WO1999026628A1 - Utilisation de composes 11-phenyl-dibenzazepine substitues dans le traitement ou la prevention de la drepanocytose, des maladies inflammatoires caracterisees par une proliferation cellulaire anormale et par la diarrhee chez l'homme et l'animal - Google Patents

Utilisation de composes 11-phenyl-dibenzazepine substitues dans le traitement ou la prevention de la drepanocytose, des maladies inflammatoires caracterisees par une proliferation cellulaire anormale et par la diarrhee chez l'homme et l'animal Download PDF

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Publication number
WO1999026628A1
WO1999026628A1 PCT/US1998/024967 US9824967W WO9926628A1 WO 1999026628 A1 WO1999026628 A1 WO 1999026628A1 US 9824967 W US9824967 W US 9824967W WO 9926628 A1 WO9926628 A1 WO 9926628A1
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WIPO (PCT)
Prior art keywords
group
aryl
compounds
halogen
trihalomethyl
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PCT/US1998/024967
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English (en)
Inventor
Carlo Brugnara
Jose Halperin
Emile M. Bellot, Jr.
Mark Froimowitz
Richard John Lombardy
John J. Clifford
Ying-Duo Gao
Reem M. Haidar
Eugne W. Kelleher
Falguni M. Kher
Adel M. Moussa
Yesh P. Sachdeva
Minghua Sun
Heather N. Taft
Wayne I. Lencer
Seth Alper
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Children's Medical Center Corporation
President And Fellows Of Harvard College
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Priority claimed from US09/159,333 external-priority patent/US6291449B1/en
Priority claimed from US09/159,337 external-priority patent/US20030134842A1/en
Application filed by Children's Medical Center Corporation, President And Fellows Of Harvard College filed Critical Children's Medical Center Corporation
Priority to IL13620998A priority Critical patent/IL136209A0/xx
Priority to EP98960380A priority patent/EP1032394A1/fr
Priority to CA002310773A priority patent/CA2310773A1/fr
Priority to JP2000521830A priority patent/JP2001523720A/ja
Priority to AU15987/99A priority patent/AU1598799A/en
Publication of WO1999026628A1 publication Critical patent/WO1999026628A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/14—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D223/18—Dibenzazepines; Hydrogenated dibenzazepines
    • C07D223/20—Dibenz [b, e] azepines; Hydrogenated dibenz [b, e] azepines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to aromatic organic compounds which are specific, potent and safe inhibitors of the Ca 2+ -activated potassium channel (Gardos channel) of erythrocytes, of mammalian cell proliferation, and/or of secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells.
  • the compounds are generally substituted 11 -phenyl-dibenzazepine compounds.
  • the compounds can be used to reduce sickle erythrocyte dehydration and/or delay the occurrence of erythrocyte sickling or deformation in situ as a therapeutic approach towards the treatment or prevention of sickle cell disease.
  • the compounds can also be used to inhibit mammalian cell proliferation in situ as a therapeutic approach towards the treatment or prevention of diseases characterized by abnormal cell proliferation.
  • the compounds can be used to inhibit chloride secretion as a therapeutic approach towards the treatment of diarrhea and scours.
  • Hb S sickle hemoglobin
  • Hb S hemoglobin S
  • 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.. 1976, Blood 47:621).
  • the delay time is dependent upon the oxygenation state of the hemoglobin, with deoxygenation shortening the delay time.
  • the delay time is between about 1 and 15 seconds, the red cell will likely sickle in the veins.
  • the delay time is less than about 1 second, red cells will sickle within the capillaries.
  • red cells that sickle within the capillaries a number of possible consequent events exist, ranging 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 destruction of the red cell.
  • cytoplasm of the normal erythrocyte comprises approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds; however, the 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. Thus, the physiological mechanisms that maintain the water content of a normal erythrocytes and the pathological conditions that cause loss of water from erythrocytes in the blood circulation are critically important.
  • Clotrimazole Another approach towards therapeutically treating dehydrated sickle cells involves the administration of imidazole, nitroimidazole and triazole antimycotic agents such as Clotrimazole (U.S. Patent 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 et al, 1993, J. Clin. Invest. 92:520-526; De Franceschi et al , 1994, J. Clin. Invest.
  • Clotrimazole When combined with a compound which stabilizes the oxy conformation 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, 1994, J. Haematol. 86:820-823).
  • 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. Each of these compounds is a known antimycotic.
  • Other imidazole-containing compounds have been found to be incapable of inhibiting the Gardos channel and preventing loss of potassium.
  • reducing sickle erythrocyte dehydration via blockade of the Gardos channel is a powerful therapeutic approach towards the treatment and/or prevention of sickle cell disease.
  • Compounds capable of inhibiting the Gardos channel as a means of reducing sickle cell dehydration are highly desirable, and are therefore an object of the present invention.
  • Cell proliferation is a normal part of mammalian existence, necessary for life itself. However, cell proliferation is not always desirable, and has recently been shown to be the root of many life-threatening diseases such as cancer, certain skin disorders, inflammatory diseases, fibrotic conditions and arteriosclerotic conditions.
  • Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments, and is associated with the synthesis of DNA. Binding of specific polypeptide growth factors to specific receptors in growth-arrested cells triggers an array of early ionic signals that are critical in the cascade of mitogenic events eventually leading to DNA synthesis (Rozengurt, 1986, Science 234:161-164). These include (1) a rapid increase in cystolic Ca 2+ , mostly due to rapid release of Ca 2+ from intracellular stores; (2) capacitative Ca 2+ influx in response to opening of ligand-bound and hyperpolarization- sensitive Ca 2+ channels in the plasma membrane that contribute further to increased intracellular Ca 2+ concentration (Tsien and Tsien, 1990, Annu. Rev. Cell Biol.
  • Clotrimazole has been shown to inhibit the Ca 2+ -activated potassium channel of erythrocytes.
  • Clotrimazole inhibits voltage- and ligand-stimulated Ca 2+ influx mechanisms in nucleated cells (Villalobos et al , 1992. FASEB J. 6:2742-2747; Montero et al, 1991. Biochem. J. 277:73-79) and inhibits cell proliferation both in vitro and in vivo (Benzaquen et al, 1995, Nature Medicine 1:534-540).
  • Clotrimazole and other imidazole-containing antimycotic agents capable of inhibiting Ca 2+ -activated potassium channels have been shown to be useful in the treatment of arteriosclerosis (U.S. Patent No. 5,358,959 to Halperin et al), as well as other disorders characterized by unwanted or abnormal cell proliferation.
  • inhibiting mammalian cell proliferation via alteration of ionic fluxes associated with early mitogenic signals is a powerful therapeutic approach towards the treatment and/or prevention of diseases characterized by unwanted or abnormal cell proliferation.
  • Compounds capable of inhibiting mammalian cell proliferation are highly desirable, and are therefore also an object of the present invention.
  • Acute and chronic diarrheas represent a major medical problem in many areas of the world. Diarrhea is both a significant factor in malnutrition and the leading cause of death (5,000,000 deaths/year) in children less than five years old. Secretory diarrheas are also a dangerous condition in patients of acquired immunodeficiency syndrome (AIDS) and chronic inflammatory bowel disease (IBD). 16 million travelers to developing countries from industrialized nations every year develop diarrhea, with the severity and number of cases of diarrhea varying depending on the country and area of travel. The major medical consequences of diarrheal diseases include dehydration, acidosis, death and impaired growth.
  • AIDS acquired immunodeficiency syndrome
  • IBD chronic inflammatory bowel disease
  • Diarrhea in barn animals and pets such as cows, pigs and horses, sheep, goats, cats and dogs, also known as scours, is a major cause of death in these animals. Diarrhea can result from any major transition, such as weaning or physical movement.
  • One form of diarrhea is characterized by diarrhea in response to a bacterial or viral infection and generally occurs within the first few hours of the animal's life. Although the major consequences of diarrheal diseases are very similar, there are numerous causes of diarrhea. Secretory and exudative diarrhea are primarily caused by bacterial or viral infections. The most common diarrheal causing bacteria is enterotoxogenic E-coli (ETEC) having the K99 pilus antigen.
  • ETEC enterotoxogenic E-coli
  • Common viral causes of diarrhea include rotavirus and coronavirus. Other infectious agents include cryptosporidium, giardia lamblia, and salmonella, among others.
  • Diarrhea which is found in travelers to industrialized nations (travelers diarrhea) frequently is caused by bacterial pathogens which are acquired through ingestion of fecally contaminated food and/or water. Approximately 50-75% of these cases are attributed to ETEC. Although traveler's diarrhea is painful, it is generally not life-threatening and often the symptoms last only 3-5 days. The symptoms include urgent diarrhea, abdominal cramps, nausea and fever. The most effective course of treatment for traveler's diarrhea is the administration of antibiotics in conjunction with oral rehydration. It has been shown that prophylactic administration of antibiotics drastically reduces the number of travelers experiencing symptoms of diarrhea. However, routine administration of antibiotics is not suggested as it may cause resistant strains of a bacteria to develop. Other treatment methods include administration of bismuth subsalicylate, often taken in the form of Pepto-Bismal, diphenoxylate and loperamide.
  • AIDS enteropathy is a disorder characterized by diarrhea without the involvement of secondary infections. It is caused by the human immunodeficiency virus (HIV) infection of the small bowel mucosal cells and colonic mucosal cells. The most common infective agent causing diarrhea due to enteric infection in AIDS patients in cryptosporidium.
  • the methods for treating diarrhea in AIDS patients include administration of antibiotics and administration of immunoglobulins or an immunoglobulin enriched fraction of bovine colostrum. Colostrum, which is the first milk produced by mammals after birthing is enriched with antibodies.
  • Acute diarrhea or scours is a main cause of death in many newborn barn animals such as calves and pigs. Scours is often caused by ETEC with a K99 pilus antigen. Infection with the ETEC causes hypersecretion of fluid and electrolytes. Hypersecretion in turn causes dehydration and pH imbalance which may result in death of the newborn calf or pig.
  • Newborn barn animals are also susceptible to viral infectious agents causing scours. Infections with rotavirus and coronavirus are common in newborn calves and pigs. Rotavirus infection often occurs within 12 hours of birth. Symptoms of rotaviral infection include excretion of watery feces, dehydration and weakness. Coronavirus which causes a more severe illness in the newborn animals, has a higher mortality rate than rotaviral infection. Often, however, a young animal may be infected with more than one virus or with a combination of viral and bacterial microorganisms at one time. This dramatically increases the severity of the disease. Generally the best protection for a newborn barn animal from viral or bacterial infection is the consumption of colostrum.
  • a common method of treatment includes administration of a concentrated colostrum solution or an immunoglobulin fraction isolated from a colostrum solution. This oral treatment may be combined with rehydration salts. Although these methods have improved the morbidity and mortality rate of newborn animals having scours, there still exists a need for more effective treatments.
  • Certain imidazoles such as clotrimazole are agents which have been used both topically and systemically as antifungals. More recently, studies have identified other uses for such imidazoles.
  • U.S. patent no. 5,273,992 revealed that these imidazoles regulate Ca ++ activated K + channels in erythrocytes, and are thus useful in treating sickle cell anemia, which involves the inhibition of potassium transport. These imidazoles have also been found to be effective in inhibiting endothelial and/or vascular smooth muscle cell proliferation. The results of this finding are described in U.S. patent no. 5,358,959 and U.S. serial no. 08/018,840, which discloses using clotrimazole for treating atherosclerotic and angiogenic conditions, respectively. Nonimidazole metabolites and analogs of the foregoing compounds also have been described as useful in treating the foregoing conditions (see U.S. serials nos. 08/307,874 and 08/307,887).
  • the present invention provides a class of organic compounds which are potent, selective and safe inhibitors of the Ca 2+ -activated potassium channel (Gardos channel) of erythrocytes, of mammalian cell proliferation and/or of secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells.
  • the compounds can be used to reduce sickle erythrocyte dehydration and/or delay the occurrence of erythrocyte sickling or deformation in situ as a therapeutic approach towards the treatment or prevention of sickle cell disease.
  • the compounds can also be used to inhibit mammalian cell proliferation in situ as a therapeutic approach towards the treatment or prevention of diseases characterized by abnormal cell proliferation.
