Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Laminitis also termed founders is inflammation of the laminae of the foot—the soft tissue that attaches the coffin or pedal bone of the foot to the hoof wall. Laminitis occurs especially in horses, but is also found in other ungulates, e.g., dairy cows.
• Laminitis is regarded as the most important claw disease of cattle. Laminitis, and the accompanying local trauma, has been made responsible for the development of sole ulcers.
• the immune system is vulnerable to inflammatory episodes; for example, the dairy cow is very prone especially around the time of birth (35 days before calving to 70 days after calving) because of massive changes in its metabolism caused by hormonal changes. Because of diagnostic uncertainties, and therefore limited possibilities to diagnose in practice, basic data for the frequency of the appearance of laminitis are missing in dairy cows during this period. [0004] Concerning the pathogenesis of laminitis, the contacts of the lamellae become loose from the epidermis lamella, which physiologically grip into each other like a zipper. Local edema formation and swelling as a result of inflammatory signals from an activated immune system cause a higher outlet of tissue liquid and blood cells, results in high pressure between the coffin bone and the hoof wall.
• laminitis cortisone
• laminitis e.g., colic, enteritis, lumbago, thyroid diseases, and Cushing’s syndrome.
• causes of laminitis are types of housing of the animals and their stress levels, allergies, toxins generated through too high of a protein supply, too much starch, or different kinds of sugars in high concentrations (e.g., fructans), consuming poisonous plants, pesticides, fertilizer, or overdoses of medicaments.
• More frequent factors that can induce laminitis include unnatural preserving agents and additives to nutrients that are not of the natural habitat of the animals in question.
• laminitis In relation to bovine, previous systemic diseases like rumen acid anemia and endometritis are known triggers for laminitis, which causes functional and morphological changes in the capsule due to the creation of an inflammatory cascade of protease enzymes and certain cytokines.
• laminitis In addition to bovine, laminitis also occurs commonly in other ungulates from families like equine, deer, ovis, and capra.
• Metalloproteinases and serine proteases are naturally occurring enzymes present in many tissues of the equine body and in mammals in general. These enzymes act to degrade proteins, normally in a controlled and specific manner.
• MMPs Metalloproteinases
• the metalloproteinase family can be subdivided into five groups according to their structural and functional properties: (i) the collagenases (metalloproteinases-1, 8, and 13); (ii) gelatinases A and B (metalloproteinase-2 and metalloproteinase-9); (iii) stromelysins 1 and 2 (metalloproteinase-3 and metalloproteinase-10); (iv) matrilysin (MMP-7), enamelysin (MMP-20), macrophage metalloelastase (MMP-12) and MMP-19 (making up the classical metalloproteinases); and (v) membrane-type metalloproteinases (MT-MMP-1 to 4, stromelysin-3, and MMP-11).
• MMP-7 matrilysin
• MMP-20 macrophage metalloelastase
• MMP-19 membrane-type metalloproteinases
• metalloproteinases share a common multi-domain structure but are glycosylated to different extents and at different sites. According to sequence alignments, the assembly of these domains might have been an early evolutionary event, followed by diversification. [0009] Collectively, metalloproteinases can degrade all the major components of the extracellular matrix (ECM). The homeostasis of the ECM is controlled by a delicate balance between the synthesis of ECM proteins, the production of ECM-degrading extracellular matrix metalloproteinases, and the presence of metalloproteinase tissue inhibitors. [0010] One family of metalloproteinase inhibitor peptides is the tissue inhibitors of metalloproteinases (TIMPs).
• TIMPs tissue inhibitors of metalloproteinases
• the TIMP family is comprised of at least four distinct members (TIMP-1 to 4) that possess 12 conserved cysteine residues and express metalloproteinase inhibitory activity by forming non-covalent complexes with metalloproteinase enzymes.
• TIMPs bind to the highly conserved active zinc-binding site of metalloproteinases in a 1:1 stoichiometry, but can also bind at other domains of metalloproteinase-2.
• WO 2010126544 describes the use of mast cell stabilizers to prevent, treat, or mitigate the severity of laminitis.
• US 20140144109 discloses a boot for treating laminitis in horses wherein the boot has a hoof casing for snugly receiving and supporting the hoof wall of the laminitic hoof and a sole pivotally attached to the hoof casing such that the laminitic hoof may pivot with respect to the sole while the sole is planted on the ground, thereby reducing stress on the inflamed laminae.
• EP 2497475 describes the use of specific anti- platelet drugs for the treatment and/or prevention of laminitis.
• the prior art in this field has not been able to effectively prevent or cure laminitis.
