WO2003059267A2 - SELECTIVE 11β-HSD INHIBITORS AND METHODS FOR USE THEREOF - Google Patents

SELECTIVE 11β-HSD INHIBITORS AND METHODS FOR USE THEREOF Download PDF

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WO2003059267A2
WO2003059267A2 PCT/US2002/041356 US0241356W WO03059267A2 WO 2003059267 A2 WO2003059267 A2 WO 2003059267A2 US 0241356 W US0241356 W US 0241356W WO 03059267 A2 WO03059267 A2 WO 03059267A2
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reduced
keto
subject
hsdl
dehydro
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PCT/US2002/041356
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WO2003059267A3 (en
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David J. Morris
Andrew S. Brem
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Rhode Island Hospital
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Priority to AU2002361861A priority patent/AU2002361861A1/en
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Publication of WO2003059267A3 publication Critical patent/WO2003059267A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01146—11-Beta-hydroxysteroid dehydrogenase (1.1.1.146)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Glucocorticoids are steroid hormones.
  • One example of a common glucocorticoid is cortisol. Modulation of glucocorticoid activity is important in regulating physiological processes in a wide range of tissues and organs. High levels of glucocorticoids may result in excessive salt and water retention by the kidneys, which may lead high blood pressure.
  • Glucocorticoids play an important role in the regulation of vascular tone and blood pressure. Glucocorticoids can bind to and activate the glucocorticoid receptor (GR) and, possibly, the mineralocorticoid receptor (MR) to potentiate the vasoconstrictive effects of both catecholamines and angiotensin II (Ang II).
  • GR glucocorticoid receptor
  • MR mineralocorticoid receptor
  • Tissue glucocorticoid levels are regulated by two isoforms of the enzyme 11 ⁇ -hydroxysteroid dehydrogenase (l l ⁇ -HSD).
  • 11 ⁇ -HSD converts glucocorticoids into 11 -keto metabolites that are unable to bind to mineralocorticoid receptors (Edwards C R et al. (1988) Lancet 2:986-9; Funder et al, (1988) Science 242, 583,585).
  • the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject.
  • the method includes administering to the subject an effective amount of a 1 l ⁇ -HSDl reductase inhibitor, e.g., 11-keto- testoterone, 11-keto-androsterone, 11-keto-pregnenolone, 11 -keto-dehydro- epiandrostenedione, 3 ⁇ , 5 ⁇ -reduced-l l-ketoprogesterone, 3 ⁇ , 5 ⁇ -reduced-l l-keto- testosterone, 3 ⁇ , 5 ⁇ -reduced-l 1 -keto-androstenedione, or 3 ⁇ ,5 ⁇ -tetrahydro-l 1 ⁇ - dehydro-corticosterone.
  • a 1 l ⁇ -HSDl reductase inhibitor e.g., 11-keto- testoterone, 11-keto-androster
  • the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject, by administering to said subject an effective amount of a 1 l ⁇ -HSDl reductase inhibitor, wherein the inhibitor is a nucleic acid
  • the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject, by administering to the subject an effective amount of a 1 l ⁇ -HSDl reductase inhibitor in combination with an effective amount of a 17 ⁇ -hydroxylase inhibitor, a 17-HSD inhibitor, 20 ⁇ -reductase inhibitor, or a 20 ⁇ -reductase inhibitor.
  • the invention pertains, at least in part, to a method for increasing the concentration of glucocorticoids in a tissue of a subject.
  • the method includes administering to the subject an effective amount of a 1 l ⁇ -HSDl dehydrogenase inhibitor, such as, for example, 3 ⁇ , 5 ⁇ -reduced-l 1 ⁇ -OH-testosterone, 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ -OH-progesterone, 3 ⁇ , 5 ⁇ -reduced-l 1 ⁇ -OH-testosterone, 3 ⁇ , 5 ⁇ -reduced-l l ⁇ -OH- androstendione, 3 ⁇ , 5 ⁇ -reduced-corticosterone, 3 ⁇ , 5 ⁇ -reduced-aldosterone, 3 ⁇ , 5 ⁇ - reduced-progesterone, 3 ⁇ , 5 ⁇ -reduced testosterone, 3 ⁇ , 5 ⁇ -reduced-chenodeoxycholic acid, or 11 ⁇ -OH testosterone.
  • the 11 ⁇ -dehydrogenase inhibitor is a nucleic acid.
  • the invention pertains, at least in part, to a method for increasing the concentration of glucocorticoids in a tissue of a subject. The method includes administering to the subject an effective amount of a 1 l ⁇ -HSDl dehydrogenase inhibitor in combination with an effective amount of a 17 ⁇ -hydroxylase inhibitor, a 17- HSD inhibitor, a 20 ⁇ -reductase inhibitor or a 20 ⁇ -reductase inhibitor.
  • the invention pertains, at least in part, to a method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 l ⁇ -HSDl dehydrogenase inhibitor, such that the concentration of glucocorticoids in said tissue are increased, wherein said
  • I l ⁇ -HSDl dehydrogenase inhibitor is 3 ⁇ , 5 ⁇ -reduced-l 1 ⁇ -OH-progesterone, 3 ⁇ , 5 ⁇ - reduced- 11 ⁇ -OH-testosterone, 3 ⁇ , 5 ⁇ -reduced-l 1 ⁇ -OH-androstendione, 3 ⁇ , 5 ⁇ -reduced-
  • the 11 ⁇ - HSD1 dehydrogenase inhibitor is a nucleic acid.
  • the invention also pertains, at least in part, to a method for treating hypertension in a subject, by administering to the subject an effective amount of a 1 l ⁇ -HSDl reductase inhibitor, such as, for example, 11-keto- progesterone, 11 -keto-testosterone, 11-keto-androsterone, 11 -keto-pregnenolone, 11- keto-dehydro-epiandrostenedione, 3 ⁇ , 5 ⁇ -reduced-l l-keto-progesterone, 3 ⁇ , 5 ⁇ - reduced-11-keto-testosterone, 3 ⁇ , 5 ⁇ -reduced-l 1-keto-androstenedione, 3 ⁇ ,5 ⁇ - tetrahydro- 11 -dehydro-corticosterone, 3 ⁇ , 5 ⁇ -reduced-l 1-keto-pregnenolone, or 3 ⁇ , 5 ⁇ - reduced- 11 -keto-dehydro-epiandros
  • the invention also pertains, at least in part, to a method for treating hypertension in a subject, by administering to the subject an effective amount of a 1 l ⁇ -HSDl reductase inhibitor, such as, for example, a nucleic acid.
  • a 1 l ⁇ -HSDl reductase inhibitor such as, for example, a nucleic acid.
  • the invention pertains, at least in part, to a method for treating hypertension in a subject, by administering to the subject an effective amount of a 11 ⁇ -HSD 1 reductase inhibitor in combination with an effective amount of a 17 ⁇ -hydroxylase inhibitor, a 17-HSD inhibitor, a 20 ⁇ -reductase inhibitor or a 20 ⁇ - reductase inhibitor.
  • the invention pertains, at least in part, to a method for increasing insulin sensitivity of a tissue in a subject.
  • the method includes administering an effective amount of a 11 ⁇ -HSD 1 reductase inhibitor to the subject.
  • the 1 l ⁇ -HSDl reductase inhibitor include nucleic acids, 11-keto- progesterone, 11-keto-testosterone, 11-keto-androsterone, 11 -keto-pregnenolone, 11- keto-dehydro-epiandrostenedione, 3 ⁇ , 5 ⁇ -reduced-l l-ketoprogesterone, 3 ⁇ , 5 ⁇ -reduced- 11 -keto-testosterone, 3 ⁇ , 5 ⁇ -reduced-l 1-keto-androstenedione, 3 ⁇ ,5 ⁇ -tetrahydro-l l- dehydro-corticosterone, 3 ⁇ , 5 ⁇ -reduced-l 1-keto-pregnenolone
  • the invention pertains, at least in part, to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of 11 ⁇ -OH-progesterone, 11 ⁇ -OH-testosterone, 3 ⁇ ,5 ⁇ -reduced-11 ⁇ -OH-progesterone, 3 ⁇ ,5 ⁇ -reduced-l 1 ⁇ -OH- testosterone, chenodeoxycholic acid, 3 ⁇ , 5 ⁇ -reduced-pregnenolone, 3 ⁇ , 5 ⁇ -reduced- dehydro-epiandrostenedione, 3 ⁇ ,5 ⁇ -reduced-l 1 ⁇ -OH-progesterone, 3 ⁇ ,5 ⁇ -reduced- 11 ⁇ -OH-testosterone, 3 ⁇ ,5 ⁇ -reduced-l 1 ⁇ -OH-androstenedione, 11 -keto-progesterone, 11 -keto-testosterone, 11 -keto-androstenedione, 3 ⁇ ,5 ⁇ -reduced-l 1 -
  • I l ⁇ -HSDl reductase inhibitor wherein said 1 l ⁇ -HSDl reductase inhibitor is an siRNA.
  • the invention pertains to a composition
  • a composition comprising an
  • I I ⁇ -HSD2 dehydrogenase inhibitor wherein said 11 ⁇ -HSD2 dehydrogenase inhibitor is an siRNA.
  • Figure 1 is a bar graph which shows that the exposure of rat aortic rings to corticosterone and 11 ⁇ -HSD2 antisense resulted in a statistically significant increase in the contractile response to phenylephrine.
  • Figure 2 is a bar graph which shows that in aortic rings treated with 1 l ⁇ -HSDl antisense, the contractile responses to all concentrations of phenylephrine were significantly increased compared to aortic rings treated with corticosterone and nonsense oligomers.
  • Figure 3 is a bar graph which illustrates that 11 -dehydro-corticosterone amplifies the contractile responses to phenylephrine in rat aortic rings.
  • Figure 4 is a bar graph which shows that the conversion of corticosterone to 11 - dehydrocorticosterone was lower than in aortic rings incubated with corticosterone and l l ⁇ -HSDl nonsense oligomers.
  • Figures 5A-5D are representative HPLC chromatograms showing the metabolism of 3 H-11 -dehydrocorticosterone (11-dehydroB) by rat aortic rings.
  • the analysis of the tissue is shown for 1 l ⁇ -HSDl nonsense and 1 l ⁇ - HSDl Antisense, respectively.
  • Figures 5C and 5D the analysis of the incubation media is shown for 1 l ⁇ -HSDl nonsense and 1 l ⁇ -HSDl antisense, respectively.
  • Glucocorticoids can affect vascular tone by modifying the actions of several vasoactive substances. Glucocorticoids amplify the vasoconstrictive actions of adrenergic catecholamines and angiotensin II on vascular smooth muscle cells. It has been reported that glucocorticoids decrease the biosynthesis of both nitric oxide and prostaglandin I, and attenuate the vasorelaxant actions of atrial natriuretic peptide in vascular tissue. Thus, the multiple effects of glucocorticoids in vascular tissue operate to increase vascular tone. Since vascular smooth muscle cells contain both glucocorticoid and mineralocorticoid receptors it is possible that glucocorticoids mediate their effects in vascular tissue via either or both of these receptor types.
  • Glucocorticoids are metabolized in vascular and other tissue by two isoforms of 11 ⁇ -hydroxy steroid dehydrogenase (11 ⁇ -HSD).
  • 11 ⁇ -HSD2 is unidirectional and metabolizes glucocorticoids to their respective inactive 11-dehydro derivatives, using NAD + as a co-factor.
  • 1 l ⁇ -HSDl is bi-directional and possesses both dehydrogenase activity as well as reductase activity.
  • the reductase activity of 11 ⁇ - HSD1 regenerates active glucocorticoids from the inactive 11-dehydro derivatives.
  • 1 l ⁇ - HSDl uses NADP + as a co-factor.
  • glucocorticoids In vascular tissue, glucocorticoids amplify the pressor responses to catecholamines and angiotensin II and down-regulate certain depressor systems such as nitric oxide and prostaglandins. Both 11 ⁇ -HSD2 and 1 1 ⁇ - HSD1 are believed to regulate glucocorticoid levels in vascular tissue and are part of additional mechanisms that control vascular tone.
  • Glucocorticoids are known to play an important role in the regulation of vascular tone and blood pressure.
  • Glucocorticoid receptors and mineralocorticoid receptors are present in aorta, mesenteric arteries and rat vascular smooth muscle cells in culture.
  • Glucocorticoids can bind to and activate glucocorticoid receptors (and possibly mineralocorticoid receptors) to potentiate the vasoconstrictive effects of both catecholamines and Ang II.
  • Human and rat vascular endothelial cells contain both 11 ⁇ - HSD2 and 11 ⁇ -HSD 1.
  • 1 1 ⁇ -HSD2 operates to protect both mineralocorticoid receptors and glucocorticoid receptors from excessive stimulation by glucocorticoids. It has been noted that glucocorticoids further amplify the contractile effects of phenylephrine and Ang II when 1 1 ⁇ -HSD enzyme activity is inhibited.
  • Rat vascular smooth muscle cells contain only 11 ⁇ -HSD 1. Under “physiologic conditions,” 1 l ⁇ -HSDl acts largely as a reductase generating active corticosterone from inactive 11 -dehydro-corticosterone.
  • I l ⁇ -HSDl reductase has an important role as a generator of active glucocorticoids in vascular tissue.
  • 11 ⁇ -HSD inactivates glucocorticoid molecules, allowing lower circulating levels of aldosterone to maintain renal homeostasis.
  • Human and rat vascular endolethial cells contain both 11 ⁇ -HSD 1 and 11 ⁇ -HSD2.
  • I I ⁇ -HSD2 operates to protect both mineralocorticoid receptors and glucocorticoid receptors from excessive stimulation by glucocorticoids. It has also been shown that glucocorticoids further amplify the contractile effects of phenylephrine (PE) and Ang II when 11 ⁇ -HSD 1 or 2 dehydrogenase enzyme activity is inhibited.
  • PE phenylephrine
  • Ang II Methods of Treating Glucocorticoid Associated States
  • the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject.
  • the method includes administering to the subject an effective amount of a 1 l ⁇ -HSDl reductase modulating compound, such that the subject is treated.
  • glucocorticoid associated states include states which are associated with the presence or absence of aberrant amounts of glucocorticoids, particularly local levels in target tissues. It includes states which can be treated by modulating, e.g., inhibiting, the activating of a l l ⁇ -HSDl reductase, or, alternatively, l l ⁇ -HSDl dehydrogenase or 1 l ⁇ -HSD2 dehydrogenase. The term includes 1 l ⁇ -HSDl reductase associated states. Examples of glucocorticoid associated states include blood pressure disorders, obesity, diabetes mellitus, interocular pressure, lung disorders, and neurological disorders. The glucocorticoid associated states may also include states associated with undesirable levels of glucocorticoids in adipose tissue, epithelial tissue in the eye, and interocular pressure.
  • 1 l ⁇ -HSDl reductase associated state includes states which can be treated by the administration of an 1 l ⁇ -HSDl reductase modulating compound, e.g., an 1 l ⁇ -HSDl reductase inhibitor. In certain embodiments, these states may be characterized by undesirable amounts of glucocorticoids in a tissue, fluid, or elsewhere in the subject.
  • blood pressure disorders include disorders which are associated with or characterized by abnormal or undesirable blood pressure.
  • blood pressure disorders include, but are not limited to, high blood pressure, congestive heart failure, chronic heart failure, left ventricular hypertrophy, acute heart failure, myocardial infarction, cardiomyopathy, and hypertension, e.g., arterial hypertension and pulmonary hypertension.
  • lung disorders include disorders caused by or related to the presence or absence of glucocorticoids which can be treated by the compounds of the invention, for example, 1 l ⁇ -HSDl reductase inhibitors.
  • the lung contains considerable 1 l ⁇ -HSDl activity (Nicholas and Lugg, J Steroid Biochem 17:113-118, 1982).
  • Glucocorticoids which can be treated by the compounds of the invention.
  • the lung contains considerable 1 l ⁇ -HSDl activity (Nicholas and Lugg, J Steroid Biochem 17:113-118, 1982).
  • fetal there is little reductase activity but enzymatic activity increases significantly during lung maturation following birth.
  • there is a predisposition to pulmonary hypertension with an increase in pulmonary artery wall thickness Cras et al.
  • glucocorticoids enhance endothelin receptor expression in lung (ShimaJRe / ⁇ tr Surg 35:203-207, 2000), a factor contributing to increased vascular resistance in the pulmonary arteries.
