WO2007093840A2 - Utilisation de conjugués spécifiques de cellule pour traiter des maladies inflammatoires des voies gastro-intestinales - Google Patents

Utilisation de conjugués spécifiques de cellule pour traiter des maladies inflammatoires des voies gastro-intestinales Download PDF

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WO2007093840A2
WO2007093840A2 PCT/IB2006/001488 IB2006001488W WO2007093840A2 WO 2007093840 A2 WO2007093840 A2 WO 2007093840A2 IB 2006001488 W IB2006001488 W IB 2006001488W WO 2007093840 A2 WO2007093840 A2 WO 2007093840A2
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compound
formula vii
disease
inflammatory
group
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PCT/IB2006/001488
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WO2007093840A3 (fr
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Mladen Mercep
Milan Mesic
Linda Tomaskovic
Stribor Markovic
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Glaxosmithkline Istrazivacki Centar Zagreb D.O.O.
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Publication of WO2007093840A3 publication Critical patent/WO2007093840A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to the use for the treatment and prevention of inflammatory diseases, disorders, and conditions of the gastrointestinal tract in a subject of a conjugate or its pharmaceutically acceptable salt, prodrug or solvates which has low bioavailability when administered orally or when otherwise delivered to the gastrointestinal mucosa of a conjugate of (i) a macrolide subunit which preferentially accumulates in immune system cells and (ii) an anti-inflammatory drug.
  • the anti-inflammatory drugs can be selected from one or more of anti-inflammatory corticosteroids or nonsteroidal antiinflammatory drugs (NSAIDs).
  • NSAIDs nonsteroidal antiinflammatory drugs
  • the low orally-bioavailable conjugate compounds of the invention are represented by formula VII below.
  • this invention relates to the long-term maintenance treatment of inflammatory bowel diseases which are in remission totally or partially and which respond to treatment with glucocorticoids or NSAIDs (e.g., Crohn's disease, ulcerative colitis, and celiac disease).
  • glucocorticoids or NSAIDs e.g., Crohn's disease, ulcerative colitis, and celiac disease.
  • IBD Human inflammatory bowel disease
  • Crohn's disease is a chronic inflammatory disease of unknown etiology that can affect any part of the bowel. Although lesions may start superficially, the inflammatory process extends thorough the bowel wall to draining lymph nodes. The course of the disease may be relapsing or continuous, mild or severe. It cannot be cured by resection of the involved segment of the bowel. Most patients with Crohn's disease undergo surgery at some point, but subsequent relapse is common and continuous medical treatment is the norm.
  • Ulcerative colitis is also a chronic inflammatory disease of unknown etiology but it affects only the large bowel and is limited to the bowel mucosa except in very severe cases. The course of this disease may also be relapsing or continuous, mild or severe. It can be cured by total colectomy, which may be necessary for acute severe disease or chronic unremitting disease cases. Most patients with ulcerative colitis are managed medically rather than surgically.
  • glucocorticoid steroids are the treatment of choice, but only until remission is achieved, after which they should be discontinued. If the disease does not satisfactorily remit, glucocorticoids are used to maintain control of symptoms. Sulfasalazine is used in less severe cases, especially if the disease involves the colon. For symptomatic treatment of Crohn's disease, analgesics for pain and opiates for diarrhea control are also used. However, many patients eventually require surgery.
  • glucocorticoid steroids prednisolone, prednisolone acetate or budesonide
  • Talder CJ and GN Tytgat are used for the treatment of acute attacks of ulcerative colitis.
  • New drugs have recently been developed like 5-amino- salicylic acid or compounds having a 5-aminosalicylic acid moiety, and are as effective as sulfasalazine but without the side effects associated with sulfapyridine class of drugs.
  • these new drugs do have their own side effects, such as diarrhea (Ardizzone S and GB Porro, Drug Saf. 2002, 25:561-82).
  • Celiac disease is a chronic intestinal disease that represents reaction to gluten present in wheat and rye proteins, and leads to changes in the small intestinal mucosa and impaired absorption of nutrients in patients who do not tolerate gluten.
  • IBD IBD
  • it has certain symptomatology in common, e.g., local infiltration of inflammatory cells, malabsorption and malnutrition due to colonic tissue damage, elevated mediators of inflammation in the local tissue.
  • Many patients today are treated with a gluten- free diet high in proteins and normal in fat. However, some patients do not respond to such treatment. These patients are treated with glucocorticoids such as hydrocortisone, prednisone or prednisolone.
  • M represents a macrolide subunit possessing the property of accumulation in inflammatory cells
  • A represents an anti-inflammatory subunit that can be steroid or nonsteroid
  • L represents a linker molecule linking M and A
  • M represents a macrolide subunit possessing the property of accumulation in inflammatory cells
  • A represents an anti-inflammatory subunit that can be steroid or nonsteroid
  • L represents a linker molecule linking M and A
  • M represents a macrolide subunit possessing the property of accumulation in inflammatory cells
  • A represents an anti-inflammatory subunit that can be steroid or nonsteroid
  • L represents a linker molecule linking M and A
  • M represents a macrolide subunit (macrolide moiety) derived from macrolide possessing the property of accumulation in inflammatory cells
  • S represents a steroid subunit derived from a steroid drug with anti-inflammatory activity
  • L represents a linker molecule linking M and S to their pharmaceutically acceptable salts and solvates processes and intermediates for their preparation and to their use in the treatment of inflammatory diseases and conditions in humans and animals.
  • M represents a macrolide subunit (macrolide moiety) derived from macrolide possessing the property of accumulation in inflammatory cells
  • V represents an antiinflammatory steroid or non steroid subunit or an anti neoplastic or antiviral subunit
  • L represents a linking group covalently linking M and V to their pharmaceutically acceptable salts and solvates processes and intermediates for their preparation and to their use in the treatment of inflammatory diseases and conditions in humans and animals.
  • M represents a macrolide subunit (macrolide moiety) derived from macrolide possessing the property of accumulation in inflammatory cells
  • D represents a nonsteroidal subunit (nonsteroidal moiety) derived from a nonsteroid drug with anti-inflammatory, analgesic and/or antipyretic activity (NSAID)
  • L represents a linking group covalent linking M and D to their pharmaceutically acceptable salts and solvates processes and intermediates for their preparation and to their use in the treatment of inflammatory diseases and conditions in humans and animals.
  • WO 04/005310 A2 describes conjugate steroid-macrolide compounds of Formula II having the steroid subunit linked through the 17 ⁇ -0H group with the macrolide subunit at the position C/ 11 or N/9a of a macrolide having a modified or eliminated dimethylamino group of desosamine sugar; and also conjugate steroid-macrolide compounds with the macrolide subunit linked to the steroid through position C/6 of the macrolide lactonic ring, or through position C/4" of the cladinose sugar, or through the amine functionality at position C/3' of the desosamine sugar.
  • M represents a macrolide subunit (macrolide moiety) derived from macrolide possessing the property of accumulation in inflammatory cells
  • D represents a nonsteroidal subunit (nonsteroidal moiety) derived from a non-steroid drug with anti-inflammatory, analgesic and/or antipyretic activity (NSAID)
  • L represents a linking group covalently linking M and D; (b) their pharmacologically acceptable salts, prodrugs and solvates, (c) processes and intermediates for their preparation, and (d) their use in the treatment of inflammatory diseases and conditions in humans and animals.
  • Patent Application Serial Number PCT/IB2005/003213 herein incorporated by reference in its entirety describes yet further conjugate compounds having a steroid or non-steroidal anti-inflammatory subunit Z linked via the chain L to position 11 of an aglycone type macrolide subunit and further conjugate compounds having a aglycone type macrolide subunits M linked via the chain L to position 17alpha of a steroid subunit.
  • conjugates having low bioavailability when administered by oral or enteral route where the conjugates comprises (i) an immune cell specific macrolide compound combined with (ii) an anti-inflammatory corticosteroids or nonsteroidal anti- inflammatory drugs (NSAIDs) and pharmaceutically acceptable salts, prodrugs and solvates thereof for the maintenance treatment and prevention of recurrence of inflammatory diseases, disorders, and conditions of the gastrointestinal tract has hitherto not been described.
  • an immune cell specific macrolide compound combined with (ii) an anti-inflammatory corticosteroids or nonsteroidal anti- inflammatory drugs (NSAIDs) and pharmaceutically acceptable salts, prodrugs and solvates thereof for the maintenance treatment and prevention of recurrence of inflammatory diseases, disorders, and conditions of the gastrointestinal tract has hitherto not been described.
  • NSAIDs nonsteroidal anti- inflammatory drugs
  • the present invention is directed to methods for the prevention of recurrence and for the treatment (including not only acute-phase treatment but also maintenance treatment) of inflammatory diseases, disorders, and conditions of the gastrointestinal tract by administering to a patient in need of such treatment a conjugate compound according to Formula VII, or pharmaceutically acceptable salts, prodrugs, or solvates thereof having low oral bioavailability:
  • M represents a macrolide subunit possessing the property of accumulation in inflammatory cells
  • T represents an anti-inflammatory subunit that can be a steroid or a nonsteroid (nonsteroidal moiety) derived from a non-steroid drug with anti-inflammatory, analgesic and/or antipyretic activity (NSAID)
  • L represents a linker covalently linking M and T, and continuing said administration into or through the maintenance phase of treatment of such disorders.
  • NSAID conjugates of the present invention by not being substantially absorbed are suitable for long term maintenance therapy in preventing or delaying relapse.
  • the modified steroid conjugates of the present invention are also not substantially absorbed, unlike standard glucocorticoids, and thus they show systemic effect and be appropriate for long term therapy and thus useful for maintenance therapy to prevent or delay relapse.
