US20040102425A1 - Selective glucocorticoid receptor agonists - Google Patents

Selective glucocorticoid receptor agonists Download PDF

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US20040102425A1
US20040102425A1 US10/415,711 US41571103A US2004102425A1 US 20040102425 A1 US20040102425 A1 US 20040102425A1 US 41571103 A US41571103 A US 41571103A US 2004102425 A1 US2004102425 A1 US 2004102425A1
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compound
formula
cells
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Alexander Barr
Michael McKervey
Hughes Miel
David Ray
Andrew Brass
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MATRIX THERAPEUTICS Ltd
University of Manchester
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Priority claimed from GB0115161A external-priority patent/GB0115161D0/en
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Publication of US20040102425A1 publication Critical patent/US20040102425A1/en
Assigned to MATRIX THERAPEUTICS LTD. reassignment MATRIX THERAPEUTICS LTD. A CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNOR NAME ON REEL 014811 FRAME 0266 Assignors: BRASS, ANDREW MICHAEL, RAY, DAVID WILLIAM, BARR, STEPHEN ALEXANDER, MCKERVEY, MICHAEL ANTHONY, MIEL, HUGHES JEAN-PIERRE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
    • C07J5/0046—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa
    • C07J5/0061—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa substituted in position 16
    • C07J5/0092—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa substituted in position 16 by an OH group free esterified or etherified
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J21/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having an oxygen-containing hetero ring spiro-condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J21/001—Lactones
    • C07J21/003—Lactones at position 17
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J3/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by one carbon atom
    • C07J3/005—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by one carbon atom the carbon atom being part of a carboxylic function
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • C07J41/0011—Unsubstituted amino radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0038—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 with an androstane skeleton, including 18- or 19-substituted derivatives, 18-nor derivatives and also derivatives where position 17-beta is substituted by a carbon atom not directly bonded to a further carbon atom and not being part of an amide group

Definitions

  • Glucocorticoids have been used therapeutically for many years to treat inflammation, as immunosuppressants and in the treatment of lymphoproliferative disorders and certain other malignancies 1;2 . These compounds are the most potent and effective anti-inflammatory agents known.
  • the use of GCs has been limited by their multiple and often devastating side effects.
  • This invention concerns improvements to GCs such that the therapeutic effects of the selected agonists is maintained but the potential for the compounds to cause harmful side-effects is significantly reduced.
  • GCs were first introduced into medicine in the 1950's. Early enthusiasm for these drugs, due to success in controlling inflammation in a wide range of diseases, was soon tempered by the realisation that these compounds cause a very wide range of side effects that were often serious, often irreversible and in many cases more serious than the inflammatory condition under treatment.
  • the diseases in which GCs have been shown to have a pronounced anti-inflammatory effect include inflammatory arthritides such as rheumatoid arthritis, ankylosing spondylitis and psoriatic arthropathy, other rheumatoid diseases such as systemic lupus erythematosis, scleroderma, vasculitides including temporal arteritis and polyarteritis nodosa, inflammatory bowel disease such as Crohns disease and ulcerative colitis, lung diseases such as asthma and chronic obstructive airways disease, as well as many other conditions such as polymyalgia rheumatica.
  • inflammatory arthritides such as rheumatoid arthritis, ankylosing spondylitis and psoriatic arthropathy
  • other rheumatoid diseases such as systemic lupus erythematosis, scleroderma, vasculitides including temporal arte
  • GCs have also been used very extensively for their immunosuppressive properties in the prevention and treatment of transplant rejection. Finally GCs have been used for their anti-tumour effects in a number of malignancies. The activity of GCs is in the treatment of lymphoproliferative and other malignances is thought to be due to the ability of GCs to induce apoptosis 3;4 .
  • GCs particularly in inflammatory disease
  • a number of approaches have been taken to overcome the side effects of the drugs.
  • the most frequently adopted approach has been to apply the steroid locally to the site of inflammation.
  • Target organs where this approach has been adopted include the lung with oral inhalation for the treatment of asthma and chronic obstructive airways disease; the nose with local installation for the treatment of allergic rhinitis; the eye with local installation for the treatment of a number of serious inflammatory eye conditions such as uveitis; in large joints with intra-articular injection of steroids to treat inflammation; and on the skin for the treatment of eczema, psoriasis and a range of other conditions of the skin.
  • Soft steroids such as fluticasone for topical application. These soft steroids are inactivated rapidly by metabolism following absorption into the systemic circulation thus minimising systemic side effects. Local application of soft steroids, however, is still associated with significant local side effects such as skin thinning. Soft steroids are of no use when systemic administration of the drug is required in diseases such as temporal arteritis or polymyalgia rheumatica.