  • the compounds can also be used to inhibit chloride secretion in intestinal cells as a therapeutic approach towards the treatment of diarrhea and scours.
  • the compounds are generally substituted 11 -phenyl-dibenzazepine compounds.
  • the compounds capable of inhibiting the Gardos channel, mammalian cell proliferation and/or secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells according to the invention are compounds having the structural formula (I):
  • R is -R, (C 6 -C 20 ) aryl or substituted (C 6 -C 20 ) aryl;
  • R 2 is -R, -OR, -SR, halogen or trihalomethyl
  • R 3 is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with R 4 , is (C 6 -
  • R 4 is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with R 3 , is (C 6 - C 20 ) aryleno; each of R 5 , Rg, R 7 , R 8 , R 9 , R 10 , R ⁇ , R) 2 , Rn and R M is independently selected from the group consisting of -R, halogen and trihalomethyl;
  • R 15 is -R", -C(O)R", -C(S)R", -C(O)OR", -C(S)OR", -C(0)SR", -C(S)SR", -C(O)N(R") 2 , -C(S)N(R") 2 , -C(O)C(O)R", -C(S)C(0)R", -C(0)C(S)R", -C(S)C(S)R", -C(O)C(O)OR", -C(S)C(O)OR", -C(O)C(S)OR", -C(O)C(0)SR", -C(S)C(S)OR", -C(O)C(0)SR", -C(S)C(S)OR", -C(O)C(0)SR", -C(S)C(S)OR", -C(O)C(0)SR", -C(S)C(S)OR", -C(O)C
  • the chalcogens in the compounds of formula (I) are each oxygen.
  • the compounds are those of structure (I) wherein the halogens are each independently -F, -Cl, -Br or -I.
  • the alkyl, alkenyl and alkynyl groups are each independently (C,-C 3 ) and/or the aryl groups are phenyl and/or the aryleno groups are benzeno.
  • R 5 , R ⁇ R 7 , R 9 , R 10 , R ⁇ and R 13 are each independently -R.
  • the substituted aryl and alkaryl are mono- substituted.
  • R ls is -R", -C(0)R", -C(0)OR", -C(0)N(R") 2 , -C(O)C(O)R", -C(0)C(0)OR” or -C(O)C(0)N(R") 2 .
  • the compounds are those of structural formula (I) wherein: R, is -R or (C 6 -C 20 ) aryl; R 2 is -R or -OR; R 3 is -R' or -OR' or, when taken together with R 4 , is (C 6 -C 20 ) aryleno; R 4 is -R or -OR' or, when taken together with R 3 , is (C 6 -C 20 ) aryleno; each of R 5 , R f ,, R 7 , R 8 , R 9 , R 10 , R n , R 12 , R 13 and R 14 is independently selected from the group consisting of -R' and halogen; R 15 is -R", -C(O)R", -C(O)OR", -C(0)N(R") 2 , -C(0)C(0)R", -C(0)C(0)OR” or -C(O)C(O)N(R") 2 ;
  • the compounds are those of formula (I) wherein: R, is -R or (C 6 -C 10 ) aryl; R 2 is -R or -OR; R 3 is -R' or -OR or, when taken together with R 14 , is (C 6 -C !0 ) aryleno; R 4 is -R' or -OR or, when taken together with R 3 , is (C 6 -C I0 ) aryleno; each of R 5 , R f , and R 7 is -H; R 8 is -R, -F, -Cl, -Br or -I; each of R 9 , R 10 and R consider is -H; R 12 is -R, -F, -Cl, -Br or -I; R ]3 is -H; R 14 is -R, -F, -Cl, -Br or -I; R 15 is -R", -C(
  • the compounds are those of structural formula (I) wherein: R, is -R' or phenyl; R 2 is -R or -OR; R 3 is -R or -OR' or, when taken together with R 4 , is benzeno; R 4 is -R or -OR' or, when taken together with R 3 , is benzeno; each of R 5 , R 6 and R 7 is -H; R 8 is -R', -Cl or -Br; each of R 9 , R ]0 and R jail is -H; R 12 is -R, -F or -Cl; R 13 is -H; R 14 is -R' or -Cl; R I5 is -R", -C(O)R", -C(0)OR", -C(0)NHR", -C(O)C(O)R" or -C(O)C(O)OR”; each R is independently selected from the group consisting of -H,
  • the invention provides a method for reducing sickle erythrocyte dehydration and/or delaying the occurrence of erythrocyte sickling or deformation in situ.
  • the method involves contacting a sickle erythrocyte in situ with an amount of at least one compound according to the invention, or a pharmaceutical composition thereof, effective to reduce sickle erythrocyte dehydration and/or delay the occurrence of erythrocyte sickling or deformation.
  • the sickle cell dehydration is reduced and erythrocyte deformation is delayed in a sickle erythrocyte that is within the microcirculation vasculature of a subject, thereby preventing or reducing the vaso-occlusion and consequent adverse effects that are commonly caused by sickled cells.
  • the invention provides a method for the treatment and/or prevention of sickle cell disease in a subject, such as a human.
  • the method involves administering a prophylactically or therapeutically effective amount of at least one compound according to the invention, or a pharmaceutical composition thereof, to a patient suffering from sickle cell disease.
  • the patient may be suffering from either acute sickle crisis or chronic sickle cell episodes.
  • a method is provided for inhibiting unwanted cellular proliferation associated with an inflammatory disease.
  • the method includes the step of contacting a cell the proliferation of which contributes to inflammation in situ with an amount of a compound having the above described formula (I) effective to inhibit proliferation of the cell.
  • the method of administration is selected from the group consisting of oral, parenteral, intravenous, subcutaneous, transdermal and transmucosal for a living human.
  • the mammalian cell is a fibrotic cell or a lymphocyte.
  • a method for treating or preventing an inflammatory disease.
  • the method includes the step of administering to a subject in need of such treatment a therapeutically effective amount of a compound of the above-described formula (I).
  • the inflammatory disease is diarrhea.
  • the diarrhea is caused by inflammatory bowel disease.
  • the inflammatory disease is an autoimmune disease.
  • the inflammatory disease is selected from the group consisting of proliferative glomerulonephritis; lupus erythematosus; scleroderma; temporal arteritis; thromboangiitis obliterans; mucocutaneous lymph node syndrome; asthma; host versus graft; inflammatory bowel disease; multiple sclerosis; rheumatoid arthritis; thyroiditis; Grave's disease; antigen- induced airway hyperactivity; pulmonary eosinophilia; Guillain-Barre syndrome; allergic rhinitis; myasthenia gravis; human T-lymphotrophic virus type 1 -associated myelopathy; herpes simplex encephalitis; inflammatory myopathies; atherosclerosis; and Goodpasture's syndrome.
  • the administration is parenteral or per oral.
  • the invention provides a method for inhibiting mammalian cell proliferation in situ.
  • the mammalian cell proliferation is not associated with a proliferative disease selected from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
  • the method involves contacting a mammalian cell in situ with an amount of at least one compound according to the invention, or a pharmaceutical composition thereof, effective to inhibit cell proliferation.
  • the compound or composition may act either cytostatically, cytotoxically or a by a combination of both mechanisms to inhibit proliferation.
  • Mammalian cells in this manner include vascular smooth muscle cells, fibroblasts and endothelial cells.
  • the invention provides a method for treating and/or preventing unwanted or abnormal cell proliferation in a subject, such as a human.
  • the unwanted or abnormal cell proliferation is not associated with a proliferative disease selected from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
  • at least one compound according to the invention, or a pharmaceutical composition thereof is administered to a subject in need of such treatment in an amount effective to inhibit the unwanted or abnormal mammalian cell proliferation.
  • the compound and/or composition may be applied locally to the proliferating cells, or may be administered to the subject systemically.
  • the compound and/or composition is administered to a subject that has a disorder characterized by unwanted or abnormal cell proliferation, and preferably the unwanted or abnormal cell proliferation is not associated with a proliferative disease selected from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
  • a proliferative disease selected from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma.
  • disorders include, but are not limited to, non-cancerous angiogenic conditions or arteriosclerosis.
  • the invention provides a method for the treatment and/or prevention of diseases that are characterized by unwanted and/or abnormal mammalian cell proliferation.
  • the unwanted or abnormal cell proliferation is not associated with a proliferative disease selected from the group consisting of cancer, actinic keratosis, and
  • Kaposi's sarcoma The method involves administering a prophylactically or therapeutically effective amount of at least one compound according to the invention, or a pharmaceutical composition thereof, to a subject in need of such treatment.
  • Diseases that are characterized by abnormal mammalian cell proliferation include, but are not limited to, blood vessel proliferative disorders, fibrotic disorders and arteriosclerotic conditions.
  • a method for treating diarrhea of diverse etiology involves administering to a subject who is in need of such treatment, an aromatic compound of the invention in an amount effective to inhibit the diarrhea.
  • the compound is administered orally in conjunction with oral rehydration fluids.
  • the aromatic compounds useful in the invention are substituted 11-phenyl- dibenzazepine, or analogues thereof.
  • the aromatic compounds according to the invention are compounds having the above-described formula (I).
  • the subject in need of such treatment is a subject who has symptoms of diarrhea or scours.
  • the subject in need of such treatment is a subject at risk of developing diarrhea or scours.
  • diarrhea is a secretory disorder, which is caused by at least one of several mechanisms.
  • the diarrhea is an exudative form of diarrhea;
  • the diarrhea is a nonexudative form of diarrhea;
  • the diarrhea is a decreased absorption form of diarrhea;
  • the diarrhea is a non-decreased absorption form of diarrhea;
  • the diarrhea is a secretory form of diarrhea.
  • the diarrhea is a nonsecretory form of diarrhea.
  • the diarrhea is a noninflammatory form of diarrhea.
  • the present invention provides pharmaceutical compositions comprising one or more compounds according to the invention in admixture with a pharmaceutically acceptable carrier, excipient or diluent.
  • a pharmaceutically acceptable carrier excipient or diluent.
  • Such a preparation can be administered in the methods of the invention.
  • pharmaceutical preparations comprising one or more of the aromatic compounds of the invention in admixture with a pharmaceutically acceptable carrier, excipient or diluent, wherein the aromatic compound(s) of the invention is (are) in an amount effective for treating: (i) unwanted or abnormal cell proliferation, preferably not a proliferative disease selected from the group consisting of cancer, actinic keratosis, and Kaposi's sarcoma; (ii) an inflammatory disease; (iii) sickle cell disease; and (iv) diarrhea or scours.
  • the aromatic compounds useful according to the invention have the general formula (I) provided above.
  • the pharmaceutical preparations include the aromatic compounds of the invention together with a non-formula (I) agent selected from the group consisting of an anti-proliferative agent; (ii) an anti-inflammatory agent; (iii) anti-sickle cell agent; and (iv) an anti-diarrhea or anti-scours agent.
  • a non-formula (I) agent selected from the group consisting of an anti-proliferative agent; (ii) an anti-inflammatory agent; (iii) anti-sickle cell agent; and (iv) an anti-diarrhea or anti-scours agent.
  • the use of aromatic compounds of the invention in the manufacture of medicaments is provided.
  • the medicaments are useful for treating: (i) unwanted or abnormal cell proliferation, preferably not a proliferative disease that includes cancer, actinic keratosis, and Kaposi's sarcoma; (ii) an inflammatory disease; (iii) sickle cell disease; and (iv) diarrhea or scours.
  • pharmaceutical preparations are provided. These pharmaceutical preparations include the aromatic compounds of the invention together with an anti-diarrheal agent.
  • the aromatic compounds useful according to the invention have the general formula (I) provided above.
  • the aromatic compounds useful according to the invention are the preferred compounds described above.
  • the pharmaceutical, composition of the invention may be administered orally.
  • the invention also provides the aromatic compounds of the invention in the manufacture of a medicament for the treatment of diarrhea or scours.
  • the aromatic compounds useful according to the invention have the general formula (I) provided above.
  • the aromatic compounds useful according to the invention are the preferred compounds described above.
  • veterinary preparations are provided.
  • veterinary preparations include the aromatic compounds useful according to the invention together with an anti-scours preparation.
  • the aromatic compounds useful according to the invention have the general formula (I) provided above.