  • the present compounds are also less likely to damage the intestinal mucosa in a way characteristic of NSAIDs especially those used in long-term therapy, which are known to cause gastrointestinal ulceration and renal and respiratory toxicity.
  • the undesirable side effects of unmodified NSAIDs have been attributed to the inhibition of prostaglandins in the affected organ, i.e., the gastrointestinal mucosa.
  • the present application provides bioavailability data for compounds 2', 3', 4', 9', 10', and 11' as well as the bioavailability for the use of compounds 1 and 5 and DNBS induced distal colitis for compounds 1, 2, and 3.
  • Fig. IA is a bar graph showing macroscopic damage score after administration to a rat model of compounds 1', 2' and 3' or budesonide (positive control) or vehicle (negative control) to measure damage to the colon induced by TNBS. Significance of results was analyzed using Mann- Whitney test.
  • Fig. IB is a bar graph showing histological damage score after administration to a rat model of compounds 1', 2' and 3' or budesonide (positive control) or vehicle (negative control) to measure damage to the colon induced by TNBS. Significance of results was analyzed using Mann-Whitney test.
  • Fig. 2 is a bar graph showing macroscopic damage score after administration of varying dosages of compound 1' or budesonide (positive control) or vehicle (negative control) to measure damage to the colon induced by TNBS. Significance of results was analyzed using Mann- Whitney test.
  • Fig. 3 is a bar graph showing the colonic macroscopic damage score/colonic ulceration for Compounds 1, 2, and 3 as well as for a native control, vehicle control, sulfasalazine, and budesonide.
  • the present invention solves the problem of effective prevention and treatment of inflammatory diseases, disorders, and conditions of the gastrointestinal tract, including, but not limited to, irritable bowel disease (IBD) by use of conjugate compounds of the Formula VII that exhibit low bioavailability when administered orally.
  • the compounds of Formula VII that exhibit low oral bioavailability exhibit fewer side effects than standard glucocorticoids, since they are not absorbed from the gastrointestinal tract to systemic circulation. This is an advantage over the use of standard steroids.
  • the low orally- bioavailable M-L-T conjugates of Formula VII of the present invention reach the inflamed site in the bowel where they exert an anti-inflammatory effect, but they do not cause systemic side effects since they are not absorbed.
  • the conjugates of the Formula IV which exhibit low oral bioavailability can safely be used not only in acute phases of the disease but also, and more importantly, during maintenance therapy of IBD, when the use of conventional steroids and conjugates of high oral bioavailability would not be indicated or would not be treatment of choice because of the long duration of the therapy.
  • the maintenance phase of therapy begins when the disease is in partial or total remission.
  • Conventional therapy of acute phase such as acute relapse lasts 4-6 weeks if 5- aminosalicylic acid and its derivatives is used as the therapeutic agent, and 2-3 weeks if steroids like prednisone or hydrocortisone are used.
  • Responsiveness to treatment is ascertained by laboratory tests (like stool examination and blood analysis) so effectiveness is ascertained after treatment has been initiated. Unconjugated NSAIDs are not indicated for IBD.
  • the present invention provides a method for the prevention and treatment of inflammatory diseases, disorders, and conditions of the gastrointestinal tract, which comprises administering to a patient in need thereof an effective amount of a conjugate compound of Formula VII that exhibits low bioavailability when administered by the oral route, or a pharmaceutically acceptable salt, prodrug or solvate thereof.
  • the present invention also provides a method for the prevention or delay of recurrence (including maintenance phase treatment) of inflammatory bowel diseases which respond to treatment with glucocorticoids, such as, but not limited to, Crohn's disease, ulcerative colitis, and celiac disease, by administering to a patient an effective amount of a conjugate compound of the Formula VII which exhibits low oral-bioavailability, or a pharmaceutically acceptable salt, prodrug or solvate thereof.
  • glucocorticoids such as, but not limited to, Crohn's disease, ulcerative colitis, and celiac disease
  • the conjugate compound of Formula VII, or salt, prodrug, or solvate thereof exhibits less than 10% oral bioavailability, i.e., between about 0% and about 10%. In another embodiment of the present invention the conjugate compound of Formula VII, or salt, prodrug, or solvate thereof exhibits less than 5% oral bioavailability, i.e., between about 0% and about 5% .
  • the conjugate compound of Formula VII, or salt, prodrug, or solvate thereof exhibits less than 2% oral bioavailability, i.e., between about 0 % and about 2% .
  • the present invention is also directed to pharmaceutical compositions comprising at least one compound selected from conjugates of the general Formula VII which exhibit low oral-bioavailability, and pharmaceutically acceptable salts, prodrugs or solvates thereof, and a pharmaceutically acceptable carrier.
  • the composition comprises at least one compound selected from the group of conjugates of Formula VII which exhibits less than 10% oral bioavailability. In another specific embodiment, the composition comprises at least one compound selected from the group of conjugates of Formula VII which exhibits oral bioavailability of less than 5% . In a further specific embodiment, the composition comprises at least one compound selected from the group of conjugates of Formula VII which exhibit oral bioavailability of less than 2% .
  • a particular characteristic of this conjugates is their low bioavailability, which is due to very high molecular mass of such molecules (> 700). According to the "rule of five" (Lipinski CA. et al., Adv. Drug. Deliv. Rev. , 2001, 46:3-26) which is followed by majority of drugs, molecular mass of orally bioavailable drugs should be less then 500. There are few successful orally active drugs that fail to fit this rule. Additionally, bioavailability of conjugates of Formula VII could be estimated on the basis of Caco-2 in vitro model which is accepted as fairly good model for prediction of oral bioavailability (Yee S and W.W. Day, Applications of Caco-2 cells in drug discovery and development.
  • the invention provides an oral pharmaceutical composition which comprises at least one compound selected from the group of conjugates of Formula
  • the low orally-bioavailable conjugates of Formula VII, and pharmaceutically acceptable salts, prodrugs, and solvates thereof, are represented as shown below:
  • M represents a macrolide subunit selected from the group consisting of 12-, 14-, 15-, 16-, 17-, and 18-membered lactonic ring molecules wherein “membered” refers to the number of carbon atoms or heteroatoms in the lactonic ring said macrolide having the property of accumulating within mammalian immune system cells that mediate inflammatory immune responses
  • T represents an anti-inflammatory subunit that can be a steroid or nonsteroid (nonsteroidal moiety) derived from a non-steroid drug with antiinflammatory, analgesic and/or antipyretic activity (NSAID)
  • L represents a linker covalently linking M and T.
  • this invention relates to the use of compounds, represented by the Formula VII, and salts, prodrugs and solvates thereof, wherein M specifically represents a 14- or 15-member lactonic ring macrolide subunit most preferably represented by the Formula VIII:
  • Rt and Rs independently are H or alkyl (preferably methyl or H);
  • RM is OH, OR P , alkoxy or substituted alkoxy (in either Syn or Anti configurations or mixtures thereof)
  • RN is H, R p , alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, or
  • X is O or S
  • U and Y are independently H, halogen, alkyl, or hydroxyalkyl (preferably H, methyl, or hydroxymethyl);
  • S 1 is H or a sugar moiety at position C/5 (e.g., a desozamine group) of the formula:
  • R 8 and R 9 are both hydrogen or together form a bond, or R 9 is hydrogen and R 8 is -N(CHs)R y , wherein
  • R 10 is hydrogen or R p ;
  • S 2 is H or a sugar moiety (e.g., is a cladinosyl group) of the formula
  • R 2 is H, hydroxy, OR P group, alkoxy (preferably C1-C4 alkoxy, most preferably methoxy) or substituted alkoxy;
  • A is H or methyl;
  • (ix) E is H or halogen (preferably fluorine);
  • R 3 is hydroxy, OR P group or alkoxy (preferably C1-C4 alkoxy, most preferably methoxy), substituted alkoxy or R 3 is a group that can combine with R 5 to form a
  • NR N "bridge” e.g., a cyclic carbonate or carbamate
  • R 3 is a group that can combine with W or Z to form a "bridge” (e.g., a cyclic carbamate)
  • R 4 is C1-C4 alkyl (preferably methyl);
  • R 5 is H, hydroxy, OR P group, C1-C4 alkoxy, substituted alkoxy or a group that may combine with R 3 to form a bridge (e.g., a cyclic carbonate or carbamate);
  • R 6 is H or C1-C4 alkyl (preferably methyl or ethyl);
  • the subunit M has a linkage site through which it is linked to the subunit T via the linking group L, the linkage site being at one or more of the following:
  • R p groups may be independently present in the macrolide subunit of Formula VIII, wherein R p represents a protective group which may be selected from alkyl (preferably methyl), alkanoyl (preferably acetyl), alkoxycarbonyl (preferably methoxycarbonyl or tert-butoxycarbonyl), arylmethoxycarbonyl (preferably benzyloxycarbonyl), aroyl (preferably benzoyl), arylalkyl (preferably benzyl), alkylsilyl (preferably trimethylsilyl) or alkylsilylalkoxyalkyl (preferably trimethylsilylethoxymethyl).
  • R p represents a protective group which may be selected from alkyl (preferably methyl), alkanoyl (preferably acetyl), alkoxycarbonyl (preferably methoxycarbonyl or tert-butoxycarbonyl), arylmethoxycarbonyl (preferably benzyloxycarbonyl), aroyl (preferably benzoyl),
  • R 1 is hydroxyl and S 1 is a sugar moiety of the formula (R ⁇ , R ⁇ , R ⁇ are as previously defined):
  • aglycone compounds according to formula VIII is wherein S 1 is hydrogen and R 1 is hydroxyl.
  • the Linker L is
  • L can be selected to be a linking group represented by the Formula IXA or IXB:
  • Q is -NH- or -CH 2 -;
  • linking group is preferred not only for conjugates of NSAIDS and macrolides of Formula VIII but for any conjugate within Formula VII.
