IE20150034A1 - Laminitis prevention and treatment through camelid serum - Google Patents

Abstract

The present invention provides evidence that metalloprotease and other protease type enzyme peptide inhibitors present or generated by vaccination in camelid serum /plasma can be used alone or combined with other agents to prevent or treat Laminitis. The serum/ plasma and isolated or synthesised peptides listed in this patent have use in the prevention and treatment of Laminitis. In equine treatment this camel plasma and peptides outlined in this patent can be utilised to inhibit all the inflammatory protease enzymes that have been identified as the causative agents for equine laminitis.

Description

[0001] The present invention relates to protease/metalloprotease enzyme inhibitory peptides and antibodies discovered to be present in camelid serunr/plasma or additional inhibitory MMP and serine protease enzyme inhibitory antibodies which can be generated by a vaccination program utilising identified enzyme antigens known to be responsible for causing the laminitis condition.

More specifically these enzyme inhibitory peptides isolated from carnelid blood are active against equine metalloproteinases and other equine serine proteases enzymes and are effective in the treatment and or prevention of laminitis. These protease/rnetalloprotease inhibitory peptides are naturally present in camelid blood and are not present in such elevated levels or with such broad spectrum inhibition in other ruminants and can be isolated from same for use in therapeutic formulations of carnelid plasma in laminitis.

Brtckgrotmd ofihe Invention

[0002] Proteinases (rne'talloPl‘otease) and ser:irrc: Proteases are naturally oecrrnirrg enzymes present, in many tissues of‘ the equine body and in mammals in general. These enzymes act to degrade proteins, normally in a corrtrolled and specific manner. To prevent the unconnollecl destruction of target proteins and tissue such as the hoof the activity of these proteolytie enzymes are modulated by inhibitor serum peptides normally present under healthy conditions. Thus, the combined and balanced actions of proteinases and inhibitors act to control the level of biologically active or structurally important proteins of the body, thereby regulating many important physiological processes and maintaining structural integrity.

[0003]One important group of proteinases are the metalloproteinases. These enzymes are characterised by their requirement for the presence of a metal ion in order to catalyse proteolysis.

Approximately 17 different metalloproteinases have been identified and/or cloned which share significant sequence homology. The metalloproteinase family can be subdivided into five groups according to their structural and functional properties: (i) the collagenases (metalloproteinases—i , 8 and 13); (ii) the gelatinases A and B (metalloproteinase—2 and metalloproteinase~9) (iii) the stromelysins 1 and 2 (metalloproteinases— 3 and 10); (iv) matrilysin (MMP~7); enamelysin (MMP~20), macrophage metalloelastase (MMP--12), and MMP-~19 (making up the classical metalloproteinases): (V) the membrane-type metalloproteinases (MT—MMP—l to 4 and strome1ysin—3, MMP-11) . These metalloproteinases share a common multi—domain structure, but are glycosylated to different extents and at different sites. According to sequence alignment, the assembly of these domains might have been an early evolutionary event, followed by diversification. Collectively, metalloproteinases can degrade all the major components of the extracellular matrix (ECM).

[0004] The homeostasis of the ECM is controlled by a delicate balance between the synthesis of ECM proteins, production of ECM—degrading extracellular matrix metalloproteinases (MMPS), and the presence of metalloproteinase ll1l1il)l'[O1'S_.

[0005] One family of metalloproteinases inliibitor peptides are the tissue inhibitors of metalloproteinases (TIMPS). The TIMP family is comprised of at least four distinct members (TIMP-l to 4) which possess 12 conserved, cysteine residues and express metalloproteinase inhibitory activity by forming non— covalent complexes with inetalloproteinases enzymes.