  • Another example of a glucocorticoid associated state is insulin insensitivity. High concentrations of cortisol in the liver substantially reduce insulin sensitivity, which increases gluconeogenesis and raises blood sugar levels of a subject. This effect is particularly disadvantageous in subjects suffering from impaired glucose tolerance or diabetes mellitus. In Cushing's syndrome, the antagonism of insulin can provoke diabetes mellitus in subjects.
  • the 1 l ⁇ -HSDl reductase inhibitors can be used to inhibit hepatic gluconeogenesis.
  • glucocorticoid associated state is obesity (including centripetal obesity). It is thought that inhibition of the 1 l ⁇ -HSDl reductase may reduce the effects of insulin resistance in adipose tissue in subjects. Not to be limited by theory, but it is thought that by decreasing insulin resistance will result in greater tissue utilization of glucose and fatty acids, thus reducing circulating levels.
  • Glucocorticoid associated state are neurological disorders.
  • Glucocorticoid excess potentiates the action of certain neurotoxins, which leads to neuronal dysfunction and loss.
  • neurological disorders that may be treated by include neuronal dysfunction and loss due to, for example, glucocorticoid potentiated neurotoxicity.
  • Glucocorticoids may be involved in the cognitive impairment of aging with or without neuronal loss and also in dendritic attenuation. Furthermore, glucocorticoids have been implicated in the neuronal dysfunction of major depression.
  • HSD1 reductase, l l ⁇ -HSDl dehydrogenase, or 11 ⁇ -HSD2 dehydrogenase modulators, e.g., inhibitors, of the invention include both neuropsychiatric and neurodegenerative disorders such as Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette' s syndrome, multiple sclerosis, amylotropic lateral sclerosis (ALS), progressive supranuclear palsy, epilepsy, and Creutzfeldt- Jakob disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, Korsakoff s psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthy
  • subject includes subjects capable of suffering from a glucocorticoid associated states, such as mammals. Examples of mammals include dogs, cats, bears, rabbits, mice, rats, goats, cows, sheep, horses, and, preferably, humans.
  • the subject may be suffering from or at risk of suffering from a glucocorticoid associated state, e.g., a blood pressure associated disorder (e.g., hypertension), obesity, diabetes, or a neurological disorder.
  • a blood pressure associated disorder e.g., hypertension
  • treat or “treating” includes the prevention, alleviation or reduction of at least one symptom or other indication of a particular glucocorticoid associated state.
  • the associated state is a blood pressure associated disorder, e.g., hypertension, and the administration of the modulating compound modulates, e.g., reduces, the blood pressure of the subject.
  • a blood pressure associated disorder e.g., hypertension
  • the administration of the modulating compound modulates, e.g., reduces, the blood pressure of the subject.
  • the term "effective amount" of the 1 l ⁇ -HSDl reductase, 1 l ⁇ -HSDl dehydrogenase, or 1 l ⁇ -HSD2 dehydrogenase modulating compound is that amount necessary or sufficient to treat or prevent a particular glucocorticoid associated state, e.g. prevent the various morpho logical and somatic symptoms of a glucocorticoid associated state.
  • the effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular 1 l ⁇ -HSDl reductase, 1 l ⁇ -HSDl dehydrogenase, or 1 l ⁇ -HSD2 dehydrogenase modulating compound, e.g., inhibiting, compound.
  • 11 ⁇ -HSD2 dehydrogenase modulating compound may be administered in combination with a pharmaceutically acceptable carrier.
  • the invention pertains to a method for treating a blood pressure associated disorder, e.g., hypertension, in a subject, by administering to the subject an effective amount of an 11 l ⁇ -HSDl reductase, 1 l ⁇ -HSDl dehydrogenase, or
  • the invention features a method for decreasing the concentration (or amount) of glucocorticoids in a tissue of a subject.
  • the method includes administering an effective amount of a selective 1 l ⁇ -HSDl reductase inhibitor, such that the concentration of glucocorticoids in the tissue are decreased.
  • the 1 l ⁇ -HSDl reductase inhibitor is a small molecule, e.g., a steroid or a derivative thereof.
  • tissues where the concentration of glucocorticoids in a subject may be decreased include tissues which express 1 l ⁇ -HSDl or other wise contain an undesirable concentration of glucocorticoids.
  • tissues include a subject's blood, liver, eye, lung, muscle, adipose tissue, nerve tissue, brain, or vascular tissue.
  • the invention features a method for treating a blood pressure associated disorder, such as, for example, hypertension, in a subject. The method includes administering to a subject an effective amount of a 1 l ⁇ -HSDl reductase inihibitor, such that the subject is treated.
  • the 1 1 ⁇ - HSDl reductase inihibitor is a selective inhibitor.
  • the reductase inihibitor is a small molecule, e.g., a steroid or a derivative thereof.
  • the invention features a method for increasing insulin sensitivity of a tissue in a subject.
  • the method includes administering to a subject an effective amount of a selective 1 l ⁇ -HSDl reductase inhibitor, such that the insulin sensitivity of the tissue in the subject is increased.
  • tissue where increased insulin sensitivity may be desirable include, for example, the subject's liver, muscle, nerve or adipose tissue.
  • the invention features a method for increasing the concentration of glucocorticoids in a tissue of a subject.
  • the method includes administering to a subject an effective amount of a selective 1 l ⁇ -HSDl dehydrogenase inhibitor, such that the concentration of glucocorticoids in the tissue are increased.
  • the tissue may be any tissue which an increase in the concentration of glucocorticosteroids is desired.
  • tissue include, but are not limited to, subject's liver, blood, lung, eye, muscle, adipose tissue, nerve tissue, brain, and vascular tissue.
  • the invention features a method for increasing the concentration of glucocorticoids in a tissue of a subject.
  • the method includes administering to a subject an effective amount of a selective 1 l ⁇ -HSD2 dehydrogenase inhibitor, such that the concentration of glucocorticoids in the tissue are increased.
  • the tissue may be any tissue which an increase in the concentration of glucocorticoids is desired. Examples of such tissues include, but are not limited to, subject's liver, eye, blood, lung, muscle, adipose tissue, nerve tissue, brain, and vascular tissue.
  • the invention also includes a method for selectively inhibiting 1 l ⁇ -HSDl reductase.
  • the method includes contacting 1 l ⁇ -HSDl reductase with a selective 1 l ⁇ - HSDl reductase inhibitor.
  • the invention includes a method for selectively inhibiting 1 l ⁇ -HSDl dehydrogenase.
  • the method includes contacting 1 l ⁇ -HSDl dehydrogenase with a selective 1 l ⁇ -HSDl dehydrogenase inhibitor.
  • the invention features a method for selectively inhibiting
  • 11 ⁇ -HSD2 dehydrogenase The method includes contacting 11 ⁇ -HSD2 dehydrogenase with a selective 11 ⁇ -HSD2 dehydrogenase inhibitor.
  • 1 l ⁇ -HSDl reductase modulating compound include compounds and agents (e.g., oligomers, proteins, etc.) which modulate or inhibit the activity of 11 ⁇ - HSDl reductase.
  • the l l ⁇ -HSDl reductase modulating compound is an 1 l ⁇ -HSDl reductase inhibitor (also referred to as "1 l ⁇ -HSDl reductase inhibiting compound”).
  • the 1 l ⁇ -HSDl reductase modulating compound may be a small molecule, e.g., a compound with a molecular weight below 10,000 daltons.
  • the 1 l ⁇ -HSDl reductase modulating compound is a selective inhibitor of 1 l ⁇ -HSDl reductase.
  • selective 1 l ⁇ -HSDl reductase inhibitor includes compounds which selectively inhibit the reductase activity of 11 ⁇ - HSD1 as compared to the dehydrogenase activity.
  • the reductase activity is inhibited at a rate about 2 times or greater, about 3 times or greater, about 4 times or greater, about 5 times or greater, about 10 times or greater, about 15 times or greater, about 20 times or greater, about 25 times or greater, about 50 times or greater, about 75 times or greater, about 100 times or greater, about 150 times or greater, about 200 times or greater, about 300 times or greater, about 400 times or greater, about 500 times or greater, about 1 x 10 3 times or greater, about 1 x 10 4 times or greater, about 1 x 10 5 times or greater, or about 1 x 10 or greater as compared with the inhibition of the dehydrogenase activity of 11 ⁇ -HSD 1.
  • the 1 1 ⁇ -HSD 1 reductase modulating compound may be a steroid or a steroid derivative.
  • the steroid ring system is generally numbered according to IUPAC conventions, as shown below:
  • Examples of 11 ⁇ -HSD 1 reductase modulating compounds include 11-keto steroid compounds, e.g., compounds with the steroid ring system with a carbonyl functional group at the 11 -position of the steroid ring.
  • Examples of steroid compounds with an 11-keto group include, for example, 11-keto progesterone, 11 -keto-testosterone, 11 -keto-androsterone, 11-keto-pregnenolone, 11 -keto-dehydro-epiandrostenedione, 3 ⁇ , 5 ⁇ -reduced-l 1-ketoprogesterone, 3 ⁇ , 5 ⁇ -reduced-l 1-keto-testosterone, 3 ⁇ , 5 ⁇ -reduced- 11-keto-androstenedione, 3 ⁇ ,5 ⁇ -tetrahydro-l l -dehydro-corticosterone, 3 ⁇ , 5 ⁇ -reduced- 11-keto-pregnenolone, and 3 ⁇ , 5 ⁇ -reduced-l 1-keto-dehydro-epiandrostenedione.
  • 1 l ⁇ -HSDl reductase modulating compounds of the invention are compounds which conserve a least a portion of the steroid nucleus. These compounds may have additional substiuents, such as fatty acid tails at the 22 position, or other modifications (e.g., substitutions of the ring by halogens, formation of esters or other protecting groups for the hydroxyl groups of the steroids, or replacement of functional groups with others that may, for example, advantageously, lengthen the time the molecule is in its active form in a subjects body. Alternatively, the modifications can be such that the reduce the time the compound is in its active form in a subject's body.
  • Examples of 1 l ⁇ -HSDl reductase modulating compounds also include 3 ⁇ , 5 ⁇ - reduced steroid compounds.
  • Examples of 3 ⁇ , 5 ⁇ -reduced steroid compounds include 3 ⁇ , 5 ⁇ -reduced-l 1-ketoprogesterone, 3 ⁇ , 5 ⁇ -reduced-l 1-keto-testosterone, 3 ⁇ , 5 ⁇ - reduced-11-keto-androstenedione, 3 ⁇ ,5 ⁇ -tetrahydro- 11 -dehydro-corticosterone, 3 ⁇ , 5 ⁇ - reduced-11-keto-pregnenolone, and 3 ⁇ , 5 ⁇ -reduced-l l-keto-dehydro- epiandrostenedione.
  • Steroid derivatives include compounds with a steroid ring structure optionally substituted with additional substituents which allow the compound to perform its intended function. It should be noted that the steroid compounds may be converted to the active form of the modulating compound within the subject.
  • the invention includes administering compounds which are in other forms, e.g., prodrugs, and which are metabolized in vivo to yield the 1 l ⁇ -HSDl reductase modulating compounds described herein.
  • the 1 l ⁇ -HSDl reductase inhibitors possess IC 50 's less than about 0.5 ⁇ M using 600 nanoM 11 -dehydro-corticosterone substrate concentration and testicular leydig cell homogenates. Methods for testing the IC 50 's of the enzymes are described in further detail in Latif, S. A. et al. Steroids 62: 230-237, 1997.
  • the 11 ⁇ -HSD 1 reductase inhibitors have an IC 50 of 80 ⁇ M or less, or, preferrably, 15 ⁇ M or less.
  • the 1 l ⁇ -HSDl reductase inhibitors have an ICso of less than 100 ⁇ M.
  • 1 l ⁇ -HSDl reductase modulating compounds include carbenoxolone and derivatives thereof.
  • 1 l ⁇ -HSDl reductase modulating compound is a nucleic acid.
  • the 1 l ⁇ -HSDl reductase inhibitor is an antisense nucleic acid.
  • the 1 l ⁇ -HSDl reductase inhibitor is a siRNA.
  • RNA interference can be understood as a two step process (Zamore P.D., Nature Struc. Biol, 8, 9, 746-750, (2001)).
  • the dsR ⁇ A is cleaved to yield short interfering R ⁇ As (siR ⁇ As) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt).
  • the siR ⁇ As target the corresponding mR ⁇ A sequence specific for destruction (Fire A. et al, Nature, Vol 391, (1998); Hamilton AJ et al Science, 286, 950-952, (1999); Zamore PD. et al. Cell, 101, 25-33, (2000); Elbashir SM. et al, Genes & Development, 15, 188-200, (2001); Bernstein E. et al Nature 409, 363-366, 2001).
  • 21 nt siRNA duplexes specifically suppress expression of endogenous and heterologeous genes in different mammalian cell lines, including human kidney and HeLa cells (Elbashir SM. et ah, Nature, 411, 494-498, (2001)). It was discovered that no unspecific effects occurred in mammalian cells by transfection of short sequences ( ⁇ 30nt). It was suggested that 21 nt siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
  • siRNAs mediated RNAi in cell extracts and synthetic siRNAs can induce gene-specific inhibition of expression in C. elegans and in cell lines from humans and mice (Caplen, N.J. et al. PNAS 171251798, 1-6, (2001)30). It was also shown that siRNAs can have direct effects on gene expression in C. elegans and mammalian cell culture in vivo.
  • siRNAs Methods for making and using siRNAs are described in, for example, WO 01/75164, US 2002/0137210, WO 01/29058, WO 02/072762, WO 02/059300, WO 02/44321, WO 01/92513, WO 01/68836, US 2002/0173478, US 2002/0160393, US 2002/0162126, US 2002/0137709, US 2002/0132788, US 2002/0086356, and WO 99/32619; each of which is expressly incorporated herein by reference.
  • the 1 l ⁇ -HSDl reductase inhibitor is a double stranded RNA oligomer, wherein the antisense strand is complementary to at least a portion of SEQ. ID. No. 1.
  • the portion is 40 base pairs or less, 35 base pairs or less, 30 base pairs or less, 29 base pairs or less, 28 base pairs or less, 27 base pairs or less, 26 base pairs or less, 25 base pairs or less, 24 base pairs or less, 23 base pairs or less, 22 base pairs or less, 21 base pairs or less, 20 base pairs or less, 19 base pairs or less, or about 18 base pairs or less.
  • the oligomer has 10 or more base pairs, 11 or more base pairs, 12 or more base pairs, 13 or more base pairs, 14 base pairs or more, 15 base pairs or more, 16 base pairs or more, 17 base pairs or more, 18 base pairs or more, or 19 base pairs or more.
  • the 1 l ⁇ -HSDl reductase inhibitor has an antisense strand having the sequence 5'-CAT AAC TGC CGT CCA ACA GC-3' (SEQ ID NO. 2).
  • the term "11 ⁇ -HSD 1 dehydrogenase modulating compound” include compounds and agents (e.g., oligomers, proteins, etc.) which modulate or inhibit the activity of 1 l ⁇ -HSDl dehydrogenase.
  • the 1 l ⁇ -HSDl dehydrogenase modulating compound is an 1 l ⁇ -HSDl dehydrogenase inhibitor (also referred to as "1 l ⁇ -HSDl dehydrogenase inhibiting compound”).
  • the 1 l ⁇ -HSDl dehydrogenase modulating compound may be a small molecule, e.g., a compound with a molecular weight below 10,000 daltons.
  • the 1 l ⁇ -HSDl dehydrogenase modulating compound is a selective inhibitor of 1 l ⁇ -HSDl dehydrogenase.
  • selective 1 l ⁇ -HSDl dehydrogenase inhibitor includes compounds which selectively inhibit the dehydrogenase activity of 11 ⁇ -HSD 1 as compared to the reductase activity of 11 ⁇ - HSD1.
  • the dehydrogenase activity is inhibited at a rate about 2 times or greater, about 3 times or greater, about 4 times or greater, about 5 times or greater, about 10 times or greater, about 15 times or greater, about 20 times or greater, about 25 times or greater, about 50 times or greater, about 75 times or greater, about 100 times or greater, about 150 times or greater, about 200 times or greater, about 300 times or greater, about 400 times or greater, about 500 times or greater, about 1 x 10 times or greater, about 1 x 10 4 times or greater, about 1 x 10 5 times or greater, or about 1 x 10 6 or greater as compared with the inhibition of the reductase activity of 1 l ⁇ -HSDl .