  • Other linking groups can be used as long as they provide the necessary spacer.
  • Preferred linker molecules are those having a length of 1-20 carbon atoms (not counting heteroatoms in the chain) and those having a length of 2-10 carbon atoms are most preferred.
  • the llinkage of L to the macrolide moiety is effected either through the ring nitrogen atom at position 9a or through the hydroxy group at position 11 and position 6 or through the 2' hydroxy or the 3' amino group of the desosamine sugar moiety or through the 4" hydroxy group of the cladinose sugar or if the M moiety is aglycone or missing one of the desosamine and cladinose sugar groups, through the OH group created at position 5 or 3 of the macrolide ring.
  • the linker can serve to link one subunit of the Formula VII, e.g., M, with another subunit of Formula VII, e.g., T, as is well-known in the art.
  • T represents a nonsteroidal subunit derived from a nonsteroidal anti-inflammatory drug (NSAID) or a steroid subunit that can be represented by the substructure IX:
  • NSAID nonsteroidal anti-inflammatory drug
  • R a and R b are, independently of each other, hydrogen or halogen
  • R c is hydroxy, alkoxy (preferably methoxy), substituted alkoxy, alkyl, thiocarbamoyl, carbamoyl or a valence-bond attached to X 2 of chain L;
  • R d and R e are, independently of each other, hydrogen, hydroxy, methyl or C1-C4 alkoxy (preferably methoxy or n-propoxy) or each are a group that forms a 1,3- dioxolane ring with the other (optionally alkyl or alkenyl mono-or di-substituted) (preferably a 2,2-dimethyl or 2-monopropyl or trans-propenyl ring) or a valence bond attached to X 2 of chain L;
  • R j is hydrogen or chlorine
  • steroids useful as a source of steroid subunits in the present invention include, but are not limited to, corticosteroids (such as glucocorticoids and mineralocorticoids) and androgens.
  • corticosteroids such as glucocorticoids and mineralocorticoids
  • androgens Non-limiting examples of corticosteroids include
  • nonsteroidal subunits denotes subunits derived from nonsteroidal anti-inflammatory drugs (NSAIDs) and other drugs with antiinflammatory, anti-allergic and immunosuppressive properties, such as aceclofenac, acemetacin, acetaminophen, acetaminosalol, acetyl-salicylic acid, acetyl-salicylic-2-amino- 4-picoline-acid, 5-aminoacetylsalicylic acid, alclofenac, amino-profen, amfenac, anileridine, azathioprine, bendazac, benoxaprofen, bermoprofen, ⁇ -bisabolol, bromfenac, 5-bromosalicylic acid acetate, bromosaligenin, bucloxic acid, butibufen, carprofen, CC 1088 (Celgene), CC 5013 (Celgene), CDC 80
  • NSAIDs non
  • Preferred NSAIDs are acetyl salicylic acid, indomethacin, naproxen, ibuprofen, flurbiprofen, ketoprofen, sulindac, etodolac, ketorolac, suprofen, flunixin, sodium diclofenac, flufenamic acid, theophylline, meclofenamic acid, mefenamic acid and tolmetin.
  • the compounds of Formula VII may be obtained in the following way: one end of the chain L is first linked to the macrolide subunit M, and then the other end of the chain is linked to the subunit T; or, one end of the chain L is first linked to the subunit T and then the other end of the chain to the macrolide subunit M, or finally, one moiety of the chain L is linked to the macrolide subunit M, whereas the other moiety of the chain is linked to the subunit T, with the ends of the chain parts being then chemically linked to form the chain L.
  • acyl groups such as alkanoyl, alkoxycarbonyl and aroyl groups, may be removed by solvolysis, e.g., by hydrolysis under acidic or basic conditions.
  • Arylmethoxycarbonyl groups may be cleaved by hydrogenolysis in the presence of a catalyst such as palladium-on-charcoal .
  • conjugate compounds within Formula VII can be prepared by the processes set forth in International Publication Nos. WO 02/055531, WO 04/005310, and WO 04/005309, and in Wegn Patent Application HR 20030324 (and its U.S. counterpart, U.S. Published Application 2005 008003), each of which is incorporated herein by reference in its entirety.
  • Alkyl means a linear or branched saturated monovalent hydrocarbon radical of one to ten carbon atoms, more preferably one to six carbon atoms
  • the preferred straight-chain or branched-chain alkyls include methyl, ethyl, propyl, iso-p ⁇ opyl, butyl, sec-butyl and tert-butyl. Methyl is most preferred.
  • Alkyl groups may be substituted with one up to five substituents including halogen (preferably fluorine or chlorine), hydroxy, alkoxy (preferably methoxy or ethoxy), acyl, acylamino cyano, amino, N-(Cl- C4)alkylamino (preferably N-methylamino or N-ethylamino), N,N-di(Cl-C4-alkyl)amino (preferably dimethylamino or diethylamino), aryl (preferably phenyl) or heteroaryl, thiocarbonylamino, acyloxy, amino, amidino, alkyl amidino, thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aryl, heteroaryl, aryloxy, aryloxyaryl, nitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, carboxyl- cycl
  • Alkenyl means a linear or branched monovalent hydrocarbon radical of two to ten and preferably two to six carbon atoms which has at least one double carbon- carbon bond. Alkenyl groups may be substituted with the same groups as alkyl and such optionally substituted alkenyl groups are encompassed within the term "alkenyl.” Ethenyl, propenyl, butenyl and cyclohexenyl are preferred.
  • Alkynyl means a linear or branched monovalent hydrocarbon radical, having a straight-chain or a branched-chain of two to ten, and preferably two to six carbon atoms and containing at least one and preferably no more than three triple carbon-carbon bonds.
  • Alkynyl groups can be substituted with the same groups as alkyl, and the substituted groups are within the present definition of alkynyl. Ethynyl, propynyl and butynyl groups are preferred.
  • Cycloalkyl means a cyclic group having 3-8 carbon atoms having a single ring optionally fused to an aryl or heteroaryl group.
  • the cycloalkyl groups can be substituted as specified for "aryl” below, and the substituted cycloalkyl groups are within the present definition of "cycloalkyl”.
  • Preferred cycloalkyls are cyclopentyl and cyclohexyl.
  • Aryl means an unsaturated aromatic carbocyclic group having 6-14 carbon atoms having a single ring such as phenyl or multiple fused rings such as naphthyl.
  • Aryl may optionally be further fused to an aliphatic or aryl group or can be substituted with one or more substituents such as halogen (fluorine, chlorine and/or bromine), hydroxy, Ci-C? alkyl, C1-C7 alkoxy or aryloxy, C1-C7 alkylthio or arylthio, alkylsulfonyl, cyano or primary or nonprimary amino.
  • Heteroaryl means a monocyclic or a bicyclic aromatic hydrocarbon ring having from 2 to 10 carbon atoms and from 1 to 4 heteroatoms, such as O, S or N.
  • the heteroaryl ring may optionally be fused to another heteroaryl ,aryl or aliphatic cyclic group. Examples of this type are furan, thiophene, pyrrole, imidazole, indole, pyridine, oxazole, thiazole, pyrrole, pyrazole, tetrazole, pyrimidine, pyrazine and triazine, with fiiran, pyrrole, pyridine and indole being preferred.
  • the term includes groups that are substituted with the same substituents as specified for aryl above.
  • Heterocyclic means a saturated or unsaturated group having a single or multiple rings and from 1 to 10 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulphur or oxygen, wherein in a fused ring system the oilier ring or rings can be aryl or heteroaryl. Heterocyclic groups can be substituted as specified for alkyl groups and the thus substituted heterocyclic groups are within the present definition.
  • Halogen means a halogen atom which may be: flourine, chlorine or bromine (most preferably fluorine or chlorine)
  • salts can include acid addition salts or addition salts of free bases.
  • acids which may be employed to form pharmaceutically acceptable acid addition salts include but are not limited to salts derived from nontoxic inorganic acids such as nitric, phosphoric, sulfuric, or hydrobromic, hydroiodic, hydrofluoric, phosphorous, as well as salts derived from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy 1 alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and acetic, maleic, succinic, or citric acids.
  • Non-limiting examples of such salts include napadisylate, besylate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like.
  • salts of amino acids such as arginate and the like and gluconate, galacturonate (see, for example, Berge S. M. et al. "Pharmaceutical Salts,” J. of Pharma. ScL, 1977; 66:1).
  • the acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner.
  • the free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner.
  • the free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.
  • Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid.
  • compositions of the invention refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., human).
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in mammals, and more particularly in humans.
  • carrier applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which an active compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. However, since memantine is highly soluble, aqueous solutions are preferred. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin, 18th Edition.
  • the present invention are carriers suitable for immediate-release, i.e., release of most or all of the active ingredient over a short period of time, such as 60 minutes or less, and make rapid absorption of the drug possible.
  • the present invention also encompasses prodrugs of the Formula VII compounds, i.e., compounds which release an active parent drug according to Formula (VII) in vivo when administered to a mammalian subject.
  • Prodrugs of a compound of Formula VII are prepared by modifying functional groups present in the compound of Formula VII in such a way that the modifications may be cleaved in vivo to release the parent compound.
  • Prodrugs include compounds of Formula VII wherein a hydroxy, amino, or carboxy group of a Formula VII compound is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino or carboxy group, respectively.
  • Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives) of compounds of Formula VII or any other derivative which upon being brought to the physiological pH or through enzyme action is converted to the active parent drug.
  • the present invention also encompasses solvates of the compounds . of Formula VII or their salts.
  • Preferred solvates are hydrates.
  • the compounds of Formula VII have one or more chirality centers and, depending on the nature of individual substituents, they can also have geometrical isomers. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has a chiral center, a pair of enantiomers is possible.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R-- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomer respectively).