  • Osteoporosis growth impairment; avascular osteonecrosis; proximal myopathy; impaired glucose tolerance or frank diabetes; fluid retention and oedema; hypertension; hypokalaemia; Cushingoid faces; weight gain; obesity; euphoria; psychosis; insomnia; raised intracranial pressure; aggravation of epilepsy; memory impairment; hippocampal atrophy; peptic ulceration; pancreatitis; suppression of the hypothalamic pituitary axis; raised introcular pressure; glaucoma; papiloedema; skin thinning; reduced resistance to infection; impaired wound healing.
  • GCs act via specific glucocorticoid receptors (GR), members of the nuclear receptor superfamily. Hormone binding promotes receptor dimerisation, DNA binding, and transcriptional activation. This mechanism of GC action is well-defined in-vitro, and is critical for regulation of the hypothalamic-pituitary-adrenal axis and gluconeogenesis in-vivo 5-8 . Hormone bound receptor is also able to influence gene transcription in a dimerisation-independent manner by interfering with the activity of transcription factors, such as AP-1 and NFkB, which are critically involved in the inflammatory reaction.
  • transcription factors such as AP-1 and NFkB
  • the induction of apoptosis in T lymphocytes may be important to the immunosuppressive activity of GCs.
  • this same mechanism may be important in the anti-inflammatory effects of GCs with the deletion of clonogenic memory T cells responsible for the induction of a response to an antigen 14 .
  • the inflammatory processes may, at least in part, depend on failure of apoptosis in inflammatory cells. This has been shown to be the case with neutrophils in inflammatory bowel disease 15 .
  • the GR migrates from the cytoplasm of the cell to the nucleus, and binds to glucocorticoid response elements in the regulatory region of target genes.
  • the activated GR then recruits co-factors, including the glucocorticoid receptor interacting protein 1 (GRIP-1) and steroid receptor coactivator 1 (SRC1). These accessory proteins bind to the receptor and link the GR with the general transcription machinery 16-22 .
  • GRIP-1 glucocorticoid receptor interacting protein 1
  • SRC1 steroid receptor coactivator 1
  • Glucocorticoid effects on transcription may be mediated by both the direct binding of activated GR to target DNA, homodimerisation and recruitment of co-activators but also by GR interfering with other transcription factor function, including AP-1, NFkB and NUR77 23-31 . These two modes of receptor activity are dissociable, that is negative effects on NFkB activity retained but with loss of transactivation. It appears that this second mechanism is largely responsible for mediating the therapeutically desirable anti-inflammatory activity of the GR 28;31-33 .
  • cytokines expressed at the site of inflammation may induce relative glucocorticoid resistance, possibly by activating AP-1 or NFkB 19;23;31;34-37 . This is of importance as the pro-inflammatory cytokines signal by activation of NFkB, and the majority of the anti-inflammatory actions of GCs are thought to be mediated by opposing NFkB action.
  • a method for treating an inflammatory condition, treating haematological and other malignancies, causing immunosuppression or preventing or treating transplant rejection in man or other animals which comprises administering to a patient a compound that has the structure of Formula I or Formula II as defined below, or a pharmaceutically acceptable derivative thereof or pro-drug therefor
  • R NH 2 , NHR 1 , NHOR 2 , NHNHR 2 ⁇ , NHCOR 2 ,
  • R 1 C (1-4) alkyl, C( 3-6) cycloalkyl,
  • R 2 methyl,ethyl
  • R 3 alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl, aryl, heteroaryl, substituted aryl, or substituted hetreoaryl;
  • R 4 ,R 5 C (1-4) alkyl.
  • alkyl groups of any of any of R 1 to R 5 may be straight or branched chain.
  • R 6 and R 7 are any of H, CH 3 CO, CH 3 CH 2 CO, CH 3 CH 2 CH 2 CO provided that R 6 and R 7 are not both H,
  • R 8 and R 9 are any of H, CH 3 CO, CH 3 CH 2 CO or CH 3 CH 2 CH 2 CO.
  • pro-drugs are useful as pro-drugs.
  • pro-drug is meant a compound that undergoes a chemical conversion to become an active drug when metabolised by the body. This normally results in increased drug effectiveness through an increase of absorption by the body, a prolongation of the duration of action in the body or through a reduction of certain side effects.
  • a compound according to Formula I or II as defined above or a pharmaceutically acceptable derivative thereof or pro-drug therefor, or a compound according to Formula III or IV, for use as a medicine.