• a method for treating or preventing a disorder associated with undesirable immune inflammatory activity leading to protease activity in a subject in need thereof comprising administering a therapeutically or prophylactically effective amount of a 21-aminosteroid (also known as a lazaroid), or a therapeutically acceptable salt thereof to the subject.
• a method for treating or preventing a disorder associated with undesirable immune inflammatory activity leading to protease activity in a subject in need thereof comprising administering to a subject a therapeutically or prophylactically effective amount of a 21-aminosteroid or a therapeutically acceptable salt thereof.
• a method for treating or preventing equine chronic lung disease in an ungulate in need thereof the method comprising administrating to the ungulate a therapeutically or prophylactically effective amount of a 21-aminosteroid or a therapeutically acceptable salt thereof.
• a method for treating or preventing equine septic joint disease in an ungulate in need thereof the method comprising administrating to the ungulate a therapeutically or prophylactically effective amount of a 21-aminosteroid or a therapeutically acceptable salt thereof.
• a method for treating or preventing equine chronic obstructive pulmonary disease in an ungulate in need thereof the method comprising administrating to the ungulate a therapeutically or prophylactically effective amount of a 21-aminosteroid or a therapeutically acceptable salt thereof.
• a method for treating or preventing equine Crohn’s disease in an ungulate in need thereof the method comprising administrating to the ungulate a therapeutically or prophylactically effective amount of a 21-aminosteroid or a therapeutically acceptable salt thereof.
• a method for preventing or treating a condition associated with a gastrointestinal injury, disease, or ulcer in an animal in need thereof comprising administering to the animal a therapeutically or prophylactically effective amount of a 21- aminosteroid or a therapeutically acceptable salt thereof.
• SEQ ID NO:1 Sequence of anti-hemorrhagic peptide (15 amino acids).
• SEQ ID NO:2 Sequence of anti-hemorrhagic peptide (10 amino acids).
• DETAILED DESCRIPTION [0027] The inventor has demonstrated that 21-aminosteroids (lazaroids) are useful to inhibit the cell-damaging effects of protease enzymes, inflammatory cytokines, and oxidative radicals produced by these.
• the 21-aminosteroid, or a therapeutically acceptable salt thereof is chosen from a compound of Formula I: or a therapeutically acceptable salt thereof, wherein W is chosen from C and CR 4 ; Z is CR 6 ; R 1 is heteroaryl, optionally substituted by one or more R 2 groups; each R 2 is independently chosen from heterocycloalkyl and amino, either of which may be optionally substituted by one or more R 3 groups; each R 3 is independently chosen from alkyl and allyl; R 4 is chosen from H, hydroxyl, and alkyl; R 5 is chosen from H and alkyl; R 6 is chosen from H, hydroxyl, and oxo; R 7 is chosen from H, halo, and alkyl; R 8 is chosen from H and hydroxyl; L is chosen from -CH2-, -C(O)-, and -C(CH3)H-; and n is chosen from 1, 2, or 3.
• R 1 is chosen from pyridinyl and pyrimidinyl, either of which may be optionally substituted by one or two R 2 groups.
• R 1 is pyrimidinyl, substituted by one or two R 2 groups.
• R 2 is independently chosen from pyrrolidinyl, morpholinyl, diethylamino, amino, dimethylamino, piperazinyl, 4-methyl-1-piperazinyl, piperidinyl, allylamino, and ethylamino.
• each R 2 is pyrrolidinyl.
• each R 3 is independently chosen from C1-C3 alkyl and allyl.
• R 4 is chosen from H, hydroxyl, and C1-C3 alkyl.
• R 4 is H.
• R 5 is H.
• R 5 is chosen from H and C1-C3 alkyl.
• R 5 is methyl.
• R 7 is chosen from H, halo, and C1-C3 alkyl.
• R 6 , R 7 , and R 8 are H, L is -C(O)-, and n is 1.
• the 21-aminosteroid is U-74389G/Methylated Tirilazad or a therapeutically acceptable salt thereof. In some embodiments, the 21-aminosteroid is the maleate salt of Methylated Tirilazad. [0043] In some embodiments, the 21-aminosteroid is Tirilazad or a therapeutically acceptable salt of Tirilazad. In some embodiments, the 21-aminosteroid is the mesylate salt of Tirilazad.
• 21-aminosteroid or other drug or compound described herein may be known in the art by alternate names; these alternate names are intended to be encompassed with the scope of the present disclosure.
• the 21-aminosteroid may be administered alone, but will typically be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent, or carrier selected depending on the intended route of administration, which is usually intravenous (IV).
• the 21-aminosteroid may be provided at a concentration ranging from about 0.01 ⁇ g/ml to about 100 mg/ml in the formulation.