  • the aromatic compounds useful according to the invention are the preferred compounds described above.
  • FIG. 1 is a general reaction scheme for synthesizing certain compounds according to the invention
  • FIG. 2 is a general reaction scheme for synthesizing certain compounds according to the invention.
  • FIG. 3 is a bar graph depicting the effect of clotrimazole in the inhibition of cAMP and Ca ++ dependent Cl " secretion in T84 cells;
  • FIG. 4 is a graph showing the effect of clotrimazole on the inhibition of base line and Ca ++ - stimulated 86 Rb efflux from T84 monolayers.
  • Clotrimazole in addition to inhibiting the Gardos channel of erythrocytes, also modulates ionic mitogenic signals and inhibits cell proliferation both in vitro and in vivo.
  • Clotrimazole inhibits the rate of cell proliferation of normal and cancer cell lines in a reversible and dose-dependent manner in vitro (Benzaquen et al. 1995 Nature Medicine 1:534-540). Clotrimazole also depletes the intracellular Ca 2+ stores and prevents the rise in cystolic Ca 2+ that normally follows mitogenic stimulation.
  • the present invention provides a new class of organic compounds that are capable of inhibiting the Gardos channel of erythrocytes, mammalian cell proliferation, particularly mitogen-induced cell proliferation, and/or Cl " secretion from intestinal cells.
  • the compounds of the invention do not contain an imidazole or imidazole-like moiety.
  • the imidazole or imidazole-like moiety is well-recognized as the essential functionality underlying the antimycotic and other biological activities of Clotrimazole and the other above-mentioned anti-mycotic agents.
  • the 11 -phenyl dibenzazepine compounds of the invention provide an entirely new class of compounds that are capable of effecting inhibition of the Gardos channel of erythrocytes, mammalian cell proliferation, particularly mitogen-induced cell proliferation, and/or Cl " secretion from intestinal cells.
  • the invention provides a method of reducing sickle cell dehydration and/or delaying the occurrence of erythrocyte sickling in situ as a therapeutic approach towards the treatment of sickle cell disease.
  • the method involves only a single step — the administration of at least one pharmacologically active compound of the invention, or a composition thereof, to a sickle erythrocyte in situ in an amount effective to reduce dehydration and/or delay the occurrence of cell sickling or deformation.
  • the invention is also directed to methods of treating or preventing sickle cell disease.
  • an effective amount of one or more compounds according to the invention, or a pharmaceutical composition thereof is administered to a patient suffering from sickle cell disease.
  • the methods may be used to treat sickle cell disease prophylactically to decrease intracellular Hb S concentration and/or polymerization, and thus diminish the time and duration of red cell sickling and vaso-occlusion in the blood circulation.
  • the methods may also be used therapeutically in patients with acute sickle cell crisis, and in patients suffering chronic sickle cell episodes to control both the frequency and duration of the crises.
  • the invention provides methods of inhibiting mammalian cell proliferation as a therapeutic approach towards the treatment or prevention of diseases characterized by unwanted or abnormal cell proliferation.
  • the method involves only a single step — the administration of an effective amount of at least one pharmacologically active compound according to the invention to a mammalian cell in situ.
  • the compound may act cytostatically, cytotoxically, or by a combination of both mechanisms to inhibit cell proliferation.
  • Mammalian cells treatable in this manner include vascular smooth muscle cells, fibroblasts, endothelial cells, various pre-cancer cells and various cancer cells.
  • cell proliferation is inhibited in a subject suffering from a disorder that is characterized by unwanted or abnormal cell proliferation. Such diseases are described more fully below.
  • the invention is also directed to methods of treating or preventing diseases characterized by abnormal cell proliferation.
  • an effective amount of at least one compound according to the invention, or a pharmaceutical composition thereof is administered to a patient suffering from a disorder that is characterized by abnormal cell proliferation.
  • administration of an appropriate amount of a compound according to the invention to a subject inhibits cell proliferation by altering the ionic fluxes associated with early mitogenic signals.
  • Such alteration of ionic fluxes is thought to be due to the ability of the compounds of the invention to inhibit potassium channels of cells, particularly Ca 2+ -activated potassium channels.
  • the method can be used prophylactically to prevent unwanted or abnormal cell proliferation, or may be used therapeutically to reduce or arrest proliferation of abnormally proliferating cells.
  • the compound, or a pharmaceutical formulation thereof can be applied locally to proliferating cells to arrest or inhibit proliferation at a desired time, or may be administered to a subject systemically to arrest or inhibit cell proliferation.
  • Diseases which are characterized by abnormal cell proliferation that can be treated or prevented by means of the present invention include blood vessel proliferative disorders, fibrotic disorders, arteriosclerotic disorders and various cancers.
  • Blood vessel proliferation disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels.
  • Other examples of blood vessel proliferative disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage and ocular diseases such as diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness and neovascular glaucoma.
  • neovascularization is that associated with solid tumors. It is now established that unrestricted growth of tumors is dependent upon angiogenesis and that induction of angiogenesis by liberation of angiogenic factors can be an important step in carcinogenesis. For example, basic fibroblast growth factor (bFGF) is liberated by several cancer cells and plays a crucial role in cancer angiogenesis. The demonstration that certain animal tumors regress when angiogenesis is inhibited has provided the most compelling evidence for the role of angiogenesis in tumor growth. Other cancers that are associated with neovascularization include hemangioendotheliomas, hemangiomas and Kaposi's sarcoma.
  • bFGF basic fibroblast growth factor
  • Proliferation of endothelial and vascular smooth muscle cells is the main feature of neovascularization.
  • the invention is useful in inhibiting such proliferation, and therefore in inhibiting or arresting altogether the progression of the angiogenic condition which depends in whole or in part upon such neovascularization.
  • the invention is particularly useful when the condition has an additional element of endothelial or vascular smooth muscle cell proliferation that is not necessarily associated with neovascularization.
  • psoriasis may additionally involve endothelial cell proliferation that is independent of the endothelial cell proliferation associated with neovascularization.
  • a solid tumor which requires neovascularization for continued growth may also be a tumor of endothelial or vascular smooth muscle cells.
  • the invention is also useful for the treatment of fibrotic disorders such as fibrosis and other medical complications of fibrosis which result in whole or in part from the proliferation of fibroblasts.
  • Medical conditions involving fibrosis include undesirable tissue adhesion resulting from surgery or injury.
  • arteriosclerosis is a term used to describe a thickening and hardening of the arterial wall.
  • An arterio sclerotic condition as used herein means classical atherosclerosis, accelerated atherosclerosis, atherosclerotic lesions and any other arteriosclerotic conditions characterized by undesirable endothelial and/or vascular smooth muscle cell proliferation, including vascular complications of diabetes.
  • Proliferation of vascular smooth muscle cells is a main pathological feature in classical atherosclerosis. It is believed that liberation of growth factors from endothelial cells stimulates the proliferation of subintimal smooth muscle which, in turn, reduces the caliber and finally obstructs the artery.
  • the invention is useful in inhibiting such proliferation, and therefore in delaying the onset of, inhibiting the progression of, or even halting the progression of such proliferation and the associated atherosclerotic condition.
  • Proliferation of vascular smooth muscle cells produces accelerated atherosclerosis, which is the main reason for failure of heart transplants that are not rejected. This proliferation is also believed to be mediated by growth factors, and can ultimately result in obstruction of the coronary arteries.
  • the invention is useful in inhibiting such obstruction and reducing the risk of, or even preventing, such failures.
  • vascular injury can also result in endothelial and vascular smooth muscle cell proliferation.
  • the injury can be caused by any number of traumatic events or interventions, including vascular surgery and balloon angioplasty. Restenosis is the main complication of successful balloon angioplasty of the coronary arteries. It is believed to be caused by the release of growth factors as a result of mechanical injury to the endothelial cells lining the coronary arteries.
  • the compounds described herein can be used to delay, or even avoid, the onset of restenosis.
  • Atherosclerotic conditions which can be treated or prevented by means of the present invention include diseases of the arterial walls that involve proliferation of endothelial and/or vascular smooth muscle cells, such as complications of diabetes, diabetic glomerulosclerosis and diabetic retinopathy.
  • Cancers which can be treated by means of the present invention include, but are not limited to, biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute and chronic lymphocytic and myelogenous leukemia, multiple myeloma, AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mes
  • the compounds of the invention are useful with hormone dependent and also with nonhormone dependent cancers. They also are useful with prostate and nonprostate cancers and with breast and nonbreast cancers. They further are useful with multidrug resistant strains of cancer.
  • the invention is also useful in treating or preventing dermatological diseases including keloids, hypertrophic scars, seborrheic dermatosis, papilloma virus infection (e.g., producing verruca vulgaris, verruca plantaris, verruca plan, condylomata, etc.), and eczema and epithelial precancerous lesions such as actinic keratosis. It also is useful with pathologies mediated by growth factors such as uterine leiomyomas.
  • an "inflammatory disease associated with cellular proliferation" as used herein is a disease in which lymphoproliferation contributes to tissue or organ damage leading to disease. For instance, excessive T cell proliferation at the site of a tissue or organ will cause damage to the tissue or organ. Inflammatory disease are well known in the art and have been described extensively in medical textbooks (See, e.g., Harrison's Principles of Experimental Medicine, 13th Edition, McGraw-Hill, Inc., NN.).
  • Inflammatory diseases associated with cellular proliferation include but are not limited to proliferative glomerulonephritis; lupus erythematosus; scleroderma; temporal arteritis; thromboangiitis obliterans; mucocutaneous lymph node syndrome; asthma; host versus graft; inflammatory bowel disease; multiple sclerosis; rheumatoid arthritis; thyroiditis; Grave's disease; antigen-induced airway hyperactivity; pulmonary eosinophilia; Guillain-Barre syndrome; allergic rhinitis; myasthenia gravis; human T-lymphotrophic virus type 1- associated myelopathy; herpes simplex encephalitis; inflammatory myopathies; atherosclerosis; and Goodpasture's syndrome.
  • Some examples of inflammatory diseases associated with cellular proliferation as well as animal models for testing and developing the compounds are set forth in Table 1 below. Table 1
  • the compounds and methods of the invention provide myriad advantages over agents and methods commonly used to treat cell proliferative disorders. For example, many of the compounds of the invention are more potent than Clotrimazole in in vitro tests, and therefore may provide consequential therapeutic advantages in clinical settings.
  • the compounds of the invention have reduced toxicity as compared with these other agents.
  • Clotrimazole it is well-known that the imidazole moiety is responsible for inhibiting a wide range of cytochrome P-450 isozyme catalyzed reactions, which constitutes their main toxicological effects (Pappas and Franklin, 1993, Toxicology 80:27-35; Matsuura et al, 1991. Biochemical Pharmacology 41 : 1949-1 56). Analogues and metabolites of Clotrimazole do not induce cytochrome P-450 (Matsuura et al, 1991, Biochemical Pharmacology 41 :1949-1956). and therefore do not share Clotrimazole's toxicity.
  • the invention in another aspect also involves methods and products for reducing the symptoms of diarrhea or preventing diarrhea in a subject at risk for developing diarrhea, using the compounds of the invention.
  • the aromatic compounds useful according to the invention may be provided in a pharmaceutical preparation or a veterinary preparation.
  • the aromatic compounds of the invention are also useful in a method for treating diarrhea and scours as well as a method for preventing diarrhea and scours.
  • Diarrhea indicates a medical syndrome which is characterized by the symptoms of diarrhea or scours. In general, diarrhea is a disorder resulting in a secretory imbalance.
  • diarrhea is divided into three categories based on the underlying mechanism: exudative, decreased absorption, and secretory and the term diarrhea as used herein encompasses each of these categories.
  • Exudative diarrheas result from inflammatory processes leading to impaired colonic absorption, and outpouring of cells and colloid caused by such disorders as ulcerative colitis, shigellosis, and amebiasis.
  • Disorders of decreased absorption include osmotic, anatomic derangement, and motility disorders.
  • Osmotic diarrhea can occur as a result of digestive abnormalities such as lactose intolerance.
  • Anatomic derangement results in a decreased absorption surface caused by such procedures as subtotal colectomy and gastrocolic fistula.