  • These compounds are particularly effective in the treatment of an inflammatory condition, for treating haematological and other malignancies, causing immunosuppression or the prevention or treatment of transplant rejection.
  • a fourth aspect of the present invention there is provided the use of a compound according to Formula I or II as defined above, or a pharmaceutically acceptable derivative thereof or pro-drug therefor, or a compound according to Formula III or IV, in the manufacture of a medicament for the treatment of an inflammatory condition, for treating haematological and other malignancies, causing immunosuppression or preventing or treating transplant rejection.
  • a pharmaceutical composition which comprises a compound according to Formula I or II as defined above, or a pharmaceutically acceptable derivative thereof or pro-drug therefor, or a compound according to Formula III or IV, and a pharmaceutically acceptable carrier.
  • a composition has particular use in the treatment of an inflammatory condition, for treating haematological and other malignancies, causing immunosuppression or the prevention or treatment of transplant rejection.
  • the sixth aspect of the present invention is directed to the use of such a composition in the manufacture of a medicament for the treatment of an inflammatory condition, for treating haematological and other malignancies, causing immunosuppression or preventing or treating transplant rejection.
  • a method of inducing apoptosis in target cells which comprises administering to the target cells or to the vicinity in which the target cells are located a Compound according to Formula I or II as defined above or a pharmaceutically acceptable derivative thereof or pro-drug therefore, or a compound according to Formula III or IV.
  • the eighth aspect of the present invention is directed to the use of compound according to Formula I or II as defined above or a pharmaceutically acceptable derivative thereof or pro-drug therefor, or a compound according to Formula III or IV, in the manufacture of a medicament for inducing apoptosis in target cells.
  • the target cells are generally pro-inflammatory cells or malignant cells.
  • this aspect of the invention is useful for treating inflammation and to treat haematological and other malignancies.
  • the preferred compounds of Formula I used according to the invention include those wherein R is NHR 1 and R 1 is methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl or
  • R 3 is straight or branched chain alkyl or substituted alkyl, preferably C (1-6) alkyl or substituted alkyl, most preferably propyl or hexyl, or where R 3 is a benzyl or a substituted benzyl group, preferably halobenzyl, most preferably fluorobenzyl, or R 3 is the substituted alkyl group
  • Preferred compounds having a structure according to Formula II include those where R 4 and/or R 5 is methyl and/or ethyl.
  • Examples of the particularly preferred compounds used according to the present invention, in addition to the novel compounds of Formula III and IV, are designated as “Compound A”, “Compound G” and “Compound H” “Compound I”, “Compound K”, “Compound M”, “Compound N,” Compound S”, “Compound T”, “Compound 2”, “Compound 3”, “Compound 4”, “Compound 5” and “Compound 7”, the structures of which are shown in FIGS. 2A and 2B of the accompanying drawings.
  • the most preferred compound used in accordance with the present invention is Compound G in view of its superior potency and selectivity for transrepression over transactivation.
  • compositions according to the invention may take a number of different forms depending, in particular on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, liposome or any other suitable form that may be administered to a person or animal.
  • vehicle of the composition of the invention should be one which is well tolerated by the subject to whom it is given and enables delivery of the compounds to the target tissue.
  • the compounds used according to this invention may be administered by any route including but not limited to oral inhalation, local installation into the nose or eye, local injection into joints or muscles, orally in the form of capsules, tablets, liquids or suspensions, by injection into a vein, muscle or under the skin and topically to the skin.
  • the vehicle will usually form from 0.1% to 99.9%, preferably 90% to 99.9% by weight of the composition and can, in the absence of other adjuncts, form the balance of the composition.
  • A. Compound G 500 micorgram tablets A. Compound G 500 micorgram tablets Compound G 0.5% w/w Lactose Ph Eur 89.5% w/w Povidone (K25) Ph Eur 5.0% w/w Collloidal Silicon Dioxide Ph Eur 1.0% w/w Purified Water Ph Eur 20% w/w* Magnesium Stearate Ph Eur 1.0% w/w Sodium Starch Glycollate Ph Eur 3.0% w/w
  • the tertiary structure GR ligand binding domain (LBD) containing 251 amino acids (Q527-K777) was predicted using SWISS-MODEL. The result included structural alignment against templates and 3D co-ordinates. It used the 1.8 ⁇ PR LBD dimer (1A28) as a template and the identity between these two LBDs is >50%.
  • the ligand binding domain of the human GR is outlined by helices 5,7,11 and 12, the ⁇ turn and loops L6-7 and L11-12.
