When not identical, is represented as C^*The carbon atom of (a) constitutes a chiral center; x¹And X²One of which is amino, nitro, alkyl of 1 to 6 carbon atoms or NH-Z, X¹Or X²Is hydrogen; r¹And R²Each independently is hydroxy or NH-Z; r³Is alkyl of 1 to 6 carbon atoms, halogen or hydrogen; z is hydrogen, aryl or alkyl or acyl of 1-6 carbon atoms; and n has a value of 0, 1 or 2; and salts thereof.

Specific examples include, but are not limited to, 4-carbamoyl-4- {4- [ (furan-2-yl-methyl) -amino ] -1, 3-dioxo-1, 3-dihydro-isoindol-2-yl } -butyric acid, 4-carbamoyl-2- {4- [ (furan-2-yl-methyl) -amino ] -1, 3-dioxo-1, 3-dihydro-isoindol-2-yl } -butyric acid, 2- {4- [ (furan-2-yl-methyl) -amino ] -1, 3-dioxo-1, 3-dihydro-isoindol-2-yl } -4-phenylcarbamoyl-butyric acid, and 2- {4- [ (furan-2-yl-methyl) -amino ] -1, 3-dioxo-1, 3-dihydro-isoindol-2-yl } -glutaric acid, which have the following structures, and pharmaceutically acceptable salts, solvates, prodrugs, and stereoisomers thereof, respectively:

and

other specific examples of compounds have the formula:

wherein X¹And X²One of which is nitro or NH-Z, X¹Or X²Is hydrogen;

R¹and R²Each independently is hydroxy or NH-Z;

R³is alkyl of 1 to 6 carbon atoms, halogen or hydrogen;

z is hydrogen, phenyl, acyl of 1 to 6 carbon atoms or alkyl of 1 to 6 carbon atoms; and is

n has a value of 0, 1 or 2;

provided that if X is¹And X²Is nitro and n is 1 or 2, then R¹And R²Are not hydroxyl; and

if-COR¹And- (CH)₂)_nCOR²Is different, then denoted as C^*The carbon atom(s) constituting the chiral center. Other representative compounds have the formula:

wherein X¹And X²One is an alkyl group having 1 to 6 carbon atoms;

R¹and R²Each independently is hydroxy or NH-Z;

R³is alkyl of 1 to 6 carbon atoms, halogen or hydrogen;

z is hydrogen, phenyl, acyl of 1 to 6 carbon atoms, or alkyl of 1 to 6 carbon atoms; and

n has a value of 0, 1 or 2; and

if-COR²And- (CH)₂)_nCOR¹Is different, then denoted as C^*The carbon atom(s) constituting the chiral center.

Other specific immunomodulatory compounds of the invention include, but are not limited to: isoindolin-1-ones and isoindolin-1, 3-diones substituted at the 2-position with a 2, 6-dioxo-3-hydroxypiperidin-5-yl group, which are described in U.S. Pat. No. 6,458,810, which is incorporated herein by reference. Representative compounds have the formula:

wherein:

is represented as C^*The carbon atom of (a) constitutes a chiral center;

x is-C (O) -or-CH₂-；

R¹Is an alkyl group having 1 to 8 carbon atoms or-NHR³；

R²Is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen;

and

R³is a hydrogen atom, and is,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxy of 1 to 8 carbon atoms, halogen, amino, or alkylamino of 1 to 4 carbon atoms,

a cycloalkyl group having 3 to 18 carbon atoms,

phenyl unsubstituted or substituted by alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halogen, amino, or alkylamino of 1 to 4 carbon atoms,

benzyl unsubstituted or substituted by alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halogen, amino, or alkylamino of 1 to 4 carbon atoms, or-COR⁴Wherein

R⁴Is a hydrogen atom, and is,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxy of 1 to 8 carbon atoms, halogen, amino, or alkylamino of 1 to 4 carbon atoms,

a cycloalkyl group having 3 to 18 carbon atoms,

phenyl unsubstituted or substituted by alkyl of 1 to 8 carbon atoms, alkoxy of 1 to 8 carbon atoms, halogen, amino, or alkylamino of 1 to 4 carbon atoms,

and benzyl which is unsubstituted or substituted by alkyl with 1-8 carbon atoms, alkoxy with 1-8 carbon atoms, halogen, amino, or alkylamino with 1-4 carbon atoms.

The compounds of the present invention are commercially available or can be prepared according to the methods described in the patents or patent publications disclosed herein. In addition, optically pure compounds can be asymmetrically synthesized or resolved using known resolving agents or chiral columns, as well as other standard organic chemical synthesis techniques.

The term "pharmaceutically acceptable salts" as used herein, unless otherwise indicated, includes non-toxic acid and base addition salts of the compounds to which the term refers. Acceptable non-toxic acid addition salts include those derived from organic and inorganic acids or bases known in the art, including, for example, hydrochloric, hydrobromic, phosphoric, sulfuric, methanesulfonic, acetic, tartaric, lactic, succinic, citric, malic, maleic, sorbic, aconitic, salicylic, phthalic, embonic, heptanoic, and the like.

