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Compositions for the treatment of blood disorders

Compositions for the treatment of blood disorders description/claims

This patent application is a continuation of U.S. patent application Ser. No. 08/470,830, filed Jun. 6, 1995, entitled “Compositions for the Treatment of Blood Disorders,” which is a continuation-in-part of abandoned U.S. patent application Ser. No. 08/040,173, filed Mar. 31, 1993, and U.S. patent application Ser. No. 08/142,908, filed Oct. 29, 1993.

This invention was made with support from the United States government under grant numbers HL-37118, HL-45940, HL-20895 and HL-15157, awarded by the National Institutes of Health, and grant number 000831, awarded by the United States Food & Drug Administration, and the United States government has certain rights in the invention.

The invention relates to compositions useful in the treatment and prevention of blood disorders such as anemia, thalassemia and sickle cell disease. Compositions comprise proteins or chemicals that stimulate the specific expression of a globin protein or the proliferation or development of hemoglobin expressing or other myeloid cells. The invention also relates to methods and medical aids which utilize these compositions to ameliorate symptoms associated with blood disorders.

Hematopoiesis, or the formation of blood cells, begins in the developing human embryo as clusters of stem cells called blood islands. These cells appear in the yolk sac at about the third week of development and, at about the third month, migrate to the developing liver which becomes the principal site of blood cell formation. Although the spleen, lymph nodes and bone marrow all make small contributions to blood cell development, not until the fourth month does the bone marrow become the principal site of hematopoiesis. At birth, virtually all blood cells originate from the bone marrow. Although small foci of blood-forming cells sometimes persist in the liver for longer periods of time, hepatic blood cell formation has decreased to a trickle. At this time, all of the marrow is actively forming blood cells and continues to do so until after puberty when, at about 18 years of age, the principal sites of blood cell formation become the marrow of the vertebrae, ribs, sternum, skull, pelvis and the proximal epiphyseal regions of the femur and humerus. These areas represent only about half of the available marrow. The cavities which remain are filled with yellow-fatty tissues.

In the adult, hematopoiesis involves the bone marrow, the lymph nodes and the spleen. These organs and associated tissues are traditionally divided into myeloid and lymphoid tissue-types. Myeloid tissues and the cells derived from the myeloid tissue include the erythrocytes, platelets, granulocytes and monocytes. Lymphoid and lymphoid-derived tissues include the thymus, lymph nodes and spleen. The myeloid/lymphoid division is somewhat artificial as these two types of tissues are believed to originate from a single pluripotent stem cell.

Lymphoid and myeloid stem cells, formed from division of the pluripotent cell, are precursors for all subsequent cell types (FIG. 1). The committed cell-types for the lymphoid stem cell include the pro-T cells which form mature T cells and the pro-B cells which differentiate into plasma cells. Intermediate cell types can be distinguished based on cell-surface phenomenon such as the expression of immunoglobulin heavy and light chain, Ia protein and other cell surface markers. The three committed cell-types for the myeloid stem cell include E/mega cells which differentiate into the erythrocyte-burst forming unit (BFU-E) followed by the erythrocyte-colony forming unit cells (CFU-E) and megakaryocyte-CFU cells (CFU-mega), granulocyte/macrophage-CFU cells (CFU-G/M) which differentiate into CFU-G and CFU-M cells, and the eosinophil-CFU cells (CFU-Eo) which ultimately form mature eosinophils. Although these committed cell types reside mainly in the marrow, some circulate throughout the body in the blood stream.

The bone marrow provides a unique environment for pluripotent and committed cells. It contains both structural and humoral components that have yet to be successfully duplicated in culture. The marrow cavity itself is a network of thin-walled sinusoids lined with endothelial cells. Between the walls of bone are clusters of hematopoietic cells and fat cells constantly fed by mature blood cells entering through the endothelium. Differentiated cells ready to function within the circulatory system depart the cavity in a similar fashion.