Specifically TIMPs bind to the highly conserved active zinc-binding site of the metalloproteinases in a 1:1 stoichiometry, but can also bind at other domains of metalloproteinase-2

[0006] The prior in this field has not been able to overcome the disease to an extent and it is necessary to provide for a method to effectively treat this disease. Charles F Owen in WO 2010126544 A] has disclosed mast cell stabilizers used to prevent, treat, or mitigate severity of laminitis. i._\5li_cj.i9lle_ Burij has in US 20140144109 A1 disclosed a boot for treating laminitis in horses which has a hoof casing for snugly receiving an supporting a horny 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 ground, thereby reducing stress on the inflamed laminae. _S_ab_ii_1—e_ Efileij in El’ 2497475 A1 has disclosed use of specific antiplatelet drugs for the treatment and/or prevention of Larninitis. Though, the prior art in this field has not been able to effectively cure the disease. Also the cost in using the above mentioned inventions and other similar methods is very high.

[0007] It is an aspect of the present invention to overcome or at least alleviate one or more of the difiiculties or deficiencies related to the prior art by providing a plentiful source of metalloproteinase inhibitor peptides and serine protease inhibitory proteins from camelid seruml plasma and methods for purifying these inhibitor peptides from the camelid blood. It is also the aim of this invention to provide for a cost effective treatment of laminitis. Also it has been found that by inoculating camelids with purified equine metalloproteinase enzymes and serine Proteases and adjuvant that enzyme inhibitory antibodies generated in the inoculated camelicl have the ability to inhibit imetalloprotease erizyrnatic activity and elastase and this enhances the ability of the cameiid serum to inhibit these enzymes in the laminitis hoof and lead to rapid healing .1-'<‘urtl1e1‘more, compositions including inhibitors are disclosed as well as methods for treating various conditions and diseases using the naturally occurring metalloprotease inhibitory peptides in camelid serum or coupled with specific inhibitory antibodies generated by the camelid immune system following vaccination with specific Equine metalloprotease enzymes and equine serine protease. Described herein. These specific carnelid inhibitory antibodies generated as a result of vaccination and the protease inhibitory peptides naturally present in camelid seruml plasma may be isolated for therapeutic use in larninitis and other veterinary diseases of animals where elevated tissue Proteases are responsible for the disease indication.

Summary of the Invention

[0012] The first aspect the present invention provides a composition derived directly or indirectly from the blood/serum/Plasma of a camelid species, the composition comprising an inhibitor of a equine metalloproteinase enzymes and other equine serine protease enzymes not requiring a metal group for activity. This invention outlines that camelid seium/plasma contains effective inhibitory amounts of equine metalloproteinase and general serine protease inhibitor peptides in large concentration unlike any other ruminant serum tested.

[0013] In a second aspect, the present invention provides a method for treating, preventing or ameliorating disorders associated with undesirable metalloproteinase enzyme or general protease enzyme activity, the method including administering to an animal or animal in need thereof an effective amount of a composition comprising a metalloproteinase enzyme inhibitor peptide or a general serine protease enzyme inhibitor peptide present ,generated following a vaccination program and isolated from carnelid serum/ plasma or utilised incorporated in the camelid plasma.

I)etat'Ied description oftlze inventioir

[0014] In a first aspect the present invention provides a composition derived directly or indirectly from tine camelid serum and or plasma the composition comprising a peptide inhibitor of a human metalloproteinase enzyme and or serine protease enzymes capable when administered intravenously to prevent or bring rapid treatment and relief to animals suffering with Laminitis.

Applicants have demonstrated that naturally occurring serum peptides in camelid serum/plasma are useful as a source of equine metalloproteinase enzyme inhibitors and of more general protease enzyme inhibitors.The unexpected finding of these inhibitors in camelid serum/plasma provides a plentiful, renewable source of equine metalloproteinase enzyme inhibitor peptides and non~metal protease enzyme inhibitor peptides for laminitis treatment. As used herein the term "metalloproteinase" includes proteases that proteolytically degrade a component of the extracellular matrix. The term metalloproteinases includes but is not limited to (i) the collagenases (metalloproteinases—l , 8 and 13); (ii) the gelatinases A and B (rnetalloproteinase—2 and metalloproteinase-9); (iii) the stromelysins 1 and 2 (metalloproteinases-3 and 10); (iv) matrilysin (MMP-7); enamelysin (MMP~ 20), macrophage metalloelastase (MMPIZ), and Ml\/[P-19 (making up the classical metalloproteinases) and (V) the niembrane-‘type metalloproteinases (MT~MMP—1 to 4 and stromeIysin—3, MMP- ll ).