  • a chiral compound can exist as either an individual enantiomer or as a mixture of enantiomers. A mixture containing equal proportions of the enantiomers is called a "racemic mixture".
  • the present invention encompasses all individual isomers of compounds of Formula VII.
  • the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. Methods for the determination of stereochemistry and the resolution of stereoisomers are well-known in the art.
  • the present invention also encompasses stereoisomers of the syn-anti type, and mixtures thereof encountered when an oxime or similar group is present.
  • the group of highest Calm Ingold Prelog priority attached to one of the terminal doubly bonded atoms of the oxime, is compared with hydroxyl group of the oxime.
  • a “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable excipient” as used in the present application includes both one and more than one such excipient.
  • Low orally-bioavailable and “low oral bioavailability” means that the conjugate of Formula VII exhibits a bioavailability after its administration by oral, enteral or other mucosal delivery that results in absorption of less than 10% of the administered drug through the gastric mucosa into the blood or plasma, preferably less than 5%, and most preferably less than 2% .
  • Oral bioavailability is preferably measured through comparing the bioavailability of the compound after oral administration with the bioavailability after intravenous administration of the drug. Equations used to calculate pharmacokinetic parameters including oral bioavailability are as follows:
  • AUC area under the curve
  • Vd The apparent volume of distribution at the terminal phase based on drug concentration in plasma. This is calculated as:
  • Vd Dose/ AUC»* ⁇ z, normalized by animal weight
  • the oral bioavailability can be estimated if a compound displays low AUC, as defined above.
  • a compound with an estimated oral bioavailability of less than 10% will have an AUC of less than 0.5 ⁇ g*h/mL. None of the compounds possessing bioavailability less then 10%, thus entering the scope of the present invention have AUC values above 0,5 ⁇ g*h/mL.
  • molecules defined by Formula VII, having an AUC below 0.5 ⁇ g*h/mL will have a bioavailability of less than 10% .
  • Treating” or “treatment” of a state, disorder or condition includes:
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • ulcerative colitis e.g. ulcerative colitis or Crohn's disease
  • Responsiveness of a subject to a treatment is assessed by whether a selected drug used in the acute phase causes the reduction of one or more clinical signs and symptoms described below.
  • preventing is used with reference to maintenance therapy for the prevention of recurrence of a symptom for the disease or any measure of inflammation of the colon, such a marker for inflammation.
  • prevention can be demonstrated in animals that spontaneously develop IBD (e.g. IL-IO deficient mice, TNF ⁇ ARE or SAMPl/Yit mice) and includes the avoidance or the delay of occurrence of disease in treated animals (compared to untreated controls).
  • An example of "relieving" a subclinical symptom is the observation in a treated individual of abatement in the number of immune cells that secrete pro inflammatory cytokines or lymphokines or a decrease in the mRNA encoding such lymphokines or cytokines.
  • Mainntenance therapy is therapy during a phase of the disease, disorder or condition following the achievement of remission (total or partial) of one or more symptoms of the disease until the next flare-up of the disease.
  • Partial remission is the disappearance or alleviation of one or more of the symptoms normally associated with the disease state.
  • the hallmarks of the acute phase include symptoms like nausea, diarrhea, vomiting, fever, abdominal tenderness, pain, cramps, in some cases anemia and malnutrition signs. Anal fistulas can appear.
  • Stools can be bloody or occult bleeding can occur and be determined on assay.
  • White blood cells are moderately elevated, sedimentation rate is often elevated and can be used to monitor the transition from active to remission phase.
  • Hypokalemia, hypoalbuminemia, and hypocalcemia can occur during acute phase.
  • X-ray examination of abdomen and barium enema are used to find lesions in mucosa and inflamed tissue; CT scans and ultrasound can be used for the same purpose.
  • Maintenance therapy starts in the moment when abnormal symptoms previously determined to be present return to normal values.
  • Acute phase therapy usually lasts from 2 to 6 weeks, depending on the patient, and the therapy used .
  • Length of maintenance treatment comprising administration of the compounds of the present invention during maintenance phase typically lasts from the induction of remission until the appearance of disease flare-up or indefinitely if disease is controlled. Perhaps it could be possible to discontinue the administration of the compounds of the present invention after several years of adequate disease control but signs of disease reappearance should carefully be observed.
  • "Responder" refers to a patient that has previously responded to a treatment for ulcerative colitis or Crohn's disease involving administration of a particular active agents (or combination of active agents) in particular amount or amounts
  • Patient refers to mammals, preferably humans or domestic animals, more preferably humans.
  • a “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a state, disorder or condition, is sufficient to effect such treatment.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.
  • Symptoms and signs of disorders and conditions of gastrointestinal tract such as celiac disease and inflammatory bowel disease (Crohn's disease, and ulcerative colitis) include pain, diarrhea, constipation, rectal bleeding, fever, and joint pain malabsorption, chronic vitamin and nutrient deficiency.
  • Subclinical symptoms include without limitation diagnostic markers for inflammation the appearance of which may precede the manifestation of clinical symptoms.
  • One class of subclinical symptoms is immunological symptoms, such as the invasion or accumulation in an organ or tissue of pro-inflammatory lymphoid cells or the presence locally or peripherally of activated pro-inflammatory lymphoid cells and/or presenting a pro-inflammatory lymphokine or cytokine profile or liberating other mediators of inflammation.
  • compositions for use in accordance with the present invention may be in the form of orally or enterally administered (or other mucosally administered) suspensions, capsules or tablets, which may be formulated in conventional manner using one or more pharmaceutically acceptable carriers or excipients.
  • compositions are slow, delayed or positioned release
  • This release profile can be achieved without limitation by use of a coating resistant to conditions within the stomach but releasing the contents in the colon or other portion of the GI tract wherein a lesion or inflammation site has been identified. Or a delayed release can be achieved by a coating that is slow to disintegrate. Or the two (delayed and position release) profiles can be combined in a single formulation by choice of one or more appropriate coatings. Such formulations constitute a further feature of the present invention.
  • Suitable compositions for delayed release and/or enteric coated oral formulations include tablet formulations film coated with materials that are water resistant, pH sensitive, digested or emulsified by intestinal juices or sloughed off at a slow but regular rate when moistened.
  • Suitable coating materials include, but are not limited to, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, polymers of metacrylic acid and its esters, and combinations thereof.
  • Plasticizers such as, but not limited to polyethylene glycol, dibutylphthalate, triacetin and castor oil may be used.
  • a pigment may also be used to color the film.
  • Suppositories are be prepared by using carriers like cocoa butter, suppository bases such as Suppocire C, and Suppocire NA50 (supplied by Gattefosse GmbH, D-Weil am Rhein, Germany) and other Suppocire type excipients obtained by interesterification of hydrogenated palm oil and palm kernel oil (C8-C18 triglycerides), esterif ⁇ cation of glycerol and specific fatty acids, or poly glycosylated glycerides, and whitepsol (hydrogenated plant oils derivatives with additives).
  • Enemas are formulated by using the appropriate active compound according to the present invention and solvents or excipients for suspensions.
  • Suspensions are produced by using micronized compounds, and appropriate vehicle containing suspension stabilizing agents, thickeners and emulsifiers like carboxymethylcellulose and salts thereof, polyacrylic acid and salts thereof, carboxyvinyl polymers and salts thereof, alginic acid and salts thereof, propylene glycol alginate, chitosan, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, methylcellulose, polyvinyl alcohol, polyvinyl pyrolidone, N-vinylacetamide polymer, polyvinyl methacrylate, polyethylene glycol, pluronic, gelatin, methyl vinyl ether-maleic anhydride copolymer, soluble starch, pullulan and a copolymer of methyl acrylate and 2-ethylhexyl acrylate lecithin, lecithin derivatives, propylene glycol fatty acid esters, glycerin fatty acid esters,
  • materials may be incorporated into the matrix of the tablet e.g. hydroxypropyl methylcellulose, ethyl cellulose or polymers of acrylic and metacrylic acid esters. These latter materials may also be applied to tablets by compression coating.
  • compositions can be prepared by mixing a therapeutically effective amount of the active substance with a pharmaceutically acceptable carrier that can have different forms, depending on the way of administration.
  • Pharmaceutical compositions can be prepared by using conventional pharmaceutical excipients and methods of preparation.
  • the forms for oral administration can be capsules, powders or tablets where usual solid vehicles including lactose, starch, glucose, methylcellulose, magnesium stearate, di-calcium phosphate, mannitol may be added, as well as usual liquid oral excipients including, but not limited to, ethanol, glycerol, and water. All excipients may be mixed with disintegrating agents, solvents, granulating agents, moisturizers and binders.
  • compositions e.g., starch, sugar, kaolin, binders disintegrating agents
  • preparation can be in the form of powder, capsules containing granules or coated particles, tablets, hard gelatin capsules, or granules without limitation, and the amount of the solid carrier can vary (between 1 mg to Ig). Tablets and capsules are the preferred oral composition forms.
  • Suppositories for rectal or vaginal administration of the drug can be prepared by mixing a compound of the invention with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the compound.
  • compositions containing compounds of the present invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion.
  • Liquid carriers are typically used in preparing solutions, suspensions, and emulsions.
  • Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof.
  • the liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like.
  • Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols.
  • Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.
  • the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like.
  • Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.
  • a therapeutically effective amount of the compound of the present invention can be determined by methods known in the art. Since the compound of the present invention is more efficiently delivered to the desired place of action (because of low absorption) than the corresponding unconjugated anti-inflammatory drug alone, a lesser amount of the compound on a molar basis than of the unconjugated anti-inflammatory drug can in principle be administered while still achieving the same therapeutic effect. Furthermore, since administration of the compound results in fewer side effects than with the corresponding unconjugated anti-inflammatory drug the amount delivered to the locus in need of anti-inflammatory treatment can be increased. Thus, the table below serves only as a guide.