The relative proportions of cell types in the bone marrow have a myeloid/erythroid ratio of about three to one comprising about 60% granulocytes and their precursors, about 10% lymphocytes and their precursors, about 20% erythrocytes and their precursors, and about 10% unidentified cells. The predominant myeloid cell types in the marrow cavity are the myelocytes, metamyelocytes and granulocytes. The predominant cell types in the erythroid compartment are the polychromatophilic and orhtochromic normoblasts. Under conditions of normal iron metabolism, about 30% to 40% of the normoblasts contain scattered ferritin granules. These cells are referred to as sideroblasts and the iron granules they contain are reservoirs drawn from as the cells insert iron into protoporphyrin to form heme. The production of heme and the production of globin are precisely balanced within the cell. If either is hindered or depressed, for whatever reason, excess ferritin accumulates in the sideroblasts. This increased iron accumulation can be visualized in the mitochondria, the loci of heme synthesis.

The major function of red blood cells is to transport oxygen to tissues of the body. Minor functions include the transportation of nutrients, intercellular messages and cytokines, and the absorption of cellular metabolites. Anemia, or a loss of red blood cells or red blood cell capacity, can be grossly defined as a reduction in the ability of blood to transport oxygen and may be acute or chronic. Chronic blood loss may be caused by extrinsic red blood cell abnormalities, intrinsic abnormalities or impaired production of red blood cells. Extrinsic or extra-corpuscular abnormalities include antibody-mediated disorders such as transfusion reactions and erythroblastosis, mechanical trauma to red cells such as micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation. In addition, infections by parasites such as Plasmodium, chemical injuries from, for example, lead poisoning, and sequestration in the mononuclear system such as by hypersplenism can provoke red blood cell disorders.

Impaired red blood cell production can occur by disturbing the proliferation and differentiation of the stem cells or committed cells. Some of the more common diseases of red cell production include aplastic anemia, hypoplastic anemia, pure red cell aplasia and anemia associated with renal failure or endocrine disorders. Disturbances of the proliferation and differentiation of erythroblasts include defects in DNA synthesis such as impaired utilization of vitamin B12 or folic acid and the megaloblastic anemias, defects in heme or globin synthesis, and anemias of unknown origins such as sideroblastic anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HIV, hepatitis virus or other viruses, and myelophthisic anemias caused by marrow deficiencies.

Intrinsic abnormalities include both hereditary and acquired disorders. Acquired disorders are those which have been induced through, for example, a membrane defect such as paroxysmal nocturnal hemoglobinuria. Hereditary disorders include disorders of membrane cytoskeleton such as spherocytosis and elliptocytosis, disorders of lipid synthesis such as an abnormally increased lecithin content of the cellular membrane, red cell enzyme deficiencies such as deficiencies of pyruvate kinase, hexokinase, glutathione synthetase and glucose-6-phosphate dehydrogenase. Although red blood cell disorders may be caused by certain drugs and immune system disorders, the majority are caused by genetic defects in the expression of hemoglobin. Disorders of hemoglobin synthesis include deficiencies of globin synthesis such as thalassemia syndromes and structural abnormalities of globin such as sickle cell syndromes and syndromes associated with unstable hemoglobins. Hemoglobin comprises four protein chains, two alpha chains and two beta chains (α2 β2), interwoven together, each with its own molecule of iron and with a combined molecular weight of about 68 kD. The hemoglobin macromolecule is normally glycosylated and upon absorbing oxygen from the lungs transforms into oxyhemoglobin (HbO2). There are at least six distinct forms of hemoglobin, each expressed at various times during development. Hemoglobin in the embryo is found in at least three forms, Hb-Gower 1 (ζ2ε2), Hb-Gower 2 (α2ε2), and Hb-Portand (ζ7γ2). Hemoglobin in the fetus comprises nearly totally HbF (α2γ2), whereas hemoglobin in the adult contains about 96% HbA (α2 β2), about 3% HbA2 (α2δ2) and about 1% fetal HbF (α2γ2). The embryonic switch of globin expression from ζ to α and from ε to γ begins in the yolk sac. However, chains of embryonic ζ and ε have been found in the fetal liver and complete transition to the fetal form does not occur until late in fetal development. The fetal switch from γ to β begins later in erythropoiesis with the amount of γ globin produced increasing throughout gestation. At birth, β globin accounts for about 40% of non-α globin chain synthesis and thereafter continues to rapidly increase. Neither the switch from embryonic to fetal or fetal to adult appears to be controlled through cell surface or known cytokine interactions. Control seems to reside in a developmental clock with the switch occurring at times determined only by the stage of fetal development.