  • the ligand binding pocket is predicted to be lined by 18 aminoacids. Of these 15 are predicted to contribute to the hydrophobic environment of the pocket: Met 560, Leu 563, Leu566, Gly567, Trp600, Met601, Met 604, Ala605, Leu608, Phe623, Met 646, Leu732, Tyr735, Thr739, Phe749.
  • the ligand interaction with the receptor was detected using the Ligplot programme, which was also used to plot the interaction. Based on this analysis we predict that the ligand binding domain of the hGR has a three-layered, antiparallel, 12 ⁇ helical structure, which is highly homologous to that of the PR.
  • the ligand is bound by three hydrogen bonds; Arg611 and Gln570 interact with the ketone group on C3 of the steroid A ring, and Cys736 to the ketone group on C20 of the D ring. A further 15 aminoacids are found to contribute to hydrophobic interactions with the ligand.
  • Arg611, Gln570 and Cys736 are conserved residues whose importance for ligand binding has been verified by studying either natural or engineered mutants.
  • the aromatic ring of Tyr735 appeared to form a hydrophobic interaction with the ligand.
  • the distance from the aromatic ring to the C22 substituent of the steroid D ring was calculated to be 2.98 A, and to the D ring 3.98 A.
  • the hydoxyl group of tyrosine 735 was orientated away from the ligand binding pocket.
  • Tyr735 was predicted to have hydrophobic interaction with the D ring of Dexamethasone it was important to identify changes in ligand binding affinity caused by the mutations at position 735.
  • Site-directed mutagenesis to phenylalanine (Tyr735phe) resulted in no alteration in ligand binding affinity (4.3 nM compared to 4.6 nM for wild-type), in keeping with the hypothesis that the benzene ring is sufficient to generate a hydrophobic surface for ligand interaction.
  • Change to valine (Tyr735val) resulted in lower affinity binding compared to wild-type, but only to a minor degree (6 nM).
  • the change to serine (Tyr735ser), however, resulted in a two-fold reduction in ligand binding affinity (10.4 nM).
  • the physiological ligand hydrocortisone (100 nM) induced 18 fold induction of MMTV via the wild-type GR, 11 fold with the Tyr735Phe, 10.6 fold with the Tyr735Val and failed to induce the reporter via the Tyr735Ser.
  • the mutated receptors have the same rank order of activity with both agonist ligands.
  • Tyr735Ser had a minor, but consistent, increase in IC50 for this effect, with no suppression at 0.1 nM Dexamethasone. These data are compatible with the observed Kd for binding to Dexamethasone, and show the dose-response curve for transrepression to be left-shifted in comparison with transactivation. In contrast to the transactivation data Tyr735Phe and Tyr735Val performed similarly to the wild-type GR, showing that the substitution of Tyr735 results in selective impairment of transactivation in the absence of significant changes in ligand binding affinity and transrepression. Tyr735Ser has a slightly higher IC50 for transrepression compared to the other three GR molecules examined, compatible with the observation that its affinity for Dex is reduced. However, at 100 nM Dex Tyr735Ser has achieved maximal suppression which is close to that observed with the wild-type GR, in striking contrast to the results seen on transactivation.
  • the COS 7 cell line (ECACC:87021302) is derived from transformed kidney cells of the African green monkey ( Cercopithecus aethiops ). This cell line is deficient in endogenous GR and is therefore used widely as a tool to study GR function by controlled artificial expression via transfection.
  • the A549 cell line (ECACC:86012804) is derived from the human lung epithelial cells from a carcinoma. This cell line contains functional endogenous GR and is stimulated by Tumour Necrosis Factor (TNF) to produce IL-8.
  • TNF Tumour Necrosis Factor
  • the HEP G2 cell line (ECACC: 85011430) is derived from human liver epithelial cells from a well differentiated hepatocellular carcinoma. This cell line has functional endogenous GR and is stimulated by GR activation to produce tyrosine amino transferase.
  • the CEM-C7A cell line is a steroid sensitive clone of the CCRF-CEM cell line (ECACC 85112105). This is a T lymphoblastoid cell line with functional endogenous GR.
  • COS 7 cells and A549 cells were cultured in DMEM with Glutamax (Gibco BRL, Paisley, UK), and 10% fetal calf serum (FCS).
  • HEP G2 were cultured in DMEM with Glutamax (Gibco BRL), 1% non-essential amino acids (Gibco BRL) and 10% FCS.
  • CEM-C7A cells were cultured in Optimem (Gibco BRL) with 5% FCS.
  • the firefly luciferase gene catalyses the production of light from the substrate luciferin.
  • the activity of the gene can be assessed by the intensity of the luminesence.
  • the luciferase assay yields luminescence through an ATP-dependent oxidation of luciferin the amount of light produced can be measured by a luminometer and is a measure of gene activity (Davis, 1996).