• 21-aminosteroid is present at a concentration ranging from about 0.1 ⁇ g/ml to about 1000 ⁇ g/ml. More typically, the 21- aminosteroid is present at a concentration ranging from about 1 ⁇ g/ml to 500 ⁇ g/ml.
• a 21-aminosteroid described herein may be administered in combination with an anti-hemorrhagic peptide.
• the anti-hemorrhagic peptide may be obtained from opossum serum or cotton rat, or may be a recombinant form thereof. In some embodiments, the anti-hemorrhagic peptide may be artificially produced or synthesized.
• the peptide comprises, consists of, or consists essentially of one of the following sequences: [0058] Leu-Lys-Ala-Met-Asp-Pro-Thr-Pro-Pro-Leu-Trp-Ile-Lys-Thr-Glu (SEQ ID NO:1) [0059] Leu-Lys-Ala-Met-Asp-Pro-Thr-Pro-Pro-Leu (SEQ ID NO:2). [0060] In some embodiments, an anti-hemorrhagic peptide described herein has an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1 or SEQ ID NO:2.
• an anti-hemorrhagic peptide described herein may be administered simultaneously with, sequentially with, or separately to the 21-aminosteroid.
• Pharmaceutical compositions may be adapted for administration in any suitable manner.
• the composition may be adapted for intravenous (IV) or depot (e.g., slow release) administration.
• the composition may be in injectable or suppository form, or may be formulated in a gel to make application to wound surfaces more convenient.
• delivery routes are intravenous or intramuscular depot administration.
• Methods and pharmaceutical carriers for preparation of pharmaceutical compositions, including compositions for intravenous administration, are well-known in the art.
• compositions may be formulated so that they are suitable for intravenous administration.
• the compositions may include other agents conventional in the art having regard to the type of therapeutic in question, for example, those suitable for intravenous administration.
• the composition may have at least one further active ingredient selected from antibiotics, anti-inflammatories, antiseptics, and other agents, e.g. anesthetics.
• the compositions may have other molecules associated therewith to aid releasability, stability, solubility, activity and/or association with wound healing, including carriers, solubilizing agents, and growth factors.
• the composition may also include one or more secondary therapeutic agents for treatment of the disorder in question, such as laminitis.
• Laminitis can be stimulated by bacterial and/or viral infection, or by a gastrointestinal injury, disease, infection, or ulcer.
• Laminitis may be stimulated from a disease or condition such as equine chronic lung disease, equine osteoarthritis disease, equine septic joint disease, equine colic, equine chronic obstructive pulmonary disease, equine joint disease, equine ulcerative colitis, equine Crohn’s disease, or equine inflammatory bowel disease. All of these conditions can be prevented or treated by administering adequate amounts of selected 21-aminosteroid salt.
• a subject appropriate for treatment as described herein may be a mammal, such as an ungulate, in particular a hoofed ungulate.
• the subject may be an animal from a group such equidae, bovinae, suidae, deer, ovis, and capra.
• the treated subject is a horse or dairy cow of economic importance.
• a 21-aminosteroid described herein may be injected intravenously into the ungulate, for example, the hoof.
• the ungulate may be suspected of developing or may have the sequela of laminitis at the time of administration. Administration of the 21-aminosteroid may prevent laminar detachment.
• a 21-aminosteroid may be administered into the flexor digitorum profundus muscle or into the blood supply of a limb of the ungulate.
• a method for preventing or treating a condition associated with a gastrointestinal injury, disease, or ulcer including administering to the animal in need thereof an effective amount of a 21-aminosteroid salt as described herein.
• the concentration of the 21-aminosteroid present in a formulation suitable for intravenous administration should range from about 0.1 ⁇ g/ml to about 10 mg/ml, including 0.1 ⁇ g/ml, 0.2 ⁇ g/ml, 0.3 ⁇ g/ml, 0.4 ⁇ g/ml, 0.5 ⁇ g/ml, 0.6 ⁇ g/ml, 0.7 ⁇ g/ml, 0.8 ⁇ g/ml, 0.9 ⁇ g/ml, 1 ⁇ g/ml, 2 ⁇ g/ml, 3 ⁇ g/ml, 4 ⁇ g/ml, 5 ⁇ g/ml, 6 ⁇ g/ml, 7 ⁇ g/ml, 8 ⁇ g/ml, 9 ⁇ g/ml, 10 ⁇ g/ml, 15 ⁇ g/ml, 20 ⁇ g/ml, 25 ⁇ g/ml, 30 ⁇ g/ml, 35 ⁇ g/ml, 40
• the composition may be administered at any appropriate time, including prior to, during, or after the disorder has become evident. Typically, two or more doses may be administered over time, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or more doses.
