Gardos channel antagonists

Details Gardos channel antagonists Gardos channel antagonists

1999-08-31

2001-09-11

Kumar, Shailendra (Department: 1621)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Nitrogen containing other than solely as a nitrogen in an...

C564S181000, C564S182000

Reexamination Certificate

active

06288122

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to organic compounds that are specific, potent and safe inhibitors of the Ca
2+
-activated potassium channel (Gardos channel) of erythrocytes. More particularly, the invention relates to fluorine substituted triarylmethane-based inhibitors that exhibit remarkably enhanced resistance to degradation in in vitro biological media and exhibit extended in vivo half-lives relative to their non-fluorinated homologues.
BACKGROUND OF THE INVENTION
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
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 administe

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