[0015] The present invention also includes the use of a peptide isolate and or formulation of camelid serum and or plasma containing these metalloprotease enzyme inhibitory peptides have not been found or isolated from other animal serum tested for example those other serum tested are bovine,caprine, and equine species.

[0016] Preferably the peptide inhibitor present in the serum and or plasma of the camelid are used to inhibit equine metalloproteinase enzymes and/of general non--metal requiring equine protease enzymes are present at a oo_ncei.'Il:1‘ation 1'aiigii‘|g from about 0.01 pug/ml to about l0Omg/ ml in the formulation prepared for the application of this invention. More preferably the peptide inhibitor of equine metalloproteinase is present at a concentration ranging lion: about 0.1 pg/nil to about 'l000ug/ml. Even more preferably the camelid peptide inhibitor of equine metalloproteinase is present at a concentration ranging from about I pg/ml to 500ug/ml. In a highly preferred embodiment, the inhibitor peptide from the camelid serum or plasma to equine metalloproteinase or general non-metal containing protease is present at a concentration of about 11 ug/ml, or about 45ug/ml or about 50ug/ml as quantified by a fluorescence—quenching substrate assay.

[0017] in this invention the peptide inhibitor in the camelid plasma or serum is a tissue inhibitor of a metalloproteinase (TIMP). As used herein the term "tissue inhibitor of a metalloproteinase" includes but is not limited to polypeptides isolated from camelid blood which regulate the activity of equine metalloproteinases which includes TIMP-1 , TIMP-2, TlMP~3 and TlMP—4. The TIMP family is comprised of at least four distinct members (TIMP-1 to 4} which possess 12 conserved cysteine residues and express metalloproteinase inhibitory activity by forming non-covalent complexes with metalloproteinases. Specifically TlMPs bind to the highly conserved active zinc-binding site of the metalloproteinases in a ‘i :1 stoichiometry, but can also bind at other domains of metalloproteinase-‘Z and metalloproteinase-9.

[G018] Preferably the inhibitor camelid is capable of inhibiting metalloproteinase 2 and/or metalloproteinase 9. Metalloproteinase 2 is also known as gelatinase A. Metalloproteinase 2 is a proteolytic enzyme having a molecular weight of 72kDa which catalyses the degradation of collagen type IV by acting on the peptide bonds. Metalloproteinase 9 is also known as gelatinase B. Metalloproteinase 9. is a proteolytic enzyme having a molecular weight of 92kDa which catalyses the degradation of collagen type IV by acting on the peptide bonds.

[0019] The peptide inhibitor isolated from camelid semrti and or plasma is capable of’ ll1i’llbi‘lll’lg nieiultiraiie type I11EI,l.!,iX metaIlop1'oi,eiriases and ‘l‘Il')1'le'['1'l(,’.l_'f:l.l l::ea1'i1ig 3;e:ueral pi:oiea.se K“£l;,l,‘4‘\/i1‘ll-31%] Hl,_l{'«iF as elastase,

[0020] Pharmaceutical compositions according to the present invention may be adapted for administration in any suitable manner. The composition may be adapted for internal or topical administration. The composition may be in an oral, injectable, topical or suppository form or formulated in a gel to make application to wound surfaces more convenient. Preferred delivery routes include, intravenous dermal, intravaginal, intravenous, respiratory, and gastrointestinal delivery. It is to be understood that the compositions as described herein are not limited to use with horses, and include any animal that could benefit from the compositions.