Naturally occurring acidic compounds are capable of forming salts with various pharmaceutically acceptable bases. The bases which can be used for the preparation of pharmaceutically acceptable base addition salts of such acidic compounds are those which form non-toxic base addition salts, that is, salts containing pharmacologically acceptable cations, such as, but not limited to, alkali metal or alkaline earth metal salts, especially calcium, magnesium, sodium, potassium salts. Suitable organic bases include, but are not limited to, N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine, and procaine.

The term "solvate" as used herein, unless otherwise specified, refers to a compound of the invention or a salt thereof, and also includes stoichiometric or non-stoichiometric amounts of solvents bound by non-covalent intermolecular forces. If the solvent is water, then the solvate is a hydrate.

As used herein, unless otherwise indicated, the term "prodrug" refers to a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide the compound. Examples of prodrugs include, but are not limited to, derivatives of immunomodulatory compounds of the invention comprising biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. Other examples of prodrugs include those containing-NO, -NO₂-ONO or-ONO₂Derivatives of some of the immunomodulatory compounds of the invention. Prodrugs can generally be prepared by well-known methods, such as those described in Burger's Medicinal Chemistry and drug discovery, 172-178, 949-982 (edited E.Wolff, 5 th edition 1995) and Design of Prodrugs (H.Bundgaand, Elselvier, New York 1985)The method of (1).

As used herein, unless otherwise indicated, the terms "biohydrolyzable amide", "biohydrolyzable ester", "biohydrolyzable carbamate", "biohydrolyzable carbonate", "biohydrolyzable ureide", "biohydrolyzable phosphate" refer to an amide, ester, carbamate, carbonate, ureide, or phosphate, respectively, of a compound having the following properties: 1) does not interfere with the biological activity of the compound, but may confer advantageous properties to the compound in vivo, such as absorption, duration of action or onset of action; or 2) is biologically inactive, but is converted in vivo to a biologically active compound. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, lower acyloxyalkyl esters (e.g., acetoxymethyl, acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, and pivaloyloxyethyl esters), lactonyl esters (e.g., phthalidyl and thiophthalidyl esters), lower alkoxyacyloxyalkyl esters (e.g., methoxycarbonyloxymethyl, ethoxycarbonyloxyethyl, and isopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters, and acylaminoalkyl esters (e.g., acetamidomethyl esters). Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, alpha-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines.

The term "stereoisomer" as used herein, unless otherwise indicated, includes all enantiomerically/stereomerically pure and enantiomerically/stereomerically enriched compounds of the present invention.

As used herein, unless otherwise indicated, the term "stereomerically pure" or "enantiomerically pure" means that a compound includes one stereoisomer and is substantially free of the opposite stereoisomer or enantiomer of the compound. For example, a compound is stereoisomerically or enantiomerically pure when it contains 80%, 90%, or 95% or more of one stereoisomer and 20%, 10%, or 5% or less of the opposite stereoisomer. In certain instances, when a compound of the invention is about 80% ee (enantiomeric excess) or greater, preferably equal to or greater than 90% ee, more preferably 95% ee relative to a particular chiral center, then the compound is considered optically active or stereoisomerically/enantiomerically pure (i.e., substantially R-form or substantially S-form) relative to the chiral center.

As used herein, unless otherwise indicated, the term "stereomerically enriched" or "enantiomerically enriched" includes racemic as well as other mixtures of stereoisomers of the compounds of the invention (e.g., R/S ═ 30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35, and 70/30). Various immunomodulatory compounds of the invention contain one or more chiral centers and may exist as racemic or diastereomeric mixtures of enantiomers. The invention includes the use of stereomerically pure forms of such compounds as well as the use of mixtures of those forms. For example, mixtures containing equal or unequal amounts of enantiomers of particular immunomodulatory compounds of the invention may be used in methods and compositions of the invention. These isomers can be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, for example: jacques, j. et al, entiinoers, Racemates and solutions (Wiley-Interscience, New York, 1981); wilen, s.h. et al, Tetrahedron 33: 2725 (1977); eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., Tables of solving Agents and Optical solutions, p.268 (eds. E.L.Eliel, Univ.of Notre Dame Press, Notre Dame, IN, 1972).

It should be noted that if there is a difference between the structure shown and the name of the structure, the structure shown should be the subject of the difference. Furthermore, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be understood as encompassing all stereoisomers of it.

5.3. Pharmaceutical compositions and dosage forms

The pharmaceutical compositions may be used to prepare individual single unit dosage forms. Pharmaceutical compositions and dosage forms of the invention comprise an immunomodulatory compound of the invention, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. The pharmaceutical compositions and dosage forms of the present invention may also comprise one or more excipients.