  • the 1 l ⁇ -HSDl dehydrogenase inhibitor is a small molecule, such as a steroid or a derivative thereof.
  • the steroid is 3 ⁇ , 5 ⁇ - reduced.
  • 3 ⁇ ,5 ⁇ -reduced steroids include 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ -OH- progesterone, 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ -OH-testosterone, chenodeoxycholic acid, 3 ⁇ , 5 ⁇ - reduced-pregnenolone, 3 ⁇ , 5 ⁇ -reduced-dehydro-epiandrostenedione, 3 ⁇ , 5 ⁇ -reduced- progesterone, 3 ⁇ , 5 ⁇ -testosterone, and deoxy-corticosterone.
  • the 1 l ⁇ -HSDl dehydrogenase inhibitor is a 3 ⁇ , 5 ⁇ - reduced steroid.
  • steroids include 3 ⁇ , 5 ⁇ -reduced-l 1 ⁇ -OH- progesterone, 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ -OH-testosterone, 3 ⁇ , 5 ⁇ -reduced-l l ⁇ -OH- androstendione, 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ -OH-pregnenolone, 3 ⁇ , 5 ⁇ -reduced-l l ⁇ -OH- dehydro-epiandrostenedione, 3 ⁇ , 5 ⁇ -reduced-corticosterone, 3 ⁇ , 5 ⁇ -reduced- aldosterone, 3 ⁇ , 5 ⁇ -reduced-pregnenolone, 3 ⁇ , 5 ⁇ -reduced-progesterone, 3 ⁇ , 5 ⁇ -reduced testosterone, and 3 ⁇ , 5 ⁇ -reduced-chenodeoxycholic acid.
  • the 1 l ⁇ -HSDl dehydrogenase inhibitor has an IC 50 of 0.5 ⁇ M or less. In another embodiment, the 1 l ⁇ -HSDl dehydrogenase inhibitor has an IC 50 of 100 ⁇ M or less, 80 ⁇ M or less, or 20 ⁇ M or less (using using 100 nM corticosterone substrate concentration and testicular Leydig cell homogenates).
  • the term "1 l ⁇ -HSD2 dehydrogenase inhibitor” includes agents which inhibit or decrease the dehydrogenase activity of 1 l ⁇ -HSD2.
  • the 11 ⁇ -HSD2 dehydrogenase inhibitor is a small molecule, such as a steroid or a derivative thereof. In one embodiment, the steroid is 3 ⁇ , 5 ⁇ - reduced.
  • Examples of 11 ⁇ -HSD2 dehydrogenase inhibitors include, but are not limited to, 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ -OH-progesterone, 3 ⁇ , 5 ⁇ -reduced-l l ⁇ -OH-testosterone, 3 ⁇ , 5 ⁇ - reduced-l l ⁇ -OH-androstenedione, 3 ⁇ , 5 ⁇ -reduced- 11 -keto-progesterone, 3 ⁇ , 5 ⁇ - reduced- 11 -dehydro-corticosterone, 3 ⁇ , 5 ⁇ -reduced-corticosterone, 3 ⁇ , 5 ⁇ -reduced- 11 ⁇ - OH-pregnenolone, 3 ⁇ , 5 ⁇ -reduced-l l ⁇ -OH-dehydro-epiand
  • 11 ⁇ -HSD2 dehydrogenase inhibitors include 11 ⁇ -OH- progesterone, 11 ⁇ -OH-pregnenolone, 11 ⁇ -OH-dehydro-epiandrostenedione, 11 ⁇ -OH- testosterone, 11 -keto-progesterone, 5 ⁇ -dihydro-corticosterone, and 3 ⁇ , 5 ⁇ -reduced deoxy- corticosterone.
  • 11 ⁇ -HSD2 dehydrogenase modulating compound is a nucleic acid.
  • the 1 l ⁇ -HSD2 dehydrogenase inhibitor is an antisense nucleic acid.
  • the 11 ⁇ -HSD2 dehydrogenase inhibitor is a siRNA.
  • the 11 ⁇ -HSD2 dehydrogenase inhibiting compounds have IC JO 'S less than 2.5 ⁇ M (using 50 nM coricosterone substrate concentration and sheep kidney microsomes). In another embodiment, the 11 ⁇ -HSD2 dehydrogenase inactive compounds have an IC 50 of less than lO ⁇ M.
  • the 11 ⁇ -HSD2 dehydrogenase inhibitor is a double stranded RNA oligomer, wherein the antisense strand is complementary to at least a portion of SEQ. ID. No. 3.
  • the portion is 40 base pairs or less, 35 base pairs or less, 30 base pairs or less, 29 base pairs or less, 28 base pairs or less, 27 base pairs or less, 26 base pairs or less, 25 base pairs or less, 24 base pairs or less, 23 base pairs or less, 22 base pairs or less, 21 base pairs or less, 20 base pairs or less, 19 base pairs or less, or about 18 base pairs or less.
  • the oligomer has 10 or more base pairs, 11 or more base pairs, 12 or more base pairs, 13 or more base pairs, 14 base pairs or more, 15 base pairs or more, 16 base pairs or more, 17 base pairs or more, 18 base pairs or more, or 19 base pairs or more.
  • the 1 l ⁇ -HSD2 dehydrogenase inhibitor has an antisense strand having the sequence 5'-AGG CCA GCG CTC CAT GAC TT- 3' (SEQ ID NO 4).
  • the invention also pertains to each of the nucleic acids described herein as well as pharmaceutical compositions comprising these nucleic acids.
  • the invention also pertains to administering to the subject a 17 ⁇ -hydroxylase inhibitor, a 17-HSD inhibitor, a 20 ⁇ -reductase inhibitor and/or a 20 ⁇ -reductase inhibitor, in combination with the methods described above.
  • the inhibitors can be any compound or substance known to inhibit any one of these enzymes.
  • the 17 ⁇ -hydroxylase, 17- HSD, 20 ⁇ -reductase and or 20 ⁇ -reductase inhibitors are administered in combination with the compounds of the invention described herein.
  • the language "in combination with” another agent includes co-administration of the compound of the invention and the agent, administration of the compound of the invention first, followed by the other agent and administration of the other agent first, followed by the compound of the invention.
  • the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase inhibitors can be found using assays for screening candidate or test compounds which bind to or modulate the activity of a 17 ⁇ -hydroxylase, 17-HSD, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein or polypeptide or biologically active portion thereof.
  • the sequences for 17 ⁇ - hydroxylase is shown in SEQ ID No. 5.
  • the sequence for the 17-HSD is shown in SEQ ID No. 6.
  • the polypeptide sequence for 17-HSD is shown in SEQ ID. No. 7.
  • the 17 ⁇ -hydroxylase inhibitor, the 17-HSD inhibitor, the 20 ⁇ -reductase, and/or the 20 ⁇ reductase inhibitors are nucleic acid oligomers.
  • the nucleic acid oligomers are siRNA and comprise at least a portion of SEQ ID No. 5 or SEQ ID No. 6.
  • the test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145).
  • Determining the ability of a 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ - reductase protein to bind to or interact with a target molecule can be accomplished by determining direct binding.
  • Determining the ability of the 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to bind to or interact with a target molecule can be accomplished, for example, by coupling the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein with a radioisotope or enzymatic label such that binding of the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to a target molecule can be determined by detecting the labeled 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein in a complex.
  • 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase proteins can be labeled with 125 ⁇ 35g s 14 ⁇ 0 r - ⁇ H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase proteins can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • an assay of the present invention is a cell-free assay in which a 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ - reductase protein or biologically active portion thereof is determined. Binding of the test compound to the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein can be determined either directly or indirectly.
  • the assay may include contacting the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein or biologically active portion thereof with a known compound which binds 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ - reductase or 20 ⁇ -reductase to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein, wherein determining the ability of the test compound to interact with a 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein comprises determining the ability of the test compound to preferentially bind to 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ - reductase or 20 ⁇ -reductase protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of al7 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein can be accomplished, for example, by determining the ability of the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to bind to a target molecule. Determining the ability of the 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to bind to a target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal Chem.
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • determining the ability of the test compound to modulate the activity of a 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein can be accomplished by determining the ability of the 17 ⁇ -hydroxylase, 17- HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to further modulate the activity of a target molecule.
  • the cell-free assay involves, contacting a 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein or biologically active portion thereof with a known compound which binds the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein, wherein determining the ability of the test compound to interact with the 17 ⁇ - hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein comprises determining the ability of the 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein to preferentially bind to or modulate the activity of a target molecule.
  • binding of a test compound to a 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein, or interaction of a 17 ⁇ -hydroxylase, 17-HSD, 20 ⁇ -reductase or 20 ⁇ -reductase protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • the invention pertains to the 17 ⁇ -hydroxylase, 17-HSD, the 20 ⁇ -reductase, and the 20 ⁇ -reductase inhibiting compounds which are found using the above described methods.
  • the invention pertains to a pharmaceutical composition for the treatment of a glucocorticoid associated state.
  • the composition includes an effective amount of an 1 l ⁇ -HSDl reductase, 1 l ⁇ -HSDl dehydrogenase, or 1 l ⁇ -HSD2 dehydrogenase modulating, e.g., inhibiting, compound and a pharmaceutically acceptable carrier.
  • the glucocorticoid associated state is a blood pressure disorder.
  • the pharmaceutical compositions may also comprise an inhibitor of 17 ⁇ -hydroxylase, 17- HSD, 20 ⁇ -reductase or 20 ⁇ -reductase.
  • the invention pertains, at least in part, to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of 11 ⁇ -OH-progesterone, 11 ⁇ -OH-testosterone, 3 ⁇ ,5 ⁇ -reduced- 11 ⁇ -OH-progesterone, 3 ,5 ⁇ -reduced-l l ⁇ -OH- testosterone, chenodeoxycholic acid, 3 ⁇ , 5 ⁇ -reduced-pregnenolone, 3 ⁇ , 5 ⁇ -reduced- dehydro-epiandrostenedione, 3 ⁇ ,5 ⁇ -reduced-l 1 ⁇ -OH-progesterone, 3 ⁇ ,5 ⁇ -reduced- 1 1 ⁇ -OH-testosterone, 3 ⁇ ,5 -reduced-l l ⁇ -OH-androstenedione, 11 -keto-progesterone, 11 -keto-testosterone, 11 -keto-androstenedione, 3 ⁇ ,5 ⁇ -reduced-l
  • phrases "pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal, pulmonary and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, poly vinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders,
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emuls
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Sprays also can be delivered by mechanical, electrical, or by other methods known in the art.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial, antiparasitic and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle. The compositions also may be formulated such that its elimination is retarded by methods known in the art.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route.
  • they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration or administration via inhalation is preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • suitable routes of administration including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • Other methods for administration include via inhalation.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a glucocorticoid associated state.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a compound of the present invention While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
  • certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids.
  • pharmaceutically acceptable salts is art recognized and includes relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J Farm. SCI. 66:1-19).
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts in these instances includes relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • prodrug includes compounds with moieties which can be metabolized in vivo to a hydroxyl group or other functional group and moieties which may advantageously remain in vivo.
  • the prodrugs moieties are metabolized in vivo.
  • Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
  • the prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid.
  • prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alky 1 esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alky 1 amides, di- lower alkyl amides, and hydroxy amides.
  • the invention also
  • Each well contained 1 mL of DMEM/F12 containing 1% fetal bovine serum, streptomycin (100 ⁇ g/ml), penicillin (100 units/ml) and amphotericin (0.25 ⁇ g/ml). Aortic rings were incubated for 24 hours prior to contractility measurements with the following combinations of steroids, and anti sense/nonsense oligonucleotides (3 ⁇ mol/L):
  • Antisense phosphorothioate oligonucleotides targeted to block either 11 ⁇ -HSD2 or 1 l ⁇ -HSDl gene expression, were obtained from Research Genetics, Huntsville AL. Antisense oligomers complementary to 20 bp sequences spanning the ribosome binding/translation start site were used. Oligomer sequences were: 5'-CAT AAC TGC CGT CCA ACA GC-3' (SEQ ID NO. 2) for 1 l ⁇ -HSDl Antisense and 5'-AGG CCA GCG CTC CAT GAC TT- 3' (SEQ ID NO 4) for 1 l ⁇ -HSD2 antisense. In control experiments the corresponding sense sequence was used as the nonsense oligomer.
  • Antisense and nonsense oligomers were added directly to each well at 20 ⁇ g/10: 1 sterile H 2 0 per well for a final concentration of 3 ⁇ mol/L.
  • aortic rings were suspended by tungsten wires with 1 g of tension and placed in a vessel bath containing serum free DMEM/F12 media at 37°C aerated with 95% O 2 -5% CO, at pH 7.4. Vessels were equilibrated for 20 minutes and then tested with phenylephrine (1 nmol/L - lO ⁇ mol/L).
  • phenylephrine is structurally not a catecholamine, it is considered to be a functional catecholamine as it activates both ⁇ and ⁇ adrenoceptors. Due to its favorable stability characteristics, it is widely used as a catecholamine substitute in experiments of this nature.
  • the intensity of contraction was assessed by use of a Narishige micromanipulator and model FT03 force transducer (Grass Instrument Co. West Warwick, Rl). Measurements were recorded on computer using the Labview 4.1 Virtual Instrument System (National Instruments, Austin, TX). Adhering to this protocol, test vessel viability by demonstrating the ability of the vessel to vigorously contract when exposed to known vasoconstrictors and relax back to baseline after treatment with acetylcholine.
  • both vascular endothelial and smooth muscle cells contain 11 ⁇ -HSD 1.
  • 11 ⁇ -HSD 1 antisense oligomers had an effect on the ability of corticosterone to amplify the contractile responses to phenylephrine in vascular tissue. Rings were incubated for 24-hours with corticosterone (10 nmol/L) and either 1 l ⁇ - HSDl antisense oligomers (3 ⁇ mol/L) or nonsense oligomers (3 ⁇ mol/L).
  • 1 l ⁇ -HSDl antisense oligomers attenuated the ability of 11 ⁇ -dehydro-corticosterone to amplify the contractile response to all concentrations of phenylephrine compared to 11- dehydro-corticosterone plus 1 l ⁇ -HSDl nonsense oligomers.
  • Statistically significant decreases were observed at 100 nmol/L and 1 ⁇ mol/L phenylephrine (Fig 3). In aortic rings incubated (24-hours) with corticosterone (10 nmol/L) and 11 ⁇ -
  • HSD2 antisense (3 ⁇ mol/L)
  • the contractile response to graded concentrations of phenylephrine (PE: 10 nmol/L - 1 ⁇ mol/L) were significantly (P ⁇ 0.05) increased compared to rings incubated with corticosterone and 1 1 ⁇ -HSD2 nonsense.
  • 11 ⁇ -HSD 1 antisense oligomers also enhanced the ability of corticosterone to amplify the contractile response to phenylephrine.
  • 1 l ⁇ -HSDl acts predominantly as a reductase in vascular tissue
  • 1 l ⁇ -HSDl antisense oligomers also enhanced the ability of corticosterone to amplify the contractile effects of phenylephrine in rat aortic rings.
  • This observation suggests that 1 l ⁇ -HSDl -dehydrogenase, in addition to 1 l ⁇ -HSD2, also operates to protect GR and MR from over-activation by glucocorticoids in vascular tissue.
  • Further experiments to determine whether antisense oligomers down-regulate mRNA and protein expression of their respective 11 ⁇ -HSD isoform under conditions in which they enhance contractile responses in aortic rings will be done.
  • the example confirms that 11 -dehydro-corticosterone also amplifies the contractile actions of catecholamines in rat aortic rings. Since 11-dehydro- glucocorticoids do not bind to GR (or MR) to any major extent, it is proposed that 11- dehydro-corticosterone is metabolized back to corticosterone by 1 l ⁇ -HSDl -reductase in vascular smooth muscle and/or endothelial cells.
  • the examples demonstrate that 1 l ⁇ -HSDl antisense oligomer also attenuates the ability of 11 -dehydro-corticosterone to amplify the contractile responses of phenylephrine indicating that the down-regulation of 1 l ⁇ - HSDl gene expression can affect the regeneration of active glucocorticoid (from 1 1 - dehydro-glucocorticoid) in vascular tissue.
  • the examples show that 1 l ⁇ -HSDl antisense can significantly reduce the metabolism of 11 -dehydro-corticosterone back to corticosterone in aortic ring preparations.
