IE20140274A1 - Camelid blood serum or plasma and generated enzyme inhibitory homodimer antibodies their peptide isolates and/or synthetic peptide sequences for laminitis prevention and treatment and the treatment of other equine diseases created due to elevated metalloprotease and elastase (serine protease) enzyme activity. - Google Patents

Abstract

Camelid blood serum or plasma and generated enzyme inhibitory homodimer antibodies their peptide isolates and/or synthetic peptide sequences for laminitis prevention and treatment and the treatment of other equine diseases created due to elevated metalloprotease and elastase (serine protease) enzyme activity. The present invention provides evidence that metalloprotease and other protease type enzyme peptide inhibitors found in camelid serum/plasma can be used alone or combined with other agents to prevent or treat laminitis. The serum/plasma and isolated or synthesized 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 utilized to inhibit all the inflammatory protease enzymes that have bee identified as the causative agents for equine laminitis.

Description

Title Camelid blood serum or plasma and generated enzyme antibodies their peptide isolates and/ or synthetic peptide sequences for laminitis prevention and treatment and the treatment of other equine diseases created due to elevated metalloprotease and elastase (serine protease) enzyme activity.

Description Metalloproteinase and Protease Inhibitor peptides and homodimer antibodies generated from Camelid serum/Plasma for prevention and therapy of equine diseases caused by elevated metalloprotease and other protease enzyme activity.

Field of the Invention The present invention relates to protease/metalloprotease enzyme inhibitory peptides discovered to be present in camelid serum/plasma or additional inhibitory MMP and serine protease enzyme inhibitory homodimer antibodies which can be generated by a vaccination program utilising identified equine enzyme antigens known to be responsible for causing the laminitis and other conditions in equine disease. More specifically these enzyme inhibitory peptides isolated from camelid blood are active against equine metalloproteinases and other equine serine proteases enzymes and are effective in the treatment and or prevention of laminitis and other disease conditions in the horse associated with elevated metalloprotease and other protease activity . These protease/metalloprotease 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 camelid plasma to inhibit elevated protease enzymatic activity associated with equine disease states such as laminitis, chronic lung disease, osteoarthritis, chronic obstructive pulmonary disease and IBD (irritable bowel disease) Background of the Invention Proteinases (metalloprotease) 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. To prevent the uncontrolled destruction of target proteins and tissue such as the hoof,lung and gastrointestinal tract the activity of these proteolytic enzymes are modulated by inhibitor serum peptides normally present under healthy conditions. -2Thus, the combined and balanced actions of proteinases and peptide 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.

One important group of proteinases is 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-1 , 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-1 to 4 and stromelysin3, 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) in the animal body.

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 inhibitor peptides.

One family of metalloproteinases inhibitor peptides are the tissue inhibitors of metalloproteinases (TIMPs). 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 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 -3 It is an aspect of the present invention to overcome or at least alleviate one or more of the difficulties or deficiencies related to the prior art by providing a plentiful source of metalloproteinase inhibitor peptides and serine protease inhibitory proteins from camelid serum/ plasma and methods for purifying these inhibitor peptides from the camelid blood. Also it has been found that by inoculating camelids with purified equine metalloproteinase enzymes and serine Proteases combined with adjuvant that enzyme inhibitory homodimer antibodies generated in the inoculated camelid have the ability to inhibit metalloprotease enzymatic activity and elastase and this enhances the ability of the camelid serum to inhibit these protease enzymes in the laminitis hoof and other organs such as lung and gastrointestinal tract and lead to rapid healing of these disease indications following therapy as per this invention. Furthermore, compositions including inhibitors are disclosed as well as methods for treating various conditions and diseases using the naturally occurring metalloprotease inhibitory peptides present in camelid serum or coupled with specific inhibitory homodimer antibodies generated by the camelid immune system following vaccination with specific Equine metalloprotease enzymes and equine serine protease described herein. These specific camelid inhibitory homodrmer antibodies generated as a result of vaccination and the protease inhibitory peptides naturally present in camelid serum/ plasma may be isolated for therapeutic use in laminitis and other veterinary diseases of animals where elevated tissue Proteases are responsible for these disease indications.

Summary of the Invention The first aspect of 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 serum/plasma contains effective inhibitory amounts of equine metalloproteinase and general serine protease inhibitor peptides in large concentration unlike any other ruminant serum tested.

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 -4metalloproteinase enzyme inhibitor peptide or a general serine protease enzyme inhibitor peptide present naturally in camelid plasma or generated following a vaccination program and isolated from camelid serum/ plasma or utilised incorporated in the camelid plasma. These metalloprotease and other protease inhibitor peptides present or generated within a camelid according to this patent may be produced recombinantly utilizing methods currently available in the art.