The pharmaceutical compositions and dosage forms of the present invention may also comprise one or more additional active agents. Accordingly, the pharmaceutical compositions and dosage forms of the invention comprise the active agents (e.g., immunomodulatory compounds and second active agents) disclosed herein. Optional second or additional active ingredients are disclosed (see, e.g., section 5.1).

The single unit dosage forms of the invention are suitable for administration to a patient orally, mucosally (e.g., nasally, sublingually, vaginally, buccally or rectally), or parenterally (e.g., subcutaneously, intravenously, by bolus injection, intramuscularly or intraarterially), topically (eye drops or other ophthalmic formulations), transdermally or transdermally. Examples of dosage forms include, but are not limited to: a tablet; a caplet; capsules, such as elastic soft gelatin capsules; a cachet; keeping in mouth; a lozenge; a dispersant; suppositories; a powder agent; aerosols (e.g., nasal sprays or inhalants); gelling agent; liquid dosage forms suitable for oral or transmucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; eye drops or other ophthalmic preparations suitable for topical administration; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

The composition, shape and type of dosage forms of the present invention generally vary depending on their application. For example, a dosage form for the acute treatment of a disease may contain a greater amount of one or more active agents than a dosage form for the chronic treatment of the same disease. Similarly, parenteral dosage forms may contain smaller amounts of one or more active agents than oral dosage forms used to treat the same disease. These and other ways of changing a particular dosage form of the present invention from one to another will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

Typical pharmaceutical compositions and dosage forms contain one or more excipients. Suitable excipients are well known to those of ordinary skill in the pharmaceutical arts, and non-limiting examples of suitable excipients are provided in the present specification. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including, but not limited to, the manner in which the dosage form is administered to a patient. For example, oral dosage forms (e.g., tablets) may contain excipients that are not suitable for use in parenteral dosage forms. The suitability of a particular excipient may depend on the particular active ingredient in the dosage form. For example, some excipients (e.g., lactose), or when exposed to water, may accelerate the decomposition of some active ingredients. Active ingredients containing primary or secondary amines are particularly sensitive to this accelerated decomposition. Thus, the invention includes pharmaceutical compositions and dosage forms that contain little, if any, lactose or other mono-or disaccharides. In the present invention, the term "lactose-free" is used to indicate that the amount of lactose, if any, is insufficient to substantially accelerate the rate of degradation of the active ingredient.

Lactose-free compositions of the invention may contain excipients well known in the art, such as those listed in, for example, the United States Pharmacopeia (USP)25-NF20 (2002). Typically, lactose-free compositions contain pharmaceutically compatible and pharmaceutically acceptable amounts of active agent, binder/filler and lubricant. Preferably, the lactose-free dosage form contains the active agent, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

The present invention also includes anhydrous pharmaceutical compositions and dosage forms containing the active ingredient, as water may promote the degradation of certain compounds. As water promotes the degradation of certain compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine properties of the formulation over time, such as shelf life or stability. See, e.g., jenst. carstensen, "drug stability: principles and practices (Drug Stability: Principles & Practice), second edition, Marcel Dekker, NY, NY, 1995, pages 379-80. In fact, water and heat will accelerate the decomposition of some compounds. Thus, the effect of water on the formulation is very significant, as moisture and/or humidity is often encountered during manufacture, handling, packaging, storage, shipment, and use of the formulation.

The anhydrous pharmaceutical compositions and dosage forms of the invention can be manufactured with anhydrous or low moisture content ingredients and under low humidity conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active agent comprising a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity is expected during manufacture, packaging, and/or storage.

Anhydrous pharmaceutical compositions should be prepared and stored in a manner that maintains their anhydrous nature. Accordingly, anhydrous compositions are preferably packaged with materials known to prevent exposure to water, so that they can be packaged in suitable formulation boxes. Examples of suitable packaging include, but are not limited to, sealed films, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

The invention also includes pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate of decomposition of the active ingredient. Such compounds are referred to herein as "stabilizers" and include, but are not limited to, antioxidants (such as ascorbic acid), pH buffers, or salt buffers.

As with the amount and type of excipient, the type and amount of a particular active ingredient in a dosage form may vary depending upon a variety of factors including, but not limited to, the route of administration. However, a typical dosage form of the invention contains from about 0.10 to about 150mg of an immunomodulatory compound of the invention, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. Typical dosage forms contain about 0.1, 1,2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150, or 200mg of an immunomodulatory compound of the invention, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. In one embodiment, preferred dosage forms contain about 1,2, 5, 10, 25, or 50mg of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione (i.e., α - (3-aminophthalimido) glutarimide). In one embodiment, preferred dosage forms contain about 5, 10, 25 or 50mg of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione. Typical dosage forms contain from about 1 to about 1000mg, from about 5mg to about 500mg, from about 10mg to about 350mg, from about 50mg to about 200mg of the second active ingredient. The specific amount of the anti-cancer drug will, of course, depend on the specific active ingredient used, the type of cancer being treated or controlled, and the amounts of the immunomodulatory compound and any optional additional active ingredients being co-administered to the patient.