  • Incubation media was collected, ran through a Sep-Pak and eluted with 3 mis of methanol, the eluate was then dried under nitrogen and reconstituted in 500:1 methanol.
  • the aortic rings were dried and weighed.
  • the steroids present in the eluate were separated by high-pressure liquid chromatography with a Dupont Zorbax C8 column eluted at 44°C at a flow rate of 1 mL/min using 55% methanol for 10 minutes.
  • Steroids were observed by monitoring radioactivity on-line with a Packard Radiomatic Flo- One/Beta Series A-500 counter connected to a Dell Optiflex 425 S/L computer. Corticosterone and 11 -dehydro-corticosterone were identified by comparing their retention times with that of known standards.
  • Corticosterone and phenylephrine were obtained from Sigma (St Louis, MO), 11 -dehydrocorticosterone from Research Plus (Bayonne, NJ) and H-steroids from New England Nuclear (Boston, MA). Where appropriate, data were expressed as mean ⁇ SE and analyzed using ANOVA and the Student's t test with Bonferroni modification. P values of less than 0.05 are considered significant.
  • the amount of radioactivity in the tissue was approximately 2-3% of the total radioactivity in the incubation media.
  • the production of 3 H-corticosterone in aortic rings incubated with 11 ⁇ -HSD 1 antisense was again markedly lower that that in rings incubated with 1 l ⁇ -HSDl nonsense oligomers (see HPLC chromatograms, Figures 5 A - 5D).
  • the levels of H-l 1 -dehydrocorticosterone metabolism measured in the incubate and in the aortic tissue were very similar ( Figures 5A-5D). This indicates that measuring steroid content in the media does not under-represent the level of steroid metabolism in the tissue compartment.
  • glucocorticoids have been reported to not only amplify the contractile effects of catecholamines in vascular tissue but to also diminish the effects of certain vasorelaxation pathways (glucocorticoids decrease nitric oxide and prostaglandin I 2 synthesis); such actions would serve to further enhance the effects of glucocorticoids on increasing catecholamine-induced vasoconstriction and may explain how small changes in glucocorticoid levels can have profound effects on vascular tone.
  • 1 l ⁇ -HSD2 and 1 l ⁇ -HSDl antisense also decreased the metabolism of corticosterone to 11 -dehydro-corticosterone.
  • 11 -dehydro-corticosterone (100 nmol/L) also amplified the contractile response to phenylephrine in aortic rings (P ⁇ 0.01), most likely due to the generation of active corticosterone by 1 l ⁇ -HSDl - reductase; this effect was significantly attenuated by 1 l ⁇ -HSDl antisense.
  • 1 l ⁇ -HSDl antisense also caused a marked decrease in the metabolism of 11 -dehydro-corticosterone back to corticosterone by 1 l ⁇ -HSDl- reductase.
  • the examples demonstrate that both 1 l ⁇ -HSD2 and 1 l ⁇ -HSDl regulate local glucocorticoid concentrations in vascular tissue with 1 l ⁇ -HSD2 and 1 l ⁇ -HSDl - dehydrogenase working to decrease- and 1 l ⁇ -HSDl -reductase increase the amount of glucocorticoid that can access GR and MR in vascular smooth muscle.
  • Physiological concentrations of both free corticosterone and 11 -dehydrocorticosterone are similar over the course of the day in rodents.
  • glucocorticoids amplify catecholamine and angiotensin II pressor responses and may inhibit the effects of some vasorelaxant pathways, a possible mechanism that may increase vascular tone and induce hypertension includes a decrease in 1 l ⁇ -HSD2 activity.
  • glucocorticoids amplify catecholamine and angiotensin II pressor responses and may inhibit the effects of some vasorelaxant pathways
  • a possible mechanism that may increase vascular tone and induce hypertension includes a decrease in 1 l ⁇ -HSD2 activity.
  • many patients with essential hypertension also demonstrate decreased " 11 ⁇ -HSD2 activity as assessed by altered plasma and urinary cortisol/cortisone ratios.

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Abstract

Methods for treating glucocorticoid associated states using selective 11β-HSD1 -dehydrogenase, 11β-HSD1 -reductase and 11β-HSD2 dehydrogenase modulating compounds are described.

Description

SELECTIVE llβ-HSD INHIBITORS AND METHODS OF USE THEREOF
Related Applications:
This application claims priority to U.S. Provisional Patent Application Serial No. 60/342,693, entitled "11 β-HSD 1-Reductase Inhibiting Compounds and Methods of Use Thereof," filed December 21, 2001, the entire contents of which are hereby incorporated herein by reference.
Background: Glucocorticoids are steroid hormones. One example of a common glucocorticoid is cortisol. Modulation of glucocorticoid activity is important in regulating physiological processes in a wide range of tissues and organs. High levels of glucocorticoids may result in excessive salt and water retention by the kidneys, which may lead high blood pressure. Glucocorticoids play an important role in the regulation of vascular tone and blood pressure. Glucocorticoids can bind to and activate the glucocorticoid receptor (GR) and, possibly, the mineralocorticoid receptor (MR) to potentiate the vasoconstrictive effects of both catecholamines and angiotensin II (Ang II). Tissue glucocorticoid levels are regulated by two isoforms of the enzyme 11 β-hydroxysteroid dehydrogenase (l l β-HSD). 11 β-HSD converts glucocorticoids into 11 -keto metabolites that are unable to bind to mineralocorticoid receptors (Edwards C R et al. (1988) Lancet 2:986-9; Funder et al, (1988) Science 242, 583,585).
Summary of the Invention: In an embodiment, the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject. The method includes administering to the subject an effective amount of a 1 lβ-HSDl reductase inhibitor, e.g., 11-keto- testoterone, 11-keto-androsterone, 11-keto-pregnenolone, 11 -keto-dehydro- epiandrostenedione, 3α, 5α-reduced-l l-ketoprogesterone, 3α, 5α-reduced-l l-keto- testosterone, 3α, 5α-reduced-l 1 -keto-androstenedione, or 3α,5α-tetrahydro-l 1 β- dehydro-corticosterone.
In another embodiment, the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject, by administering to said subject an effective amount of a 1 lβ-HSDl reductase inhibitor, wherein the inhibitor is a nucleic acid In yet another embodiment, the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject, by administering to the subject an effective amount of a 1 lβ-HSDl reductase inhibitor in combination with an effective amount of a 17α-hydroxylase inhibitor, a 17-HSD inhibitor, 20α-reductase inhibitor, or a 20β-reductase inhibitor.
In another embodiment, the invention pertains, at least in part, to a method for increasing the concentration of glucocorticoids in a tissue of a subject. The method includes administering to the subject an effective amount of a 1 lβ-HSDl dehydrogenase inhibitor, such as, for example, 3α, 5β-reduced-l 1 β-OH-testosterone, 3α, 5α-reduced- 11 β-OH-progesterone, 3α, 5α-reduced-l 1 β-OH-testosterone, 3α, 5α-reduced-l lβ-OH- androstendione, 3α, 5α-reduced-corticosterone, 3α, 5α-reduced-aldosterone, 3α, 5α- reduced-progesterone, 3α, 5α-reduced testosterone, 3α, 5α-reduced-chenodeoxycholic acid, or 11 β-OH testosterone. In another embodiment, the 11 β-dehydrogenase inhibitor is a nucleic acid. In yet another embodiment, the invention pertains, at least in part, to a method for increasing the concentration of glucocorticoids in a tissue of a subject. The method includes administering to the subject an effective amount of a 1 lβ-HSDl dehydrogenase inhibitor in combination with an effective amount of a 17α-hydroxylase inhibitor, a 17- HSD inhibitor, a 20α-reductase inhibitor or a 20β-reductase inhibitor. In yet another embodiment, the invention pertains, at least in part, to a method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 lβ-HSDl dehydrogenase inhibitor, such that the concentration of glucocorticoids in said tissue are increased, wherein said
I lβ-HSDl dehydrogenase inhibitor is 3α, 5α-reduced-l 1 β-OH-progesterone, 3α, 5α- reduced- 11 β-OH-testosterone, 3α, 5α-reduced-l 1 β-OH-androstendione, 3α, 5α-reduced-
I I β-OH-pregnenolone, 3α, 5α-reduced-l lβ-OH-dehydro-epiandrostenedione, 3α, 5α- reduced-corticosterone, 3α, 5α-reduced-aldosterone, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-dehydro-epiandrostenedione, 11 β-OH progesterone, 1 1 β-OH testosterone, 11 β-OH-pregnenolone, 11 β-OH-dehydro-epiandrostenedione, 3α, 5α-reduced- progesterone, 3α, 5α-reduced testosterone, 3α, 5α-reduced-chenodeoxycholic acid, or a pharmaceutically acceptable prodrug or salt thereof. In another embodiment, the 11 β- HSD1 dehydrogenase inhibitor is a nucleic acid.
In yet another embodiment, the invention also pertains, at least in part, to a method for treating hypertension in a subject, by administering to the subject an effective amount of a 1 lβ-HSDl reductase inhibitor, such as, for example, 11-keto- progesterone, 11 -keto-testosterone, 11-keto-androsterone, 11 -keto-pregnenolone, 11- keto-dehydro-epiandrostenedione, 3α, 5α-reduced-l l-keto-progesterone, 3α, 5α- reduced-11-keto-testosterone, 3α, 5α-reduced-l 1-keto-androstenedione, 3α,5α- tetrahydro- 11 -dehydro-corticosterone, 3α, 5α-reduced-l 1-keto-pregnenolone, or 3α, 5α- reduced- 11 -keto-dehydro-epiandrostenedione.
In yet another embodiment, the invention also pertains, at least in part, to a method for treating hypertension in a subject, by administering to the subject an effective amount of a 1 lβ-HSDl reductase inhibitor, such as, for example, a nucleic acid.
In an alternate embodiment, the invention pertains, at least in part, to a method for treating hypertension in a subject, by administering to the subject an effective amount of a 11 β-HSD 1 reductase inhibitor in combination with an effective amount of a 17α-hydroxylase inhibitor, a 17-HSD inhibitor, a 20α-reductase inhibitor or a 20β- reductase inhibitor.
In yet another embodiment, the invention pertains, at least in part, to a method for increasing insulin sensitivity of a tissue in a subject. The method includes administering an effective amount of a 11 β-HSD 1 reductase inhibitor to the subject. Examples of the 1 lβ-HSDl reductase inhibitor include nucleic acids, 11-keto- progesterone, 11-keto-testosterone, 11-keto-androsterone, 11 -keto-pregnenolone, 11- keto-dehydro-epiandrostenedione, 3α, 5α-reduced-l l-ketoprogesterone, 3α, 5α-reduced- 11 -keto-testosterone, 3α, 5α-reduced-l 1-keto-androstenedione, 3α,5α-tetrahydro-l l- dehydro-corticosterone, 3α, 5α-reduced-l 1-keto-pregnenolone, or 3α, 5α-reduced-l 1- keto-dehydro-epiandrostenedione.
In another embodiment, the invention pertains, at least in part, to a pharmaceutical composition comprising an effective amount of 11 β-OH-progesterone, 11 β-OH-testosterone, 3α,5β -reduced-11 β-OH-progesterone, 3α,5β-reduced-l 1 β-OH- testosterone, chenodeoxycholic acid, 3α, 5β-reduced-pregnenolone, 3α, 5β-reduced- dehydro-epiandrostenedione, 3α,5α-reduced-l 1 β-OH-progesterone, 3α,5α-reduced- 11 β-OH-testosterone, 3α,5α-reduced-l 1 β-OH-androstenedione, 11 -keto-progesterone, 11 -keto-testosterone, 11 -keto-androstenedione, 3α,5α-reduced-l 1 -keto-progesterone, 3α,5α-reduced-l 1-keto-testosterone, 3α, 5α-reduced- 11 β-OH-pregnenolone, 3α, 5α- reduced- 11 β-OH-dehydro-epiandrostenedione, 11 β-OH-pregnenolone, 11 β-OH- dehydro-epiandrostenedione, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-dehydro- epiandrostenedione, 3α,5α-reduced-l 1-keto-androstenedione, 3α,5α-tetrahydro- 11 -dehydro-corticosterone, 3α,5α-reduced-corticosterone, 5α-dihydro-corticosterone, 3α, 5α-reduced aldosterone, and pharmaceutically acceptable salts thereof, in combination with a 17α-hydroxylase inhibitor, a 17-hydroxy steroid dehydrogenase (17- HSD), a 20α-reductase inhibitor, or a 20β-reductase inhibitor. In another embodiment, the invention pertains to a composition comprising a
I lβ-HSDl reductase inhibitor, wherein said 1 lβ-HSDl reductase inhibitor is an siRNA.
In another embodiment, the invention pertains to a composition comprising an
I I β-HSD2 dehydrogenase inhibitor, wherein said 11 β-HSD2 dehydrogenase inhibitor is an siRNA.
Brief Description of the Drawings:
Figure 1 is a bar graph which shows that the exposure of rat aortic rings to corticosterone and 11 β-HSD2 antisense resulted in a statistically significant increase in the contractile response to phenylephrine.
Figure 2 is a bar graph which shows that in aortic rings treated with 1 lβ-HSDl antisense, the contractile responses to all concentrations of phenylephrine were significantly increased compared to aortic rings treated with corticosterone and nonsense oligomers. Figure 3 is a bar graph which illustrates that 11 -dehydro-corticosterone amplifies the contractile responses to phenylephrine in rat aortic rings.
Figure 4 is a bar graph which shows that the conversion of corticosterone to 11 - dehydrocorticosterone was lower than in aortic rings incubated with corticosterone and l lβ-HSDl nonsense oligomers. Figures 5A-5D are representative HPLC chromatograms showing the metabolism of 3H-11 -dehydrocorticosterone (11-dehydroB) by rat aortic rings. In Figures 5 A and 5B, the analysis of the tissue is shown for 1 lβ-HSDl nonsense and 1 lβ- HSDl Antisense, respectively. In Figures 5C and 5D, the analysis of the incubation media is shown for 1 lβ-HSDl nonsense and 1 lβ-HSDl antisense, respectively.
Detailed Description of the Invention:
I . Glucocorticoids and 11 β-HSD 1 Reductase, 11 β-HSD 1 Dehydrogenase and 11 β-
DSD2 Dehydrogenase Glucocorticoids can affect vascular tone by modifying the actions of several vasoactive substances. Glucocorticoids amplify the vasoconstrictive actions of adrenergic catecholamines and angiotensin II on vascular smooth muscle cells. It has been reported that glucocorticoids decrease the biosynthesis of both nitric oxide and prostaglandin I, and attenuate the vasorelaxant actions of atrial natriuretic peptide in vascular tissue. Thus, the multiple effects of glucocorticoids in vascular tissue operate to increase vascular tone. Since vascular smooth muscle cells contain both glucocorticoid and mineralocorticoid receptors it is possible that glucocorticoids mediate their effects in vascular tissue via either or both of these receptor types.
Glucocorticoids are metabolized in vascular and other tissue by two isoforms of 11 β-hydroxy steroid dehydrogenase (11 β-HSD). 11 β-HSD2 is unidirectional and metabolizes glucocorticoids to their respective inactive 11-dehydro derivatives, using NAD+ as a co-factor. 1 lβ-HSDl is bi-directional and possesses both dehydrogenase activity as well as reductase activity. The reductase activity of 11 β- HSD1 regenerates active glucocorticoids from the inactive 11-dehydro derivatives. 1 lβ- HSDl uses NADP+ as a co-factor. In vascular tissue, glucocorticoids amplify the pressor responses to catecholamines and angiotensin II and down-regulate certain depressor systems such as nitric oxide and prostaglandins. Both 11 β-HSD2 and 1 1 β- HSD1 are believed to regulate glucocorticoid levels in vascular tissue and are part of additional mechanisms that control vascular tone.
Glucocorticoids are known to play an important role in the regulation of vascular tone and blood pressure. Glucocorticoid receptors and mineralocorticoid receptors are present in aorta, mesenteric arteries and rat vascular smooth muscle cells in culture. Glucocorticoids can bind to and activate glucocorticoid receptors (and possibly mineralocorticoid receptors) to potentiate the vasoconstrictive effects of both catecholamines and Ang II. Human and rat vascular endothelial cells contain both 11 β- HSD2 and 11 β-HSD 1. It is generally understood that 1 1 β-HSD2 operates to protect both mineralocorticoid receptors and glucocorticoid receptors from excessive stimulation by glucocorticoids. It has been noted that glucocorticoids further amplify the contractile effects of phenylephrine and Ang II when 1 1 β-HSD enzyme activity is inhibited. Rat vascular smooth muscle cells contain only 11 β-HSD 1. Under "physiologic conditions," 1 lβ-HSDl acts largely as a reductase generating active corticosterone from inactive 11 -dehydro-corticosterone.
