Sickle cell disease has been recognized within West Africa for several centuries. Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first genetic disease to be understood at the molecular level. It is recognized today as the morphological and clinical result of a glycine to valine substitution at the No. 6 position of the beta globin chain (Ingram, Nature 178: 792-794 (1956)). The origin of the amino acid change and of the disease state is the consequence of a single nucleotide substitution (Marotta et al., J. Biol. Chem. 252:5040-5053 (1977)).
The major source of morbidity and mortality of patients suffering from sickle cell disease is vascular occlusion caused by the sickled cells, which causes repeated episodes of pain in both acute and chronic form and also causes ongoing organ damage with the passage of time. It has long been recognized and accepted that the deformation and distortion of sickle cell erythrocytes upon complete deoxygenation is caused by polymerization and intracellular gelation of sickle hemoglobin, hemoglobin S (Hb S). The phenomenon is well reviewed and discussed by Eaton et al., Blood 70:1245 (1987). The intracellular gelatin and polymerization of Hb S can occur at any time during an erythrocyte's journey through the vasculature. Thus, erythrocytes in patients with sickle cell disease containing no polymerized hemoglobin S may pass through the microcirculation and return to the lungs without sickling, may sickle in the veins or may sickle in the capillaries.
The probability of each of these events is determined by the delay time for intracellular gelation relative to the appropriate capillary transit time (Eaton, et al., Blood 47: 621(1976)). In turn, the delay time is dependent upon the oxygenation state of the hemoglobin, with deoxygenation shortening the delay time. If it is thermodynamically impossible for intracellular gelation to take place, or if the delay time at venous oxygen pressures is longer than about 15 seconds, cell sickling will not occur. If the delay time is between about 1 and 15 seconds, the red cell will likely sickle in the veins. If the delay time is less than about 1 second, red cells will sickle within the capillaries.
For red cells that sickle within the capillaries, a number of consequent events are possible. These range from no effect on transit time, to transient occlusion of the capillary, to a more permanent blockage that may ultimately result in ischemia or infarction of the surrounding cells, and in the subsequent destruction of the red cell.
Normal erythrocytes are comprised of approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds. Loss of cell water causes an exponential increase in cytoplasmic viscosity as the mean cell hemoglobin concentration (MCHC) rises above about 32 g/dl. Since cytoplasmic viscosity is a major determinate of erythrocyte deformability and sickling, the dehydration of the erythrocyte has substantial rheological and pathological consequences. Regulation of erythrocyte dehydration is recognized as an important therapeutic approach for treating sickle cell disease. Since cell water follows any osmotic change in intracellular ion concentration, maintaining the red cell's potassium concentration is of particular importance (Stuart et al., Brit J. Haematol. 69:1-4 (1988)).
Many approaches to therapeutically treating dehydrated sickle cells (thus decreasing polymerization of hemoglobin S by lowering the osmolality of plasma) have been tried with limited success, including the following approaches: intravenous infusion of distilled water (Gye et al., Am. J. Med. Sci. 266: 267-277(1973)); administration of the antidiuretic hormone vasopressin together with a high fluid intake and salt restriction (Rosa et al., M. Eng. J. Med. 303:1138-1143 (1980)); Charache et al., Blood 58: 892-896 (1981)); the use of monensin to increase the cation content of the sickle cell (Clark et al., J. Clin. Invest. 70:1074-1080 (1982)); Fahim et al., Life Sciences 29:1959-1966 (1981)); intravenous administration of cetiedil citrate (Benjamin et al., Blood 67: 1442-1447 (1986)); Berkowitz et al., Am. J. Hematol. 17: 217-223 (1984)); Stuart et al., J. Clin. Pathol. 40:1182-1186 (1987)); and the use of oxpentifylline (Stuart et al., supra).
Another approach towards therapeutically treating dehydrated sickle cells involves altering erythrocyte potassium flux by targeting a calcium-dependent potassium channel (Ishi et al., Proc. Natl. Acad. Sci. 94(21): 11651-6 (1997)). This calcium activated potassium channel is also referred to as the Gardos channel (Brugnara et al, J. Clin. Invest. 92: 520-526 (1993)). Recently, a cloned human intermediate conductance calcium activated potassium channel, hIK1, was shown to be substantially similar to the Gardos channel in terms of both its biophysical and pharmacological properties (Ishi, supra).