[0021] Methods and pharmaceutical carriers for preparation of phannaceutical compositions, including compositions for topical administration are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Coinpaiiy, Easton, Pennsylvania, U

[0022] Compositions of the present invention may be formulated so that they are suitable for oral administration. The compositions may be presented as discrete units such as capsules, sachets or tablets or in bandages each containing a predetermined amount of the active protease inhibitor peptide; as a powder or granules or gel, as a solution or a suspension in an aqueous or non- aqueous liquid; as a mouthwash or as an oil-in-water liquid emulsion or a water—in—oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

[0023] It should be understood that in addition to the ingredients particularly mentioned above, the composinons of this invention may include other agents conventional in the a.1‘l:'haV:i11g regarrl to the type of therapeutic in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. [0024} In a preferred form of the invention the compositions of the present invention include a carrier selected from the group consisting of a synthetic or biological polymer, glycosaminoglycan, or extracellular matrix molecule including fibrin, collagen, gelatin, a synthetic polymer, agarose, an alginate, methylcellulose, hyaluronic acid, a hydrocolloid, an alginate, saline solution, powder, ointment, salve or incorporated or impregnated into a dressing (absorbable and non-absorbable), a transdermal patch or releasable dressing associated with gauze, a bandage, suture, plaster, staple, prosthetic device, screw or plate (biodegradable or non- biodegradable), toothpaste, gum or resin for chewing, mouth wash or gel. The skilled artisan will be familiar with the appropriate carrier to use depending on the route or means for administration.

[0025] In another preferred {Orin the composition has at least one fiirther active ingredient selected from the group including antibiotics, anti~inflammatories, antiseptics, other agent eganaesthetics. The compositions described herein 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 as discussed above.

[0026] In the above methods for treating skin or ocular infections of animals camelid serum or plasma or isolates thereof or compositions of the present invention may be applied directly to wounds in a biologically acceptable carrier to ensure sustained release at sufiieient concentration in the wound environment. In treating a wound, the metalloprotease and non metal protease enzyme camclid peptide inhibitors may be associated with a wousnd support, gel or suitable solution. As used herein the term "wound support" includes any means wliich. is used to S1l_'{)pD.'I.'i' or secure a wound and includes a surgical securing means. The term includes plasters, dressings, sutures, staples and the like. The wound to be supported may be a wound created by surgery, or the result of accident or other injury. The camelid serum and or plasma or isolate thereof‘ or composition may be present on the surface of the wound support. or may be impregnated in the Wound support/ gel and is able to be released therefrom.

[0027] The wound to be treated according to this invention may be an ulcer caused by pressure, vascular disease, diabetes, autoimmune disease, sickle cell diseases or hemophilia; a result of surgery; therapeutically induced; associated with disorders of the central nervous system, and resulting from any exfoliative disease of the skin; a associated with either local or systemic infection or a corneal injuiy to the eye; a pathological wound; a traumatic or accidental wound; or a burn.

[0028] In a preferred method the concentration of the metalloproteinase inhibitor peptide isolated or present in the camelid serum and or plasma is from about O.l ng/ml to about l0itg/ml of fluid in the local environment at the wound or disease site. Even more preferably the concentration of the eamelid metalloproteinase peptide inhibitor present in the camelid serum and or plasma is from about 1 ng/ml to about 1 pg/ml of fluid in the local environment at the wound site.

[0029] The present invention also provides a method for preventing, ameliorating or treating a condition associated with a gastrointestinal injury, disease or ulcer, the method including administering to a human or animal in need thereof an effective amount of composition as described herein. In a preferred method the concentration of the camelid metalloproteinase/ protease peptide inhibitor present in the medication should range from about 0.1 pg/ml to about 10mg /' ml. In the context of the invention, the term "effective amoun1;" as used herein means an amount siiffieient: to elicit a statistica.lly significant response at a 95% contiric-:1ice level (psi? Preferably, an effective amount is that amount to at least partially attain the desired response of a. reduction in metalloproteinase and or other protease enzymatic activity at the disease tissue site.

[0030] The compositions may be administered in therapeutically effective amounts. A therapeutically eifective amount means the amount required at least partly to attain the desired effect, ie to alleviate or prevent the symptoms of undesirable metalloproteinase /protease enzymatic activity in the wound or tissue or alternatively to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the undesirable metalloproteinase / protease activity, or to reduce Inetalloproteinase / protease activity. Preferably the term "therapeutically efiective amount" as used herein means amount sufiicient to elicit a statistically significant response at a 95% confidence level.