  • a threshold therapeutically effective amount of the compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof is generally equal to or less than a therapeutically effective amount of the steroid or nonsteroidal anti-inflammatory drug or conjugate on a molar basis.
  • Broad and preferred effective amounts of the compound, a pharmaceutically salt thereof, a solvate thereof, or a prodrug thereof are shown in the table below.
  • the dosage range for treatment of inflammatory bowel diseases and celiac disease is about 1 to about 500 mg per day, depending on the condition of the patient and disease severity.
  • the dose per day is 10-120 mg/day per person, once or twice a day
  • for NSAID conjugates disclosed herein is 50-500 mg once or twice a day in the acute phase of the disease.
  • the maintenance phase dose can be decreased to a fraction, e.g., 50 % or lower of the acute phase treatment, and/or the number of applications can be decreased, e.g. once every second or third day instead of every day or twice a day.
  • the dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art.
  • the duration of the treatment can range from weeks to months to years as long as benefits persist and/or side-effects are tolerated.
  • the compound of Formula VII where T is a steroid is prepared by a reaction of the steroid subunit of Formula IX and the amino group of the macrolide subunit of the structure VIII whereby an amide bond is prepared.
  • the starting steroid subunits of the structure X may be obtained by the action of a corresponding halogenalkanoylchloride (preferably 3- chlorpropionylchloride) on the steroid subunit of Formula VII wherein R d is an OH group (Phillipps G. et al., J. Med. Chem. , 1994, 37, 3717-3729).
  • Compounds of Formula VII may generally be obtained in the following way: one end of the linker chain L is first linked to the macrolide subunit M, and then the other end of the chain is joined to the steroid subunit; or, one end of the chain L is first linked to the steroid subunit T and then the other end of the chain to the macrolide subunit M, or finally, one part of the chain is linked to the macrolide subunit M, whereas the other part of the chain is linked to the steroid subunit T, with the ends of the chain parts being then chemically linked to form the chain L.
  • Compounds within Formula VII can be prepared by the following processes. a) Compounds of Formula VII, where X 2 in linker L is -NH-, can be formed by reacting (i) a steroid anti-inflammatory subunit represented by Formula XI:
  • L 1 represents a leaving group (such as hydroxy), and (ii) a free amino group of a macrolide subunit represented by Formula XII:
  • the reaction is generally performed with acid derivatives which have the ability to activate the carboxylic acid group of steroidal anti-inflammatory subunit, such as halogenides, mixed anhydrides and especially carbodiimides (such as -(3- dimethylaminopropyl)-3-ethyl-carbodiimide (EDC)) and benzotriazoles.
  • acid derivatives which have the ability to activate the carboxylic acid group of steroidal anti-inflammatory subunit, such as halogenides, mixed anhydrides and especially carbodiimides (such as -(3- dimethylaminopropyl)-3-ethyl-carbodiimide (EDC)) and benzotriazoles.
  • EDC -(3- dimethylaminopropyl)-3-ethyl-carbodiimide
  • benzotriazoles benzotriazoles.
  • the reaction proceeds in the presence of a base, such as an organic base (e.g., triethylamine), at room temperature under
  • the compound of Formula VII can be formed by derivatizing an NH group on the macrolide ring to an -N-K-(NH 2 )- group and reacting the derivatized macrolide with a steroid anti-inflammatory subunit represented by Formula IX:
  • This process may also be performed when the NH group in the macrolide is attached at the 3' position of a sugar ring S 1 (i.e., a desosamine sugar) of the macrolide:
  • the reaction is generally performed with acid derivatives which have the ability to activate the carboxylic acid group such as halogenides, mixed anhydrides and especially carbodiimides and benzotriazoles.
  • the reaction is typically performed at room temperature under an inert atmosphere such as nitrogen or argon. The reaction may require several hours to several days to come to completion.
  • the starting macrolide subunits of the structure VIII are known compounds or may be obtained according to the procedures described for analogous compounds, such as those described in Costa A.M. et al., Tetrahedron Letters, 2000, 4i:3371-3375, which is hereby incorporated by reference in its entirety. See, also Bright U.S. Patent No. 4,474,768 and Bright, G.M., et al, J. Antibiot. , 1988, 41:1029-1047, both incorporated in their entirety by reference.
  • the compound of Formula VII can be formed by (1) derivatizing an NH group on a macrolide to an N-K-OH group and (2) reacting the derivatized macrolide with the free carboxylic acid group on a steroid T:
  • the linkage group -K-OH can be attached to the secondary nitrogen atom of the macrolide subunit as follows.
  • the reduction is typically performed at a low temperature and preferably at 0° C or lower.
  • This process can also be performed when the NH group is attached at the 3' position of a sugar ring in the macrolide (such as a sugar at the 3 position of the macrolide).
  • this process can be performed when acriloyl group is attached at the C/6 or C/4" position of the macrolide subunit with steroid subunit represented by Formula: .
  • the reactant macrolide subunit can be formed by oxidizing the corresponding macrolide having a hydroxy substituent at the 4" position on cladinose sugar
  • Compounds of Formula VII can be prepared by reacting a macrolide subunit having a leaving group L 2 (such as Br), and a steroid as shown below.
  • L 2 such as Br
  • the starting macrolide subunit can be prepared by cleaving the sugar group attached at the 3-position of the macrolide ring and then reacting the macrolide with a reagent of Formula I ⁇ L-L 1 , where L 2 is a leaving group.
  • Compounds of Formula VII can be prepared by reacting a macrolide subunit having a leaving group L 2 (such as Br), and a steroid as shown below.
  • L 2 such as Br
  • the salts of the compounds represented by Formula VII may be prepared by applying generally known procedures such as, e.g., a reaction of the compounds of the structure VII with a corresponding base or acid in a suitable solvent or mixture of solvents e.g. ethers (diethyl ether) or alcohols (ethanol, propanol or z ' .y ⁇ ?-propanol).
  • a suitable solvent or mixture of solvents e.g. ethers (diethyl ether) or alcohols (ethanol, propanol or z ' .y ⁇ ?-propanol).
  • the 16-membered ring macrolides are traditionally divided into sub-families based upon the substitution patterns of their aglycones.
  • the principal prototypes of this family can be represented by leucomycin, spiramycin and tylosin.
  • Tylosin is a representative of 16-membered macrolides, which possesses a highly substituted aglycone with two double bonds (tylonolide) and a third saccharide substituent ( ⁇ -D-mycinose) in addition to the disaccharide attached to the 5-hydroxyl group. Hydrolysis of mycarose from disaccharide yielded desmycarosyl-tylosin (desmycosin).
  • 16-membered ring macrolide hybrid could be prepared by reductive animation of the C-20 aldehyde group.
  • This reaction could be used also for 17-membered azalides like 8a-aza- homodesmycosins and its derivatives (such as di- and tetrahydro derivatives).
  • R -.13 is hydrogen or hydroxy
  • an appropriate reactant such as a diamine
  • ketone in position 9 may be modified by hydroxylamine hydrochloride to yield oxime and then reduced to amine.
  • the same linkage schemes can be used.
  • the steroid subunit may be linked to the macrolide through the 21 hydroxy group in steroids that have such a group.
  • Beginning with a 21 -hydroxy steroid cyclic ketal is reacted with an appropriate carboxylic acid halide or an anhydride, preferably in a solvent such as methylene chloride in the presence of a tertiary amine base or pyridine at a reduced temperature (-50 C-300 C).
  • the intermediate so produced is reacted with H2N-L-M to form compounds of Formula VII
  • the steroid subunit may also be linked to the macrolide through the 17 position on the steroid subunit.
  • One method for preparing such a compound is as follows:
  • the compound of Formula VII can be formed by derivatizing an NH group on the macrolide ring to an -N-K-(NIt)- group and reacting the derivatized macrolide with a steroid anti-inflammatory subunit represented by Formula Sa:
  • This process may also be performed when the NH group in the macrolide is attached at the 3' position of a sugar ring S 1 (i.e., a desosamine sugar) of the macrolide:
  • the carboxylic acid group at the 17 position of the starting steroid subunit may be modified prior to the reaction with NHb-L-M.
  • the carboxylic acid group at the 17 position of the starting steroid subunit can also be protected prior to the reaction with NEh-L-M and deprotected after the reaction with NH2-L-M or the esterification step.
  • T is a nonsteroidal antiinflammatory subunit
  • NSAID compounds having a hydroxyl group may alternatively be derivatized by the action of succinic anhydride in the presence of pyridine followed by reaction of the intermediate so produced with triethylamine, 4-pyrrolopyridine in methylene chloride to produce NSAID having free carboxylic acid group(Huang CM. et al. Chem.&Biol. 2000,7,453-461, Hess S. et al. Bioorg.&Med. Chem. 2001, 9, 1279-
  • the NSAID derivatives so produced may be coupled either to a linker macrolide compound such as formula XII or directly to a macrolide.
  • NSAID compounds having an amino group may alternatively be derivatized by the action of sodium hydride and tert-butyliodoacetate in N,N-dimethylformamide to produce a (butoxy carbonyl derivative of the NSAID which is then reacted with (trifluoracetic acid in methylene chloride to produce NSAID having free carboxylic acid group (Hess S. et al., Bioorg ⁇ Med. Chem. , 2001, 9:1279-91).
  • the NSAID derivatives so produced may be coupled either to a linker macrolide compound such as formula XII or directly to a macrolide.
  • NSAID compounds having an amino group may be derivatized according to Scheme III by the action of succinic anhydride in the presence of dimethylaminopyridine, N,N'-diisopropylethylamine in dimethylformamide to produce NSAID having free carboxylic acid group (Pandori M.W. et al., Chem.& Biol , 2002, 9:567-73).