If one or more compounds of the invention are administered to an individual with a cytokine, the cytokine may be in any pharmaceutically acceptable dosage form, or acceptable concentration, as described elsewhere herein. Typically, for example, Neupogen is administered as a bolus injection at a dose of about 4 to about 8 micrograms/kg/day until a neutrophil count of 10,000/mm is reached³. Ancestim (recombinant methylthio human stem cell factor) is usually given by subcutaneous injection (not intravenous injection) at 1-20 microgram/kg/day for 9-12 days; the recombinant human stem cell factor can be administered in similar doses. Sagnathist is usually administered at a rate of up to about 250 micrograms/m²The dose is administered intravenously or subcutaneously per day until the white blood cell count exceeds about 50,000/mm³. Pegylated filgrastim (Neulasta) when required^TM) Usually administered subcutaneously in a dose of about 6 mg. The appropriate dosage of cytokines that affect the amount of leukocytes in the blood can be determined on a per patient basis by measuring the number of specific leukocytes, or the total leukocyte count. Recombinant IL-3 can be derived, for example, from R&D Systems, inc. (Minneapolis, MN). Recombinant IL-3 and in vitro ED thereof₅₀From about 0.1 to about 0.4ng/ml, the same in vivo concentrations can be used. Recombinant human Stem Cell Factor (SCF) is available, for example, from BioSource International (A)Camarillo, CA). Recombinant SCF and in vitro ED thereof₅₀From about 2 to about 5ng/ml, the same in vivo concentration may be used. Recombinant human Fms-like tyrosine kinase-3 ligand (Flt-3L) may be obtained, for example, from prosepec-Tany TechnoGene LTD (Rehovot, Israel) or u.s.biological (Swampscott, MA). Recombinant human Flt-3L and in vitro ED thereof₅₀From about 1 to about 10ng/ml, the same in vivo concentration can be used. The actual working concentration of any of the above can be determined by measuring the change over time of white blood cells or red blood cells in the culture medium or in a blood sample taken from the individual, depending on the individual, by practices known in the art. CD34 may be analyzed using well-known techniques⁺Differentiation of cells along the red blood cell pathway and expression of fetal hemoglobin genes (e.g., fetal hemoglobin transcription or PCR-mediated or antibody-mediated detection of fetal hemoglobin).

Erythropoietin (e.g., Epogene)) Usually about 12.5U/kg to 525U/kg, usually about 100U/kg or less dose intravenous or subcutaneous administration. Variant of erythropoietin, Aranesp^TMUsually administered in similar doses. For erythropoietin and erythropoietin analogs, a suitable dosage is one that provides a hematocrit of about 10g/dL to about 12g/dL and avoids an increase of more than 1.0g/dL over any 2 week period.

5.3.1. Oral dosage form

Pharmaceutical compositions of the invention suitable for oral administration may be formulated in discrete dosage forms such as, but not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of the active ingredient and may be prepared by pharmaceutical methods well known to those of ordinary skill in the art. See generally, Remington's pharmaceutical sciences, 18th edition, Mack Publishing, Easton PA (1990).

Typical oral dosage forms are prepared by intimately mixing the active agent with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients may take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules using solid excipients represent the most advantageous oral unit dosage form. If desired, the tablets may be coated by standard aqueous or non-hydrated techniques. Such dosage forms may be prepared by any pharmaceutical method. Pharmaceutical compositions and dosage forms are generally prepared by: the active agent is intimately mixed with a liquid carrier, a well-dispersed solid carrier, or both, and the product is then shaped as desired.

For example, tablets may be made by compression or compression molding. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free-flowing form, for example, as a powder or granules, optionally mixed with excipients. Compression molded tablets may be prepared by compression molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Examples of excipients that may be used in the oral dosage forms of the present invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, carboxymethylcellulose sodium), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methylcellulose (e.g., nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Microcrystalline fibreSuitable forms of the element include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. One specific binder is a mixture of microcrystalline cellulose and sodium carboxymethylcellulose sold as AVICELRC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103^TMAnd Starch 1500 LM.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, cellulose powder, dextrates (dextrates), kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. The binder or filler in the pharmaceutical compositions of the present invention is present in an amount of about 50% to about 99% by weight of the pharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to provide tablets that disintegrate upon exposure to an aqueous environment. Tablets containing too much disintegrant may disintegrate in storage, while tablets containing too little may not disintegrate at the desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that does not significantly alter the release of the active agent, either too much or too little, should be used to form the solid oral dosage form of the present invention. The amount of disintegrant used varies with the type of formulation and is readily determined by one skilled in the art. Typical pharmaceutical compositions contain from about 0.5% to about 15% by weight of disintegrant, preferably from about 1% to about 5% by weight of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or sweet potato starch, other starches, pregelatinized starch, other starches, clays, other alginates, other celluloses, gums, and mixtures thereof.