  • Light intensity was measured in a Luminometer (LB 9501, Berthold). The light intensity should be proportional to luciferase concentration in the range of 10 ⁇ 16 M (10 pg/L) to 10 ⁇ 8 M (1 mg/L).
  • the GR was artificially expressed in a cell line lacking endogenous GR and a glucocorticoid response element (GRE) “driving” a luciferase reporter gene was used to measure ligand dependent activation.
  • GRE glucocorticoid response element
  • COS7 cells were transfected with 1 ⁇ g pcDNA3-GR and 3 ⁇ g AH3-Luc per 10 cm tissue culture dish. The cells were plated out in to 24 well plates after transfection and the steroids were added immediately. After 18 hour incubation the cells were lysed and a luciferase assay was performed.
  • SRC-1 is an important co-activator of GR action that associates with the GR in a ligand dependant manner.
  • the ability of SRC-1 to associate with the GR in this manner can be used as a measure of potency of ligand mediated transactivation.
  • plasmids that generated VP16-GR ligand binding domain (LBD) and GAL4-SRC-1 fusion proteins and a GAL4 binding luciferase reporter gene construct were transfected into in a cell line lacking GR and deficient in endogenous SRC-1.
  • the GAL4SRC-1 fusion protein could bind to specific sites upstream of the luciferase reporter gene construct.
  • This reaction also requires pyridoxal phosphate (PLP) as a co-factor.
  • PBP pyridoxal phosphate
  • Strong alkali conditions can then be used to convert pHPP to p-hydroxybenzaldehyde (pHBA) and oxalate.
  • pHBA p-hydroxybenzaldehyde
  • the amount of pHBA can then be assessed by measuring its absorbance at 331 nm (extinction coefficient 19,900M ⁇ 1 cm ⁇ 1 ) this figure is then assumed to be directly proportional to TAT activity.
  • HEPG2 cells were cultivated till confluent on a 10 cm plate ( ⁇ 10 7 cells). These cells were then scraped off and suspended in 500 ⁇ l of ice cold 0.14M KCl.
  • the cells were lysed by 3 cycles of 10 second burst sonication at full power (50 W, 20 kHz Sonicator; Jencons, Leighton Buzzard, UK). The cells were kept on ice between each burst. The debris in the lysed cell suspension was then spun down (13000 rpm, 20 mins; microfuge) and the protein concentration of the supernatant was assayed using the Bradford method. 100 ⁇ g of protein was then used to assay the TAT activity.
  • GR and p65 were artificially expressed in a cell line lacking GR and deficient in endogenous P65.
  • the ability of P65 to activate an NFkB response element (NRE) “driving” a luciferase reporter gene could then be repressed by GR in a ligand dependent manner and, therefore, used to measure transrepression.
  • NRE NFkB response element
  • COS7 cells were transfected with 1 ⁇ g pcDNA3-GR and 3 ⁇ g NRE-Luc and 200 ng p65 per 10 cm tissue culture dish. The cells were plated out in to 24 well plates after transfection and left for 18 hours to recover before the steroids of FIGS. 1 and 2A were added. After another 18 hour incubation the cells were lysed and a luciferase assay was performed.
  • TNF was used to stimulate NFkB activity in a cell line that expressed endogenous GR.
  • the ability of NFkB to activate an NRE “driving” a luciferase reporter gene could then be repressed by GR in a ligand dependent manner and, therefore, used to measure transrepression.
  • COS7 cells were transfected with 3 ⁇ g NRE-Luc per 10 cm tissue culture dish. The cells were plated out in to 24 well plates after transfection and left for 18 hours to recover before the steroids of FIGS. 1 and 2 were added. Next TNF was added to the samples after 3 hours incubation with the steroids and after another 15 hours the cells were lysed and a luciferase assay was performed.
  • TNF was used to stimulate NFkB activity in a cell line that expressed endogenous GR.
  • the ability of NFkB to induce IL-8 gene transcription could then be repressed by GR in a ligand dependent manner and, therefore, used to measure transrepression.
  • COS7 cells were transfected with 3 ⁇ g NRE-Luc per 10 cm tissue culture dish. The cells were plated out in to 24 well plates after transfection and left for 18 hours to recover before the steroids of FIGS. 1 and 2A were added. After incubation for 3 hours with the steroids, TNF was added to the samples and after another 15 hours an IL-8 assay (R&D systems) was performed on the cell supernatants according to the manufacturers instructions.