[0031] Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, and individual patient 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.

[0032] The composition may be administered at any appropriate time including prior to, during or after the gastrointestinal injury, disease or ulcer has become evident.

[0033] The condition 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 p:'enratni‘i‘lv; a diairheal eondil:ion.', a food inI:olerance; 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 aftects the gut.

[0034] In a fifth aspect the present invention is provided a method for preventing, ameliorating and/or treating disorders associated with undesirable metalloproteinase /protease enzymatic activity such as laminitis. The method including administering to a animal in need thereof an effective amount of the protease inhibitory peptide present in the camelid serum and or plasma or the isolated peptide or antibody thereof or composition described herein. As used herein the term "disorders associated with metalloproteinase activity” inciudes the following: [0O35} For all methods of treatment described herein the daily dosage 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, as well as the severity of the patienfs symptoms. In general a suitable dose of the inhibitor of the invention will be in the range of about 0.1 pg to about l00mg per kilogram body weight of the recipient per day, preferably in the range of about 1 gig to about 50mg per kilogram body weight per day. However, the dose wili also depend on the formulation and purity of the camelid serum and or plasma used and the cone. of inhibitory protease peptide present.

[0036] The present invention also provides a method for at least partially purifying or enriching a metalloproteinase enzyme inhibitor camelid peptide or antibody the method including the steps of isolating from the camelid serum /plasma thereof, and subjecting the camelid serum and or plasma to one or more treatment steps selected from the group consisting of centrifiigation, micro~ filtration, ultra-filtration, ion-exchange chromatography, molecular sieve chromatography, affinity chromatography, reverse-phase high peiformanee liquid chromatography and transient acidification. l. Equine metalloprotease inhibitory peptide Sequence Description 1: Leu Lys Ala Met Asp Pro Thr Pro Pro Lcu Trp Ilc Lys Thr Giu 2. Collection of Sequences: Phe—Leu-His Trp-Leu—Phe Trp-Leu—Try Trp-Leu—Arg Trp-Leu-His Phe—].,eu—Phe Phe—Leu-Try Phe—Leu-Arg = Peptide 1 = Peptide 2 = Peptide 3 = Peptide4 Peptide 5 = Peptide 6 = Peptide 7 = "Peptide 8 3. Collection of Sequences Coupled to Hydroxamate: Phe-Leu~His = Peptidel Trp~Leu—Phe = Peptide 2 Trp-Leu~T1'y = Peptide 3 Trp-Leu-Arg = Peptide 4 Coupled to hydroxamate Trp-Leu—His = Peptide 5 Phe-Leu-Phe = Peptide 6 Phe-Leu-Tiy = Peptide 7 Phe~L.eu—Arg = Peptide 8 ‘K ,/Om” 0 Ci“---H Cm. N \ / /2 \\ /C -N\ O\\ /O—H Ox /O‘-H C—N \ C —- N / \ Peptide 3 Peptide 1 Peptide 4

Claims (1)

1. Claims I. A method for prophylactically treating to prevent the laminitis condition occurring. in any limb of a horse comprising: intravenously administering a therapeutically effective amount of camelid plasma or specific antigen directed Hyperimmune camelid plasma sufficient to prevent a laminitis condition occuning in an affected equine limb. The method in claim 1 having the capability of treating a horse for laminitis and comprising of intravenously administering a therapeutically effective amount of camelid plasma or specific antigen directed Hyperimmune camelid plasma sufficient to cause rapid proteolytic enzyme inhibition in the effected hoof and cause healing. A method in Claim 2 wherein said camel plasma is obtained from a healthy camelid. A method in claim 3 wherein said Camelid plasma is obtained after an inoculation regime that produces specific antigen directed Hyperimmune plasma containing inhibitory antibodies and peptides capable of inhibiting disintegrin and metalloprotease enzymes particularly ADAM-TS