Detailed description of the invention In a first aspect the present invention provides a composition derived directly or indirectly from true camelid serum and or plasma the composition comprising a peptide inhibitor of a equine metalloproteinase enzyme and or serine protease enzymes capable when administered intravenously to prevent or bring rapid treatment and relief to animals suffering with Laminitis or other disease states resultant from uncontrolled metalloprotease or other protease activity. 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 in the therapy of horses. 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 and for the treatment of other disease states occurring in horses as a result of uncontrolled metalloprotease and other protease activity. 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-1 , 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 (MMP12), and MMP-19 (making up the classical metalloproteinases) and (v) the membrane-type metalloproteinases (MT-MMP-1 to 4 and stromelysin3, MMP-11 ).

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 -5peptides which have not been found or isolated from other animal serum tested for example those other serum tested are bovine,caprine, and equine species.

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 and present at a concentration ranging from about 0.01 pg/ml to about 100mg/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 from about 0.1 pg/ml to about 1000pg/ml. Even more preferably the camelid peptide inhibitor of equine metalloproteinase is present at a concentration ranging from about 1 pg/ml to 500pg/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 pg/ml, or about 45pg/ml or about 50pg/mi as quantified by a fluorescence-quenching substrate assay.

In this invention the peptide inhibitor of metalloprotease enzyme activity 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 down regulates the activity of equine metalloproteinases which includes TIMP-1 , TIMP-2, TIMP-3 and TIMP-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 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 and metalloproteinase-9.

Preferably the inhibitor camelid peptide 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 also called gelatinase A which catalyses the degradation of collagen 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. -6The peptide inhibitor isolated from camelid serum and or plasma is capable of inhibiting membrane bound type matrix metalloproteinases and non-metal bearing general protease enzymes such as elastase.

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 .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 to inhibit a disease the causation of which is the presence of elevated metaloprotease and other protease enzymatic activity.

Methods and pharmaceutical carriers for preparation of pharmaceutical compositions, including compositions for administration are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.

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 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-inwater liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

It should be understood that in addition to the ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard 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 or enteric coatings.

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, -7glycosaminoglycan, 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, or gel. The skilled artisan will be familiar with the appropriate carrier to use depending on the route or means for administration for the disease state concerned.

In another preferred form the composition has at least one further active ingredient selected from the group including antibiotics, anti-inflammatories, antiseptics, other agent eg.anaesthetics. The compositions described herein may have other molecules associated therewith to aid releasability, stability, solubility, activity and/or association with targeting specific tissue types affected .including carriers, solubilizing agents, and growth factors as discussed above.

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 organ in a biologically acceptable carrier to ensure sustained release at sufficient concentration in the environment.

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

In a preferred method the concentration of the metalloproteinase inhibitor peptide isolated or present in the camelid serum and or plasma is from about 0.1 ng/ml to about 10 pg/ml of fluid in the local environment of the disease tissue.. Even more preferably the concentration of the camelid 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 disease site. -8The 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 the 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 amount as used herein means an amount sufficient to elicit a statistically significant response at a 95% confidence level (p--> 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.

The compositions may be administered in therapeutically effective amounts. A therapeutically effective 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 metalloproteinase / protease activity. Preferably 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 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.

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

The condition can be a dental or oral wound; peptic ulceration of the duodenum, stomach or oesophagus; inflammatory bowel disease; an ulcer associated with stress conditions; damage to the lining of the alimentary tract; inadequate gut function or -9damage to the gut associated with prematurity; a diarrheal condition; a cancer of the gastrointestinal tract; surgically induced damage to the gut; damage due to oesophageal 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 such as irritable bowel syndrome.

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 homodimer antibody thereof or composition described herein. As used herein the term disorders associated with metalloproteinase activity includes the following: For all methods of treatment described herein the daily dosage can be routinely determined by the attending veterinarian. Generally the dosage will vary according to the age, weight, and response of the animal, as well as the severity of the symptoms. In general a suitable dose of the inhibitor of the invention will be in the range of about 0.1 pg to about 100mg per kilogram body weight of the recipient per day, preferably in the range of about 1 pg to about 50mg per kilogram body weight per day. However, the dose will also depend on the formulation and purity of the camelid serum and or plasma used and the cone, of inhibitory protease peptide present.

The present invention also provides a method for at least partially purifying or enriching a metalloproteinase enzyme inhibitor camelid peptide or homodimer 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 centrifugation, micro- filtration, ultrafiltration, ion-exchange chromatography, molecular sieve chromatography, affinity chromatography, reverse-phase high performance liquid chromatography and transient acidification. - ΙΟΙ. Sequence Description 1: Leu Lys Ala Met Asp Pro Thr Pro Pro Leu .