Methods that have been used to inhibit the Gardos channel include the administration to erythrocytes of imidazole, nitroimidazole and triazole antimycotic agents such as clotrimazole (U.S. Pat. No. 5,273,992 to Brugnara et al.). Clotrimazole, an imidazole-containing antimycotic agent, has been shown to be a specific, potent inhibitor of the Gardos channel of normal and sickle erythrocytes, and prevents Ca.sup.2+ -dependent dehydration of sickle cells both in vitro and in vivo (Brugnara, supra; De Franceschi et al., J. Clin. Invest. 93: 1670-1676 (1994)). When combined with a compound which stabilizes the oxyconformation of Hb S, clotrimazole induces an additive reduction in the clogging rate of a micropore filter and may attenuate the formation of irreversibly sickled cells (Stuart et al., J. Haematol. 86:820-823 (1994)). Other compounds that contain a heteroaryl imidazole-like moiety believed to be useful in reducing sickle erythrocyte dehydration via Gardos channel inhibition include miconazole, econazole, butoconazole, oxiconazole and sulconazole. Although these compounds have been demonstrated to be effective at reducing sickle cell dehydration, other imidazole compounds have been found incapable of inhibiting the Gardos channel and preventing loss of potassium.
Since sickle cell anemia is a chronic disease, agents designed for treating it will ideally exhibit certain characteristics that are less essential in drugs for treating resolvable illnesses (e.g., fungal infections). A clinically useful Gardos channel inhibitor will exhibit extremely low toxicity over a prolonged course of administration, will have an excellent bioavailability, will be highly specific for the Gardos channel and will be potent in its interactions with this channel.
Although clotrimazole and certain related compounds have been shown to inhibit the Gardos channel and prevent loss of potassium, these compounds are less than ideal clinical agents for the treatment of sickle cell anemia. Of primary concern is the fact that prolonged administration of imidazole antimycotics has been demonstrated to result in hepatotoxicity (see, for example, Rodriguez el al., Toxicology 6: 83-92 (1995); Findor et al., Medicina 58: 277-81 (1998); and Rodriguez et al., J. Biochem. Toxicol. 11: 127-31 (1996)). The trend towards toxicity of an agent must be balanced with other characteristics such as its bioavailability, target selectivity and potency.
Presently known Gardos channel inhibitors have low in vivo half lives and low bioavailabilities. These deficiencies are of particular concern in conjunction with these drugs, as they must be regularly administered over a significant portion of a person's lifetime. With such drugs, patient compliance with the dosage regimen is crucial, and the simpler the regimen, the more likely a patient will comply with the regimen. Gardos channel inhibitors having low bioavailabilities must be frequently administered, raising the risk of missed doses and consequent plasma drug levels inadequate to prevent the dehydration of erythrocytes. In addition to frequent dosing, agents having low bioavailabilities must generally be administered in higher dosages than analogous agents with better bioavailabilities. At higher dosages, undesirable side effects and toxicity become a very real concern.
In addition to their low bioavailability, known Gardos channel inhibitors such as clotrimazole also have relatively low potency in their interaction with the Gardos channel. The low potency of the compounds is exacerbated by their low bioavailability and rapid systemic clearance. A further shortcoming of many known Gardos channel inhibitors is the non-specific nature of their interactions with calcium activated potassium channels: these agents readily interact with calcium activated potassium channels other than the Gardos channel. Taken together, the low potency, low specificity and low bioavailability of known Gardos channel inhibitors mandate higher and more frequent dosing, thereby increasing the risk of undesirable side effects and toxicity.
In view of the above-described shortcomings of currently known Gardos channel inhibitors a substantial advance in the treatment of sickle cell anemia is expected from the discovery of Gardos channel inhibitors that do not contain imidazole as a structural component, are appreciably bioavailable, slowly metabolized and excreted and are both potent and specific in their interactions with the Gardos channel. Quite surprisingly, the present invention provides Gardos channel inhibitors having these characteristics.
USPTO Backgrounds
USPTO Backgrounds