  • Motility disorders result from decreased contact time resulting from such diseases as hyperthyroidism and irritable bowel syndrome.
  • Secretory diarrhea is characterized by the hypersecretion of fluid and electrolytes from the cells of the intestinal wall. In classical form, the hypersecretion is due to changes which are independent of the permeability, absorptive capacity and exogenously generated osmotic gradients within the intestine. As discussed above, however, all forms of diarrhea may actually manifest a secretory component.
  • the methods and products of the invention are particularly useful in treating diarrhea which is secretory.
  • the methods and products of the invention may also be used in combination with other treatment methods which are known in the art to treat diarrhea caused by decreased absorption or inflammation.
  • the compounds of the invention are involved in regulating Cl " secretion and can function alone or when used in combination with other treatment methods to decrease net fluid secretion even when this is due primarily to abnormalities in absorption or inflammation.
  • the methods and products of the invention are useful in preventing diarrhea and scours in subjects at risk of developing these disorders.
  • Subjects at risk of developing diarrhea and scours are those subjects which have a high likelihood of exposure to the bacterial and viral microorganisms which cause these symptoms. For example, approximately 1/3 of travelers to developing countries will develop diarrhea; infection with rotavirus is one of the leading causes of death in infants in developing countries; subjects with HIV have a greater than 50% chance of developing diarrhea, and many newborn calves and pigs develop scours.
  • Subjects with inflammatory bowel disease develop recurrent diarrhea.
  • the methods and products of the invention are also useful in treating subjects who already exhibit the symptoms of diarrhea and scours.
  • the symptoms of diarrhea include bowel irregularity, fecal fluid rich in sodium or potassium, fluid feces, dehydration, fever, loss of body weight, headache, anorexia, vomiting, malaise and myalgia.
  • the symptoms of scours include a loss of body weight or failure to grow, dehydration, malodorous feces, fluid feces, feces containing pieces of partially digested milk or semisolid material, and feces of a yellow- white or gray color.
  • the Compounds which are potent, selective and safe inhibitors of Ca 2+ -activated potassium channel (Gardos channel) of erythrocytes, particularly sickle erythrocytes, mammalian cell proliferation, particularly mitogen-induced cell proliferation, and or secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells according to the invention are generally substituted 11 -phenyl dibenzazepine compounds.
  • the compounds capable of inhibiting the Gardos channel, mammalian cell proliferation and/or chloride secretion in intestinal cells according to the invention are compounds having the structural formula (I):
  • R is -R', (C 6 -C 20 ) aryl or substituted (C 6 -C 20 ) aryl;
  • R 2 is -R, -OR, -SR, halogen or trihalomethyl;
  • R 3 is -R', -OR, -SR', halogen or trihalomethyl or, when taken together with R 4 , is (C 6 - C 20 ) aryleno;
  • R 4 is -R', -OR', -SR', halogen or trihalomethyl or, when taken together with R 3 , is (C 6 -
  • each of R 5 , R ⁇ , R 7 , R 8 , Rg, R 10 , R n , R 12 , R ]3 and R 14 is independently selected from the group consisting of -R, halogen and trihalomethyl;
  • R 15 is -R", -C(0)R", -C(S)R", -C(0)OR", -C(S)OR", -C(0)SR", -C(S)SR", -C(O)N(R") 2 , -C(S)N(R") 2 , -C(O)C(O)R", -C(S)C(O)R", -C(O)C(S)R", -C(O)C(O)OR", -C(S)C(0)OR", -C(0)C(O)SR", -C(S)C(S)OR", -C(0)C(O)SR", -C(S)C(S)OR", -C(0)C(O)SR", -C(S)C(S)OR", -C(0)C(S)SR", -C(S)C(S)SR", -C(0)C(O)N(R") 2 , -C(S)C(O)N(R") 2 ,
  • the compounds are those of structure (I) wherein the chalcogens are each oxygen.
  • the compounds are those of structure (I) wherein the halogens are each independently -F, -Cl, -Br or -I.
  • the alkyl, alkenyl and alkynyl groups are each independently (C r C 3 ) and/or the aryl groups are phenyl and/or the aryleno groups are benzeno.
  • R 5 , Rg, R 7 , R 9 , R, 0 , R ⁇ and R 13 are each independently -R.
  • the substituted aryl and alkaryl are mono- substituted.
  • R ]5 is -R", -C(0)R", -C(O)OR", -C(0)N(R") 2 , -C(O)C(O)R", -C(0)C(O)OR” or -C(0)C(O)N(R") 2 .
  • the compounds are those of structural formula (I) wherein: R, is -R or (C 6 -C 20 ) aryl;
  • R 2 is -R' or -OR
  • R 3 is -R' or -OR' or, when taken together with R 4 , is (C 6 -C 20 ) aryleno;
  • R 4 is -R or -OR or, when taken together with R 3 , is (C 6 -C 20 ) aryleno; each of R 5 , R ⁇ , R 7 , R 8 , Rg, R, 0 , Rn, R i2 , R ⁇ 3 and R 14 is independently selected from the group consisting of -R and halogen;
  • R 15 is -R", -C(O)R", -C(O)OR", -C(O)N(R") 2 , -C(0)C(O)R", -C(O)C(0)OR” or -C(O)C(O)N(R") 2 ; each R is independently selected from the group consisting of -H, (C,-C 6 ) alkyl, (C,-C 6 ) alkenyl and (C,-C 6 ) alkynyl; each R" is independently selected from the group consisting of -H, (C,-C 6 ) alkyl,
  • the compounds are those of formula (I) wherein:
  • R is -R' or (C 6 -C 10 ) aryl;
  • R 2 is -R or -OR;
  • R 3 is -R or -OR or, when taken together with R, 4 , is (C 6 -C 10 ) aryleno;
  • R 4 is -R or -O or, when taken together with R 3 , is (C 6 -C 10 ) aryleno;
  • each of R 5 , Rg and R 7 is -H;
  • R 8 is -R, -F, -Cl, -Br or -I;
  • each of R 9 , R 10 and R ⁇ is -H;
  • R I2 is -R, -F. -Cl, -Br or -I;
  • R 13 is -H;
  • R 14 is -R', -F, -Cl, -Br or -I;
  • R 15 is -R", -C(O)R", -C(O)OR", -C(0)N(R") 2 , -C(0)C(O)R", -C(O)C(0)OR" or
  • each R is independently selected from the group consisting of -H, (C,-C 3 ) alkyl, (C,-C 3 ) alkenyl and (C,-C 3 ) alkynyl; each R" is independently selected from the group consisting of -H, (C r C 3 ) alkyl, (C,-C 3 ) alkenyl, (C,-C 3 ) alkynyl, (C 6 -C 10 ) aryl, substituted (C 6 -C, 0 ) aryl, (C 6 -C 13 ) alkaryl or substituted (C 6 -C 13 ) alkaryl; and the aryl and alkaryl substituents are each independently selected from the group consisting of -OR,-NO 2 , -NR'R', -F, -Cl, -Br, -I, (C,-C 3 ) alkyl, (C,
  • Ri is -R' or phenyl
  • R 2 is -R or -OR
  • R 3 is -R' or -OR or, when taken together with R 4 , is benzeno;
  • R ⁇ is -R or -OR or, when taken together with R 3 , is benzeno; each of R 5 , Rg and R 7 is -H;
  • R 8 is -R', -Cl or -Br; each of R 9 , R 10 and R H is -H;
  • R 12 is -R, -F or -Cl;
  • R 13 is -H;
  • R 14 is -R or -Cl
  • R 15 is -R", -C(0)R", -C(0)OR", -C(0)NHR", -C(0)C(O)R” or -C(0)C(0)OR”; each R' is independently selected from the group consisting of -H, (C,-C 3 ) alkyl, (C,-C 3 ) alkenyl and (C,-C 3 ) alkynyl; each R" is independently selected from the group consisting of -H, (C r C 3 ) alkyl, (C r C 3 ) alkenyl, (C,-C 3 ) alkynyl, (C 6 -C 10 ) aryl, mono-substituted (C 6 -C 10 ) aryl, (C 6 -C 13 ) alkaryl or mono-substituted (C 6 -C 13 ) alkaryl; and the aryl and alkaryl substituents are each independently selected from the group consisting of -OR, -N0 2
  • alkyl refers to a saturated branched, straight chain or cyclic hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, pentyl, isopentyl, cyclopentyl, hexyl, cyclohexyl and the like.
  • heterocycloalkyl refers to a saturated cyclic hydrocarbon radical wherein one or more of the carbon atoms is replaced with another atom such as Si, Ge, N, O, S or P.
  • Typical heterocycloalkyl groups include, but are not limited to, morpholino, thiolino, piperidyl, pyrrolidinyl, piperazyl, pyrazolidyl, imidazolidinyl, and the like.
  • alkenyl refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond.
  • the radical may be in either the cis or trans conformation about the double bond(s).
  • Typical alkenyl groups include ethenyl, propenyl, isopropenyl, cyclopropenyl, butenyl, isobutenyl, cyclobutenyl, tert- butenyl, pentenyl, hexenyl and the like.
  • alkynyl refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon triple bond.
  • Typical alkynyl groups include ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like.
  • alkoxy refers to an -OR radical, where R is alkyl, alkenyl or alkynyl, as defined above.
  • aryl refers to an unsaturated cyclic hydrocarbon radical having a conjugated ⁇ electron system.
  • Typical aryl groups include, but are not limited to, penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, indacenyl, and the like.
  • heteroaryl refers to an aryl group wherein one or more of the ring carbon atoms is replaced with another atom such as N, O or S.
  • Typical heteroaryl groups include, but are not limited to,furanyl, imidazole, pyridinyl, thiophenyl, indolyl, imidazolyl, quinolyl, thienyl, indolyl, pyrrolyl, pyranyl, pyridyl, pyrimidyl, pyrazyl, pyridazyl, and the like.
  • heteroarylium refers to a heteroaryl group wherein one or more hydrogens has been added to any position of the neutral parent ring.
  • Typical heteroarylium groups include, but are not limited to, pyridinium, pyrazinium, pyrimidinium, pyridazinium, 1,3,5-triazinium, and the like.
  • in situ refers to and includes the terms “in vivo,” “ex vivo,” and “in vitro” as these terms are commonly recognized and understood by persons ordinarily skilled in the art. Moreover, the phrase “in situ” is employed herein in its broadest connotative and denotative contexts to identify an entity, cell or tissue as found or in place, without regard to its source or origin, its condition or status or its duration or longevity at that location or position.
  • the compounds of the invention are as follows:
  • the compounds are those of structural formula (I), with the proviso that when R, and R 15 are each -R, at least one of R R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R],, R 12 , R 13 or R 14 is other than -R', R 8 is other than -R or halogen and at least three of R 2 , R 3 , R 4 and R 5 are other than -OR'.
  • the compounds are those of structural formula (I), with the proviso that when R, and R 15 are each -R, at least one of R R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , R 10 , R],, R 12 , R 13 or R 14 is other than -R', R 8 is other than -R or halogen and at least three of R 2 , R 3 , R 4 and R 5 are other than -OR'.
  • the compounds are those
  • the compounds of the invention are not 11-phenyl- 5,6-dihydro-l lH-dibenz[b,e]azepine, 1 l-phenyl-9-halo-5,6-dihydro-l lH-dibenz[b,e]azepine, 1 l-phenyl-9-chloro-5,6-dihydro-l lH-dibenz[b,e]azepine, 1 l-phenyl-5,6-dihydro- 1,2,3- trialkoxy-1 lH-dibenz[b,e]azepine, 1 l-phenyl-5,6-dihydro-l,2,3-trimethoxy-l ⁇ H- dibenz[b,e]azepine, l l-phenyl-5,6-dihydro-2,3,4-trialkoxy-l lH-dibenz[b,e]azepine and/or 11-phenyl- 5,6
  • the chemical formulae referred to herein may exhibit the phenomena of tautomerism or conformational isomerism.
  • the formulae drawings within this specification can represent only one of the possible tautomeric or conformational isomeric forms, it should be understood that the invention encompasses any tautomeric or conformational isomeric forms which exhibit biological or pharmacological activity as described herein.