  • R&D systems IL-8 assay
  • triplicates of 100 ⁇ l of a 1:2 dilution of cell supernatant and 100 ⁇ l of a known dilution series of IL-8 were added to the wells of a microtitre plate coated with a monoclonal IL-8 antibody, the samples were incubated with a polyclonal biotinylated IL-8 antibody for 1 hour at room temperature and washed. Streptavidin horse radish peroxidase solution was then added to all wells and incubated for 30 mins before being washed.
  • Tetramethyl benzidine (TMB; a chromagen) was added to all the wells and incubated in the dark for 10 mins, the reaction was stopped by the addition of H 2 SO 4 and the absorbance of each well was read on a spectrophotometer using 450 nm as the primary wavelength. Regression analysis was performed on the standard data and the unknowns were expressed as pg/ml of IL-8 in comparison to the standards.
  • the un-liganded GR is present in the cytoplasm of cells in association with heat shock proteins (HSPs).
  • HSPs heat shock proteins
  • a type I antagonist ligand can bind to the GR but cannot translocate to the nucleus.
  • a type II antagonist ligand can bind to the GR, translocate to the nucleus and bind to DNA but cannot activate gene transcription. Therefore, the ability of a putative ligand to translocate the GR helps to determine its status as an agonist or antagonist.
  • COS 7 cells were transfected with 5 ⁇ g of a green fluorescent protein (GFP) tagged GR expression vector.
  • GFP green fluorescent protein
  • these cells were split into 6 well plates with sterile microscope cover slips placed at the bottom of each well. The cells were allowed to recover for 18 hours and then the medium was removed, the cells were washed three times with sterile phosphate buffered saline (PBS) and new medium was added containing charcoal stripped FCS in place of normal FCS. The cells were incubated for 18 hours and then the steroids of FIGS. 1 and 2 A were added to make a final concentration of 100 nM. The cells were incubated for a further 3 hours and then the medium was aspirated and the cells were fixed to the glass coverslips by adding ice cold methanol and incubating at ⁇ 20° C. for 30 mins.
  • PBS sterile phosphate buffered saline
  • the coverslips with the transfected cells fixed to them were mounted on slides and the cellular localisation of the GFP tagged GR was analysed by fluorescent microscopy using an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and KS300 v3.0 analysis software (Zeiss).
  • a type II antagonist should be able to compete agonist activity. As an antagonist has no ability to transactivate, competition can be measured by inhibition of an agonist in a transactivation assay.
  • RU486 is a type II antagonist of GR function and can compete against the agonist dexamethasone for ligand binding, this interaction can be used as a positive control for this analysis.
  • the GR was artificially expressed in a cell line lacking endogenous GR and a GRE “driving” a luciferase reporter gene was used to measure ligand dependent activation.
  • COS7 cells were transfected with 1 ⁇ g pcDNA3-GR and 3 ⁇ g AH3-Luc per 10 cm tissue culture dish. The cells were plated out in to 24 well plates after transfection and the steroids were added immediately. All wells except the negative control were treated with 50 ⁇ M dexamethasone to generate a final concentration of 100 nM. Certain steroids from FIG. 2A were added at the same concentration to all sample wells. After 18 hour incubation the cells were lysed and a luciferase assay was performed.
  • the PR was artificially expressed in a cell line lacking endogenous PR and a GRE “driving” a luciferase reporter gene was used to measure ligand dependent activation.
  • COS7 cells were transfected with 1 ⁇ g pSEO-PRB and 3 ⁇ g AH3-Luc per 10 cm tissue culture dish. The cells were plated out in to 24 well plates after transfection and the steroids of FIGS. 1 and 2A were added immediately to generate a final concentration of 100 nM. After 18 hour incubation the cells were lysed and a luciferase assay was performed.
  • Apoptosis (programmed cell death) is mediated by a protein called Bax the transcription of which is primarily controlled by the GR in a ligand dependant manner.
  • Bax the transcription of which is primarily controlled by the GR in a ligand dependant manner.
  • a suitable cell line was incubated with ligand and a viable cell count was performed after three days.
  • CEM-C7A cells were plated out on to a 96 well plate at 5 ⁇ 10 5 /well.
  • the steroids of FIGS. 1 and 2A were added immediately to generate a final concentration of 100 nM.
  • the live cell number in each well was assessed using Celltiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). This assay is a colorimetric method for determining the number of viable cells in proliferation or chemosensitivity assays.
  • the assay is composed of solutions of a novel tetrazolium compound (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS) and an electron coupling reagent (phenazine methosulfate; PMS).
  • MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium.
  • the absorbance of the formazan at 490 nm can be measured directly from 96 well assay plates without additional processing.