I lβ-HSDl reductase has an important role as a generator of active glucocorticoids in vascular tissue. 11 β-HSD inactivates glucocorticoid molecules, allowing lower circulating levels of aldosterone to maintain renal homeostasis. Human and rat vascular endolethial cells contain both 11 β-HSD 1 and 11 β-HSD2.
I I β-HSD2 operates to protect both mineralocorticoid receptors and glucocorticoid receptors from excessive stimulation by glucocorticoids. It has also been shown that glucocorticoids further amplify the contractile effects of phenylephrine (PE) and Ang II when 11 β-HSD 1 or 2 dehydrogenase enzyme activity is inhibited. II. Methods of Treating Glucocorticoid Associated States
In an embodiment, the invention pertains, at least in part, to a method for treating a glucocorticoid associated state in a subject. The method includes administering to the subject an effective amount of a 1 lβ-HSDl reductase modulating compound, such that the subject is treated.
The term "glucocorticoid associated states" include states which are associated with the presence or absence of aberrant amounts of glucocorticoids, particularly local levels in target tissues. It includes states which can be treated by modulating, e.g., inhibiting, the activating of a l lβ-HSDl reductase, or, alternatively, l lβ-HSDl dehydrogenase or 1 lβ-HSD2 dehydrogenase. The term includes 1 lβ-HSDl reductase associated states. Examples of glucocorticoid associated states include blood pressure disorders, obesity, diabetes mellitus, interocular pressure, lung disorders, and neurological disorders. The glucocorticoid associated states may also include states associated with undesirable levels of glucocorticoids in adipose tissue, epithelial tissue in the eye, and interocular pressure.
"1 lβ-HSDl reductase associated state" includes states which can be treated by the administration of an 1 lβ-HSDl reductase modulating compound, e.g., an 1 lβ-HSDl reductase inhibitor. In certain embodiments, these states may be characterized by undesirable amounts of glucocorticoids in a tissue, fluid, or elsewhere in the subject. The term "blood pressure disorders" include disorders which are associated with or characterized by abnormal or undesirable blood pressure. Examples of blood pressure disorders include, but are not limited to, high blood pressure, congestive heart failure, chronic heart failure, left ventricular hypertrophy, acute heart failure, myocardial infarction, cardiomyopathy, and hypertension, e.g., arterial hypertension and pulmonary hypertension.
The term "lung disorders" include disorders caused by or related to the presence or absence of glucocorticoids which can be treated by the compounds of the invention, for example, 1 lβ-HSDl reductase inhibitors. The lung contains considerable 1 lβ-HSDl activity (Nicholas and Lugg, J Steroid Biochem 17:113-118, 1982). During fetal development, there is little reductase activity but enzymatic activity increases significantly during lung maturation following birth. In circumstances where excess glucocorticoids are present in lung, there is a predisposition to pulmonary hypertension with an increase in pulmonary artery wall thickness (Cras et al. Am J Physiol Lung Cell Mol Physiol 278:L822-829, 2000) and collagen accumulation (Poiani et al Am J Respir Cr it Care Med 149:994-999, 1994). Moreover glucocorticoids enhance endothelin receptor expression in lung (ShimaJRe /αtr Surg 35:203-207, 2000), a factor contributing to increased vascular resistance in the pulmonary arteries. Another example of a glucocorticoid associated state is insulin insensitivity. High concentrations of cortisol in the liver substantially reduce insulin sensitivity, which increases gluconeogenesis and raises blood sugar levels of a subject. This effect is particularly disadvantageous in subjects suffering from impaired glucose tolerance or diabetes mellitus. In Cushing's syndrome, the antagonism of insulin can provoke diabetes mellitus in subjects. The 1 lβ-HSDl reductase inhibitors can be used to inhibit hepatic gluconeogenesis.
Another example of a glucocorticoid associated state is obesity (including centripetal obesity). It is thought that inhibition of the 1 lβ-HSDl reductase may reduce the effects of insulin resistance in adipose tissue in subjects. Not to be limited by theory, but it is thought that by decreasing insulin resistance will result in greater tissue utilization of glucose and fatty acids, thus reducing circulating levels.
Another example of a glucocorticoid associated state are neurological disorders. Glucocorticoid excess potentiates the action of certain neurotoxins, which leads to neuronal dysfunction and loss. Examples of neurological disorders that may be treated by include neuronal dysfunction and loss due to, for example, glucocorticoid potentiated neurotoxicity. Glucocorticoids may be involved in the cognitive impairment of aging with or without neuronal loss and also in dendritic attenuation. Furthermore, glucocorticoids have been implicated in the neuronal dysfunction of major depression. Other examples of neurological disorders which may be treatable using the 11 β-
HSD1 reductase, l lβ-HSDl dehydrogenase, or 11 β-HSD2 dehydrogenase modulators, e.g., inhibitors, of the invention, include both neuropsychiatric and neurodegenerative disorders such as Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette' s syndrome, multiple sclerosis, amylotropic lateral sclerosis (ALS), progressive supranuclear palsy, epilepsy, and Creutzfeldt- Jakob disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, Korsakoff s psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affective neurological disorders, e.g., migraine and obesity, cognitive impairment of old age, and traumatic brain injury. The term "subject" includes subjects capable of suffering from a glucocorticoid associated states, such as mammals. Examples of mammals include dogs, cats, bears, rabbits, mice, rats, goats, cows, sheep, horses, and, preferably, humans. The subject may be suffering from or at risk of suffering from a glucocorticoid associated state, e.g., a blood pressure associated disorder (e.g., hypertension), obesity, diabetes, or a neurological disorder.
The term "treat" or "treating" includes the prevention, alleviation or reduction of at least one symptom or other indication of a particular glucocorticoid associated state.
In one embodiment, the associated state is a blood pressure associated disorder, e.g., hypertension, and the administration of the modulating compound modulates, e.g., reduces, the blood pressure of the subject.
The term "effective amount" of the 1 lβ-HSDl reductase, 1 lβ-HSDl dehydrogenase, or 1 lβ-HSD2 dehydrogenase modulating compound is that amount necessary or sufficient to treat or prevent a particular glucocorticoid associated state, e.g. prevent the various morpho logical and somatic symptoms of a glucocorticoid associated state. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular 1 lβ-HSDl reductase, 1 lβ-HSDl dehydrogenase, or 1 lβ-HSD2 dehydrogenase modulating compound, e.g., inhibiting, compound. In a further embodiment, the 1 lβ-HSDl reductase, 1 lβ-HSDl dehydrogenase, or
11 β-HSD2 dehydrogenase modulating compound may be administered in combination with a pharmaceutically acceptable carrier.
In a further embodiment, the invention pertains to a method for treating a blood pressure associated disorder, e.g., hypertension, in a subject, by administering to the subject an effective amount of an 11 lβ-HSDl reductase, 1 lβ-HSDl dehydrogenase, or
1 lβ-HSD2 dehydrogenase modulating, e.g., inhibiting, compound.
In another embodiment, the invention features a method for decreasing the concentration (or amount) of glucocorticoids in a tissue of a subject. The method includes administering an effective amount of a selective 1 lβ-HSDl reductase inhibitor, such that the concentration of glucocorticoids in the tissue are decreased. In a further embodiment, the 1 lβ-HSDl reductase inhibitor is a small molecule, e.g., a steroid or a derivative thereof.
Examples of tissues where the concentration of glucocorticoids in a subject may be decreased include tissues which express 1 lβ-HSDl or other wise contain an undesirable concentration of glucocorticoids. Examples of such tissues include a subject's blood, liver, eye, lung, muscle, adipose tissue, nerve tissue, brain, or vascular tissue. In another embodiment, the invention features a method for treating a blood pressure associated disorder, such as, for example, hypertension, in a subject. The method includes administering to a subject an effective amount of a 1 lβ-HSDl reductase inihibitor, such that the subject is treated. In a further embodiment, the 1 1 β- HSDl reductase inihibitor is a selective inhibitor. In another embodiment, the reductase inihibitor is a small molecule, e.g., a steroid or a derivative thereof.
In another embodiment, the invention features a method for increasing insulin sensitivity of a tissue in a subject. The method includes administering to a subject an effective amount of a selective 1 lβ-HSDl reductase inhibitor, such that the insulin sensitivity of the tissue in the subject is increased. Examples of tissue where increased insulin sensitivity may be desirable include, for example, the subject's liver, muscle, nerve or adipose tissue.
In yet another embodiment, the invention features a method for increasing the concentration of glucocorticoids in a tissue of a subject. The method includes administering to a subject an effective amount of a selective 1 lβ-HSDl dehydrogenase inhibitor, such that the concentration of glucocorticoids in the tissue are increased.
The tissue may be any tissue which an increase in the concentration of glucocorticosteroids is desired. Examples of such tissues include, but are not limited to, subject's liver, blood, lung, eye, muscle, adipose tissue, nerve tissue, brain, and vascular tissue.
In another embodiment, the invention features a method for increasing the concentration of glucocorticoids in a tissue of a subject. The method includes administering to a subject an effective amount of a selective 1 lβ-HSD2 dehydrogenase inhibitor, such that the concentration of glucocorticoids in the tissue are increased. The tissue may be any tissue which an increase in the concentration of glucocorticoids is desired. Examples of such tissues include, but are not limited to, subject's liver, eye, blood, lung, muscle, adipose tissue, nerve tissue, brain, and vascular tissue.
The invention also includes a method for selectively inhibiting 1 lβ-HSDl reductase. The method includes contacting 1 lβ-HSDl reductase with a selective 1 lβ- HSDl reductase inhibitor.
In yet another embodiment, the invention includes a method for selectively inhibiting 1 lβ-HSDl dehydrogenase. The method includes contacting 1 lβ-HSDl dehydrogenase with a selective 1 lβ-HSDl dehydrogenase inhibitor. In another embodiment, the invention features a method for selectively inhibiting
11 β-HSD2 dehydrogenase. The method includes contacting 11 β-HSD2 dehydrogenase with a selective 11 β-HSD2 dehydrogenase inhibitor. HI. 11 β-HSD 1 Reductase Modulating Compounds, 11 β-HSD 1 -Dehydrogenase Modulating Compounds and 11 β-HSD2 Dehydrogenase Modulating Compounds
The term "1 lβ-HSDl reductase modulating compound" include compounds and agents (e.g., oligomers, proteins, etc.) which modulate or inhibit the activity of 11 β- HSDl reductase. In an advantageous embodiment, the l lβ-HSDl reductase modulating compound is an 1 lβ-HSDl reductase inhibitor (also referred to as "1 lβ-HSDl reductase inhibiting compound"). The 1 lβ-HSDl reductase modulating compound may be a small molecule, e.g., a compound with a molecular weight below 10,000 daltons.
In a further embodiment, the 1 lβ-HSDl reductase modulating compound is a selective inhibitor of 1 lβ-HSDl reductase. The term "selective 1 lβ-HSDl reductase inhibitor" includes compounds which selectively inhibit the reductase activity of 11 β- HSD1 as compared to the dehydrogenase activity. In a further embodiment, the reductase activity is inhibited at a rate about 2 times or greater, about 3 times or greater, about 4 times or greater, about 5 times or greater, about 10 times or greater, about 15 times or greater, about 20 times or greater, about 25 times or greater, about 50 times or greater, about 75 times or greater, about 100 times or greater, about 150 times or greater, about 200 times or greater, about 300 times or greater, about 400 times or greater, about 500 times or greater, about 1 x 103 times or greater, about 1 x 104 times or greater, about 1 x 105 times or greater, or about 1 x 10 or greater as compared with the inhibition of the dehydrogenase activity of 11 β-HSD 1.
In a further embodiment, the 1 1 β-HSD 1 reductase modulating compound may be a steroid or a steroid derivative. The steroid ring system is generally numbered according to IUPAC conventions, as shown below:
Figure imgf000011_0001
Examples of 11 β-HSD 1 reductase modulating compounds include 11-keto steroid compounds, e.g., compounds with the steroid ring system with a carbonyl functional group at the 11 -position of the steroid ring. Examples of steroid compounds with an 11-keto group include, for example, 11-keto progesterone, 11 -keto-testosterone, 11 -keto-androsterone, 11-keto-pregnenolone, 11 -keto-dehydro-epiandrostenedione, 3α, 5α-reduced-l 1-ketoprogesterone, 3α, 5α-reduced-l 1-keto-testosterone, 3α, 5α-reduced- 11-keto-androstenedione, 3α,5α-tetrahydro-l l -dehydro-corticosterone, 3α, 5α-reduced- 11-keto-pregnenolone, and 3α, 5α-reduced-l 1-keto-dehydro-epiandrostenedione. Other examples of 1 lβ-HSDl reductase modulating compounds of the invention are compounds which conserve a least a portion of the steroid nucleus. These compounds may have additional substiuents, such as fatty acid tails at the 22 position, or other modifications (e.g., substitutions of the ring by halogens, formation of esters or other protecting groups for the hydroxyl groups of the steroids, or replacement of functional groups with others that may, for example, advantageously, lengthen the time the molecule is in its active form in a subjects body. Alternatively, the modifications can be such that the reduce the time the compound is in its active form in a subject's body.
Examples of 1 lβ-HSDl reductase modulating compounds also include 3α, 5α- reduced steroid compounds. Examples of 3α, 5α-reduced steroid compounds include 3α, 5α-reduced-l 1-ketoprogesterone, 3α, 5α-reduced-l 1-keto-testosterone, 3α, 5α- reduced-11-keto-androstenedione, 3α,5α-tetrahydro- 11 -dehydro-corticosterone, 3α, 5α- reduced-11-keto-pregnenolone, and 3α, 5α-reduced-l l-keto-dehydro- epiandrostenedione.
Steroid derivatives include compounds with a steroid ring structure optionally substituted with additional substituents which allow the compound to perform its intended function. It should be noted that the steroid compounds may be converted to the active form of the modulating compound within the subject. The invention includes administering compounds which are in other forms, e.g., prodrugs, and which are metabolized in vivo to yield the 1 lβ-HSDl reductase modulating compounds described herein.
In one embodiment, the 1 lβ-HSDl reductase inhibitors possess IC50's less than about 0.5 μM using 600 nanoM 11 -dehydro-corticosterone substrate concentration and testicular leydig cell homogenates. Methods for testing the IC50's of the enzymes are described in further detail in Latif, S. A. et al. Steroids 62: 230-237, 1997. In another embodiment, the 11 β-HSD 1 reductase inhibitors have an IC50 of 80 μM or less, or, preferrably, 15 μM or less. In another embodiment, the 1 lβ-HSDl reductase inhibitors have an ICso of less than 100 μM.
Other examples of 1 lβ-HSDl reductase modulating compounds include carbenoxolone and derivatives thereof. In other embodiments, 1 lβ-HSDl reductase modulating compound is a nucleic acid. In another embodiment, the 1 lβ-HSDl reductase inhibitor is an antisense nucleic acid. In another embodiment, the 1 lβ-HSDl reductase inhibitor is a siRNA.
The basic mechanism of RNA interference can be understood as a two step process (Zamore P.D., Nature Struc. Biol, 8, 9, 746-750, (2001)). First, the dsRΝA is cleaved to yield short interfering RΝAs (siRΝAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt). Then, the siRΝAs target the corresponding mRΝA sequence specific for destruction (Fire A. et al, Nature, Vol 391, (1998); Hamilton AJ et al Science, 286, 950-952, (1999); Zamore PD. et al. Cell, 101, 25-33, (2000); Elbashir SM. et al, Genes & Development, 15, 188-200, (2001); Bernstein E. et al Nature 409, 363-366, 2001).
It has been demonstrated that chemically synthesized 21 nt siRNA duplexes specifically suppress expression of endogenous and heterologeous genes in different mammalian cell lines, including human kidney and HeLa cells (Elbashir SM. et ah, Nature, 411, 494-498, (2001)). It was discovered that no unspecific effects occurred in mammalian cells by transfection of short sequences (<30nt). It was suggested that 21 nt siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
It was also found that siRNAs mediated RNAi in cell extracts and synthetic siRNAs can induce gene-specific inhibition of expression in C. elegans and in cell lines from humans and mice (Caplen, N.J. et al. PNAS 171251798, 1-6, (2001)30). It was also shown that siRNAs can have direct effects on gene expression in C. elegans and mammalian cell culture in vivo.