  • the compounds of the invention may be in the form of free acids, free bases or pharmaceutically effective salts thereof. Such salts can be readily prepared by treating a compound with an appropriate acid.
  • Such acids include, by way of example and not limitation, inorganic acids such as hydrohalic acids (hydrochloric, hydrobromic, etc.), sulfuric acid, nitric acid, phosphoric acid, etc.; and organic acids such as acetic acid, propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid, propandioic acid, butandioic acid, etc.
  • inorganic acids hydrohalic acids (hydrochloric, hydrobromic, etc.), sulfuric acid, nitric acid, phosphoric acid, etc.
  • organic acids such as acetic acid, propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid, propandioic acid, butandioic acid, etc.
  • the salt can be converted into the free base form by treatment with alkali.
  • the invention may employ, where applicable, solvated as well as unsolvated forms of the compounds (e.g. hydrated forms).
  • the compounds described herein may be prepared by any processes known to be applicable to the preparation of chemical compounds. Suitable processes are well known in the art. Preferred processes are illustrated by the representative examples. Additional methods are described in copending application Serial Number unknown, entitled “SYNTHESIS OF 1 l-ARYL-5,6-DIHYDRO-l lH[b,e]AZEPINES, filed Nov. 20, 1997, and US application serial no. 08/975,594, filed November 20, 1997, entitled “Method for the Treatment or Prevention of Sickle Cell Disease with Substituted Phenyl-Dibenzazepine Compounds" which are both incorporated herein by reference in its entirety. Necessary starting materials may be obtained commercially or by standard procedures of organic chemistry. Moreover, many of the compounds are commercially available.
  • An individual compound's relevant activity and potency as an agent to affect sickle cell dehydration or deformation, mammalian cell proliferation and/or secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells may be determined using standard techniques.
  • a compound is subject to a series of screens to determine its pharmacological activity.
  • the active compounds of the invention exhibit three pharmacological activities: inhibition of the Gardos channel of erythrocytes, inhibition of secretagogue- stimulated transepithelial electrogenic chloride secretion in intestinal cells and inhibition of mammalian cell proliferation.
  • the compounds of the invention may exhibit only one of these pharmacological activities. Any compound encompassed by formula (I) which exhibits at least one of these pharmacological activities is considered to be within the scope of the present invention.
  • the active compounds of some embodiments of the invention are those which induce at least about 25% inhibition of the Gardos channel of erythrocytes (measured at about 10 ⁇ M), at least about 25% inhibition of secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells (measured at about 10 ⁇ M) and/or about 25% inhibition of mammalian cell proliferation (measured at about 10 ⁇ M), as measured using in vitro assays that are commonly known in the art (see, e.g., Brugnara et al, 1993, J. Biol. Chen 268(12): 8760-8768; Benzaquen et al , 1995. Nature Medicine 1 :534-540).
  • the active compounds of the invention generally will have an IC 50 (concentration of compound that yields 50% inhibition) for inhibition of the Gardos channel of erythrocytes of less than about 10 ⁇ M, an IC 50 for secretagogue-stimulated transepithelial electrogenic chloride secretion in intestinal cells of less than about 10 ⁇ M, and/or an IC 50 for inhibition of cell proliferation of less than about 10 ⁇ M, as measured using in vitro assays that are commonly known in the art (see, e.g., Brugnara et al, 1993, J. Biol. ChenL 268(12): 8760-8768; Benzaquen et al , 1995. Nature Medicine 1 :534-540) and the Examples section below. Other assays for assessing the activity and/or potency of an agent with respect to the uses of the invention are described below with respect to an effective amount of the compounds.
  • Representative active compounds according to the invention include Compounds 1 through 35, as illustrated above.
  • compounds which exhibit only one pharmacological activity, or a higher degree of one activity may be preferred.
  • the compound when the compound is to be used in methods to treat or prevent sickle cell disease, or in methods to reduce sickle cell dehydration and/or delay the occurrence of erythrocyte sickling or deformation in situ, it is preferred that the compound exhibit at least about 75% Gardos channel inhibition (measured at about 10 ⁇ M) and/or have an IC 50 of Gardos channel inhibition of less than about 1 ⁇ M, with at least about 90% inhibition and/or an IC 50 of less than about 0.1 ⁇ M being particularly preferred.
  • Exemplary preferred compounds for use in methods related to Gardos channel inhibition and sickle cell disease include Compounds 1, 2, 3, 4, 6, 9, 18, 29 and 35, with Compounds 2, 3, 4, 6, 9, 29 and 35 being particularly preferred.
  • the compound When the compound is to be used in methods to treat or prevent diarrhea and/or scours, it is preferred that the compound exhibit at least about 75% inhibition of Cl " secretion from intestinal cells (measured at about 10 ⁇ M) and/or have an IC 50 of inhibition of Cl " secretion from intestinal cells of less than about 1 ⁇ M, with at least about 90% inhibition and/or an IC 50 of less than about 0.1 ⁇ M being particularly preferred.
  • the compound When the compound is to be used in methods to treat or prevent disorders characterized by abnormal cell proliferation or in methods to inhibit cell proliferation in situ, it is preferable that the compound exhibit at least about 75% inhibition of mitogen-induced cell proliferation (measured at about 10 ⁇ M) and/or have an IC 50 of cell proliferation of less than about 3 ⁇ M, with at least about 90% inhibition and/or an IC 50 of less than about l ⁇ M being particularly preferred. Even more preferred compounds meet both the % inhibition and IC 50 criteria.
  • Exemplary preferred compounds for use in methods of inhibiting mammalian cell proliferation or for the treatment or prevention of diseases characterized by abnormal cell proliferation include Compounds 2, 3, 4, 7, 8, 9, 13, 14, 16, 17, 18, 19, 20, 21, 22, 26, 27, 28, 29, 30, 31, 32, 33 and 34, with Compounds 14, 26, 28, 29, 30 and 31 being particularly preferred.
  • Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.
  • the compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof may be administered singly, in combination with other compounds of the invention, and/or in cocktails combined with other therapeutic agents.
  • therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated.
  • a subject as used herein means humans, primates, horses, cows, sheep, pigs, goats, cats and dogs.
  • the compounds when administered to subjects undergoing cancer treatment, may be administered in cocktails containing other anti-cancer agents and/or supplementary potentiating agents.
  • the compounds may also be administered in cocktails containing agents that treat the side-effects of radiation therapy, such as anti-emetics, radiation protectants, etc.
  • Anti-cancer drugs that can be co-administered with the compounds of the invention include, e.g., Aminoglutethimide; Asparaginase; Bleomycin; Busulfan; Carboplatin; Carmustine (BCNU); Chlorambucil; Cisplatin (cis-DDP); Cyclophosphamide; Cytarabine HC1; dacarbazine; Dactinomycin; Daunorubicin HC1; Doxorubicin HC1; Estramustine phosphate sodium; Etoposide (VP-16); Floxuridine; Fluorouracil (5-FU); Flutamide; Hydroxyurea (hydroxycarbamide); Ifosfamide; Interferon Alfa-2a, Alfa 2b, Lueprolide acetate (LHRH-releasing factor analogue); Lomustine (CCNU); Mechlorethamine HC1 (nitrogen mustard); Melphalan; Mercaptopurine; Mesna; Methotrexate (
  • Tamoxifen citrate Thioguanine; Thiotepa; Vinblastine sulfate; Vincristine sulfate; Amsacrine (m-AMSA); Azacitidine; Hexamethylmelamine (HMM); Interleukin 2; Mitoguazone (methyl- GAG; methyl glyoxal bis-guanylhydrazone; MGBG); Pentostatin; Semustine (methyl- CCNU); Teniposide (VM-26); paclitaxel and other taxanes; and Vindesine sulfate.
  • m-AMSA Amsacrine
  • HMM Hexamethylmelamine
  • Mitoguazone methyl- GAG; methyl glyoxal bis-guanylhydrazone; MGBG
  • Pentostatin Semustine (methyl- CCNU); Teniposide (VM-26); paclitaxel and other taxanes; and Vindesine sulfate.
  • Supplementary potentiating agents that can be co-administered with the compounds of the invention include, e.g., Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca ++ antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Amphotericin (e.g., Tween 80 and perhexiline maleate); Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thio
  • the active compound(s) may be administered per se or in the form of a pharmaceutical composition wherein the active compound(s) is in admixture with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichloroflu
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection or a transdermal patch.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • One product of the invention is a veterinary preparation of an aromatic compound of the invention, used alone or combined with an anti-scours agent.
  • An anti-scours agent is a composition which is known to be useful in preventing or inhibiting the symptoms of scours.
  • Known compositions include, for example, colostral extracts, such as those described in U.S. patent no. 4,377,569 and Canadian patent no. 1,175,352 and widely commercially available (e.g. Soluble Colostrum Powder, by VedCo, Inc., St.
  • bovine immunoglobulin fraction prepared from bovine plasma or clear bovine serum such as the fraction described in U.S. patent no. 3,984,539
  • oral rehydration fluids and/or replacement electrolyte compositions which are widely commercially available in the form of dry compositions or liquid solutions prepared for oral or intravenous administration
  • Electrolyte H by Agri-Pet Inc., Aubrey TX
  • Electrolyte Powder 8x by Phoenix Pharmaceutical Inc, St. Joseph MO
  • Electrolyte Solution Rx by Lextron Inc., Greeley CO, ProLabs LTD, St.
  • the veterinary preparation is a dry preparation of the aromatic compound of the invention and an antiscours agent.
  • the dry preparation may be administered directly or may be hydrated and/or diluted in a liquid solution prior to administration.
  • the veterinary preparation is a liquid solution of the compound of the invention and an anti-scours agent.
  • Another product of the invention is a pharmaceutical preparation of an aromatic compound of the invention and an anti-diarrheal agent.
  • An anti-diarrheal agent includes, for example, an immunoglobulin preparation from bovine colostrum; lomotil; an intravenous or oral rehydration fluid; a dry rehydration composition salt; an electrolyte replacement composition (in dry or liquid form); an oral or intravenous sugar-electrolyte solution or dry composition; an antibiotic such as tetracycline, trirmethoprim or sulfamethoxazole; a quinolone drug such as norfloxacin or ciprofloxacin, bismuth subsalicylate, diphenoxylate; and loperamide.
  • the pharmaceutical preparation is a dry preparation of the aromatic compound of the invention and an anti-diarrheal agent.
  • the dry preparation may be administered directly or may be hydrated and/or diluted in a liquid solution prior to administration.
  • the pharmaceutical preparation is a liquid solution of the aromatic compound of the invention and an anti-diarrheal agent.
  • the time of administration of the aromatic compounds useful according to the invention varies depending upon the purpose of the administration.
  • the compounds of the invention When the compounds of the invention are administered in order to prevent the development of diarrhea in subjects traveling to areas with high risk of exposure to infectious agent or subjects otherwise exposed to diarrhea causing agents, the compounds should be administered at about the time that the subject is exposed to the risk or the high risk area.
  • the veterinary preparation When the compounds are administered to subjects in order to prevent the development of scours, the veterinary preparation should be administered within the first 12 hours after birth, and preferably within the first 4 hours after birth.
  • the compounds of the invention When the compounds of the invention are used to treat subjects having symptoms of diarrhea or scours, the compounds may be administered at any point while the subject is experiencing symptoms, and preferably as soon as the symptoms develop. Other considerations will be apparent when the compounds are used to treat inflammatory diseases, proliferative diseases, etc.
  • the formulations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions.
  • Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% W/N); citric acid and a salt (1-3% W/N); boric acid and a salt (0.5-2.5% W/N); and phosphoric acid and a salt (0.8-2% W/N).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03%) W/N); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/N) and thimerosal (0.004-0.02% W/N).
  • the active compounds of the present invention may be pharmaceutical compositions having a therapeutically effective amount of an aromatic compound of the general formula provided above in combination with a non-formula (I) active agent, optionally included in a pharmaceutically-acceptable carrier.
  • the active compounds of the present invention also may be veterinary compositions having a therapeutically effective amount of an aromatic compound of the general formula provided above in combination with a non- formula (I) active agent, optionally included in a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier as used herein means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being commingled with the compound of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • a common administration vehicle e.g., pill, tablet, bolus, powder or solution for dilution, pig pump, implant, injectable solution, etc.