Lubricants useful in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. Other lubricants include, for example, syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, MD), solidified aerosol of synthetic silica (sold by Degussa Co. of Plano, TX), CAB-O-SIL (a sintered silica product sold by Cabot Co. of Boston, MA), and mixtures thereof. Lubricants, if used at all, are generally used in amounts less than about 1% by weight of the pharmaceutical composition or dosage form into which they are incorporated.

Preferred solid oral dosage forms comprise an immunomodulatory compound of the invention, anhydrous lactose, microcrystalline cellulose, polyvinylpyrrolidone, stearic acid, colloidal anhydrous silicon dioxide, and gelatin.

5.3.2. Sustained release dosage form

The active agents of the present invention may be administered by controlled release devices or delivery devices known to those skilled in the art. Examples include, but are not limited to, those described in the following patents: U.S. Pat. nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms may be used to provide sustained or controlled release of one or more active ingredients by using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, isotonic systems, multilayer coatings, microparticles, liposomes, microspheres or combinations thereof to produce the desired release profile in varying proportions. Suitable controlled release formulations include those described herein, which are well known to those skilled in the art and are readily selected for use with the active agents of the present invention. Thus, the present invention encompasses single unit dosage forms suitable for controlled release and for oral administration, including but not limited to tablets, capsules, gelcaps, and caplets.

All controlled release drug products share the following common objectives: the curative effect of the medicine is improved to exceed the curative effect of the non-released product. Ideally, the use of optimally designed controlled release formulations in medical treatment is characterized by: the disease is cured or controlled in the shortest time with the least amount of medicine. Advantages of controlled release formulations include prolonged drug activity, reduced dosing frequency and improved patient compliance. In addition, controlled release formulations may be used to affect the time at which the effect begins or other characteristics, such as blood levels of the drug, and thereby affect the incidence of side effects (e.g., adverse side effects).

Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that immediately produces the desired therapeutic effect, and gradually and continuously release amounts of other drugs to maintain that level of therapeutic or prophylactic effect over an extended period of time. In order to maintain a constant level of drug in the body, the drug must be released from the dosage form at a rate that will compensate for the amount of drug that is metabolized and excreted from the body. Controlled release of the active agent can be stimulated by a variety of conditions, including but not limited to pH, temperature, enzymes, water, or other physiological conditions or compounds.

5.3.3 parenteral dosage forms

Parenteral dosage forms can be administered to a patient by a variety of routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial routes. Since their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, dry products that can be dissolved or suspended in a pharmaceutically acceptable carrier for injection, injectable suspensions, and emulsions.

Suitable carriers that can be used to provide the parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: USP water for injection; aqueous carriers such as, but not limited to, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, and lactated ringer's injection; water-miscible carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the active ingredients disclosed herein may also be incorporated into the parenteral dosage forms of the invention. For example, cyclodextrins and derivatives thereof can be used to increase the solubility of immunomodulatory compounds and derivatives thereof. See, for example, U.S. Pat. No. 5,134,127, which is incorporated herein by reference.

5.3.4 topical and transmucosal dosage forms

Topical and transmucosal administration forms of the present invention include, but are not limited to, sprays, aerosols, solutions, emulsions, suspensions, or other forms known to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 16 th and 18th editions, Mack Publishing, Easton PA (1980 & 1990); and Introduction to Pharmaceutical DosageForms, 4 th edition, Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissue in the oral cavity can be formulated as mouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materials that may be used in the preparation of the topical and transmucosal dosage forms of the present invention are well known to those skilled in the art of pharmacy and depend on the particular tissue to which a given pharmaceutical composition or dosage form is to be administered. In fact, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1, 3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form a non-toxic and pharmaceutically acceptable solution, emulsion, or gel. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms, if desired. Examples of such other ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16 th and 18th edition, Mack Publishing, Easton PA (1980 & 1990).

The pH of the pharmaceutical composition or dosage form may also be adjusted to enhance delivery of one or more active agents. Similarly, the polarity of the solvent vehicle, its ionic strength, or tonicity can be adjusted to enhance delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active agents to enhance delivery. In this regard, stearates can be used as lipid carriers, emulsifiers or surface active agents, as well as delivery or penetration enhancers for the formulation. Different salts, hydrates or solvates of the active agent may also be used to adjust the properties of the resulting composition.

5.3.5. Reagent kit

It is generally preferred that the active ingredients of the invention are not administered at the same time or by the same route of administration. Thus, the present invention includes kits that, when used by medical personnel, can simplify the administration of an appropriate amount of an active agent to a patient.