Methods for making and using siRNAs are described in, for example, WO 01/75164, US 2002/0137210, WO 01/29058, WO 02/072762, WO 02/059300, WO 02/44321, WO 01/92513, WO 01/68836, US 2002/0173478, US 2002/0160393, US 2002/0162126, US 2002/0137709, US 2002/0132788, US 2002/0086356, and WO 99/32619; each of which is expressly incorporated herein by reference.
In one embodiment, the 1 lβ-HSDl reductase inhibitor is a double stranded RNA oligomer, wherein the antisense strand is complementary to at least a portion of SEQ. ID. No. 1. In one embodiment, the portion is 40 base pairs or less, 35 base pairs or less, 30 base pairs or less, 29 base pairs or less, 28 base pairs or less, 27 base pairs or less, 26 base pairs or less, 25 base pairs or less, 24 base pairs or less, 23 base pairs or less, 22 base pairs or less, 21 base pairs or less, 20 base pairs or less, 19 base pairs or less, or about 18 base pairs or less. In another embodiment, the oligomer has 10 or more base pairs, 11 or more base pairs, 12 or more base pairs, 13 or more base pairs, 14 base pairs or more, 15 base pairs or more, 16 base pairs or more, 17 base pairs or more, 18 base pairs or more, or 19 base pairs or more. In another embodiment, the 1 lβ-HSDl reductase inhibitor has an antisense strand having the sequence 5'-CAT AAC TGC CGT CCA ACA GC-3' (SEQ ID NO. 2).
The term "11 β-HSD 1 dehydrogenase modulating compound" include compounds and agents (e.g., oligomers, proteins, etc.) which modulate or inhibit the activity of 1 lβ-HSDl dehydrogenase. In an advantageous embodiment, the 1 lβ-HSDl dehydrogenase modulating compound is an 1 lβ-HSDl dehydrogenase inhibitor (also referred to as "1 lβ-HSDl dehydrogenase inhibiting compound"). The 1 lβ-HSDl dehydrogenase modulating compound may be a small molecule, e.g., a compound with a molecular weight below 10,000 daltons.
In a further embodiment, the 1 lβ-HSDl dehydrogenase modulating compound is a selective inhibitor of 1 lβ-HSDl dehydrogenase. The term "selective 1 lβ-HSDl dehydrogenase inhibitor" includes compounds which selectively inhibit the dehydrogenase activity of 11 β-HSD 1 as compared to the reductase activity of 11 β- HSD1. In a further embodiment, the dehydrogenase activity is inhibited at a rate about 2 times or greater, about 3 times or greater, about 4 times or greater, about 5 times or greater, about 10 times or greater, about 15 times or greater, about 20 times or greater, about 25 times or greater, about 50 times or greater, about 75 times or greater, about 100 times or greater, about 150 times or greater, about 200 times or greater, about 300 times or greater, about 400 times or greater, about 500 times or greater, about 1 x 10 times or greater, about 1 x 104 times or greater, about 1 x 105 times or greater, or about 1 x 106 or greater as compared with the inhibition of the reductase activity of 1 lβ-HSDl . In one embodiment, the 1 lβ-HSDl dehydrogenase inhibitor is a small molecule, such as a steroid or a derivative thereof. In a further embodiment, the steroid is 3α, 5β- reduced. Examples of 3α,5β-reduced steroids include 3α, 5 β-reduced- 11 β-OH- progesterone, 3α, 5 β-reduced- 11 β-OH-testosterone, chenodeoxycholic acid, 3α, 5β- reduced-pregnenolone, 3α, 5β-reduced-dehydro-epiandrostenedione, 3α, 5β-reduced- progesterone, 3α, 5β-testosterone, and deoxy-corticosterone.
In another embodiment, the 1 lβ-HSDl dehydrogenase inhibitor is a 3α, 5α- reduced steroid. Examples of such steroids include 3α, 5α-reduced-l 1 β-OH- progesterone, 3α, 5α-reduced- 11 β-OH-testosterone, 3α, 5α-reduced-l lβ-OH- androstendione, 3α, 5α-reduced- 11 β-OH-pregnenolone, 3α, 5α-reduced-l lβ-OH- dehydro-epiandrostenedione, 3α, 5α-reduced-corticosterone, 3α, 5α-reduced- aldosterone, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-progesterone, 3α, 5α-reduced testosterone, and 3α, 5α-reduced-chenodeoxycholic acid. Other examples of steroids which can be used as 1 lβ-HSDl dehydrogenase inhibitors include 11 β-OH progesterone, 11 β-OH testosterone, 11 β-OH-pregnenolone, and 11 β-OH-dehydro- epiandrostenedione.
In one embodiment, the 1 lβ-HSDl dehydrogenase inhibitor has an IC50 of 0.5 μM or less. In another embodiment, the 1 lβ-HSDl dehydrogenase inhibitor has an IC50of 100 μM or less, 80 μM or less, or 20 μM or less (using using 100 nM corticosterone substrate concentration and testicular Leydig cell homogenates). The term "1 lβ-HSD2 dehydrogenase inhibitor" includes agents which inhibit or decrease the dehydrogenase activity of 1 lβ-HSD2.
In one embodiment, the 11 β-HSD2 dehydrogenase inhibitor is a small molecule, such as a steroid or a derivative thereof. In one embodiment, the steroid is 3α, 5α- reduced. Examples of 11 β-HSD2 dehydrogenase inhibitors include, but are not limited to, 3α, 5α-reduced- 11 β-OH-progesterone, 3α, 5α-reduced-l l β-OH-testosterone, 3α, 5α- reduced-l lβ-OH-androstenedione, 3α, 5α-reduced- 11 -keto-progesterone, 3α, 5α- reduced- 11 -dehydro-corticosterone, 3α, 5α-reduced-corticosterone, 3α, 5 α -reduced- 11 β- OH-pregnenolone, 3α, 5α-reduced-l lβ-OH-dehydro-epiandrostenedione, 3α, 5α- reduced-pregnenolone, 3α, 5α-reduced-dehydro-epiandrostenedione, and 3α, 5α- aldosterone. Other examples of 11 β-HSD2 dehydrogenase inhibitors include 11 β-OH- progesterone, 11 β-OH-pregnenolone, 11 β-OH-dehydro-epiandrostenedione, 11 β-OH- testosterone, 11 -keto-progesterone, 5α-dihydro-corticosterone, and 3α, 5α-reduced deoxy- corticosterone. In other embodiments, 11 β-HSD2 dehydrogenase modulating compound is a nucleic acid. In another embodiment, the 1 lβ-HSD2 dehydrogenase inhibitor is an antisense nucleic acid. In another embodiment, the 11 β-HSD2 dehydrogenase inhibitor is a siRNA.
In one embodiment, the 11 β-HSD2 dehydrogenase inhibiting compounds have ICJO'S less than 2.5 μM (using 50 nM coricosterone substrate concentration and sheep kidney microsomes). In another embodiment, the 11 β-HSD2 dehydrogenase inactive compounds have an IC50 of less than lOμM.
In one embodiment, the 11 β-HSD2 dehydrogenase inhibitor is a double stranded RNA oligomer, wherein the antisense strand is complementary to at least a portion of SEQ. ID. No. 3. In one embodiment, the portion is 40 base pairs or less, 35 base pairs or less, 30 base pairs or less, 29 base pairs or less, 28 base pairs or less, 27 base pairs or less, 26 base pairs or less, 25 base pairs or less, 24 base pairs or less, 23 base pairs or less, 22 base pairs or less, 21 base pairs or less, 20 base pairs or less, 19 base pairs or less, or about 18 base pairs or less. In another embodiment, the oligomer has 10 or more base pairs, 11 or more base pairs, 12 or more base pairs, 13 or more base pairs, 14 base pairs or more, 15 base pairs or more, 16 base pairs or more, 17 base pairs or more, 18 base pairs or more, or 19 base pairs or more. In another embodiment, the 1 lβ-HSD2 dehydrogenase inhibitor has an antisense strand having the sequence 5'-AGG CCA GCG CTC CAT GAC TT- 3' (SEQ ID NO 4). The invention also pertains to each of the nucleic acids described herein as well as pharmaceutical compositions comprising these nucleic acids.
Examples of 1 lβ-HSDl -reductase, 1 lβ-HSDl -dehydrogenase and 1 lβ-HSD2 dehydrogenase modulating compounds are described in Table 1.
TABLE 1
Figure imgf000017_0001
Figure imgf000018_0001
IV. 17α-Hydroxylase Inhibitor, 17-HSD-Inhibitors, 20α-Reductase Inhibitors and 20β-Reductase Inhibitors The invention also pertains to administering to the subject a 17α-hydroxylase inhibitor, a 17-HSD inhibitor, a 20α-reductase inhibitor and/or a 20β-reductase inhibitor, in combination with the methods described above. The inhibitors can be any compound or substance known to inhibit any one of these enzymes. The 17α-hydroxylase, 17- HSD, 20α-reductase and or 20β-reductase inhibitors are administered in combination with the compounds of the invention described herein.
The language "in combination with" another agent includes co-administration of the compound of the invention and the agent, administration of the compound of the invention first, followed by the other agent and administration of the other agent first, followed by the compound of the invention. The 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase inhibitors can be found using assays for screening candidate or test compounds which bind to or modulate the activity of a 17α-hydroxylase, 17-HSD, 17-HSD, 20α-reductase or 20β-reductase protein or polypeptide or biologically active portion thereof. The sequences for 17α- hydroxylase is shown in SEQ ID No. 5. The sequence for the 17-HSD is shown in SEQ ID No. 6. The polypeptide sequence for 17-HSD is shown in SEQ ID. No. 7. In another embodiment, the 17α-hydroxylase inhibitor, the 17-HSD inhibitor, the 20α-reductase, and/or the 20β reductase inhibitors are nucleic acid oligomers. In a further embodiment, the nucleic acid oligomers are siRNA and comprise at least a portion of SEQ ID No. 5 or SEQ ID No. 6. The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et a (1994). J Med. Chem. 37:2678; Cho et al. (1993) Science 261 :1303; Carrell et a (1994) Angew. Chem. Int. Ed. Engl 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J Mol. Biol. 222:301-310).
Determining the ability of a 17α-hydroxylase, 17-HSD, 20α-reductase or 20β- reductase protein to bind to or interact with a target molecule (e.g., a steroid substrate) can be accomplished by determining direct binding. Determining the ability of the 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein to bind to or interact with a target molecule can be accomplished, for example, by coupling the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein with a radioisotope or enzymatic label such that binding of the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein to a target molecule can be determined by detecting the labeled 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein in a complex. For example, 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase proteins can be labeled with 125^ 35gs 14^ 0r -^H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase proteins can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In yet another embodiment, an assay of the present invention is a cell-free assay in which a 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β- reductase protein or biologically active portion thereof is determined. Binding of the test compound to the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein can be determined either directly or indirectly. The assay may include contacting the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein or biologically active portion thereof with a known compound which binds 17α-hydroxylase, 17-HSD, 20α- reductase or 20β-reductase to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein, wherein determining the ability of the test compound to interact with a 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein comprises determining the ability of the test compound to preferentially bind to 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase or biologically active portion thereof as compared to the known compound.
In another embodiment, the assay is a cell-free assay in which a 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the 17α-hydroxylase, 17-HSD, 20α- reductase or 20β-reductase protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of al7α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein can be accomplished, for example, by determining the ability of the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein to bind to a target molecule. Determining the ability of the 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein to bind to a target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol 5:699-705. As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In an alternative embodiment, determining the ability of the test compound to modulate the activity of a 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein can be accomplished by determining the ability of the 17α-hydroxylase, 17- HSD, 20α-reductase or 20β-reductase protein to further modulate the activity of a target molecule.
In yet another embodiment, the cell-free assay involves, contacting a 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein or biologically active portion thereof with a known compound which binds the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein, wherein determining the ability of the test compound to interact with the 17α- hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein comprises determining the ability of the 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein to preferentially bind to or modulate the activity of a target molecule.
It may be desirable to immobilize either 17α-hydroxylase, 17-HSD, 20α- reductase or 20β-reductase or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein, or interaction of a 17α-hydroxylase, 17-HSD, 20α-reductase or 20β-reductase protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
In one embodiment, the invention pertains to the 17α-hydroxylase, 17-HSD, the 20α-reductase, and the 20β-reductase inhibiting compounds which are found using the above described methods.
V. Pharmaceutical Compositions
In yet another embodiment, the invention pertains to a pharmaceutical composition for the treatment of a glucocorticoid associated state. The composition includes an effective amount of an 1 lβ-HSDl reductase, 1 lβ-HSDl dehydrogenase, or 1 lβ-HSD2 dehydrogenase modulating, e.g., inhibiting, compound and a pharmaceutically acceptable carrier. In a further embodiment, the glucocorticoid associated state is a blood pressure disorder. In another embodiment, the pharmaceutical compositions may also comprise an inhibitor of 17α-hydroxylase, 17- HSD, 20α-reductase or 20β-reductase. In another embodiment, the invention pertains, at least in part, to a pharmaceutical composition comprising an effective amount of 11 β-OH-progesterone, 11 β-OH-testosterone, 3α,5 β-reduced- 11 β-OH-progesterone, 3 ,5β-reduced-l lβ-OH- testosterone, chenodeoxycholic acid, 3α, 5β-reduced-pregnenolone, 3α, 5β-reduced- dehydro-epiandrostenedione, 3α,5α-reduced-l 1 β-OH-progesterone, 3α,5α-reduced- 1 1 β-OH-testosterone, 3α,5 -reduced-l l β-OH-androstenedione, 11 -keto-progesterone, 11 -keto-testosterone, 11 -keto-androstenedione, 3α,5α-reduced-l 1 -keto-progesterone, 3α,5 -reduced-l 1-keto-testosterone, 3α, 5α-reduced-l 1 β-OH-pregnenolone, 3α, 5α- reduced-1 lβ-OH-dehydro-epiandrostenedione, 11 β-OH-pregnenolone, 1 lβ-OH- dehydro-epiandrostenedione, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-dehydro- epiandrostenedione, 3α,5α-reduced-l 1-keto-androstenedione, 3α,5α-tetrahydro- 11 -dehydro-corticosterone, 3α,5α-reduced-corticosterone, 5α-dihydro-corticosterone, 3α, 5α-reduced aldosterone, and pharmaceutically acceptable salts thereof, in combination with a 17α-hydroxylase inhibitor, a 20α-reductase inhibitor, or a 20β- reductase inhibitor.
The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal, pulmonary and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent. Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste. In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, poly vinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert dilutents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Sprays also can be delivered by mechanical, electrical, or by other methods known in the art. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial, antiparasitic and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle. The compositions also may be formulated such that its elimination is retarded by methods known in the art.
Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration or administration via inhalation is preferred.
The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Other methods for administration include via inhalation.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a glucocorticoid associated state.
If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
As set out above, certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" is art recognized and includes relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J Farm. SCI. 66:1-19).
In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances includes relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
The term "prodrug" includes compounds with moieties which can be metabolized in vivo to a hydroxyl group or other functional group and moieties which may advantageously remain in vivo. Preferably, the prodrugs moieties are metabolized in vivo. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alky 1 esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alky 1 amides, di- lower alkyl amides, and hydroxy amides. The invention also pertains to any one of the methods described supra further comprising administering to the subject a pharmaceutically acceptable carrier.