• the composition may be administered for any period of time deemed appropriate to alleviate or prevent symptoms of laminitis, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more.
• the daily dosage can be routinely determined. Generally, the dosage will vary according to the age, weight, and response of the individual, as well as the severity of the symptoms.
• a suitable dose of a 21- aminosteroid described herein will be in the range of about 0.1 ⁇ g to about 100 mg per kilogram body weight of the subject or recipient per day, such as including, but not limited to, 0.1 ⁇ g, 0.2 ⁇ g, 0.3 ⁇ g, 0.4 ⁇ g, 0.5 ⁇ g, 0.6 ⁇ g, 0.7 ⁇ g, 0.8 ⁇ g, 0.9 ⁇ g, 1 ⁇ g, 2 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 150 ⁇ g, 200
• a suitable dose of a 21-aminosteroid will be in the range of about 1 ⁇ g to about 50 mg per kilogram body weight per day.
• a method for treating or preventing a disorder associated with undesirable protease activity in a subject in need thereof comprising administering to the subject in need thereof an effective amount of a 21-aminosteroid salt as described herein in combination with an effective amount of an anti-hemorrhagic peptide described herein.
• a method for preventing or treating a condition associated with a gastrointestinal injury, disease, or ulcer including administering to the subject in need thereof an effective amount of a 21-aminosteroid salt as described herein in combination with an effective amount of an anti-hemorrhagic peptide described herein.
• the anti-hemorrhagic peptide is obtained from opossum serum or cotton rat, or is a recombinant form thereof.
• the anti- hemorrhagic peptide is selected from a peptide disclosed herein, such as SEQ ID NO:1 or SEQ ID NO:2, the following or SEQ ID NO:2.
• an anti-hemorrhagic peptide may be one or more of peptides 1-8 disclosed herein. In some embodiments, any combinations of these peptides may be used as described herein. [0077] In some embodiments, the concentration of an anti-hemorrhagic peptide described herein may be in a dose range of from about 0.1 ⁇ g/ml to about 10 mg/ml, including, but not limited to, 0.1 ⁇ g, 0.2 ⁇ g, 0.3 ⁇ g, 0.4 ⁇ g, 0.5 ⁇ g, 0.6 ⁇ g, 0.7 ⁇ g, 0.8 ⁇ g, 0.9 ⁇ g, 1 ⁇ g, 2 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 6 ⁇ g, 7 ⁇ g, 8 ⁇ g, 9 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50
• an anti-hemorrhagic peptide described herein may be administered at any appropriate time, including prior to, during, or after the disorder has become evident. Any number of doses of an anti-hemorrhagic peptide may be administered as deemed appropriate. Typically, two or more doses may be administered over time. [0079]
• the daily dosage for an anti- hemorrhagic peptide can be routinely determined by the attending physician or veterinarian. Generally, the dosage will vary according to the age, weight, and response of the individual patient or subject, as well as the severity of the patient’s symptoms.
• a suitable dose of an anti-hemorrhagic peptide described herein will be in the range of about 0.1 ⁇ g to about 100 mg per kilogram body weight of the subject or recipient per day, preferably in the range of about 1 ⁇ g to about 50 mg per kilogram body weight per day.
• a disorder appropriate for treatment as described herein can be a dental or oral wound; peptic ulceration of the duodenum, stomach or esophagus; inflammatory bowel disease; an ulcer associated with stress conditions; damage to the lining of the alimentary tract; inadequate gut function or damage to the gut associated with prematurity; a diarrheal condition; a food intolerance; a cancer of the gastrointestinal tract; surgically induced damage to the gut; damage due to esophageal reflux; a condition associated with loss of gut barrier function; a congenital condition resulting in inadequate gastrointestinal function or damage; or an autoimmune disease that affects the gut.
• Tirilazad refers to 16 ⁇ -methyl-21-[4-[2,6-bis(1-pyrrolidinyl)-4-pyrimidinyl]-1- piperazinyl]pregna-1,4,9(11)-triene-3,20-dione, which has the following structure.
• Methylated tirilazad or U-74389G refers to 21-[4-(2,6-bis(l-pyrrolidinyl)-4- pyrimidinyl)-l-piperazinyl]pregna-l,4, 9(ll)-triene-3, 20-dione, which has the following structure:
• alkyl refers to a straight-chain or branched-chain alkyl radical containing from 1 to 20 carbon atoms. In certain embodiments, said alkyl will comprise from 1 to 10 carbon atoms. In further embodiments, said alkyl will comprise from 1 to 8 carbon atoms.