  • the conversion of MTS into aqueous, soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells.
  • the quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
  • Batch 2 Compounds G to T inclusive.
  • dexamethasone the structure of which is shown in FIG. 1, was included as an active control.
  • FIGS. 11 and 12 The ability of the compounds of FIG. 2A to transrepress TNF activation in A549 cells is shown in FIGS. 11 and 12.
  • the ability of compound G to repress TNF activation of A549 cells was examined using an NRE reporter gene and 1, 10, 100 and 500 nM of compound. It was found that compound G repressed at least as well as dexamethasone and also had a “left shifted” response with greater maximal repression. Compounds A D and H were also assessed in this assay. All compounds exhibited transrepression.
  • FIGS. 13 and 14 The ability of the compounds of FIG. 2A to transrepress IL-8 activation in A549 cells is shown in FIGS. 13 and 14.
  • the ability of 100 nM compound G to repress TNF activation of A549 cells was also examined using IL-8 production as an endogenous end point. It was found that compound G repressed IL-8 production better than dexamethasone (p 0.0076, two tailed t-test). Compounds A, D and H exhibited transrepression in this assay.
  • CEM C7A cells are a human T lymphoblast cell line which undergoes apoptosis in response to glucocorticoid.
  • the exact mechanism is uncertain, but likely relates to the lymphocytolytic action of glucocorticoid in vivo, and may model aspects of glucocorticoid mediated anti-inflammation.
  • Dexamethasone and compound A were the most potent inducers of apoptosis.
  • RU24858 impaired proliferation but did not induce significant apoptosis (FIG. 20). We have extensive previous data using RU24858, and it consistently fails to induce apoptosis.
  • Binding of compound G to the glucocorticoid receptor was investigated using a standard competition assay. The conditions were as follows: Source Human HeLa 53 cells Ligand 6 nM 3 H Dexamethasone Vehicle 0.4% DMSO Incubation time/temp 2 hours @25° C. Incubation buffer RPMI 1640, 10 nM Hepes, pH 7.2 Non-specific ligand 20 ⁇ M Dexamethasone Kd 5 nM B max 61000 R/cell Specific binding 75% Quantitation method Radiological binding Significance criteria >50% of max stimulation or inhibition.
  • IC 50 values were determined by a non-linear, least squares regression.
  • the K i values were calculated using the equation of Cheng and Prusoff using the observed IC 50 of the test compound, the concentration of radioligand employed in the assay, and the historic values for the K d for the ligand.
  • Compound G bound to the glucocoticoid receptor.
  • the binding curve was right shifted when compared to dexamethasone. This was a surprising finding as, in the transrepression assays, Compound G was at least as effective as dexamethasone. It is possible to speculate that the binding in this assay is more reflective of the transactivational activity of the compounds than the transrepressive activity of the compounds.
  • Compound G was also shown not to bind to the progesterone receptor in a classic receptor binding assay (data not shown). Compound G was also shown not to bind to other members of the steroid receptor super family. Interestingly, in a classic receptor binding assay (FIG. 19), Compound G appeared to be a less potent ligand than dexamethasone for the GR so the findings in both the in-vitro and in-vivo assays were surprising, with Compound G showing the same or greater activity in transrepression.
  • mice Male outbred Swiss albino mice (18-20 g body weight) were purchased from Bantin and Kingman (T.O. strain; Hull, Humberside) and maintained on a standard chow pellet diet with tap water ad libitum and a 12:00 h light/dark cycle. All animals were housed for 3 days prior to experimentation to allow body weight to reach 20-22 g. On the day of experiment mice ranged from 24-26 g.
  • Air-pouches were formed on the back of mice by air injection (2.5 ml s.c.) on day 0 and day 3 (Perretti and Flower, 1993).
  • mice were subjected to vehicle (300 ⁇ l, groups A and D), or treated with dexamethasone (3 ⁇ g, group B) or compound G (3 ⁇ g, group C) at ⁇ 1 h injected intra-peritoneal (i.p.), group E was left untreated.
  • Compound G and Dexamethasone were prepared by diluting stock solutions in DMSO in paraffin oil (final DMSO concentration, less than 0.01%).
  • Zymosan A was allowed to defrost at room temperature for 30 min prior to being added to sterile PBS at a concentration of 2 mg/ml forming a homogenous suspension.
  • mice received either 0.5 ml of zymosan (1 mg) (group A, B, C) injected locally into the air-pouch. Group D and E were left untreated at this time-point. At 4 h post-zymosan the animals were placed in CO 2 . Air-pouches were then washed with 2 ml of PBS containing 3 mM EDTA, and livers were recovered and snap frozen for later analysis.