  • a common administration vehicle e.g., pill, tablet, bolus, powder or solution for dilution, pig pump, implant, injectable solution, etc.
  • the present invention provides pharmaceutical or veterinary compositions, for medical or veterinary use, which comprise the active compounds of the invention together with one or more pharmaceutically acceptable carriers thereof and other therapeutic ingredients.
  • a variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the condition being treated and the dosage required for therapeutic efficacy.
  • the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • Such modes of administration include oral, rectal, topical, nasal, transdermal or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous and intramuscular routes are not particularly suited for long term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic treatment because of the convenience to the subject as well as the dosing schedule.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3 -butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the active compounds of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • long-term sustained release implant may be particularly suitable for treatment of diarrhea in immunodeficient subjects, who need continuous administration of the compositions of the invention.
  • Long-term release as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. Effective Dosages
  • compositions suitable for use with the present invention include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose.
  • a therapeutically effective amount i.e., in an amount effective to achieve its intended purpose.
  • the actual amount effective for a particular application will depend, wter alia, on the condition being treated.
  • such compositions when administered in methods to reduce sickle cell dehydration and/or delay the occurrence of erythrocyte sickling or distortion in situ, such compositions will contain an amount of active ingredient effective to achieve this result.
  • When administered in methods to inhibit cell proliferation, such compositions will contain an amount of active ingredient effective to achieve this result.
  • compositions When administered to subjects suffering from disorders characterized by abnormal cell proliferation, such compositions will contain an amount of active ingredient effective to, inter alia, prevent the development of or alleviate the existing symptoms of, or prolong the survival of, the subject being treated.
  • a therapeutically effective amount further includes that amount of compound which arrests or regresses the growth of a tumor. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.
  • Target plasma concentrations will be those concentrations of active compound(s) that are capable of inducing at least about 25% inhibition of the Gardos channel, at least about 25% inhibition of Cl " secretion in intestinal cells and/or at least about 25% inhibition of cell proliferation in cell culture assays, depending, of course, on the particular desired application.
  • Target plasma concentrations of active compound(s) that are capable of inducing at least about 50%, 75%, or even 90% or higher inhibition of the Gardos channel, of Cl " secretion in intestinal cells, and/or of cell proliferation in cell culture assays are preferred.
  • the percentage inhibition of the Gardos channel, of Cl " secretion in intestinal cells, and/or cell proliferation in the subject can be monitored to assess the appropriateness of the plasma drug concentration achieved, and the dosage can be adjusted upwards or downwards to achieve the desired percentage of inhibition.
  • Therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a circulating concentration that has been found to be effective in animals.
  • Useful animal models for diseases characterized by abnormal cell proliferation are well-known in the art.
  • a particularly useful animal model for sickle cell disease is the SAD mouse model (Trudel et al, 1991, EMBO J. 11 :3157-3165).
  • Useful animal models for diseases characterized by abnormal cell proliferation are well-known in the art.
  • the following references provide suitable animal models for cancer xenografts (Corbett et al. , 1996, J. Exp. Ther. Oncol. 1:95-108; Dykes et al. , 1992, Contrib. Oncol. Basel. Karger 42: 1 -22), restenosis (Carter et al. , 1994, L Am. Coll. Cardiol. 24(5): 1398-1405). atherosclerosis (Zhu et al, 1994. Cardiology 85f6):370- 377) and neovascularization (Epstein et al, 1987, Cornea 6(4):250-257).
  • the dosage in humans can be adjusted by monitoring Gardos channel inhibition and/or inhibition of cell proliferation and adjusting the dosage upwards or downwards, as described above.
  • a therapeutically effective dose can also be determined from human data for compounds which are known to exhibit similar pharmacological activities, such as
  • Clotrimazole and other antimycotic agents see, e.g., Brugnara et al, 1995, JPET 273:266- 272; Benzaquen et al, 1995, Nature Medicine 1:534-540; Brugnara et al, 1996, J. Clin. Invest. 97(5):1227-1234).
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound as compared with Clotrimazole. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • the systemic circulating concentration of administered compound will not be of particular importance.
  • the compound is administered so as to achieve a concentration at the local area effective to achieve the intended result.
  • a circulating concentration of administered compound of about 0.001 ⁇ M to 20 ⁇ M is considered to be effective, with about 0.1 ⁇ M to 5 ⁇ M being preferred.
  • Subject doses for oral administration of the compounds described herein typically range from about 80 mg/day to 16,000 mg/day, more typically from about 800 mg/day to 8000 mg/day, and most typically from about 800 mg/day to 4000 mg/day.
  • typical dosages range from about 1 to 200 mg/kg/day, more typically from about 10 to 100 mg/kg/day, and most typically from about 10 to 50 mg/kg/day.
  • typical dosages range from about 40 to 8000 mg/m 2 /day, more typically from about 400 to 4000 mg/m 2 /day, and most typically from about 400 to 2000 mg/m 2 /day.
  • a circulating concentration of administered compound of about 0.001 ⁇ M to 20 ⁇ M is considered to be effective, with about 0.1 ⁇ M to 5 ⁇ M being preferred.
  • Subject doses for oral administration of the compounds described herein for the treatment or prevention of cell proliferative disorders typically range from about 80 mg/day to 16,000 mg/day, more typically from about 800 mg/day to 8000 mg/day, and most typically from about 800 mg/day to 4000 mg/day. Stated in terms of subject body weight, typical dosages range from about 1 to 200 mg/kg/day, more typically from about 10 to 100 mg/kg/day, and most typically from about 10 to 50 mg/kg/day. Stated in terms of subject body surface areas, typical dosages range from about 40 to 8000 mg/m 2 /day, more typically from about 400 to 4000 mg/m 2 /day, and most typically from about 400 to 2000 mg/m 2 /day.
  • dosage amount and interval can be adjusted individually to provide plasma levels of the administered compound effective for the particular clinical indication being treated.
  • a compound according to the invention can be administered in relatively high concentrations multiple times per day.
  • the compounds can be administered before, during or after surgical removal of the tumor.
  • the compounds can be administered to the tumor via injection into the tumor mass prior to surgery in a single or several doses.
  • the tumor, or as much as possible of the tumor may then be removed surgically. Further dosages of the drug at the tumor site can be applied post removal.
  • surgical removal of as much as possible of the tumor can precede administration of the compounds at the tumor site.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular subject.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular subject.
  • many factors are important in determining a therapeutic regimen suitable for a particular indication or subject. Severe indications such as cancer may warrant administration of higher dosages as compared with less severe indications such as sickle cell disease.
  • the formulations of the invention are also administered in effective amounts when treating diarrhea or scours.
  • An effective amount is one sufficient to inhibit or prevent diarrhea or scours and is thus sufficient to inhibit the Cl " secretion of intestinal epithelial cells.
  • An amount which is sufficient to inhibit the Cl " secretion of intestinal epithelial cells thereby effectively decreases the secretory response, thereby resulting in a decrease in diarrhea or scours and/or the symptoms thereof. Effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual subject parameters including age, physical condition, size and weight; concurrent treatment; frequency of treatment; and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • An effective amount for an individual compound may be assesed using any method known in the art which reliably determines the amount of Cl " secretion from intestinal cells.
  • a compound may be subject to a series of standard assays or screens to determine its pharmacological activity and effective amounts.
  • the active compounds of the invention are those which induce at least about 25% inhibition of the Cl " secretion, as measured using in vitro assays that are commonly known in the art (see, e.g., Example 4).
  • the active compounds of the invention generally will have an IC 50 (concentration of compound that yields 50% inhibition) for inhibition of the Cl " secretion of less than about 10 ⁇ M as measured using in vitro assays.
  • a maximum dose that is, the highest safe dose according to sound medical judgment, particularly if acute diarrhea or scours are the dominant clinical manifestation. Dosage may be adjusted appropriately to achieve desired drug plasma levels. Generally, daily oral doses of active compounds will be from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral doses in the range of 50 to 500 milligrams/kg, in one or several administrations per day, will yield the desired results. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that subject tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds. Toxicity
  • the ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be expressed as the ratio between LD 50 (the amount of compound lethal in 50% of the population) and ED 50 (the amount of compound effective in 50% of the population).
  • Compounds which exhibit high therapeutic indices are preferred.
  • Therapeutic index data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage of such compounds preferably lies within a range of plasma concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition. (See e.g. Fingl et al , 1975, In: The Pharmacological Basis of Therapeutics. Ch. 1 pi).
  • Clotrimazole which is outside of the scope of the present claims is used to exemplify how the compounds of the present invention are tested for certain conditions.
  • the compounds of the present invention are structurally distinct from the structure of clotrimazole. Nevertheless, the compounds of the present invention act on chloride secretion in the same manner as clotrimazole and, therefore, are useful in the methods and products of the present invention.
  • This example provides a general method for synthesizing substituted 1 l-aryl-5,6- dihydro-1 lH-dibenz[b,e]azepine compounds according to the invention.
  • a general reaction scheme is provided in FIG. 1.
  • R 2 -R ⁇ 5 are as previously defined for structural formula (I).
  • a mixture of an appropriately substituted 2-aminobenzophenone 100 (1 equivalent), an appropriately substituted benzyl chloride 102 (1 equivalent), potassium carbonate (2 equivalents) and sodium iodide (1 equivalent) in acetonitrile is refluxed for 12 hours.
  • the reaction mixture is cooled to room temperature and water added.
  • the mixture is extracted with ethyl acetate.
  • the combined ethyl acetate extracts are washed with water then dried over sodium sulfate. Evaporation of the solvent followed by column chromatography gives the substituted N-alkyl-2-aminobenzophenone derivative 104 in about 55-80% yield.
  • the substituted N-alkyl-2-aminobenzophenone derivative 104 (1 equivalent) is dissolved in a 3:1 mixture of tetrahydrofurammethanol. Sodium borohydride (10 equivalents) is slowly added and the reaction mixture is stirred at room temperature for 12 hours. The reaction is quenched by adding 2 N aqueous hydrochloric acid solution. The reaction mixture is neutralized by adding 4 N aqueous sodium hydroxide solution and extracted with ethyl acetate. The combined ethyl acetate extracts are dried over sodium sulfate. Evaporation of the solvent followed by column chromatography gives the substituted N-alkyl-2-amino- benzyl alcohol derivative 106 in about 40-60% yield.
  • substituted 1 l-aryl-5,6-dihydro-l lH-dibenz[b,e]azepines can be synthesized from appropriate starting materials according to the methods described in Sasakura and Sugasawa, 1981, Heterocycles 15 :421 -425.
  • This example provides a general method for synthesizing 11-aryl-l l-substituted-5,6- dihydro-dibenz[b,e]azepine compounds according to the invention.
  • a general reaction scheme is provided in FIG. 2.
  • RrR ⁇ 5 are as previously defined for structural formula (I).
  • the substituted N-alkyl-2-aminobenzophenone derivative 104 is prepared as described in Section 1.1, supra. To a solution (0.25 M) of an approriate grignard reagent in diethyl ether at -40 °C is added a solution (0.1 M) of the substituted N-alkyl-2- aminobenzophenone derivative 104 in diethyl ether. The mixture is stirred at -40 °C for 30 min., warmed to room temperature and quenched with water. Extraction with ethyl acetate and evaporation of the solvent gives the crude alcohol product 112 as an oil. The alcohol product 112 is purified by column chromatography to give the pure alcohol product 112 as a white solid in 85-90% yield.
  • the 11-aryl-l l-substituted-5, 6-dihydro-dibenz[b,e] azepine 114 can be converted to the corresponding N-substituted-11-aryl-l l-substituted-5,6-dihydro-dibenz[b,e]azepine 116 as described in Section I A, supra.
  • a preferred method of synthesis of N-phenoxy carbonyl- 1 l-phenyl-5,6-dihydro-l 1H- dibenz[b,e]azepine (Compound 14) is as follows: A mixture of 0.25 g (0.00092 mole) of 11- phenyl-5,6-dihydro-HH-dibenz[b,e]azepine, 0.318 g (0.0023 mole) of potassium carbonate and 0.318 g (0.002 mole) of phenyl chloroformate in 10 mL of acetonitrile, was stirred at room temperature for 2 days.