• amino refers to -NRR , wherein R and R are independently chosen from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optionally substituted. Additionally, R and R’ may combine to form heterocycloalkyl, either of which may be optionally substituted.
• halo or halogen, as used herein, alone or in combination, refers to fluorine, chlorine, bromine, or iodine.
• heteroaryl refers to a 3 to 15 membered unsaturated heteromonocyclic ring, or a fused monocyclic, bicyclic, or tricyclic ring system in which at least one of the fused rings is aromatic, which contains at least one atom chosen from N, O, and S.
• said heteroaryl will comprise from 1 to 4 heteroatoms as ring members.
• said heteroaryl will comprise from 1 to 2 heteroatoms as ring members.
• said heteroaryl will comprise from 5 to 7 atoms.
• heterocycloalkyl and, interchangeably, “heterocycle,” as used herein, alone or in combination, each refer to a saturated, partially unsaturated, or fully unsaturated (but nonaromatic) monocyclic, bicyclic, or tricyclic heterocyclic group containing at least one heteroatom as a ring member, wherein each said heteroatom may be independently chosen from nitrogen, oxygen, and sulfur.
• said hetercycloalkyl will comprise from 1 to 4 heteroatoms as ring members. In further embodiments, said hetercycloalkyl will comprise from 1 to 2 heteroatoms as ring members. In certain embodiments, said hetercycloalkyl will comprise from 3 to 8 ring members in each ring. In further embodiments, said hetercycloalkyl will comprise from 3 to 7 ring members in each ring. In yet further embodiments, said hetercycloalkyl will comprise from 5 to 6 ring members in each ring.
• Heterocycloalkyl and “heterocycle” are intended to include sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclic fused and benzo fused ring systems; additionally, both terms also include systems where a heterocycle ring is fused to an aryl group, as defined herein, or an additional heterocycle group.
• the heterocycle groups may be optionally substituted unless specifically prohibited.
• any definition herein may be used in combination with any other definition to describe a composite structural group.
• the trailing element of any such definition is that which attaches to the parent moiety.
• the composite group alkylamido would represent an alkyl group attached to the parent molecule through an amido group
• the term alkoxyalkyl would represent an alkoxy group attached to the parent molecule through an alkyl group.
• the term “optionally substituted” means the anteceding group may be substituted or unsubstituted.
• Asymmetric centers exist in the compounds disclosed herein. These centers are designated by the symbols “R” or “S,” depending on the configuration of substituents around the chiral carbon atom.
• the invention encompasses all stereochemical isomeric forms, including diastereomeric, enantiomeric, and epimeric forms,as well as d-isomers and 1-isomers, and mixtures thereof.
• Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, direct separation of enantiomers on chiral chromatographic columns, or any other appropriate method known in the art.
• Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art. Additionally, the compounds disclosed herein may exist as geometric isomers.
• the present invention includes all cis, trans, syn, anti,
• E
• Z
• compounds may exist as tautomers; all tautomeric isomers are provided by this invention.
• the compounds disclosed herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms.
• disease as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the animal to have a reduced duration or quality of life.
• disorder disorder
• condition condition
• therapeutically effective is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder or on the effecting of a clinical endpoint.
• the term “therapeutically acceptable” refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
• the compounds disclosed herein can exist as therapeutically acceptable (e.g., biologically effective) salts.
• the present invention includes compounds listed above in the form of salts, including acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable.
• salts of non-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question.
• Basic addition salts may also be formed and be pharmaceutically acceptable.
• Pharmaceutical Salts: Properties, Selection, and Use refer to Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).
• the term “therapeutically acceptable salt,” as used herein, represents salts or zwitterionic forms of the compounds disclosed herein which are water or oil-soluble or dispersible and therapeutically acceptable as defined herein.
• the salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound in the form of the free base with a suitable acid.
• Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenyl
• basic groups in the compounds disclosed herein can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
• acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
• Salts can also be formed by coordination of the compounds with an alkali metal or alkaline earth ion.
• a “therapeutically effective amount” of a compound, drug, or other agent refers to an amount that is sufficient to generate a desired therapeutic response, such as reduce, eliminate, or measurably alter outward signs or symptoms of a condition or disease described herein.
• a therapeutically effective amount may be an amount necessary to reduce, eliminate, ameliorate, or otherwise improve clinical or histopathological signs and/or symptoms of laminitis described herein.
• Histopathological symptoms of laminitis may include, but are not limited to, separation of the basement membrane, pyknosis or karyopyknosis, and presence of cytoplasmic and nuclear alteration in epidermal cells.