A typical kit of the invention comprises a dosage form of an immunomodulatory compound of the invention, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. Preferably, the immunomodulatory compound in the kit is 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione or a compound of the formula:

the kits of the invention may also comprise cytokines or cytokine derivatives, such as G-CSF, GM-CSF, Epo, Flt-3L, SCF, IFN, IL2, IL8, IL18, and the like, and/or other compounds, including but not limited to, compounds for treating anemiaAny other compound known to have or that may have beneficial effects in disease or hemoglobinopathy, oblimersen (Genasense)) Melphalan, topotecan, dacarbazine, irinotecan, docetaxel, COX-2 inhibitor, pentoxifylline, ciprofloxacin, dexamethasone, Ara-C, vinorelbine, isotretinoin, 13 cis-retinoic acid, or a pharmacologically active mutant or derivative thereof, or a combination thereof. Other compounds contained in the kit include one or more of the following: a compound that induces fetal hemoglobin; a compound that relaxes blood vessels; a compound that reduces self-aggregation of hemoglobin S when covalently bound thereto; compounds that are Gardos channel antagonists; and a compound that reduces adhesion of red blood cells. In a more specific embodiment, the second compound is hydroxyurea, a guanidino derivative, nitrous oxide, a butyrate or butyrate derivative, an aldehyde or aldehyde derivative, a plant extract having anti-sickle cell forming activity (e.g., NIPRISAN)^TM(HEMOXIN^TM) Clotrimazole, derivatives of triarylmethane, monoclonal antibodies or polyethylene glycol derivatives. Examples of additional active ingredients include, but are not limited to, those described herein (see, e.g., section 5.1).

If some of the ingredients (e.g., immunomodulatory compounds, e.g., IMiD) are administered orally during the treatment of hemoglobinopathies^TMFor example, 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione; extract) and other components are administered by another common route, e.g., intravenously or subcutaneously, then the kit of the invention may include the component or compound to be administered as an adjuvant to the immunomodulatory compound in addition to the immunomodulatory compound of the invention.

The kit of the invention may also comprise a device for administering the active ingredient. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers.

The kits of the invention may also comprise cells or blood for transplantation and a pharmaceutically acceptable carrier useful for administering one or more active ingredients. For example, if the active ingredient is in solid form and must be formulated for parenteral administration, the kit may comprise a sealed container containing a suitable carrier in which the active ingredient can be dissolved to form a sterile, particle-free solution suitable for parenteral administration. Examples of pharmaceutically acceptable carriers include, but are not limited to: USP water for injection; aqueous carriers such as, but not limited to, sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, and lactated ringer's injection; water-miscible carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-hydrated vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

6. Examples of the embodiments

6.1. Example 1: differentiation of bone marrow-derived CD34+ hematopoietic progenitor cells into dendritic cells indicates upregulation of erythrocyte-specific genes

BM-CD34⁺Cells were obtained from Cambrex (East Rutherford, NJ) and cultured for 6 days in Iscove's MDM containing BIT 95000(StemCell technologies, UK) in the presence of Stem Cell Factor (SCF), Fit3-L, granulocyte macrophage-colony stimulating factor (GM-CSF) and TNF α. To investigate the effect of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione on dendritic cell production, CD34+ progenitor cells were cultured for 6 days in the presence or absence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione. After 6 days of culture, the cells of the red blood cell markers (CD36, CD71, glycophorin a and fetal hemoglobin) were phenotypically characterized by flow cytometry. Gene expression was monitored by microarray analysis on days 1, 3 and 6 of CD34+ differentiation (fig. 1).

RNA purification and microarray analysis Using RNAeasy (Qiagen) from CD34⁺The total RNA was isolated from the cells. Gene expression analysis was performed using the Affymetrix U133A gene chip. Briefly, it used 5. mu.g of RNA to synthesize double-stranded cDNA. Biotinylated cRNA was synthesized using the MessageAmp aRNA kit (Ambion), and 15 μ g of cRNA was fragmented and hybridized to each array. The above procedure was performed twice for each RNA sample to obtain duplicate biotin-labeled probes. The results of the two chips were averaged and the fold difference was calculated.

4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione treatment up-regulated the gene expression of erythrocyte specific genes during CD34+ differentiation in the presence of SCF, Flt3-L, GM-CSF and TNF α. Importantly, once CD34 is detected⁺Cell differentiation, 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione increased fetal hemoglobin gene expression, specifically 18-fold increase in fetal hemoglobin epsilon at day 6, and 7-fold increase in hemoglobin gamma at day 6 (fig. 2).

Differentiated CD34 by flow cytometry in the presence or absence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione⁺The phenotypic characteristics of the cells show modulation of red blood cell and hemoglobin markers. The expression of glycophorin a (fig. 3) and fetal hemoglobin (fig. 4) increased in a dose-dependent manner. 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione also induced other erythrocyte specific genes (FIG. 5). It was also found that the expression of genes encoding glycophorin B, rhesus blood group-related glycoprotein, Kell blood group precursor, EDRF/AHSP (alpha hemoglobin stabilizing protein), and erythrocyte Kruppel-like transcription factor essential for normal erythropoiesis in the presence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione is expressed in differentiated CD34⁺Is also up-regulated in the cell.