EXEMPLIFICATION OF THE INVENTION
Example 1: Ability of Corticosterone and 11-Dehydro-Corticosterone to Amplify the Contractile Responses of Phenylephrine
Experimental: Male Sprague-Dawley (150-200g) rats were anesthetized with pentobarbital (50 mg/kg IP), and a median sternotomy was performed followed by the rapid removal of the thoracic aorta. The adventitia was removed, but the endothelium was left intact. The aorta was cut into 2-3 mm rings and individual rings were placed into a single well of a twenty four well culture plate and incubated at 37°C under 95%) O2- 5% CO . Each well contained 1 mL of DMEM/F12 containing 1% fetal bovine serum, streptomycin (100 μg/ml), penicillin (100 units/ml) and amphotericin (0.25 μg/ml). Aortic rings were incubated for 24 hours prior to contractility measurements with the following combinations of steroids, and anti sense/nonsense oligonucleotides (3 μmol/L):
Corticosterone (10 nmol/L) + 1 lβ-HSD2 antisense or 1 lβ-HSD2 nonsense oligomer
Corticosterone (10 nmol/L) + 1 lβ-HSDl antisense or 1 lβ-HSDl nonsense oligomer
In 11 -dehydrocorticosterone experiments with vehicle alone 11 -dehydrocorticosterone (100 nmol/L) + 1 lβ-HSDl Antisense or 1 lβ-HSDl nonsense oligomer
Antisense phosphorothioate oligonucleotides, targeted to block either 11 β-HSD2 or 1 lβ-HSDl gene expression, were obtained from Research Genetics, Huntsville AL. Antisense oligomers complementary to 20 bp sequences spanning the ribosome binding/translation start site were used. Oligomer sequences were: 5'-CAT AAC TGC CGT CCA ACA GC-3' (SEQ ID NO. 2) for 1 lβ-HSDl Antisense and 5'-AGG CCA GCG CTC CAT GAC TT- 3' (SEQ ID NO 4) for 1 lβ-HSD2 antisense. In control experiments the corresponding sense sequence was used as the nonsense oligomer. Antisense and nonsense oligomers were added directly to each well at 20 μg/10: 1 sterile H20 per well for a final concentration of 3 μmol/L. For contraction measurements, aortic rings were suspended by tungsten wires with 1 g of tension and placed in a vessel bath containing serum free DMEM/F12 media at 37°C aerated with 95% O2-5% CO, at pH 7.4. Vessels were equilibrated for 20 minutes and then tested with phenylephrine (1 nmol/L - lOμmol/L). Although phenylephrine is structurally not a catecholamine, it is considered to be a functional catecholamine as it activates both α and β adrenoceptors. Due to its favorable stability characteristics, it is widely used as a catecholamine substitute in experiments of this nature. The intensity of contraction was assessed by use of a Narishige micromanipulator and model FT03 force transducer (Grass Instrument Co. West Warwick, Rl). Measurements were recorded on computer using the Labview 4.1 Virtual Instrument System (National Instruments, Austin, TX). Adhering to this protocol, test vessel viability by demonstrating the ability of the vessel to vigorously contract when exposed to known vasoconstrictors and relax back to baseline after treatment with acetylcholine.
Results: Effect of 11 β-HSD Antisense on Vascular Contractile Response
Experiments were carried out to determine whether specific 1 1 β-HSD2 antisense oligomers affect the contractile response of vascular rings. Rat aortic rings, with endothelium intact, were incubated for 24 hours with corticosterone (10 nmol/L) and either specific 11 β-HSD2 antisense oligomers (3 μmol/L) or nonsense oligomers (3 (μmol/L). Following incubation, the contractile responses to graded concentrations of phenylephrine were determined. Previously, it had been demonstrated that the incubation of aortic rings with corticosterone resulted in amplified contractile responses to graded concentrations of phenylephrine compared to controls. The exposure of rings to corticosterone together with 1 lβ-HSD2 antisense demonstrated a statistically significant increase in the contractile response to all concentrations (1, 10, 100 nmol/L and 1 μmol/L) of phenylephrine (Fig 1).
In the rat, both vascular endothelial and smooth muscle cells contain 11 β-HSD 1. Even though this isoform operates mainly as a reductase under physiologic conditions, it was examined if 11 β-HSD 1 antisense oligomers had an effect on the ability of corticosterone to amplify the contractile responses to phenylephrine in vascular tissue. Rings were incubated for 24-hours with corticosterone (10 nmol/L) and either 1 lβ- HSDl antisense oligomers (3 μmol/L) or nonsense oligomers (3 μmol/L). In rings treated with 1 lβ-HSDl antisense the contractile responses to all concentrations of phenylephrine (10 nmol/L, 100 nmol/L and 1 μmol/L) were significantly increased compared to rings treated with corticosterone and nonsense oligomers (Fig 2). In rat vascular tissue, 1 lβ-HSDl acts predominantly as a reductase metabolizing inactive 11-dehydro-glucocorticoid back to the active parent hormone. 11 -dehydrocorticosterone (just like corticosterone) also amplifies the contractile responses to phenylephrine in rat aortic rings (Fig 3). In the rat, 1 lβ-HSDl is present in both vascular endothelial and smooth muscle cells and under physiological conditions this enzyme functions predominantly as a reductase.
Furthermore, the effect of 11 β-HSD 1 antisense oligomers on the ability of 11- dehydro-corticosterone to amplify the contractile responses to phenylephrine was studied. Rings were incubated for 24 hours with 11 -dehydro-corticosterone (100 nmol/L) and either 1 lβ-HSDl antisense (3 μmol/L) or nonsense (3 μmol/L) oligomers. 1 lβ-HSDl antisense oligomers attenuated the ability of 11 β-dehydro-corticosterone to amplify the contractile response to all concentrations of phenylephrine compared to 11- dehydro-corticosterone plus 1 lβ-HSDl nonsense oligomers. Statistically significant decreases were observed at 100 nmol/L and 1 μmol/L phenylephrine (Fig 3). In aortic rings incubated (24-hours) with corticosterone (10 nmol/L) and 11 β-
HSD2 antisense (3 μmol/L), the contractile response to graded concentrations of phenylephrine (PE: 10 nmol/L - 1 μmol/L) were significantly (P<0.05) increased compared to rings incubated with corticosterone and 1 1 β-HSD2 nonsense. 11 β-HSD 1 antisense oligomers also enhanced the ability of corticosterone to amplify the contractile response to phenylephrine.
Discussion
Earlier experiments showed that inhibitors of 11 β-HSD dehydrogenase activity enhance the ability of corticosterone to amplify the vasoconstrictive actions of phenylephrine and angiotensin II in rat aorta. The examples show that a specific 11 β- HSD2 antisense oligomer also enhances the ability of corticosterone to amplify the contractile responses of catecholamines. Since 11 β-HSD2 appears to exist only in endothelial cells, this observation supports a role for the action of glucocorticoids in affecting endothelial cell function. Although 1 lβ-HSDl acts predominantly as a reductase in vascular tissue, 1 lβ-HSDl antisense oligomers also enhanced the ability of corticosterone to amplify the contractile effects of phenylephrine in rat aortic rings. This observation suggests that 1 lβ-HSDl -dehydrogenase, in addition to 1 lβ-HSD2, also operates to protect GR and MR from over-activation by glucocorticoids in vascular tissue. Further experiments to determine whether antisense oligomers down-regulate mRNA and protein expression of their respective 11 β-HSD isoform under conditions in which they enhance contractile responses in aortic rings will be done. Using a similar protocol to the one described here, it has been shown using RT-PCR analysis, that 11 β- HSD2 antisense and 1 lβ-HSDl antisense down-regulate the expression of their respective enzyme isoforms in cultured rat vascular endothelial and smooth muscle cells.
The example confirms that 11 -dehydro-corticosterone also amplifies the contractile actions of catecholamines in rat aortic rings. Since 11-dehydro- glucocorticoids do not bind to GR (or MR) to any major extent, it is proposed that 11- dehydro-corticosterone is metabolized back to corticosterone by 1 lβ-HSDl -reductase in vascular smooth muscle and/or endothelial cells. This hypothesis is supported by the discovery that 11 -keto-progesterone, a specific inhibitor of 1 lβ-HSDl -reductase activity (backward reaction), diminished the ability of 11 -dehydro-corticosterone to amplify the contractile effects of phenylephrine and decreased the metabolism of 11 -dehydrocorticosterone back to corticosterone. The examples also demonstrate that 1 lβ-HSDl antisense oligomer also attenuates the ability of 11 -dehydro-corticosterone to amplify the contractile responses of phenylephrine indicating that the down-regulation of 1 lβ- HSDl gene expression can affect the regeneration of active glucocorticoid (from 1 1 - dehydro-glucocorticoid) in vascular tissue. Indeed, the examples show that 1 lβ-HSDl antisense can significantly reduce the metabolism of 11 -dehydro-corticosterone back to corticosterone in aortic ring preparations.
Example 2: Metabolism of Corticosterone and 11-Dehydro-Corticosterone in Vascular Tissue
Experimental:
The effects of 1 lβ-HSDl and 1 lβ-HSD2 antisense on the inter-conversion of 3H-corticosterone and 3H-11 -dehydro-corticosterone by aortic rings was also determined. Rings (2-3mm) obtained in a similar manner as those in the contraction studies, were incubated in 1 ml DMEM/F12 media containing 1% FBS at 37°C under 95% O -5% CO2 in 24-well culture plates. Rings were incubated for 24 hours with:
(i) 3H-corticosterone (10 nmol/L) ± 1 lβ-HSD2 or 1 lβ-HSDl antisense (3 μmol/L); control groups received nonsense oligomers. The amount of 3H-ll-dehydro- corticqsterone in the incubation medium after 24 hrs was then measured. The effects of 1 lβ-HSDl anti sense/nonsense were measured in quadruplicate (n = 6 aortic rings per well) and the effects of 11 β-HSD2 antisense/nonsense in duplicate (n •= 8 aortic rings per well), (ii) 3H- 11 -dehydro-corticosterone ( 10 nmol/L) ± 11 β-HSD 1 antisense (3 μmol/L); this experiment was performed in duplicate (n = 10 aortic rings per well). Control groups were incubated with the appropriate nonsense oligomer. 3H- corticosterone in the incubation medium after 24 hrs was then measured. In this experiment, aortic rings were also analyzed for 3H-corticosterone content. Rings from duplicate incubations (total n = 20) were blotted dry, pooled and homogenized in 50 % methanol using a Polytron. The homogenates were then centrifuged, extracted as below using Sep-Paks and injected onto a HPLC system for analysis.
Incubation media was collected, ran through a Sep-Pak and eluted with 3 mis of methanol, the eluate was then dried under nitrogen and reconstituted in 500:1 methanol. The aortic rings were dried and weighed. The steroids present in the eluate were separated by high-pressure liquid chromatography with a Dupont Zorbax C8 column eluted at 44°C at a flow rate of 1 mL/min using 55% methanol for 10 minutes. Steroids were observed by monitoring radioactivity on-line with a Packard Radiomatic Flo- One/Beta Series A-500 counter connected to a Dell Optiflex 425 S/L computer. Corticosterone and 11 -dehydro-corticosterone were identified by comparing their retention times with that of known standards.
Corticosterone and phenylephrine were obtained from Sigma (St Louis, MO), 11 -dehydrocorticosterone from Research Plus (Bayonne, NJ) and H-steroids from New England Nuclear (Boston, MA). Where appropriate, data were expressed as mean ± SE and analyzed using ANOVA and the Student's t test with Bonferroni modification. P values of less than 0.05 are considered significant.
Results: Effects of 11 β-HSD Antisense on Steroid Metabolism
A series of experiments were then conducted to test whether 11 β-HSD2 and l lβ- HSDl antisense oligomers did affect the enzymatic conversion of corticosterone and 11- dehydrocorticosterone. In experiments in which aortae were taken from rats (n= 4) and 6 rings cut from each aorta were incubated for 24 hrs with H-corticosterone (10 nM) plus 1 lβ-HSDl antisense (3 μM), the conversion of corticosterone to 11- dehydrocorticosterone was 21% lower than in aortic rings incubated with corticosterone and 1 lβ-HSDl nonsense oligomers (Figure 4). In a further two experiments, aortae were taken from rats (n = 2) and 8 aortic rings cut from each. Aortic ring preparations incubated for 24hrs with corticosterone and 11 β-HSD2 antisense (3 μM), demonstrated a 24%) reduction in the conversion of corticosterone to 11 -dehydrocorticosterone compared to aortic rings incubated with corticosterone and 11 β-HSD2 nonsense (Figure 4). To determine the effects of 11 β-HSD 1 antisense on 11 β-HSD 1 -reductase activity rat aortae were taken from rats( n= 2) and 10 aortic rings cut from each. These aortic rings were then incubated for 24 hours with H-l 1 -dehydrocorticosterone and either 1 lβ-HSDl antisense or nonsense and the production of corticosterone was measured. The production of 3H-corticosterone was markedly reduced in rings incubated with 1 lβ- HSDl antisense compared to rings incubated with 1 lβ-HSDl nonsense oligomers (Figure 4, representative HPLC chromatograms from these experiments are also shown in Figure 5). Thus, 1 lβ-HSDl antisense profoundly diminished the ability of the rat aortic rings to metabolize 11 -dehydro-corticosterone back to corticosterone. The aortic ring tissue in these experiments was also pooled (n = 20) and analyzed for steroid content. The amount of radioactivity in the tissue was approximately 2-3% of the total radioactivity in the incubation media. The production of 3H-corticosterone in aortic rings incubated with 11 β-HSD 1 antisense was again markedly lower that that in rings incubated with 1 lβ-HSDl nonsense oligomers (see HPLC chromatograms, Figures 5 A - 5D). The levels of H-l 1 -dehydrocorticosterone metabolism measured in the incubate and in the aortic tissue were very similar (Figures 5A-5D). This indicates that measuring steroid content in the media does not under-represent the level of steroid metabolism in the tissue compartment.
Discussion
In this example, experiments were undertaken to determine whether antisense oligomers could affect 11 β-HSD enzyme activity and, indeed, it has been demonstrated that 1 lβ-HSD2 and 1 lβ-HSDl antisense caused moderate reductions (24 and 21% respectively) in the metabolism of corticosterone. These reductions in metabolism translate to relatively small increases in residual corticosterone levels in the aortic ring tissue that would not appear to account for the relatively large increases in phenylephrine-induced vasoconstriction observed in the contractile studies. However, glucocorticoids have been reported to not only amplify the contractile effects of catecholamines in vascular tissue but to also diminish the effects of certain vasorelaxation pathways (glucocorticoids decrease nitric oxide and prostaglandin I2 synthesis); such actions would serve to further enhance the effects of glucocorticoids on increasing catecholamine-induced vasoconstriction and may explain how small changes in glucocorticoid levels can have profound effects on vascular tone.
In addition, 1 lβ-HSD2 and 1 lβ-HSDl antisense also decreased the metabolism of corticosterone to 11 -dehydro-corticosterone. 11 -dehydro-corticosterone (100 nmol/L) also amplified the contractile response to phenylephrine in aortic rings (P<0.01), most likely due to the generation of active corticosterone by 1 lβ-HSDl - reductase; this effect was significantly attenuated by 1 lβ-HSDl antisense. 1 lβ-HSDl antisense also caused a marked decrease in the metabolism of 11 -dehydro-corticosterone back to corticosterone by 1 lβ-HSDl- reductase. These findings underscore the importance of 1 lβ-HSD2 and 1 lβ-HSDl in regulating local concentrations of glucocorticoids in vascular tissue. They also indicate that decreased 11 β-HSD2 activity may be a possible mechanism in hypertension and other blood pressure associated disorders and that 1 lβ-HSDl -reductase may be a possible target for anti-hypertensive therapy.
The results of these examples underscore the importance of 1 lβ-HSD2 in regulating the access of glucocorticoids to GR and/or MR in vascular tissue and suggest that 1 lβ-HSDl -dehydrogenase may also play a role in protecting GR and MR in this tissue. In addition, they suggest that the antisense oligomers used in these experiments down-regulate 11 β-HSD gene expression and decrease glucocorticoid metabolism in vascular tissue, an effect leading to increased vascular responsiveness to catecholamines.
The examples also demonstrate that both 1 lβ-HSD2 and 1 lβ-HSDl regulate local glucocorticoid concentrations in vascular tissue with 1 lβ-HSD2 and 1 lβ-HSDl - dehydrogenase working to decrease- and 1 lβ-HSDl -reductase increase the amount of glucocorticoid that can access GR and MR in vascular smooth muscle. Physiological concentrations of both free corticosterone and 11 -dehydrocorticosterone are similar over the course of the day in rodents. Therefore, significant quantities of not only glucocorticoid, but also of 11-dehydro-glucocorticoid are available for conversion back to the glucocorticoid. Since glucocorticoids amplify catecholamine and angiotensin II pressor responses and may inhibit the effects of some vasorelaxant pathways, a possible mechanism that may increase vascular tone and induce hypertension includes a decrease in 1 lβ-HSD2 activity. Interestingly, many patients with essential hypertension also demonstrate decreased" 11 β-HSD2 activity as assessed by altered plasma and urinary cortisol/cortisone ratios. Moreover, the plasma half-life of 1 lα-3H-cortisol is prolonged in patients with essential hypertension consistent with the idea that 1 lβ-HSD2 activity is diminished in this condition. The present work also suggests that since 1 lβ-HSDl reductase generates active glucocorticoid in vascular tissue, a possible therapeutic target in the treatment of hypertension could be the specific inhibition of 1 lβ-HSDl reductase activity. EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.