• a therapeutically effective amount may also be sufficient to reduce the Obel score of an animal, e.g., a horse, indicating an improvement of signs/symptoms of laminitis.
• a therapeutically effective amount is also an amount required to at least partly attain a desired effect, i.e., to alleviate or remove the symptoms associated with undesirable immune inflammatory activity leading to protease activity, or alternatively to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the undesirable immune inflammatory activity leading to protease activity.
• the term “therapeutically effective amount” as used herein means amount sufficient to elicit a statistically significant response at a 95% confidence level. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, and individual subject parameters, including age, physical condition, size, weight, and other concurrent treatment, and will be at the discretion of the attending veterinary person. These factors are well known to those of ordinary skill in the art, and can be addressed with no more than routine experimentation. It is generally preferred that a minimum effective dose be determined according to sound veterinary judgment. [00101] An effective amount may be a prophylactically effective amount, which is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing.
• a prophylactically effective amount is an amount that prevents the development of histopathological signs/symptoms of laminitis, or otherwise provides a protective effect to the subject.
• treatment refers to any regime that can benefit a subject. References herein to “therapeutic” and “prophylactic” treatment are to be considered in their broadest context. The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
• treating or preventing laminitis refers to a reduction, improvement, lessening, elimination, reversal, or prevention of the development of signs and/or symptoms of a disease described herein.
• treating laminitis refers to an improvement or reduction in clinical signs or symptoms of laminitis, e.g., difficulty walking, improvement in gait or turning, improved ability to lift the affected leg, or a reduction in an Obel score for an evaluated animal.
• Preventing laminitis refers to the prevention of development of a clinical or histopathological indication of laminitis, e.g., separation of the basement membrane, following administration of tirilazad or a therapeutically acceptable salt thereof.
• Prophylactic administration of tirilazad may be preferred in certain instances where animals are subjected to conditions that are favorable for developing laminitis as described herein. In such instances, administration of tirilazad or another compound disclosed herein may prevent the development of cellular degeneration and other histopathological indications of laminitis, thereby preventing laminitis or development of clinical signs of disease in the animal.
• Group 1 Three horses (Group 1) will receive 6 mL/kg bodyweight of 0.9% saline solution (placebo) immediately after onset of pyrexia and/or diarrhea. Thereafter, Group 1 horses will receive fluid therapy for dehydration and shock, but no specific treatments will be administered for laminitis. Three horses (Group 2) will receive the experimental treatment of IV 21 -aminosteroid immediately after the onset of pyrexia and/or diarrhea. Three remaining horses (Group 3) will receive the experimental treatment of IV 21-aminosteroid upon achievement of an Obel lameness score ⁇ 1.
• Horses are the target species for use of the biological product to be evaluated. This study will utilize mature, random-source, light saddle breed horses, females or neutered males, and 3 to 7 years of age at the time of induction. Candidate horses must have Obel lameness scores of “0”, be sound by hoof tester examination, and exhibit no radiographic evidence of prior laminitic episodes (i.e., no ventral deviation of the third phalanx). Candidate horses will be healthy, as determined by clinical health observations and physical examination during the acclimation period and prior to initiation of the carbohydrate overload induction regimen. Pre-enrollment testing of candidates will be conducted to rule out PPID (Cushing’s Disease) and Equine Metabolic Syndrome (insulin resistance).
• PPID Cutting’s Disease
• Equine Metabolic Syndrome insulin resistance
• Candidates must have a body condition score of ⁇ 3 to ⁇ 7. Female horses may not be pregnant or lactating. [00119] At least nine candidate horses will be received at the site to begin acclimation, and any that meet inclusion criteria will be enrolled. Additional candidates may be evaluated, but ultimately, only nine will be enrolled in the study. [00120] Horses will be derived from the resident, facility herd or purchased from a commercial livestock vendor. [00121] All horses will be uniquely identified by a numbered neck band and by a complete physical description in the study record. [00122] A candidate horse will be eligible for enrollment if it meets all of the following criteria: - It conforms to the animal description in section 8.1.1 (age, gender, class, physiologic status).
• a candidate will be excluded from enrollment if: - It does not conform to inclusion criteria.
• Horses will be acclimated to the facility for at least seven days prior to initiation of the carbohydrate overload induction regimen. During the acclimation period, feed, water, housing, management, and environmental conditions will simulate those expected during the study. [00126] Medication and/or vaccination during acclimation period [00127] Candidate horses may be treated with approved pharmaceutical products prior to initiation of the acclimation period, but no medicinal products may be administered to subjects from the start of acclimation until completion of the study. Certain prior treatments are proscribed, as described in Section 8.2 and Section 8.3.