  • the number of migrated leukocytes was determined in the lavage fluid from each air-pouch, by taking an aliquot (100 ⁇ l) of the lavage fluid and diluting 1:10 in Turk's solution (0.01% crystal violet in 3% acetic acid). The samples were then vortexed and 10 ⁇ l of the stained cell solution were placed in a Neubauer haematocymometer and neutrophils numbers counted using a light microscope (Olympus B061).
  • TAT Tyrosine Amino Transferase
  • This reaction also requires pyridoxal phosphate (PLP) as a co-factor. Strong alkali conditions can then be used to convert pHPP to p-hydroxybenzaldehyde (pHBA) and oxalate. The amount of pHBA can then be assessed by measuring its absorbance at 331 nm (extinction coefficient 19,900M ⁇ 1 cm ⁇ 1 ) this figure is then assumed to be directly proportional to TAT activity. Livers were homogenized and the cells were stored on ice. The debris in the lysed cell suspension was then spun down (13000 rpm, 20 mins; microfuge) and the protein concentration of the supernatant was assayed using the Bradford method. 100 ⁇ g of protein was then used to assay the TAT activity.
  • PRP pyridoxal phosphate
  • Dexamethasone and compound G caused a significant reduction ( ⁇ 30%) of neutrophil accumulation.
  • the TAT assays showed that all animals that had received the drug vehicle ip had elevated levels of TAT compared to the untreated controls. This was due to the stress associated with an inflammatory reaction induced by the vehicle and the associated raised endogenous glucocorticoid levels. Dexamethasone caused further elevation of TAT, unlike compound G.
  • This experiment therefore shows that compound G and dexamethasone have an equivalent anti-inflammatory effect when administered at the same dose and that compound G, unlike dexamethasone, does not cause induction of the gluconeogenic enzyme, tyrosine amino transferase, at this dose. This proves that in-vivo compound G, and thus other compounds in the series shown to have selectivity in the in-vitro assays, will have the desired reduction of side effects that result from transactivation.
  • the selected series of dexamethasone analogues of the present invention having a structure according Formula I, II, III and IV as defined hereinbefore exhibit selectivity for transrepression over translocation.
  • the selected series of compounds of the present invention clearly bind to the glucocorticoid receptor and do not cause transactivation or cause significantly less transactivation than dexamethasone at concentrations which inhibit the inflammatory process. This is classically a situation where one would expect to see an antagonist activity. This is surprisingly not evident.
  • Addison's Disease which is characterised by reduced blood pressure, hypokalaemia; coma, reduced resistance to stress and infection.
  • Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 1997;90:569-80.

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4996335A (en) * 1980-07-10 1991-02-26 Nicholas S. Bodor Soft steroids having anti-inflammatory activity
US5767113A (en) * 1995-05-10 1998-06-16 The Salk Institute For Biological Studies Compounds useful for concurrently activating glucocorticoid-induced response and reducing multidrug resistance

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NL7502252A (nl) 1974-02-27 1975-08-29 Pierrel Spa Werkwijze voor het bereiden van een geneesmid- del met anti-inflammatoire werking, gevormd ge- neesmiddel verkregen volgens deze werkwijze alsmede werkwijze voor het bereiden van in het geneesmiddel gebruikte nieuwe steroiden.
US6024957A (en) 1993-06-02 2000-02-15 Research Corporation Technologies, Inc. Immunomodulators and methods for the prevention and reversal of organ transplant rejection using same
TW408127B (en) * 1993-09-17 2000-10-11 Glaxo Inc Androstenones
US5965434A (en) 1994-12-29 1999-10-12 Wolff; Jon A. Amphipathic PH sensitive compounds and delivery systems for delivering biologically active compounds
US5981517A (en) * 1996-05-09 1999-11-09 Soft Drugs, Inc. Androstene derivatives
CA2261666C (en) 1996-07-30 2010-09-14 Novartis Ag Pharmaceutical compositions for the treatment of transplant rejection, autoimmune or inflammatory conditions comprising cyclosporin a and 40-0-(2-hydroxiethyl)-rapamycin
US6235891B1 (en) * 1999-03-31 2001-05-22 South Alabama Medical Science Foundation Glucocorticoid receptor agonist and decreased PP5
US6753329B2 (en) 2001-12-03 2004-06-22 Gpc Biotech Inc. Inhibitors of cyclin-dependent kinases, compositions and uses related thereto

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* Cited by examiner, † Cited by third party
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US4996335A (en) * 1980-07-10 1991-02-26 Nicholas S. Bodor Soft steroids having anti-inflammatory activity
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