  • the NSAID derivatives so produced may be coupled either to a linker macrolide compound such as formula XII or directly to a macrolide.
  • NSAID compounds contain sulfonamide group
  • they could be derivatized according to the procedure described in Patent App. US 2003/0055012A1 using ester derivatives of chloro- and bromoacetic acid such as methyl-bromoacetate.
  • esters could be hydrolyzed in basic conditions with, for example, LiOH producing free acid which could be coupled either to a linker macrolide compound such as formula XII or directly to a macrolide.
  • This process may also be performed when the NH group in the macrolide is attached at the 3' position of a sugar ring S 1 (i.e., a desosamine sugar) of the macrolide according to Scheme V:
  • the reactant macrolide subunit can be formed by oxidizing the corresponding macrolide having a hydroxy substituent at the 4" position on cladinose sugar
  • the reaction is generally performed with acid derivatives which have the ability to activate the carboxylic acid group of the nonsteroidal anti-inflammatory subunit, such as halogenides (such as EDC), mixed anhydrides, especially carbodiimides .
  • acid derivatives which have the ability to activate the carboxylic acid group of the nonsteroidal anti-inflammatory subunit, such as halogenides (such as EDC), mixed anhydrides, especially carbodiimides .
  • the reaction is typically performed at room temperature under an inert atmosphere, such as nitrogen or argon. The reaction may require several hours to several days to come to completion.
  • the starting macrolide subunits of the structure XIII are known compounds or may be obtained according to the procedures described for analogous compounds, such as those described in Costa A.M. et al., Tetrahedron Letters, 2000, 41:3371-75, which is hereby incorporated by reference.
  • the compound of Formula VII can be formed by (1) derivatizing an >NH group on a macrolide to an > N-K-OH group and (2) reacting the derivatized macrolide with the free carboxylic acid group on a non-steroidal anti-inflammatory subunit according to Scheme VII:
  • the linkage group -K-OH can be attached to the primary or secondary nitrogen atom of the macrolide subunit as follows.
  • a metal hydride e.g.,
  • This process can also be performed when the NH group is attached at the 3' position of a sugar ring in the macrolide (such as a sugar at the 5 position of the macrolide).
  • 4" is the 4 position on a sugar S 2 , such as a cladinose sugar, and a derivatized nonsteroidal anti-inflammatory subunit having a free amino group represented by the formula:
  • the compounds of the Formula VII can be prepared by reacting a macrolide subunit having a leaving group L 2 (such as Br), and a non-steroidal antiinflammatory drug as shown below.
  • L 2 such as Br
  • the starting macrolide sutmnit can be prepared by cleaving the sugar group attached at the 3-position of the macrolide ring and then reacting the macrolide with a reagent of the Formula I ⁇ L-L 1 , where L 2 is a leaving group.
  • the compounds of Formula VII can be prepared by reacting a macrolide subunit having a leaving group L 2 (such as Br), and a non-steroidal antiinflammatory drug as shown below.
  • L 2 such as Br
  • Compounds of the Formula VII can be prepared by reacting a macrolide subunit having a leaving group L 2 (such as Br) and a non-steroidal antiinflammatory drug as shown below.
  • L 2 such as Br
  • Tablets may be prepared by known art methods, such as direct compression or wet granulation.
  • the active ingredient may be blended with excipients, such as, but not limited to, lactose, croscarmellose sodium and magnesium stearate.
  • excipients such as, but not limited to, lactose, croscarmellose sodium and magnesium stearate.
  • the resultant mix may be compressed into tablets using appropriate punches.
  • Tablets of other strengths may be prepared by altering the ratio of active ingredient to excipient(s) or the compression weight and using punches to suit.
  • the tablets may be film coated with a film coating suspension using suitable film coating equipment to give a weight of film coat of about 5 to about 10 mg.
  • the release profile of the formulation may be modified by selection of a suitable coating and/or degradable matrix. Alternatively, the release as is known in the formulation art.
  • mice Male Sprague-Dawley rats weighing approximately 200 g were used. After weighing, each rat was given an earmark and placed into a cage. The day before the beginning of the study, animals were randomly distributed into 5 groups with 5 rats/group and placed into solid bottom cages, on wood dust-free bedding (VRF-I Rodent's diet, Altromin Gmbh). During the experiment, the rats were maintained on a 12-h light-dark cycle. The rats were weighed and fasted for 12 hours prior to p.o. administration.
  • Compound 5 was administered p.o. in 0.125% carboxymethyl cellulose suspension.
  • the dissolved substance was administered oral at a dose of 30 mg/kg body weight (b.w.), using 2,5 mL syringe (BD) and metal gavage.
  • the administered volume was 10 mL/kg b.w.
  • Plasma and blood samples were obtained at the following time points after administration: 15, 30, 60, 120, 240, 360 and 480 minutes.
  • Blood samples were collected by puncturing the lateral tail vein. Prior to each blood sampling, animals were maintained in warming cabinets at 37°C for 15 minutes. At each sampling time-point 50 ⁇ L of blood were collected and added to an Eppendorf test tube (1.5 mL) containing 50 ⁇ L demi water. Blood was collected according the time points stated hi the protocol. Specimens were stored at -20°C until analysis.
  • concentrations of a compound 5 in the biological matrices were determined by a high performance liquid chromatography method using an acetonitrile and water (acidified by 0.1% formic acid) gradient as the chromatographic medium with tandem mass spectrometry detection (HPLC/MS-MS) (Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, May 2001).
  • Biological samples were prepared by deproteinization with acetonitrile, containing an internal standard. After centrifugation and evaporation of the supernatant fraction, samples were reconstituted in acetonitrile: water with addition of 0.1% formic acid.
  • MS - MS detection was performed in multi-resolution mode (MRM).
  • concentration of the administered compound in biological matrices are calculated by the peak area ratios of compound 5 to an internal standard of roxithromycin using calibration curves and quality control samples.
  • Roxithromycin is also a macrolide antibiotic (synthesized in house). Roxithromycin is commercially available from Sigma- Aldrich Milwaukee Wisconsin.
  • Non-compartmental analysis was used for pharmacokinetic analysis of the compound in different matrices Jang et al. Med red. Rev. 2001; 21:382-96.
  • the pharmacokinetic parameters were calculated using the PK solutions 2.0 software (Summit Research Services, Pharmacokinetics and Metabolism Software).
  • mice Male Wistar Han rats weighing approximately 220 g were used. After weighing, each rat was given an earmark and placed into a cage. The rats were divided in 3 groups, one with 15 rats for i.v. application, one with 5 rats (p.o.) in order to obtain blood within 24h, and one with 2 rats in order to collect feces 24 h post administration. During the experiment, the rats were maintained on a 12-h light-dark cycle and allowed free access to food (MUCEDOLA, Italy) and tap water. After grouping, the rats were held in a cage placed in racks. The rats were weighed and fasted for 12 hours prior to p.o. administration.
  • the compound shown above was administered p.o. in 0.125% carboxymethyl cellulose suspension.
  • the compound was administered i.v. in the form of its phosphate salt in phosphate buffer saline (PBS).
  • PBS phosphate buffer saline
  • Plasma and blood samples were obtained at the following time points after administration: 15, 45, 60, 120, 240, 360, 720 and 1440 minutes. Samples were collected by puncturing the tail vein. At each sampling time point 250 ⁇ L of blood was collected, from which 50 ⁇ L was added to an eppendorf test tube (1.5 mL) containing 100 ⁇ L demineralized water. Blood was collected according the time points stated in the protocol up to 24h post administration. 24 h post administration, after the last collection of blood, the rats were put into a CO2 atmosphere, and the rat's small and large intestine were removed, cleaned and placed into a Petri dish. Specimens were stored at -2O 0 C until analyzed.
  • the oral bioavailability in rats was determined for the following compounds to provide the following oral bioavailability in rats. These compounds are anticipated to have low bio-availability when administered to humans.
  • methyl cellulose 0.5% methyl cellulose was added up to 100% of the total volume.
  • the solution was applied in a volume of 10 ml/kg b.w. (P.O.) and 10 ml/kg b.w. (I.N.).
  • P.O. 10 ml/kg b.w.
  • I.N. 10 ml/kg b.w.
  • LV. administration the substance was dissolved in DMF.
  • PBS was added up to 100% of the total volume.
  • the solution was applied in a volume of 5 ml/kg b.w. (LV.)
  • mice were weighed, marked and grouped a day before the beginning of the study. After weighing each mouse was given an earmark and placed into a cage (5 mice per cage). During the experiment mice were maintained on a 12 h light-dark cycle and allowed free access to food (VRF-I Altromin, Charles River, Hungary) and tap water. Mice were weighed and fasted for 12 hours prior P.O. administration.
  • the substance was administered P.O. using a ImL syringe (BD) and gavage for oral administration. Prior to IV administration, animals were maintained in warming cabinets (SCANBOUR, Denmark) (38°C) for 15 minutes, in order to facilitate vasodilatation. After formulating the end - use item, substance was administered into the lateral tail vein, using a 1-mL syringe (BD) with a 0.4 x 19 needle.
  • BD ImL syringe
  • mice Prior each blood sampling, mice were anaesthetized using Thiopental ® ,
  • MS - MS detection was performed in multi-resolution mode (MRM).
  • the concentration of the administered compound in biological matrices are calculated by the peak area ratios of compound 1 to an internal standard of roxithromycin using calibration curves and quality control samples.
  • Roxithromycin is also a macrolide antibiotic (synthesized in house). Roxithromycin is commercially available from Sigma-
  • Non-compartmental analysis was used for pharmacokinetic analysis in plasma and tissue.