The erythrocyte specific gene enhanced by 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione has obvious effect on improving anemia. An increase in hemoglobin levels and alpha-hemoglobin stabilizing protein (AHSP) can increase oxygen carrying capacity while preventing cellular alpha-hemoglobin levels from being excessive, which can damage blood cells. IMiD generally has an effect on increased erythropoiesis, particularly on the genes described above, and is useful in overcoming the anemic effects of chemotherapy and in treating diseases where low red blood cell count is a symptom or therapeutic effect.

It is contemplated that IMiD^TMHas synergistic effect with erythropoietin. IMiD^TMEarly erythroid precursor synthesis can be induced, while erythropoietin is crucial for the proliferation, survival and differentiation of the late stage of differentiation.

6.2. Example 2: CD34⁺Differentiation of cells into erythrocytes

Differentiation of Bone Marrow (BM) CD34+ hematopoietic progenitor cells: BM-CD34+ progenitor cells were obtained from Cambrex and cultured in Iscove's MDM containing BIT 95000 (serum replacement; StemShell technologies) in the presence of growth factors. On the first 6 days, CD34⁺The cells were expanded with SCF (100ng/ml), Flt3-L (100ng/ml) and IL-3(20ng/ml), and then differentiated toward erythrocytes by culturing in the presence of SCF (50ng/ml), and Epo (2U or 4U/ml) for 6 days. To study IMiD^TMThe CD34+ progenitor cells were differentiated for 6 days in the presence or absence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione or 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione (FIG. 6).

Flow cytometry: surface antigen expression was analyzed by flow cytometry (FACScan, Coulter) after 6 days of culture. Cells were double stained (30min, 4 ℃) on day 6 with FITC and PE-coupled monoclonal antibodies (mAb). The antibodies used were: CD34-PE, CD36-FITC, CD71-FITC, and glycophorin A-PE, all obtained from BD Pharmingen (San Diego, Calif.). After 6 days of culture, cells were washed with Phosphate Buffered Saline (PBS), fixed with 2% paraformaldehyde, permeabilized with cytopermeafix (BD Pharmingen), and stained with HbF-PE (BDPharmingen, San Diego, CA), Hb ε -FITC (Cortex Biochem, San Leandro, CA) mAb and HbA-FITC (Perkin Elmer), and analyzed by flow cytometry (FACScan, Coulter or FCASAria, BD Pharmingen).

As a result: IMiD^TM4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione and 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione are potent inducers of hemoglobin F in erythrocyte precursors. CD34+ progenitor cells were first expanded with a combination of growth factors (SCF, Flt3-L and IL-3) for 6 days. After expansion, in the presence or absence of IMiD^TMIn the case of (2), CD34 cells were differentiated into erythrocytes for 6 days with SCF and Epo (FIG. 6). Surface labeling by characteristic erythrocytes: expression of glycophorin a (CD235) and transferrin receptor (CD71) to monitor the differentiation of CD34+ progenitor cells in the presence of SCF and Epo (fig. 7). With or without IMiD^TMDifferentiated CD34⁺When cells are present, the red blood cell phenotype is present. Interestingly, expression of glycophorin A in IMiD^TMLower in the treated cells, while the expression of CD71 remained at a higher level under both conditions.

The percentage of cells expressing fetal hemoglobin was monitored by flow cytometry after 6 days of culture with SCF and Epo. IMiD^TMThe expression of fetal hemoglobin was increased in a dose-dependent manner (fig. 8). Importantly, an increase in fetal hemoglobin (HbF) is associated with a decrease in adult hemoglobin (HbA). In IMiD^TMIn the presence of this, the ratio HbF/HbA increases. (FIG. 9)

In addition to the mature phenotype, quantification of hemoglobin, the proliferation status of the cells was also measured. Cell counts were performed after 6 days of culture with SCF and Epo. In IMiD^TMThe total cell count increased in the presence and correlated with the developmental stage of the colony (i.e., less maturation).

6.3. Example 3: IMID synergizes with existing fetal hemoglobin nucleotherapy

As previously describedAs described, CD34+ progenitor cells were first expanded with a combination of growth factors (SCF, Flt3-L, and IL-3) for 6 days, and then red blood cells were induced to differentiate with SCF and Epo for 6 days. During differentiation of erythrocytes, in IMiD^TMCulturing CD34 in the presence or absence, alone or in combination with one of hydroxyurea and 5-azacytidine⁺Cells to compare IMiD^TMAnd the effects of these two known fetal hemoglobin synthesis inducers. On day 6 of differentiation, the hemoglobin capacity of the cells was measured by flow cytometry. Hydroxyurea and 5-azacytidine were reported to increase fetal hemoglobin expression (fig. 10). However, IMiD induced fetal hemoglobin production more significantly than hydroxyurea or 5-azacytidine, resulting in a 10-fold induction in the presence of 10 μ M4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione. Interestingly, 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione showed a surprising synergistic effect when combined with hydroxyurea, resulting in significant reactivation of fetal hemoglobin (fig. 11).