  • a preferred method of synthesis of N-phenoxycarbonyl-1 l-(2'-chlorophenyl)-5,6- dihydro-1 lH-dibenz[b,e]azepine (Compound 26) is as follows: A mixture of 0.2 g (0.00065 mole) of l l-(2'-chlorophenyl)-5,6-dihydro-HH-dibenz[b,e]azepine, 0.18 g (0.0013 mole) of potassium carbonate and 0.204 g (0.0013 mole) of phenyl chloroformate in 10 mL of acetonitrile, was stirred at room temperature for 2 days.
  • a preferred method of synthesis of N-(4'-nitrobenzoyl)-l l-(2'-chlorophenyl)-5,6- dihydro-1 lH-dibenz[b,e]azepine (Compound 28) is as follows: A mixture of 0.2 g (0.00065 mole) of l l-(2'-chlorophenyl)-5,6-dihydro-HH-dibenz[b,e]azepine, 0.179 g (0.0013 mole) of potassium carbonate and 0.133 g (0.00072 mole) of 4-nitrobenzoyl chloride in 10 mL of acetonitrile, was stirred at room temperature for 12 hours.
  • This Example demonstrates the ability of several exemplary compounds of formula (I) to inhibit the Gardos channel of erythrocytes (Gardos channel assay) and/or mitogen-induced cell proliferation (mitogenic assay) in vitro.
  • the assays are generally applicable for demonstrating the in vitro activity of other compounds of formula (I). Methods. The percent inhibition of the Gardos channel (10 ⁇ M compound) and the
  • IC 50 were determined as described in Brugnara et al., 1993, J. Biol. Chem. 268(12):8760- 8768. The percent inhibition of mitogen-induced cell proliferation (10 ⁇ M compound) and the IC 50 were determined as described in Benzaquen et al. (1995, Nature Medicine 1:534- 540) with NIH 3T3 mouse fibroblast cells (ATCC No. CRL 1658). Other cell lines, e.g., cancer cells, endothelial cells and fibroblasts, as well as many others, may be used in the cell proliferation assay. Selection of a particular cell line will depend in part on the desired application, and is well within the capabilities of an ordinarily skilled artisan. Results. The results of the experiment are provided in Table 2, below. Clotrimazole is reported for purposes of comparison. Most of the compounds tested exhibited significant activity in both assays. All of the compounds tested exhibited significant activity in at least one of the assays.
  • Example 3 Activity In Cancer Cell Lines This Example demonstrates the antiproliferative effect of several exemplary compounds of formula (I) against a variety of cancer cell lines. The assays are generally applicable for demonstrating the antiproliferative activity of other compounds of formula (I).
  • VP-16 a standard anti-cancer agent
  • ATCC American Type Culture Collection
  • the ATCC assession numbers were as follows: HeLa (CCL-2); CaSki (CRL-1550); MDA-MB-231 (HTB-26); MCF-7 (HTB-22); A549 (CCL-185); HTB-174 (HTB-174); HEPG2 (HB-8065); DU-145 (HTB-81); SK-MEL- 28 (HTB-72); HT-29 (HTB-38); HCT-15 (CCL-225); ACHN (CRL-1611); U-118MG (HTB- 15); SK-OV-3 (HTB-77).
  • MMRU cells Stender et al., 1993. J. Dermatology 20:61 1-617 were a gift of one of the authors.
  • Tablets each containing 60 mg of active ingredient are made up as follows:
  • the active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve.
  • the granules are dried at 50°-60°C and passed through a No. 18 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules, which, after mixing are compressed by a tablet machine to yield tablets each weighing 150 mg.
  • Tablets can be prepared from the ingredients listed by wet granulation followed by compression. 4.2. Gelatin Capsules
  • Hard gelatin capsules are prepared using the following ingredients:
  • Aerosol Solution The above ingredients are mixed and filled into hard gelatin capsules in 460 mg quantities. 4.3. Aerosol Solution
  • An aerosol solution is prepared containing the following components:
  • the active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30 °C and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container. 4.4. Suppositories
  • Suppositories each containing 225 mg of active ingredient are made as follows:
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool. 4.5. Suspensions
  • the active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste.
  • the benzoic acid solution, flavor and some color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.
  • Example 5 Clotrimazole inhibits water and electrolyte secretion in intestinal epithelial cells.
  • the biochemical basis of secretory diarrhea involves intestinal Cl " secretion in intestinal crypt cells.
  • Cl " ions are maintained within intestinal crypt cells at levels above their electrochemical potential by primarily and secondarily active transport mechanisms such as the Na/K ATPase pumps and Na/K/2C1 cotransporters.
  • Cl " is transported into the lumen from the intestinal crypt cells through apical Cl " channels. Intracellular levels of K + , cAMP, cGMP, and Ca ++ are all involved in regulating the secretory response.
  • T84 cells were used to determine whether clotrimazole regulates Cl " secretion in intestinal crypt cells. T84 cells form confluent monolayers of columnar epithelia that exhibit high transepithelial resistances, polarized apical and basilateral membranes, and cAMP and Ca ++ regulated Cl " secretory pathways analogous to those found in native intestine.
  • T84 cells obtained from ATCC were cultured and passaged in equal parts of dulbecco's modified eagle's medium (DMEM), lg/lD-glucose) and Hams F-12 nutrient mixture, supplemented with 5% newborn calf serum, 15 mM HEPES, 14 mM Na HC0 3 , 40mg/l penicillin, 8mg/l ampicillin, 0.90 mg/1 streptomycin. Cells were seeded at confluent density onto 0.33 cm 2 or 5cm 2 Transwell inserts (Costar, Cambridge, MA) coated with dilute rat collagen solution as previously described (Lencer et al., J. Clin. Invest..
  • Transepithelial resistances attain stable levels (>1000 Ohms.cm 2 ) after 7 days.
  • the development of high transepithelial resistances correlated with the formation of confluent monolayers with well- developed tight junctions as assessed by morphological analysis, and with the ability of monolayers to secrete Cl " (Madara et al., Gastro. 92: 1133-1145 (1987).
  • Electrophvsiology (mesurement of electrogenic Cl " secretion). Confluent monolayers were transferred to Hanks Buffered Salt Solution (HBSS) containing 0.185 g/l CaCl 2 , 0.098 g/l MgSO 4 , 0.4 g/l KC1, 0.06 g/l KH 2 P0 4 , 8 NaCl, 0.048 g/l Na 2 HPO 4 , 1 g/l glucose, and lOnM HEPES, pH 7.4. Serosal and mucosal reservoirs were interfaced with Calomel and Ag- Ag Cl electrodes via 5% agar bridges made with Ringer's buffer.
  • HBSS Hanks Buffered Salt Solution
  • Transepithelial resistance was measured using a dual voltage clamp device to apply 25 or 50 ⁇ A current pulses.
  • Short circuit current (ISC) was calculated using Ohms law as previously described (Lencer et al., 1 Clin. Invest. 92: 2941-2951 (1993); Lencer et al. J. Cell Biol. 117: 1197-1209 (1992).
  • vasoactive intestinal peptide (VIP) and cholera toxin are cAMP mediated agonists of the K + channel
  • carbachol is a Ca ++ -dependent agonist of the Ca ++ regulated K + channels.
  • the pathway by which a particular inhibitor of Cl " secretion in T84 cells is functioning may be identified by measuring the ability of the inhibitor to modify transepithelial resistances in T84 cells which have been treated with VIP or carbachol to stimulate Cl " secretion.
  • T84 cells were grown as described above and Cl " secretion was stimulated by the addition to the media of either carbachol (lOOmM) or VIP (5nM). The cells were then treated with BaCl (3mM), charybdotoxin (lOOnM), or clotrimazole (33mM). The short circuit current (ISC) was determined for the various inhibitor treatments as a percentage of the control in the absence of inhibitor (FIG. 3). BaCl strongly inhibited the secretory response to the cAMP mediated agonist VIP, but had no apparent affect on the secretory response elicited by the Ca ++ -dependent agonist carbachol.
  • ISC short circuit current
  • clotrimazole potentiated slightly (by 5-10%) the Cl-secretory responses to either agonist, clotrimazole inhibited effectively the secretory response to cholera toxin (20 nM, a cAMP-dependent agonist) and E. Coli heat-stabile toxin (100 nm, a cGMP-agonist) (IC50 values of 10 ⁇ M and 15 ⁇ M, respectively).
  • IC50 values 10 ⁇ M and 15 ⁇ M, respectively.
  • the effect of clotrimazole on K * conductances was also examined by isotopic flux studies using 86 RB. T84 cells were grown in the presence of a cAMP agonist, VIP, or a Ca ++ mediated agonist (Thapsigargin).
  • Clotrimazole was added and 86 RB efflux was measured. Clotrimazole significantly inhibited baseline and Ca ++ stimulated 86 RB efflux in the presence of both cAMP and Ca ++ mediated agonists compared to those cells which were not treated with clotrimazole.
  • clotrimazole To determine the site of clotrimazole action, the effects of clotrimazole pretreatment were examined on monolayers stimulated with agonists that initiate Cl- secretion at sequential steps in the cAMP signalling cascade. T84 monolayers were preincubated in HBSS in the presence or absence of clotrimazole (33 ⁇ M) and then stimulated with either 5 ⁇ M VIP (which activates adenylate cyclase through heterotrimeric GTPase-linked cell surface receptors), 10 ⁇ M forskolin (which activates adenylate cyclase directly), or 3 mM 8Br-cAMP (a direct stimulator of protein kinase A). Clotrimazole inhibited the secretory response to each of these agonists. These data provide evidence that clotrimazole acts at a step distal to the activation of Protein Kinase A.
  • IP3 inositol trisphosphate
  • Downstream events may be mediated by [Ca++]i, IP3, diacylglycerol, or as yet unidentified diffusable factors (Putney and Bird, Cell 75:199-201 (1993)).
  • this signalling, cascade, T84 monolayers pretreated in the presence or absence of clotrimazole (33 ⁇ M) were stimulated with the Ca++-dependent agonists carbachol (100 ⁇ M which elicits both Ca++ and IP 3 signals), thapsigargin (5 ⁇ M, which elevates cytoplasmic Ca++ via inhibition of ER Ca++- ATPase) (Vandorpe et al., Biophys. J.
  • Clotrimazole inhibited strongly the Cl-secretory response to each to these reagents. These data suggest that clotrimazole acts at steps in the secretory response distal to the release of intracellular Ca++ stores.
  • Example 7 Clotrimazole does not affect apical membrane anion conductance or basolateral NaK2Cl cotransporters.
  • VIP vasoactive intestinal peptide
  • 5 ⁇ nM vasoactive intestinal peptide
  • thapsigargin 5 ⁇ M
  • the cell monolayer was rinsed, cut with its support from the polystyrene ring, and the residual cell-associated radioactivity was determined. Monolayers were maintained at 37° C in room air throughout the study. 125 I was counted by gamma counting and normalized to percent total uptake as previously described (Venglarik, et al, Am. J. Physiol (Cell Physiol 2SJ:C358-C364 (1990).
  • Cl- secretion might occur by blockade of apical membrane Cl-channels, or blockade of basolaterally situated NaK2Cl cotransporters.
  • clotrimazole affected ion conductance through apical membrane Cl-channels, we examined the time course of 125 I efflux from T84 monolayers pretreated in the presence or absence of clotrimazole (Venglarik, et al, Am. J. Physiol (Cell Physiol 28):C358-C364 (1990). Clotrimazole had little or no effect on the time course of 125 I efflux from monolayers treated with VIP.
  • Clotrimazole had similar lack of effect on 125 I efflux stimulated by thapsigargin.
  • Example 8 Clotrimazole inhibits Chloride secretion by inhibiting K+ efflux through basolateral K+ channels in T84 cells.
  • Clotrimazole inhibits chloride secretion by blockade of K+ transport through both Ba++- sensitive and charybdotoxin-sensitive channels- Methods: 86 Rb Efflux Studies Confluent monolayers on 5 cm 2 Transwell inserts were used 10-14 days after plating. 86 Rb flux was measured as an indicator of apical C1-, channel and basolateral K+ channel activity as previously described (Venglarik, et al, Am. J. Physiol. (Cell Physiol. 2S):C358-C364 (1990).