  • the pharmacokinetic parameters were calculated based on average values at each time point using WinNonlin Professional Software, Version 4.1 ⁇ Phar sight).
  • mice were determined for compound 2' to provide an oral bioavailability of less than 1% (below the limit of quantitation). This compound is anticipated to have a low bio-availability when administered to humans.
  • mice or rats having an AUC of less than 0.5 Dg*h/mL include the compounds in the table below:
  • These compounds therefore have an oral bioavailability of less than 10% .
  • the efficacy of the compounds of Formula VII for the treatment of inflammatory bowel diseases can be determined using different in vivo models such as the effect of steroid treatment on the inflammatory parameters of trinitrobenzene sulfonic acid (TNBS)-induced inflammatory bowel disease in rats (Yue G. et al., /. Pharmacol. Exp. Ther., 1996, 276:265-70; Palmen MJ. et al., Dig. Dis. Sci., 1998, 43:2518-25; Nakase H. et al., J. Pharmacol. Exp. Ther., 2001, 297:1122-8; Kankuri E.
  • TNBS trinitrobenzene sulfonic acid
  • mice on TNBS-induces BD in mice (Fiorucci S. et al., Proc. Natl. Acad. Sci. , 2002, 99:15770-75), on acetic acid induced acute chemical colitis in rats (Kim Y.S. et al., Arch. Pharm. Res., 1999, 22:354-60), on dextran sulfate sodium (DSS) induced colitis in mice (van Meeteren M. E. et al., Scand. J. Gastroenterol., 2000, 35:517- 21, Nakase H. et al., J. Pharmacol. Exp.
  • mice homozygous for a disrupted interleukin-10 (IL-10) gene support the hypothesis that a disregulated immune response to enteric flora can trigger IBD (Kuhn R. et al., Cell, 1993, 75:263-74). These mice, left untreated invariably develop IBD so this model is particularly suited for testing the preventive effect of compound for development of IBD but can also be used for testing the therapeutic effects on developed disease. The severity of the colitis depends on the inbred strain background in which the disrupted gene is placed (Berg DJ. et al., J. Clin. Invest. , 1996, 98:1010-20).
  • C3H/HeJBir (C3H) strain is a genetic background that is highly susceptible to several experimentally induced forms of IBD. After 10 backcrosses of a disrupted interleukin-10 (IL-10) gene colon lesions are much more severe in C3H mice than in C57BL/6J (B6) mice (Farmer M. A. et al., Proc. Natl. Acad. Sci. USA, 2001, 98:13820-25). Severe lesions of the cecum and colon can be detected in C3H.IL107 " mice as early as 4 weeks of age, whereas B6 IL107 " mice develop mild lesions that do not progress in severity.
  • IL-10 interleukin-10
  • mice are necropsied for tissue collection and analyzed for various phenotypes positively correlated with the progression of colitis.
  • the advantage of this model is that all mice with a disrupted IL-10 gene in C3H genetic background will ultimately develop severe inflammatory bowel disease with the similar intensity.
  • the effectiveness of steroid conjugates (sterolides) is tested by administering them per os from 4 weeks of age at a starting dose of 1-100 mg/kg and following the effect on disease incidence and severitiy. Their activity should be compared to that of standard steroids.
  • Macroscopic observation and histological evaluation are used to determine the degree of colonic inflammation.
  • the criteria for assessing macroscopic damage and the numerical rating score are as follows: 0, no ulcer, no inflammation; 1, no ulcer, local hyperemia; 2, ulceration without hyperemia; 3, ulceration and inflammation at one site only; 4, two or more sites of ulceration and inflammation; and 5, ulceration extending more than 2 cm.
  • the degree of inflammation on microscopic tissue sections was scored as follows: 0, no leukocyte infiltration; 1, low level of leukocyte infiltration; 2, moderate level of leukocyte infiltration; 3, high vascular density and thickening of the colon wall; and 4 transmural leukocyte infiltration, loss of goblet cells, high vascular density, and thickening of the colon wall.
  • Dextran sodium sulfate (2-5% w/v) is given is drinking water to mice.
  • the protocol was as follows:
  • the compounds are dissolved in DMSO (up to a final concentration of 2%) and 0.125% carboxymethyl cellulose and initially applied p.o. at 2 and 10 mg/kg /day for 7 days.
  • the positive control group received only vehicle.
  • DSS solution was provided as drinking fluid for the same time period, and mice were sacrificed on the day 8.
  • DSS solution is administered in drinking water for 7 days.
  • the administration of the compounds started at day 0 and continued to day 14, 21 or 28, and mice were sacrificed on the day 15, 22 or 29..
  • DSS solution is administered in drinking water for 7 days.
  • the administration of the compounds started at day 8 and continued to day 14, 21 or 28, and mice are sacrificed on the day 15, 22 or 29.
  • Severity of colitis is assessed using a disease activity index (DAI), calculated based on weight loss, stool consistency and bleeding (Cooper H. S. et al., Lab. Invest. , 1993, 69:238-49; Hartman G. et al., J. Pharm. Exp. Ther. , 2000, 292:22-30).
  • DAI disease activity index
  • No weight loss is scored as 0 points, weight loss of 1 to 5% as 1 point, of 5 to 10% as 2 points, 10 to 20% as 3 points, and weight loss >20% as 4 points.
  • For stool consistency 0 point is given for well formed pellets, 2 points for pasty and semiformed stools that do not stick to the anus, and 4 points for liquid stools that do stick to the anus.
  • Bleeding was scored 0 points for no blood in hemoccult, 2 points for positive hemoccult, and 4 points for gross bleeding.
  • the disease activity index is the sum of the combined scores of weight loss, stool consistency and bleeding divided by 3, forming a DAI that ranges from 0.0 (healthy) to 4.0 (maximal activity in colitis). Histological score is assessed in formalin fixed tissue samples of the entire colon. Sections were stained with haematoxylin/eosin and scored by a pathologist unaware of the treatments applied. Infiltration of inflammatory cells, tissue damage and cryp score are subjectively assessed by separate and combined scoring.
  • rare inflammatory cells in lamina limbal growth factor are scored as 0; increased number of inflammatory cells as 1; confluence of inflammatory cells extending into the submucosa as 2; and transmural extension of the infiltrate as 3.
  • tissue damage no mucosal damage is defined as 0, discrete lymphoepithelial lesions are defined as 1, surface mucosal erosion as 2 and extensive mucosal damage as 3.
  • Crypt scoring is performed as follows: 0, intact crypt; 1, loss of bottom one third of the crypt; 2, loss of bottom two thirds of the crypt; 3, loss of entire crypt with the surface epithelium remaining intact; 4, loss of the entire crypt and surface epithelium (erosion).
  • Clinical improvement in this model is defined by one or more of the following: improvement in DAI, decreased hi infiltration of the inflammatory cells, decreased tissue damage and decreased crypt score.. These parameters are compared to the values of the positive control group (vehicle treated mice) and animals treated with standard steroids.
  • TNBS Trinitrobenzene Sulfonic Acid
  • Colitis is induced in male Wistar rats by intracolonic administration of 10-50 mg of TNBS in 0.25 ml of 50% ethanol.
  • Two days prior to administration of TNBS animals are treated with various doses of sterolide (initially ranging from 0.5 to 10 mg/kg of the compound in 0.125% carboxymethyl cellulose in phosphate buffered saline and 1% DMSO) or vehicle control (0.125% carboxymethyl cellulose in phosphate buffered saline and 1 % DMSO) or standard steroid such as for example budesonide (1 mg/kg) for 14 days after the TNBS is applied.
  • sterolide initially ranging from 0.5 to 10 mg/kg of the compound in 0.125% carboxymethyl cellulose in phosphate buffered saline and 1% DMSO
  • vehicle control 0.125% carboxymethyl cellulose in phosphate buffered saline and 1 % DMSO
  • standard steroid such as for example bud
  • Rats were sacrificed, blood samples werewere taken (at day 7 and 15 after application of TNBS) to obtain serum samples followed by macroscopic observation and histological evaluation to determine the degree of colonic inflammation. Macroscopic and histological evaluation of tissue damage was done blindly by pathologist. The criteria for assessing macroscopic damage and the numerical rating score are as follows: 0, no damage; 1, localized hyperemia and/or edema; 2, linear ulcer ⁇ half of the width of the colon; 3, linear ulcer > half of the width of the colon; 4, small circular ulcer ( ⁇ 1 cm); 5, circular ulcer between 1 and 2 cm; 6, circular ulcer > 2 cm. Scores from individual animals were added together and divided by number of animals to get average microscopic damage.
  • each colon is blindly scored according to the following criteria: A) Ulceration: 0, no ulcer, epithelization; 1, small ulcer ⁇ 3 mm; 2, large ulcer >3 mm; B) Inflammation: 0, no inflammation; 1, mild;
  • Improvement in this experiment is defined by the lower average macroscopic damage and lower histological score, as well as lowering of LTBB4, serum ⁇ l acidic glycoprotein and MPO activity in sterolide treated then in the group of positive control (animals receiving vehicle alone).
  • Fig. IA describes the macroscopic damage score of the colon in the rat model of TNBS induced colitis (Mann-Whitney Test).
  • the macroscopic damage score of the colon is defined as 0 - no damage; 1 - localized hyperemia and/or edema; 2 - linear ulcer ⁇ half of the width of the colon; 3 - linear > half the width of the colon or small ulcer ( ⁇ lcm) 4 - circular ulcer ⁇ lcm; 5 - circular between 1 and 2 cm; and 6 - circular ulcer > 2cm.
  • the compounds 1', 2' and 3' as shown in the table below were compared against budesonide [a known drug useful for the treatment of Crohn's disease in humans] as a treatment control and just a vehicle as a no treatment control.