• Soiled bedding is replaced as necessary, usually about once weekly during acclimation, and at least daily during the overload induction phase. Facility details will be described and documented in the study record.
• Overhead incandescent lighting is available to provide illumination during late p.m. and early a.m. activities.
• the equine housing facility is under roof, but subjects are otherwise exposed to ambient environmental conditions. Climatic conditions (minimum and maximum temperature and relative humidity) will be monitored electronically on a constant basis, and daily minima and maxima will be recorded manually on a data capture form customized for the specific study.
• Each stall is equipped with a combination concentrate/hay feeder designed to offer both dietary components simultaneously. Feeders are checked daily and cleaned if necessary.
• a qualified veterinarian will conduct a physical examination during the week of acclimation (between Days -10 and -4). The examination will evaluate the physiological status of each animal by systems, including rectal temperature, eyes, cardiovascular system, respiratory system, gastrointestinal and genitourinary systems, skin and hair coat, neurologic and musculoskeletal function, and overall physical condition. Findings for individual horses will be recorded on the Physical Examination Record.
• a Body Condition Score (BCS) will be assigned to each candidate during the physical examination. Scores will range from 1 to 9, and are based on the Henneke system (Henneke et al. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Veterinary Journal 15:371-372, 1983).
• Body weight Each candidate will be weighed once between Days -10 to -4. Relevant body weights will be used to calculate appropriate quantities of oligofructose for the carbohydrate overload induction model. [00146] Body weights will be measured with a scale that has been certified by a commercial service within 6 months before the start of the study. Prior to weighing the first animal, and again after weighing the last animal, the accuracy of the scale will be verified with standard weights ranging from 45.4 kg (100 lbs) to 364 kg (800 lbs). Body weights will be measured to the nearest kg and recorded on the Body Weight Record.
• Lameness examinations Prior to enrollment, each horse will be assessed for lameness, as described in facility SOP LAM-FD-2.2. Each horse will be assessed at a walk and at a trot (if possible) to assign baseline Obel scores for each forefoot. [00149] Guidelines for Obel lameness scoring are as follows: [00150] Grade 0: No lameness observed at a walk or trot, even on hard surfaces. [00151] Grade 1: The horse may alternately lift its feet, but no lameness is observable at a walk. The horse may have a short, stilted gait when trotting in a straight line on a hard surface, and turns carefully at a walk.
• Grade 2 Moves with a stiff gait at the walk.
• the horse may have a short, stilted gait at a trot on a hard surface. Turns with great difficulty. A foot can be lifted off the ground without great difficulty.
• Grade 3 Reluctant to move at a walk on any surface. It is difficult to lift a limb. The horse may be almost non-weight bearing on one limb.
• Grade 4 The animal will not move, and is particularly reluctant to move from a soft to a hard surface. It is almost impossible to lift a limb.
• Prior to enrollment horses will be evaluated for foot pain using hoof testers.
• the hoof testers will be applied in a systemic manner to the entire sole, frog region and hoof wall to test for sensitivity/pain.
• a hoof tester score of “0” for both forefeet is required to be eligible for enrollment.
• Prior to enrollment, lateral radiographs of both forefeet of each horse will be recorded and examined for evidence of prior laminitis, defined as ventral rotation of the third phalanx (P3) in the lateral view. A written interpretation of each horse’s radiographs will be included in the study record.
• Clinical observations will be recorded once daily from Day -10 to the final day of enrollment.
• the parameters to be observed include general health, appetite, attitude and fecal consistency (Daily Health Observation Record). Findings will be recorded as “normal” or “abnormal”, with further characterization in the study record of any abnormal observation.
• Findings will be recorded as “normal” or “abnormal”, with further characterization in the study record of any abnormal observation.
• At ⁇ 6-hour intervals (+30 minutes) after administration of the final step of the oligofructose model general health observations will be conducted, along with measurement of rectal temperature, heart rate, and assessment of capillary refill time. Observations will be recorded on Data Capture Forms specifically created for the study, and abnormal observations will be further characterized in the study record.
• a venous blood sample will be collected every 2 hours for measurement of packed cell volume and total protein concentration. Heart rate, CRT, and total protein concentration will be used to assess dehydration as per ETCR SOP LAM-FD-1.x. Fluid therapy will be initiated at the discretion of the veterinarian or when percentage dehydration achieves 6% or greater.
• any abnormal health observations will qualify as Adverse Events (AE).
• AE Adverse Events
• the Clinical Investigator will report the AE to the Sponsor Monitor, and the event will be documented on the Adverse Event Record.