6.4. Example 4: increased phosphorylation of STAT5 by EPO + IMID

To further study Epo and IMiD^TMSynergistic Effect on erythrocytes Signal experiments were carried out in the UT-7 cell line, in particular, to determine IMiD^TMWhether or not there is an effect on STAT5 expression, it is known that STAT5 is activated once Epo binds to the erythropoietin receptor (EpoR). UT-7 is a human leukemia cell line, absolutely dependent on erythropoietin for proliferation, isolated from patients with acute myeloid leukemia (AML M7). EpoR expression levels in these cells were about 60%.

To study IMiD^TMRole in Epo signaling UT-7 cells were stimulated with Epo in the presence or absence of 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione, as described below. UT-7 cells were expanded in RPMI medium containing 10% FBS and GM-CSF (5 ng/ml). Cells were deprived of serum and growth factors overnight, then treated with 10. mu.M 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione orThe DMSO control was preincubated for 45 min and stimulated with Epo (10U/ml) for 10 min. 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione increased Epo-induced STAT5(Tyr694) phosphorylation by a factor of 2 (FIG. 12). This effect was detected within 10 minutes of the stimulation with Epo.

6.5. Example 5: toxicology studies

The effect of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione on cardiovascular and respiratory function was studied in anesthetized dogs. Two groups of Beagle dogs (2/gender/group) were used. One group received only three doses of vehicle and the other three ascending doses of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione (2, 10, 20 mg/kg). In all cases, 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione or carrier was administered sequentially by jugular infusion at intervals of at least 30 minutes.

The cardiovascular and respiratory changes induced by 3-4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione were minimal at all doses compared to vehicle controls. The only statistically significant difference between vehicle and test group after administration of low doses of 3- (4-amino-1-oxo-1, 3-dihydro-isoindol-2-yl) -piperidine-2, 6-dione was a small increase in arterial blood pressure (from 94mmHg to 101 mmHg). This effect lasted for approximately 15 minutes and was not observed at the higher doses. Deviations in femoral blood flow, respiratory parameters, and Qtc interval were common to both the control and treatment groups and were not considered treatment-related.

The above-described embodiments of the invention are intended to be examples only, and those embodiments may be understood or appreciated by those of ordinary skill in the art using routine experimentation, numerous equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are covered by the following claims.

7. Reference to the literature

All references cited herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference in its entirety. Any publications cited prior to the filing date are provided for public purposes only and are not to be construed as an admission that the invention is not prior art to these publications.

Claims (14)

1. Use of a compound for the preparation of a medicament for modulating CD34⁺Use of a stem cell or precursor cell in the manufacture of a medicament for differentiation into a erythroid lineage, wherein the compound has the structural formula:

wherein one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；R²Is hydrogen or a methyl group, or a mixture thereof,

or a pharmaceutically acceptable salt thereof.

2. The use of claim 1, wherein the compound is amino-substituted thalidomide.

3. The use of claim 1, wherein the compound is 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione.

4. The use of claim 1, wherein the compound is 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione.

5. The use of any one of claims 1-4, wherein the CD34⁺The stem cells or precursor cells are in vitro cells.

6. The use of any one of claims 1-4, wherein the CD34⁺The stem cell or precursor cell is an in vivo cell.

7. The use according to any one of claims 1 to 4, wherein the medicament further comprises at least one cytokine.

8. The use of claim 7, wherein the at least one cytokine is Epo, SCF, GM-CSF, Flt-3L, TNF α, IL-3, or any combination thereof.

9. Use of a compound in the manufacture of a medicament for detectably increasing Alpha Hemoglobin Stabilizing Protein (AHSP) in a subject, wherein the compound has the structural formula:

wherein one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；R²Is hydrogen or a methyl group, or a mixture thereof,

or a pharmaceutically acceptable salt thereof.

10. The use of claim 9, wherein the compound is 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione.

11. The use of claim 9, wherein the compound is 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione.

12. Use of a compound for the manufacture of a medicament for inducing the expression of one or more genes associated with or necessary for erythropoiesis or hematopoiesis in a hematopoietic stem cell, progenitor cell, or precursor cell of a subject in the presence of erythropoietin and a stem cell factor, wherein the compound has the structural formula:

wherein one of X and Y is C ═ O, and the other of X and Y is C ═ O or CH₂；R²Is hydrogen or a methyl group, or a mixture thereof,

or a pharmaceutically acceptable salt thereof.

13. The use of claim 12, wherein the compound is 4- (amino) -2- (2, 6-dioxo (3-piperidyl)) -isoindoline-1, 3-dione.

14. The use of claim 12, wherein the compound is 3- (4-amino-1-oxo-1, 3-dihydroisoindol-2-yl) -piperidine-2, 6-dione.