Trp lie Lys Thr Glu 2. Collection of Sequences Phe-Leu-His Trp-Leu-Phe Trp-Leu-Try Trp-Leu-Arg Trp-Leu-His Phe-Leu-Phe Phe-Leu-Try Phe-Leu-Arg = Peptide 1 = Peptide 2 = Peptide 3 = Peptide 4 = Peptide 5 = Peptide 6 = Peptide 7 = Peptide 8 3. Collection of Sequences Coupled to Hydroxamate: Phe-Leu-His Trp-Leu-Phe Trp-Leu-Try Trp-Leu-Arg T rp-Leu-His Phe-Leu-Phe Phe-Leu-Try Phe-Leu-Arg = Peptide 1 = Peptide 2 = Peptide 3 = Peptide 4 = Peptide 5 = Peptide 6 = Peptide 7 = Peptide 8 Coupled to hydroxamate Any combination of the above peptides sequences.

Claims (17)

Claims

1. A method for prophylactically treating to prevent thelaminitis condition occurring, in any limb of a horse comprising: intravenously administering a therapeutically effective amount of camelid plasma or Hyperimmune camelid plasma which has the proven ability to inhibit equine metalloprotease and other protease enzymatic activity sufficient to prevent a laminitis condition occurring in an affected equine limb or limbs.

2. A method for treating a horse for laminitis comprising intravenously administering a therapeutically effective amount of camelid plasma or Hyperimmune camelid plasma demonstrated to be capable of inhibitory activity towards equine metalloprotease and other protease enzymatic activity sufficient to cause rapid proteolytic enzyme inhibition in the effected hoof and cause immediate healing and pain relief.

3. A method as defined in claim land 2 above wherein said camel plasma is obtained from a healthy camelid.

4. A method as defined in claim 1 and 2 above wherein said Camelid plasma is obtained after an inoculation regime that produces Hyperimmune plasma containing inhibitory homodimer antibodies and peptides capable of inhibiting equine disintegrin and equine metalloprotease enzymes particularly ADAMTS4/5 also termed aggrecanase 1/2 and the metalloprotease enzymes (MMP) specifically but not limited too MMP-2 and MMP-9.and serine protease enzyme activity.

5. A method as defined in claims 1,2,3,4 where the camelid plasma is injected intravenously into said animal and/ or it's effected hoof suspected of developing or actually affected with Laminitis.

6. A method for preventing laminar detachment in a limb of a horse having the sequela of a laminitis condition in said limb comprising: intravenously injecting camelid plasma and /or/ homodimer metalloprotease inhibitory antibodies as outlined in claims 1,2 and 3 above in such a therapeutically effective amount -13sufficient to prevent laminar detachment into a flexor digitorum profundus muscle of said equine limb.

7. A method as defined in claim 1,-2 wherein said intravenous administration is composed of the isolated antibodies and peptides sequences and combinations as outlined herein from the camelid plasma known to cause proteolytic equine enzyme inhibition and these peptides suspended in a designated IV suitable solution.

8. A method for treating the sequela of a laminitis condition in an affected limb of a hoofed animal comprising: administering a plurality of intravenous administrations containing a pharmaceutically effective amount of camelid plasma as specified in claims 1,2,3, and 7,into the affected leg blood supply.

9. A method for treating equine chronic lung disease by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1, 2, 3, 7 above

10. A method for treating equine osteoarthritis disease condition by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies and / or the amino acid sequences outlined in this patent to be capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

11. A method for treating equine septic joint disease by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

12. A method for treating equine Colic by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies or amino acid sequences outlined in this patent capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

13. A method for treating equine chronic obstructive pulmonary disease by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies or the amino acid sequences outlined in this patent capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

14. A method for treating equine joint disease by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies or amino acid sequences outlined in this patent capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

15. A method for treating equine ulcerative colitis,Crohns disease by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies or peptide sequences outlined in this patent capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

16. A method for treating equine inflammatory bowel disease by administering a number of intravenous doses of camelid serum/ plasma or peptide isolates and/ or homodimer antibodies or peptide sequences outlined in this patent capable of inhibiting equine metaloprotease and other protease enzymes specified in claims 1,2,3,7 above

17. A method for treating a horse for laminitis comprising intravenously administering a therapeutically effective amount of camelid plasma or Hyperimmune camelid plasma or the amino acid sequences outlined in this patent demonstrated to be capable of inhibitory activity towards equine metalloprotease and other protease enzymatic activity sufficient to cause rapid proteolytic enzyme inhibition in the effected hoof and cause immediate healing and pain relief.