• the Adverse Event Record will categorize the severity of the abnormal observation, and the reporting veterinarian will speculate as to the relationship of the AE to experimental treatment as follows: Magnitude of Adverse Event
• Feed and Water Consumption [00164] Feed and hay will be provided twice daily in weighed quantities. Appetite will be characterized as: 0- consumed ⁇ 25% of hay/grain 1- consumed 25-75% of hay/grain 2- consumed 75-100% of hay/grain. [00165] Water will be provided in two, 16-L buckets per horse. Water consumption will be measured in 1/8 bucket (i.e., 2-L) increments and recorded twice daily prior to re-filling of the respective water buckets.
• a serum sample (marbled red top tube; 9.5 mL draw) will be collected from each enrolled horse on Day 0 prior to induction, and again just prior to euthanasia. Serum will be harvested from each sample and stored frozen. Serum samples will be shipped to an external laboratory for measurement of camelid antibodies by a proprietary ELISA. Methods and results will be described in a separate report to be prepared by the analytical laboratory. [00170] Removal of subject(s) from the study [00171] This protocol seeks to balance the need to generate relevant efficacy data with humane considerations. As such, horses experiencing adverse events, whether or not related to the test article, may receive veterinary care as medically appropriate and under the parameters described above.
• a participating horse may be removed from the study if it is determined that: - It is uncooperative with study procedures. - It encounters a serious adverse reaction, injury, or illness necessitating treatment with contraindicated, concomitant medications (see section above) or dictating immediate removal for humane reasons. - It dies spontaneously or is euthanatized.
• a horse will be removed from the study if any of the indicated removal criteria apply. The Clinical Investigator will consult with the Sponsor whenever possible prior to removing a horse from the study. However, the final decision whether to remove a horse from the study will rest with the Clinical Investigator. The Clinical Investigator will document the horse’s identity, the date of the removal, the reason for the removal, and the fate of the animal.
• Explant tissue samples were obtained from the dermoepidermal junction of the hoof of the right forelimb under nervous system block without the need for euthanasia. [00184] Under sterile conditions, laminar cuts (mini-explants) were made of each explant and the tissue washed with physiological solution. The mini-explants were pre-incubated for 5 hours with tirilazad and then for 24 hours with Bothrops venom.
• Culture medium was as follows: Dulbecco’s minimum essential medium (DMEM, GIBCO-Invitrogen) with 10% heat-inactivated fetal bovine serum (FBS, GIBCO-Invitrogen), L-Glutamine (29.2 mg/mL), Penicillin (10,000 units/mL), and Streptomycin (10,000 ⁇ g/mL, GIBCO-Invitrogen) as antibiotics.
• DMEM minimum essential medium
• FBS GIBCO-Invitrogen
• L-Glutamine 29.2 mg/mL
• Penicillin 10,000 units/mL
• Streptomycin 10,000 ⁇ g/mL, GIBCO-Invitrogen
• Table 4 demonstrates the results from this experiment obtained using a Car Zeiss Primo Star optical microscope. [00187] The tissue incubated only in the culture medium showed no histological alteration. [00188] Tissue incubated with Bothrops alternatus venom at a dose of 75 ⁇ g/ml showed intense separation of the basement membrane and alteration of epidermal cells. [00189] Tissue incubated with tirilazad at a dose of 1 ⁇ M did not show separation of the basement membrane, and epidermal cells were normal and showed preserved cytoplasmic and nuclear structure.
• Tissue incubated with 1 ⁇ M tirilazad and 75 ⁇ g/ml Bothrops alternatus venom did not show separation of the basement membrane, although intense presence of altered epidermal cells with nuclear pyknosis was observed.
• Tissue incubated with 3 ⁇ M tirilazad did not show separation of the basement membrane, although slight epidermal cytoplasmic and nuclear alteration was observed.
• Incubation of tissue with 3 ⁇ M tirilazad and 75 ⁇ g/ml Bothrops alternatus venom showed slight separation of the basement membrane and intense presence of altered epidermal cells, with nuclear pyknosis predominating.
• Tissue incubated with 10 ⁇ M tirilazad did not show separation of the basement membrane, and epidermal cells were normal and showed preserved cytoplasmic and nuclear structure, similar to tissue incubated with 1 ⁇ M tirilazad.
• Tissue incubated with 10 ⁇ M tirilazad and 75 ⁇ g/ml Bothrops alternatus venom did not show separation of the basement membrane. Moderate presence of altered epidermal cells was observed, predominantly moderate nuclear pyknosis.
• Tissue incubated with 20 ⁇ M tirilazad showed intense separation of the basement membrane, while epidermal cells were normal and showed preserved cytoplasmic and nuclear structure.

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