All patents, patent applications, and literature references cited herein or in Appendix A are hereby expressly incorporated by reference.

Claims

1. A method for treating a glucocorticoid associated state in a subject, comprising administering to said subject an effective amount of a 11 β-HSD 1 reductase inhibitor, such that the glucocorticoid associated state is treated, wherein said 1 lβ-HSDl reductase inhibitor is 11-keto-testoterone, 11 -keto-androsterone, 11-keto-pregnenolone, 11-keto-dehydro-epiandrostenedione, 3α, 5α-reduced-l 1-ketoprogesterone, 3α, 5α- reduced-11-keto-testosterone, 3α, 5α-reduced-l 1-keto-androstenedione, 3α,5α- tetrahydro-11 β-dehydro-corticosterone, or a pharmaceutically acceptable prodrug or salt thereof
2. A method for treating a glucocorticoid associated state in a subject, comprising administering to said subject an effective amount of a 1 lβ-HSDl reductase inhibitor, such that the glucocorticoid associated state is treated, wherein said 1 lβ-HSDl reductase inhibitor is a nucleic acid.
3. The method of claim 1 or 2, wherein said glucocorticoid associated state is a blood pressure associated disorder.
4. The method of claim 3, wherein said blood pressure associated disorder is high blood pressure, congestive heart failure, chronic heart failure, left ventricular hypertrophy, acute heart failure, myocardial infarction, cardiomyopathy, or hypertension.
5. The method of claim 1 or 2, wherein said glucocorticoid associated state is obesity, diabetes mellitus, interocular pressure, lung disorder, or a neurological disorder.
6. The method of claim 5, wherein said neurological disorder is associated with glucocorticoid potentiated neurotoxicity.
7. The method of claim 2, wherein said nucleic acid is an antisense oligomer.
8. The method of claim 2, wherein said nucleic acid is an siRNA.
9. The method of claim 8, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
10. A method for treating a glucocorticoid associated state in a subject, comprising administering to said subject an effective amount of a 1 lβ-HSDl reductase inhibitor in combination with a 17α-hydroxylase inhibitor, 17-HSD inhibitor, 20α-reductase inhibitor, or a 20β-reductase inhibitor.
1 1. The method of claim 10, wherein said 1 lβ-HSDl reductase inhibitor is a selective l lβ-HSDl reductase inhibitor.
12. The method of claim 11, wherein said 1 lβ-HSDl reductase inhibitor is a steroid or a derivative thereof.
13. The method of claim 12, wherein said steroid is an 11-keto steroid.
14. The method of claim 13, wherein said 11-keto steroid is 11 -keto-progesterone, 11 -keto-testosterone, 11 -keto-androsterone, 11 -keto-pregnenolone, 11 -keto-dehydro- epiandrostenedione, or a pharmaceutically acceptable prodrug or salt thereof.
15. The method of claim 12, wherein said steroid is 3α, 5α-reduced.
16. The method of claim 15, wherein said steroid is 3α, 5α-reduced-l 1- ketoprogesterone, 3α, 5α-reduced-l 1-keto-testosterone, 3α, 5α-reduced-l 1-keto- androstenedione, 3α,5α-tetrahydro-l 1 -dehydro-corticosterone, 3α, 5α-reduced- l 1-keto- pregnenolone, 3α, 5α-reduced-l 1-keto-dehydro-epiandrostenedione or a pharmaceutically acceptable prodrug or salt thereof.
17. The method of claim 10, wherein said 1 lβ-HSDl reductase inhibitor is a nucleic acid.
18. The method of claim 17, wherein said nucleic acid is an antisense oligomer.
19. The method of claim 17, wherein said nucleic acid is an siRNA.
20. The method of claim 19, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
21. A method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 lβ-HSDl dehydrogenase inhibitor, such that the concentration of glucocorticoids in said tissue are increased, wherein said 1 lβ-HSDl dehydrogenase inhibitor is 3α, 5 α-reduced- 11 β-OH- progesterone, 3α, 5α-reduced-l 1 β-OH-testosterone, 3α, 5α-reduced-l lβ-OH- androstendione, 3α, 5α-reduced- 11 β-OH-pregnenolone, 3α, 5α-reduced-l lβ-OH- dehydro-epiandrostenedione, 3α, 5α-reduced-corticosterone, 3α, 5α-reduced- aldosterone, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-dehydro-epiandrostenedione, 3α, 5β-reduced-progesterone, 3α, 5β-testosterone, deoxy-corticosterone, 11 β-OH progesterone, 11 β-OH testosterone, 11 β-OH-pregnenolone, 11 β-OH-dehydro- epiandrostenedione, 3α, 5α-reduced-progesterone, 3α, 5α-reduced testosterone, 3α, 5α- reduced-chenodeoxycholic acid, or a pharmaceutically acceptable prodrug or salt thereof.
22. A method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 lβ-HSDl dehydrogenase inhibitor, such that the concentration of glucocorticoids in said tissue are increased, wherein said 11 β-HSD 1 dehydrogenase inhibitor is a nucleic acid.
23. The method of claim 21 or 22, wherein said tissue is said subject's liver, eye. lung, muscle, adipose tissue, nerve tissue, brain, or vascular tissue.
24. The method of claim 22, wherein said nucleic acid is an antisense oligomer.
25. The method of claim 22, wherein said nucleic acid is an siRNA.
26. The method of claim 25, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
27. A method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 lβ-HSDl dehydrogenase inhibitor in combination with a 17α-hydroxylase inhibitor, 17HSD inhibitor, 20α-reductase inhibitor or 20β-reductase inhibitor, such that the concentration of glucocorticoids in said tissue are increased.
28. The method of claim 27, wherein said 1 1 β-HSD 1 dehydrogenase inhibitor is a nucleic acid, 3α, 5β-reduced steroid or a 3α, 5α-reduced steroid.
29. The method of claim 28, wherein said steroid is 3α, 5α-reduced- 11 β-OH- progesterone, 3α, 5α-reduced- 11 β-OH-testosterone, 3α, 5α-reduced-l lβ-OH- androstendione, 3α, 5α-reduced-l l β-OH-pregnenolone, 3α, 5α-reduced-l lβ-OH- dehydro-epiandrostenedione, 3α, 5α-reduced-corticosterone, 3α, 5α-reduced- aldosterone, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-dehydro-epiandrostenedione, 11 β-OH progesterone, 11 β-OH testosterone, 11 β-OH-pregnenolone, 11 β-OH-dehydro- epiandrostenedione, 3α, 5α-reduced-progesterone, 3α, 5α-reduced testosterone, 3α, 5α- reduced-chenodeoxycholic acid, or a pharmaceutically acceptable prodrug or salt thereof.
30. A method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 lβ-HSD2 dehydrogenase inhibitor, such that the concentration of glucocorticoids in said tissue are increased, wherein said 1 lβ-HSD2 dehydrogenase inhibitor is a nucleic acid, 3α, 5α- reduced-1 1 β-OH-progesterone, 3α, 5α-reduced-l 1 β-OH-testosterone, 3α, 5α-reduced- 11 β-OH-androstenedione, 3α, 5α-reduced- 11 -keto-progesterone, 3α, 5α-reduced-l l- dehydro-corticosterone, 3α, 5α-reduced-corticosterone, 3α, 5α-aldosterone, 1 1 β-OH- progesterone, 11 β-OH-testosterone, 11 -keto-progesterone, 5α-dihydro-corticosterone, 5α-dihydro-corticosterone, 3α, 5α-reduced deoxy- corticosteroneor a pharmaceutically acceptable prodrug or salt thereof.
31. The method of claim 30, wherein said tissue is said subject's liver, eye, lung, muscle, adipose tissue, nerve tissue, brain, or vascular tissue.
32. A method for increasing the concentration of glucocorticoids in a tissue of a subject, comprising administering to a subject an effective amount of a 1 lβ-HSD2 dehydrogenase inhibitor in combination with a 17α-hydroxylase inhibitor, 17-HSD inhibitor, 20α-reductase inhibitor, or a 20β-reductase inhibitor, such that the concentration of glucocorticoids in said tissue are increased.
33. The method of claim 21, wherein said 11 β-HSD2 dehydrogenase inhibitor is a nucleic acid, 3α, 5α-reduced steroid, 11 β-OH-progesterone, 11 β-OH-testosterone, 11- keto-progesterone, or 5α-dihydro-corticosterone, or a pharmaceutically acceptable salt or prodrug thereof.
34. The method of claim 33, wherein said steroid is 3α, 5α-reduced-l 1 β-OH- progesterone, 3α, 5α-reduced- 11 β-OH-testosterone, 3α, 5α-reduced-l lβ-OH- androstenedione, 3α, 5α-reduced- 11 -keto-progesterone, 3α, 5α-reduced- 11 -dehydrocorticosterone, 3α, 5α-reduced-corticosterone, or 3α, 5α-aldosterone.
35. A method for treating hypertension in a subject, comprising administering to said 5 subject an effective amount of a 11 β-HSDl reductase inhibitor, such that said subject is treated, wherein said 1 lβ-HSDl reductase inhibitor is 11 -keto-progesterone, 11-keto- testosterone, 11 -keto-androsterone, 11-keto-pregnenolone, 11 -keto-dehydro- epiandrostenedione, 3α, 5α-reduced-l l -keto-progesterone, 3α, 5α-reduced-l 1-keto- testosterone, 3α, 5α-reduced-l 1-keto-androstenedione, 3α,5α-tetrahydro-l l-dehydro- 10 corticosterone, 3α, 5α-reduced-l 1-keto-pregnenolone, 3α, 5α-reduced-l 1 -keto-dehydro- epiandrostenedione or a pharmaceutically acceptable prodrug or salt thereof.
36. A method for treating hypertension in a subject, comprising administering to said subject an effective amount of a 1 lβ-HSDl reductase inhibitor, such that said subject is
15 treated, wherein said 1 lβ-HSDl reductase inhibitor is a nucleic acid.
37. The method of claim 36, wherein said nucleic acid is an antisense oligomer.
38. The method of claim 36, wherein said nucleic acid is an siRNA. z 0
39. The method of claim 38, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
40. A method for treating hypertension in a subject, comprising administering to said 25 subject an effective amount of a 11 β-HSDl reductase inhibitor in combination with a
17α-hydroxylase inhibitor, a 17-HSD inhibitor, a 20α-reductase inhibitor or a 20β- reductase inhibitor, such that said subject is treated.
41. The method of claim 40, wherein said 11 β-HSD 1 reductase inhibitor is a steroid 30 or a derivative thereof.
42. The method of claim 41, wherein said steroid is an 11-keto steroid.
43. The method of claim 42, wherein said 11-keto steroid is 1 1 -keto-progesterone, 35 11-keto-testosterone, 11 -keto-androsterone, 11-keto-pregnenolone, 11-keto-dehydro- epiandrostenedione, or a pharmaceutically acceptable prodrug or salt thereof.
44. The method of claim 41, wherein said steroid is 3α, 5α-reduced.
45. The method of claim 44, wherein said steroid is 3α, 5α-reduced-l 1 -keto- progesterone, 3α, 5α-reduced- 11 -keto-testosterone, 3α, 5α-reduced-l l-keto- androstenedione, 3α,5α-tetrahydro-l 1-dehydro-corticosterone, 3α, 5α-reduced-l 1-keto- pregnenolone, 3α, 5α-reduced-l l-keto-dehydro-epiandrostenedione or a pharmaceutically acceptable prodrug or salt thereof.
46. The method of claim 40, wherein said 1 lβ-HSDl reductase inhibitor is a nucleic acid.
47. The method of claim 46, wherein said nucleic acid is an antisense oligomer.
48. The method of claim 46, wherein said nucleic acid is an siRNA.
49. The method of claim 48, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
50. A method for increasing insulin sensitivity of a tissue in a subject, comprising administering an effective amount of a 1 lβ-HSDl reductase inhibitor to said subject, such that the insulin sensitivity of said tissue in said subject is increased, wherein said 1 lβ-HSDl reductase inhibitor is a nucleic acid. 1 1 -keto-progesterone, 1 1-keto- testosterone, 11 -keto-androsterone, 11 -keto-pregnenolone, 11 -keto-dehydro- epiandrostenedione, 3α, 5α-reduced-l 1-ketoprogesterone, 3α, 5α-reduced-l l-keto- testosterone, 3α, 5α-reduced-l 1-keto-androstenedione, 3α,5α-tetrahydro-l 1-dehydro- corticosterone, 3α, 5 α-reduced-11-keto-pregnenolone, and 3α, 5α-reduced-l 1-keto- dehydro-epiandrostenedione or a pharmaceutically acceptable prodrug or salt thereof.
51. The method of claim 50, wherein said tissue is said subject's liver, muscle, nerve or adipose tissue.
52. The method of claim 50, wherein said nucleic acid is an antisense oligomer.
53. The method of claim 50, wherein said nucleic acid is an siRNA.
54. The method of claim 53, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
55. The method of any one of claims 1, 35, 36 or 40, wherein said subject is a human.
56. The method of any one of claims 1, 35, 36 or 40, further comprising administering a pharmaceutically acceptable carrier.
57. A pharmaceutical composition comprising an effective amount of
11 β-OH-progesterone, 11 β-OH-testosterone, 3α,5 β-reduced- 11 β-OH-progesterone, 3α,5β-reduced-l 1 β-OH-testosterone, chenodeoxycholic acid, 3α, 5β-reduced- pregnenolone, 3α, 5β-reduced-dehydro-epiandrostenedione, 3α,5α-reduced- 11 β-OH- progesterone, 3α,5α-reduced-l 1 β-OH-testosterone, 3α,5α-reduced-l 1 β-OH- androstenedione, 11 -keto-progesterone, 11 -keto-testosterone, 11-keto-androstenedione, 3α,5α-reduced-l 1 -keto-progesterone, 3α,5α-reduced-l 1-keto-testosterone, 3α,5α- reduced-11-keto-androstenedione, 3α,5α-tetrahydro-l 1-dehydro-corticosterone, 3α,5α- reduced-corticosterone, 5α-dihydro-corticosterone, 3α, 5α-reduced-l l β-OH- pregnenolone, 3α, 5α-reduced-l lβ-OH-dehydro-epiandrostenedione, 11 β-OH- pregnenolone, Uβ-OH-dehydro-epiandrostenedione, 3α, 5α-reduced-pregnenolone, 3α, 5α-reduced-dehydro-epiandrostenedione, 3α, 5α-reduced aldosterone, or a pharmaceutically acceptable salt or prodrug thereof, in combination with a 17α- hydroxylase inhibitor, a 17-HSD inhibitor, a 20α-reductase inhibitor, or a 20β-reductase inhibitor.
58. A composition comprising a l l β-HSDl reductase inhibitor, wherein said 11 β- HSDl reductase inhibitor is an siRNA.
59. The composition of claim 58, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 1.
60. The composition of claim 58, wherein said siRNA comprises an antisense strand having the sequence of SEQ ID. No. 2.
61. A composition comprising an 1 lβ-HSD2 dehydrogenase inhibitor, wherein said 1 lβ-HSD2 dehydrogenase inhibitor is an siRNA.
62. The composition of claim 61, wherein said siRNA has an antisense strand which is complementary to at least a portion of SEQ ID No. 3.
63. The composition of claim 61, wherein said siRNA comprises an antisense strand having the sequence of SEQ ID. No. 4.
64. A pharmaceutical composition comprising the composition of any one of claims 58-63 and a pharmaceutically acceptable carrier.
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US7345058B2 (en) 2005-04-05 2008-03-18 Hoffmann-La Roche Inc. Pyrazoles
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WO2008109531A3 (en) * 2007-03-02 2009-02-05 Ndrna Inc Nucleic acid compounds for inhibiting hsd11b1 expression and uses thereof
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US8377923B2 (en) 2008-07-03 2013-02-19 Astellas Pharma Inc. Triazole derivative or salt thereof
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AU2002361861A1 (en) 2003-07-30
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WO2003059267A3 (en) 2007-02-08
EP1587574A2 (en) 2005-10-26

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