  • the cell monolayer was rinsed, cut with its support from the polystyrene ring, and the residual cell-associated radioactivity was determined. Monolayers were maintained at 37° C in room air throughout the study. 86 Rb was counted by scintillation counting and normalized to percent total uptake as previously described (Venglarik, et al, Am. J. Physiol. (Cell Physiol. 25):C358-C364 (1990).
  • K+ channel activity was estimated by measurement of 86 Rb efflux.
  • Clotrimazole was found to significantly inhibit the rate of 86 Rb efflux after treatment with the cAMP agonist VIP (5 ⁇ M).
  • Clotrimazole inhibits chloride secretion through distinct cAMP and Ca ⁇ sensitive basolateral K + channels.
  • the Basolateral potassium conductance was measured using the technique developed by Dawson and co-workers.
  • a potassium gradient (mucosal to serosal) was first established across the monolayer using asymmetric mucosal and serosal buffers containing K + as the sole permeant ion.
  • the addition of amphotericin B (20 ⁇ M) to the mucosal reservoir forms conductive pores in the apical membrane, and thus removes all resistance to transepithelial potassium movement across this membrane.
  • the amphotericin-dependent Isc becomes a measure of the rate of the transepithelial potassium flux across basolateral membranes.
  • Changes in short circuit current (Isc) then represent changes in basolateral K + conductances (gK).
  • Isc and K + conductances were measured using calomel electrodes, 3M KCI-agar bridges, and a voltage clamp (University of Iowa, Iowa City). To generate a voltage-current channel relationships, currents were elicited by 1 sec test potentials from -80 to +80 in 10 mV increments in the asymmetrical high K + gluconate solution.
  • Basolateral K+ transport was examined in T84 monolayers permeabilized apically by pretreatment with amphotericin B.
  • Apical and basolateral buffers contained K+ as the sole permeant ion. All studies were performed with a 135 mM basolaterally directed K+ gradient. This method has been utilized previously to examine both Cl- and K+ transport in T84 cells and HT29-C1.16E cells. Briefly, ion conductances in the luminal or basolateral membranes of confluent T84 cell monolayers can be assessed separately by selectively permeabilizing the apical or basolateral membrane using the ionophore amphotericin B.
  • K+ transport was measured at baseline and after the ordered additions of cAMP- and Ca ++ -agonists.
  • the initial permeabilization with amphotericin B was associated with 49 ⁇ 19% increase in conductance. Pores formed by amphotericin B display selectivity for monovalent cations.
  • Ca ++ remained relatively impermeant as evidenced by the small steady state increase in Isc and G ⁇ caused by apical permeabilization with amphotericin B. Given this low baseline Isc and G ⁇ , both cAMP- and Ca ++ -sensitive K+ permeabilities (PK) were readily apparent after agonist stimulation.
  • Clotrimazole and 2-chlorophenyl-bs-phenyl methanol a structurally related stable metabolite, inhibit K+ transport through both cAMP- and Ca ++ -dependent K+ channels.
  • clotrimazole may inhibit directly basolateral membrane K+ channels in human intestinal T84 cells, as it does in the red cell, clotrimazole significantly inhibited the time course of K+ transport after treatment with the cAMP agonist forskolin (10 ⁇ M) and the Ca ++ agonist carbachol (100 ⁇ M).
  • Formal IV relations taken at steady state after cAMP or Ca ++ stimulation confirm that clotrimazole affected both cAMP- and Ca ++ - sensitive channels.
  • Clotrimazole targets the basolateral rather than the apical surface of T84 cells
  • clotrimazole In contrast to the clear inhibitory effects of clotrimazole on basolateral K+ conductances, however, clotrimazole had no detectable effect on either forskolin- or thapsigargin-stimulated Cl-conductances. I/N relations for Cl- transport were nearly identical in monolayers treated or not treated with clotrimazole. These data provide further evidence that clotrimazole inhibits Cl- secretion in intact T84 cell monolayers by affecting specifically basolateral K+ channels. Apical membrane Cl-channels are not inhibited.
  • Example 9 Clotrimazole inhibits Cl " secretion in vivo. 9.1. Chamber studies using rabbit colonic mucosa. Methods: 4 male, New Zealand rabbits (2.5 kg) were anesthetized by an intravenous injection of pentobarbital (0.5 ml/kg). A 15 cm length of distal colon was removed and opened longitudinally.
  • Luminal and serosal buffer solutions were interfaced via Ag-AgCl electrodes (Voltage/Current Clamp, Model VCC600, Physiologic Instruments, Inc., San Diego, CA, USA) and Ringer/agar bridge to voltage clamp device (model DVC-1000; Voltage/Current Clamp, World Precision Instruments, Inc.). Resistance (R) was calculated using Ohm's law and the Isc and is given in ⁇ x cm2.
  • mucosal preparations were incubated in the presence or absence of serosal clotrimazole (30 ⁇ M) for 30 min, and then stimulated by the addition of forskolin (10 ⁇ M) or carbachol (10 ⁇ M) to the serosal reservoir.
  • Murine model of secretotory diarrhea- Methods Treated and control, untreated, mice were gavage fed either clotrimazole (150 mg/kg/day divided in two equal doses, dissolved in peanut oil at a concentration of 20 mg/ml) or vehicle control over a 7 day loading period. Mice were then challenged by gavage with either 25 ⁇ g purified cholera toxin (Calbiochem, San Diego, CA) in PBS, vehicle control alone (PBS without cholera toxin), or cholera toxin in PBS containing 30 ⁇ M clotrimazole. Animals were sacrificed after 5 hours in an uncrowded C0 2 hood.
  • the carcass was weighed, the abdomen was opened, and ligatures were tied at the proximal duodenum and distal rectum.
  • the intestinal block was dissected free of supporting structures and removed as a single unit and weighed. Small and large intestinal segments were normalized to body weight (intestinal weight/carcass weight) for each animal.
  • mice were gavage fed 150 mg/kg/day clotrimazole, divided into two equal doses, or vehicle control every 12 h for 7 days and subsequently challenged orally with purified cholera toxin (25 ⁇ g). Five hours after treatment with cholera toxin, the mice were sacrificed and intestinal fluid secretion assessed gravimetrically. Pretreatment with clotrimazole reduced by 86% intestinal fluid secretion induced by cholera toxin. Clotrimazole had no effect on intestinal secretion in the absence of cholera toxin. Thus, clotrimazole effectively treated secretory diarrhea in vivo, presumably by inhibiting basolateral K+ channels of crypt epithelial cells.

Abstract

La présente invention porte sur des composés 11-phényl-dibenzazépine substitués, et sur leurs analogues, qui sont des inhibiteurs spécifiques, puissants et sûrs des vannes à potassium activées par Ca2+ (canal Gardos) des hématies, de la prolifération cellulaire mammalienne et/ou de la sécrétion de chlorure électrogénique transépithélial stimulée par le sécrétagogue dans les cellules intestinales. Ces composés peuvent être utilisés pour réduire la déshydratation drépanocytaire et/ou retarder l'apparition de la falciformation ou déformation des hématies in situ dans des méthodes thérapeutiques utilisées dans le traitement ou la prévention de la drépanocytose. Ces composés peuvent être également utilisés pour inhiber la prolifération des cellules mammaliennes in situ dans des méthodes thérapeutiques utilisées dans le traitement ou la prévention de maladies caractérisées par une prolifération cellulaire anormale, ainsi que pour inhiber la sécrétion de chlorure dans les cellules intestinales dans des méthodes thérapeutiques utilisées dans le traitement de la diarrhée chez l'homme et l'animal.
PCT/US1998/024967 1997-11-20 1998-11-20 Utilisation de composes 11-phenyl-dibenzazepine substitues dans le traitement ou la prevention de la drepanocytose, des maladies inflammatoires caracterisees par une proliferation cellulaire anormale et par la diarrhee chez l'homme et l'animal WO1999026628A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
IL13620998A IL136209A0 (en) 1997-11-20 1998-11-20 Use of substituted 11-phenyl-dibenzazepine compounds for the treatment or prevention of sickle cell disease, inflammatory diseases characterized by abnormal cell proliferation, diarrhea and scour
EP98960380A EP1032394A1 (fr) 1997-11-20 1998-11-20 Utilisation de composes 11-phenyl-dibenzazepine substitues dans le traitement ou la prevention de la drepanocytose, des maladies inflammatoires caracterisees par une proliferation cellulaire anormale et par la diarrhee chez l'homme et l'animal
CA002310773A CA2310773A1 (fr) 1997-11-20 1998-11-20 Utilisation de composes 11-phenyl-dibenzazepine substitues dans le traitement ou la prevention de la drepanocytose, des maladies inflammatoires caracterisees par une proliferationcellulaire anormale et par la diarrhee chez l'homme et l'animal
JP2000521830A JP2001523720A (ja) 1997-11-20 1998-11-20 鎌状赤血球病、異常な細胞増殖によって特徴づけられる炎症性疾患、下痢症および伝染性下痢症の治療または予防用の置換１１−フェニル−ジベンザゼピン化合物の使用。
AU15987/99A AU1598799A (en) 1997-11-20 1998-11-20 Use of substituted 11-phenyl-dibenzazepine compounds for the treatment or prevention of sickle cell disease, inflammatory diseases characterized by abnormal cell proliferation, diarrhea and scour

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US97559497A 1997-11-20 1997-11-20
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US09/159,333 US6291449B1 (en) 1998-09-23 1998-09-23 Use of 11-phenyl-dibenzazepine compounds to treat diarrhea or scours
US09/159,337 1998-09-23
US09/159,337 US20030134842A1 (en) 1997-11-20 1998-09-23 Methods for the treatment or prevention of inflammatory diseases characterized by abnormal cell proliferation
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Publication number Priority date Publication date Assignee Title
US6291449B1 (en) 1998-09-23 2001-09-18 Children's Medical Center Corporation Use of 11-phenyl-dibenzazepine compounds to treat diarrhea or scours
WO2003080082A1 (fr) * 2002-03-21 2003-10-02 Anadis Ltd Compositions contenant des materiaux bioactifs labiles et du colostrum de mammifere, methodes de preparation et de traitement
AU2003212098B2 (en) * 2002-03-21 2008-07-24 Anadis Ltd Compositions containing labile bioactive materials and mammalian colostrum, methods of preparation and treatment
WO2022124900A1 (fr) 2020-12-11 2022-06-16 Sanquin Innovatie B.V. Traitement et prévention de l'anémie inflammatoire

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5273992A (en) * 1992-11-02 1993-12-28 Beth Israel Hospital Assoc. Inc. Method for reducing sickle erythrocyte dehydration and delaying the occurrence of erythrocyte sickling in-situ
WO1996008242A2 (fr) * 1994-09-16 1996-03-21 Children's Medical Center Corporation Metabolites de clotrimazole contre la drepanocytose

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5273992A (en) * 1992-11-02 1993-12-28 Beth Israel Hospital Assoc. Inc. Method for reducing sickle erythrocyte dehydration and delaying the occurrence of erythrocyte sickling in-situ
WO1996008242A2 (fr) * 1994-09-16 1996-03-21 Children's Medical Center Corporation Metabolites de clotrimazole contre la drepanocytose

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SASAKURA K AND SUGASAWA T: "Synthesis of 11-Phenyl-5,6-dihydro-11H-dibenz(b,e)azepine Derivatives", HETEROCYCLES, vol. 15, no. 1, 1981, pages 421 - 425, XP002093039 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291449B1 (en) 1998-09-23 2001-09-18 Children's Medical Center Corporation Use of 11-phenyl-dibenzazepine compounds to treat diarrhea or scours
WO2003080082A1 (fr) * 2002-03-21 2003-10-02 Anadis Ltd Compositions contenant des materiaux bioactifs labiles et du colostrum de mammifere, methodes de preparation et de traitement
AU2003212098B2 (en) * 2002-03-21 2008-07-24 Anadis Ltd Compositions containing labile bioactive materials and mammalian colostrum, methods of preparation and treatment
WO2022124900A1 (fr) 2020-12-11 2022-06-16 Sanquin Innovatie B.V. Traitement et prévention de l'anémie inflammatoire

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