Defects or mutations in globin chain expression are common. Some of these genetic mutations pose no adverse or only minor consequences to the person, however, most mutations prevent the formation of an intact or normal hemoglobin molecule through a functional or structural inability to effectively bind iron, an inability of the chains or chain pairs to effectively or properly interact, an inability of the molecule to absorb or release oxygen, a failure to express sufficient quantities of one or more globin chains or a combination of these malfunctions. For example, substitutions of valine for glutamic acid at the sixth position of the β chain produces HbS and was found to occur in about 30% of black Americans. In the HbS heterozygote, only about 40% of total hemoglobin is HbS with the remainder being the more normal HbA.

Upon deoxygenation, HbS molecules undergo aggregation and polymerization ultimately leading to a morphological distortion of the red cells which acquire a sickle or holly-leaf shape. Sickling has two major consequences, a chronic hemolytic anemia and an occlusion of small blood vessels that results in ischemic damage to tissues. Further, when exposed to low oxygen tensions, polymerization converts HbS hemoglobin from a free-flowing liquid to a viscous gel. Consequently, the degree of pathology associated with sickle cell anemia can be correlated with the relative amount of HbS in the patient's system. Individuals with severe sickle cell anemia develop no symptoms until about five to six months after birth. In these infants it was determined that fetal hemoglobin did not interact with HbS and, as long as sufficient quantities were present, could modulate the effects of HbS disease. This modulating effect of β globin is also observed with other β globin disorders, such as HbC and HbD, and other mutations of the β chain. HbS polymerization is also significantly affected by the hemoglobin concentration of the cell. The higher the HbS concentration, the greater the chances for contact between two or more HbS molecules. Dehydration increases hemoglobin concentration and greatly facilitates sickling.

To some extent, sickling is a reversible phenomenon. With increased oxygen tensions, sickled cells depolymerize. This process of polymerization-depolymerization is very damaging to red cell membranes and eventually leads to irreversibly sickled cells (ISC) which retain their abnormal shape even when fully oxygenated. The average ISC survives for about 20 days in the body, as compared to the normal 120 day life span. Individuals with HbS syndromes have frequent infections, chronic hemolysis with a striking reticulocytosis and hyperbilirubinemia. The course of the disease is typically punctuated with a variety of painful crises called vaso-occlusive crises. These crises represent episodes of hypoxic injury and infarction in the organs, abdomen, chest, extremities or joints. Leg ulcers are an additional manifestation of the vaso-occlusive tendency of this disease. Central nervous system involvement is common producing seizures and even strokes. Aplastic crises, also common, represent a temporary cessation of bone marrow activity and may be triggered by infections, folic acid deficiency or both. Crises are episodic and reversible, but may be fatal. Damage from crisis episodes tends to be cumulative and even in those individuals with milder forms of sickle cell disorders, life-spans can be greatly reduced. Absent alternative intervention, patients typically die before the age of 30.

Anti-gelling compounds including clofibric acid (ClC6H5OC(CH3)2COOH), p-chloro phenoxy acetic acid (ClC6H5OCH2COOH), and phenoxy acetic acid (C6H5OCH2COOH) have been shown to prophylactically inhibit polymerization in artificially deoxygenated blood (D. J. Abraham et al., J. Med. Chem. 25:1015-17, 1982). It was speculated that these compounds may be useful in a narrow respect to prevent blood cell sickling in sickle cell disease. Such treatments may potentially decrease the frequency of symptomatic episodes caused by vaso-occlusive crises if enough of the chemical can be administered to bind all hemoglobin in the body.

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