  • the no treatment control gave a score of 4 while the treatment control, budesonide, at a dosage of lmg/kg gave a score of 2.
  • Fig. IB describes effect of conjugates on histological damage score in TNBS induced colitis in rats (Mann-Whitney Test).
  • the histological damage score is defined according to the following criteria: A) Ulceration: 0, no ulcer, epithelization; 1, small ulcer ⁇ 3 mm; 2, large ulcer > 3 mm; B) Inflammation: 0, no inflammation; 1, mild; 2, moderate; 3, severe; C) Depth of lesion: 0, none; 1, submucosa; 2, muscularis basement; 3, serosa; D) Necrosis: 0, none; 1, mild; 2, severe; E) Granulomas: No, no granulomas; Yes, granulomas present.
  • Maximum score defined as a sum of all individual scores in this scoring system is 10 + granulomas.
  • Compounds 1, 2 and 3 were compared against budesonide [a known drug useful for the treatment of Crohn's disease in humans] as a treatment control and just a vehicle as a no treatment control. The no treatment control gave a score of 7,6 while the treatment control, budesonide, at a dosage of lmg/kg gave a score of 4,5.
  • Compounds 1' (2mg/kg); T (2mg/kg) and 3' (10mg/kg) all demonstrated comparable or superior efficacy to that of the standard steroid budesonide (1 mg/kg) in the rat model of TNBS induced colitis.
  • Fig. 2 shows the effect of varying doses of compound 1' in TNBS induced colitis model in rats (Mann-Whitney Test).
  • the macroscopic damage score is as in Fig. IA as was the treatment and no treatment controls.
  • Compound 1' in dosages ranging from 4mg/kg to 0.5 mg/kg demonstrates comparable or superior efficacy to that of the standard steroid budesonide (1 mg/kg).
  • TNBS is applied in amounts of 1 to 2.5 mg per mouse to BALB/c and male Swiss Albino mice (6-8 weeks old).
  • Mice are treated with sterolide (1 to 10 mg/kg of the compound in 0.125% carboxymethyl cellulose in phosphate buffered saline and 1% DMSO), vehicle control (0.125% carboxymethyl cellulose in phosphate buffered saline and 1% DMSO) or standard steroid such as budesonide (1-10 mg/kg) or prednisolone (1-10 mg/kg) for 7 days.
  • Mice are monitored for the appearance of diarrhea, loss of body weight, and overall mortality.
  • surviving mice are sacrificed and a segment of the colon is excised for macroscopic and microscopic damage evaluation.
  • This animal model is suitable for showing prevention or delay of recurrence or relapse. Damage persists for more then 30 days if untreated, however if treated in the beginning (as described above), no recurrence will occur.
  • mice are treated therapeutically with steroids or vehicle control from day 21 to 35 after TNBS administration. Animals are killed 35 days post-TNBS injection.
  • a sample of colonic tissue located 2 cm above the anal canal is obtained, fixed in 10% buffered formalin phosphate, embedded in paraffin, sectioned, and stained with haematoxylin/eosin.
  • the degree of inflammation on microscopic cross sections is graded semiquantitatively from 0 to 4 as follows: 0, no signs of inflammation; 1, very low level of inflammation; 2 low level of leukocyte infiltration; 3, high level of leukocyte inflammation, high vascular density, and thickening of the colon wall; and 4, transmural infiltrations, loss of goblet cells, high vascular density, and thickening of the colon wall. Improvement in this model is defined by the lower levels of macroscopic damage and inflammation and damage defined by histological score, then in the group of positive control (animals receiving vehicle alone)
  • DNBS was instilled into distal colon of animals at 24 hours after the first dosing and at one hour after the second dosing on day 2.
  • One normal control group was treated without DNBS challenge.
  • the animals were sacrificed 24 hours after the final daily dosing and the colon was removed and weighed.
  • fecal occult blood and stool consistency were monitored daily.
  • adhesions between the colon and other organs were noted as was the presence of colonic ulceration after removal and weighing of each colon (a macroscopic damage score).
  • the colon-to-body weight ratio is calculated according to the formula:
  • test substance -I- DNBS [(test substance + DNBS)-(vehicle blank)] mean value of colon-to-body weight ratio
  • Rat DNBS-induced Abnormal Activity Indices were determined in all animals daily throughout the study, (i) Body weight (ii) Stool consistency (score: 0, normal stools; 1, soft stools; and 2, liquid stools) (iii) Rectal bleeding (occult fecal blood) (fecal occult blood was measured on a Hemoccult Sensa Card (HemoFEC; Boehringer Mannheim, Barcelona, Spain) score: 0- negative; 1-fecal occult blood test positive; and 2, visible rectal bleeding)
  • Rat DNBS Histology Readouts at the end of the study Criteria for Macroscopic scoring of damage include the following: (for reference see Mourelle, Gastro, 1996, 110:1093-1097; Appleyard&Wallace, AJP, 1995, 269: Gl 19-125)
  • Test substances were each administered orally once a day for 7 consecutive days and DNBS was instilled intracolonically on the second day. Groups of 10 test animals were sacrificed 24 hours after the final dosing when the colon-to-body weight ratio was recorded. Percent decrease in the colon weight per 100 g body weight relative to the vehicle-treated control was calculated.
  • a 30 percent or more (> 30%) reduction in the colon-to-body weight ratio relative to the vehicle-treated group is considered significant anti-inflammatory activity (in parenthesis).
  • the cottontop tamarin is a small new world primate that is unique among animal models of IBD in that it develops a spontaneous form of colitis which shows many similarities to the condition of ulcerative colitis in humans. Animals present clinically with chronic diarrhea and weight loss and may die from the condition if untreated. Both the pathology and response to the therapy with 5-aminosalicylic acid compounds in conjunction with in some cases corticosteroids show close similarities to the condition in humans.
  • a unique feature of the disease in the cottontop tamarin is the development of secondary complications, namely clonic adenocarcinoma and sclerosing cholangtis which are also seen in human ulcerative colitis.
  • cottontop tamarin has been used before to evaluate potential therapeutic regimens for treating ulcerative colitis, including Budesonide et al. (Watkins P.E. et al., Gut, 1997, 40:628-33 herein incorporated by reference in its entirety). Therefore, this model has been validated as predictive for the efficacy of drugs in humans.
  • Cottontop tamarins with clinically manifested ulcerative colitis (diarrhoea and weight loss) and confirmed by endoscopic examination of the colon along with histopathological examination of a rectal biopsy specimen are recruited to this study. Faecal samples are taken from the animals for culture to eliminate known faecal pathogens as cause of these symptoms.
  • the effectiveness of the conjugates of the invention is tested by administering them p.o. for up to 2 months with doses starting from 1-10 mg/kg.
  • Standard indicators of disease progression in the cottontop tamarin are used to evaluate response to treatment. These are clinical signs, body weight and histopathological examination of rectal biopsy specimens taken on days 14, 28, 42 and 56. Specimens are graded from 0 (normal) to 5 (severe active disease) on the basis of inflammatory cell infiltrate, crypt architecture and mucosal disruption (Table 1) by a pathologist.
  • Biopsy scores and body weights before and after conjugate treatment are compared using the Friedman non-parametric repeated measures test. Differences are considered significant when p ⁇ 0.05.
  • Cottontop tamarins treated with the compounds of the invention showed an improvement of at least 1 based upon the histological scoring system for either acute or chronic pathology in Table 1.

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Abstract

La présente invention concerne des méthodes de prévention et de traitement de maladies, de troubles, et de pathologies inflammatoires des voies gastro-intestinales en administrant à un patient nécessitant un tel traitement, des composés conjugués de Formule VII de faible biodisponibilité orale ou des sels, promédicaments ou solutés de ceux-ci pharmaceutiquement acceptables, M représentant une sous-unité macrolide qui possède une propriété d'accumulation dans des cellules inflammatoires, T représentant une sous-unité anti-inflammatoire qui peut être un stéroïde ou un non stéroïde (groupement non stéroïdien) dérivé d'un médicament non stéroïdien à activité anti-inflammatoire, analgésique et/ou antipyrétique (AINS), et L représentant un lieur covalent entre M et T. La présente invention concerne aussi des compositions pharmaceutiques contenant des composés conjugués de Formule VII à faible biodisponibilité orale.
PCT/IB2006/001488 2006-02-15 2006-02-15 Utilisation de conjugués spécifiques de cellule pour traiter des maladies inflammatoires des voies gastro-intestinales WO2007093840A2 (fr)

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WO2014166503A1 (fr) 2013-04-10 2014-10-16 Probiotic Pharmaceuticals Aps Dérivés antimicrobiens d'azithromycine à effet pharmaceutique non antibiotique

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WO2003070173A2 (fr) * 2002-02-15 2003-08-28 Sympore Gmbh Conjugues de composes biologiquement actifs, methodes de preparation et d'utilisation des conjugues, formulation et applications pharmaceutiques obtenues a partir desdits conjugues
WO2004094449A1 (fr) * 2003-04-24 2004-11-04 Pliva-Istrazivacki Institut D.O.O. Conjugues macrolides a activite anti-inflammatoire

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WO2003070173A2 (fr) * 2002-02-15 2003-08-28 Sympore Gmbh Conjugues de composes biologiquement actifs, methodes de preparation et d'utilisation des conjugues, formulation et applications pharmaceutiques obtenues a partir desdits conjugues
WO2004094449A1 (fr) * 2003-04-24 2004-11-04 Pliva-Istrazivacki Institut D.O.O. Conjugues macrolides a activite anti-inflammatoire

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WO2014166503A1 (fr) 2013-04-10 2014-10-16 Probiotic Pharmaceuticals Aps Dérivés antimicrobiens d'azithromycine à effet pharmaceutique non antibiotique

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