WO2007077445A1

WO2007077445A1 - Method of diagnosis of a predisposition to laminitis

Info

Publication number: WO2007077445A1
Application number: PCT/GB2007/000013
Authority: WO
Inventors: Patricia Harris; Jonathan Elliott; Simon Reeves Bailey
Original assignee: Mars Uk Limited
Priority date: 2006-01-06
Filing date: 2007-01-05
Publication date: 2007-07-12

Abstract

A method is provided for diagnosis of a predisposition to laminitis in an ungulate animal. The method comprises measuring the levels of insulin in one or more basal samples obtained from the animal when fed a basal diet, and measuring the levels of insulin in one or more subsequent samples obtained from the animal when fed said basal diet, and additionally a source of a non-hydrolysable non-structural carbohydrate or fructose. The levels of insulin in step a) and step b) are then compared.

Description

Method of diagnosis of a predisposition to laminitis

The present invention provides a method of diagnosis of a predisposition to laminitis in 5 ungulate animals, such as horses. The present invention further provides a method of determining the level of insulin resistance in a mammal, in particular an ungulate. The present invention also provides a composition comprising a non-hydrolysable nonstructural carbohydrate or fructose for use in the diagnosis of a predisposition to laminitis in an ungulate animal. In addition, the present invention relates to the use of a non- 10 hydrolysable non-structural carbohydrate or fructose in the preparation of a composition for use in determining the level of insulin resistance in a mammal, in particular an ungulate.

Laminitis is one of the most important equine diseases worldwide in terms of animal 15 suffering, mortality, loss of use and financial cost to the owner. Laminitis should be considered to be a systemic disease, which manifests as a condition of the foot. Acute ' laminitis, active pain and inflammation in the digits, follows on from a prodromal or developmental phase (in which the initiating events are occurring but clinical signs are not apparent) in the absence of any pre-existing pedal bone displacement or lamellar 20 damage. Once these secondary changes occur, leading to hyperkeratinization and necrosis in some regions of the tissue, disruption to the normal lamellar architecture and growth result in chronic instability in the foot and repeated episodes of lameness (chronic laminitis).

25 Although many animals recover after an acute episode, many are affected for the rest of their lives, resulting in loss of use to the owner and chronic or recurrent pain for the animal. According to a general survey in the USA, apart from colic, laminitis is the most common reason a horse or pony will be presented for veterinary treatment (USDA- NAHMS. In: NAHMS, editor.: National Animal Health Monitoring System, Fort Collins,

30 CO; 2000.) and 13% of horse owners/operations reported problems with laminitis in their horses over the previous 12 months with approximately 5% of those affected by laminitis dying or being euthanized. Higher fatalities have been reported in veterinary teaching

551914V1 hospital and referral centre surveys (Cripps PJ, Eustace RA. Equine Vet J. 1999 Sep;31:433-42.) For example, 20% died or were euthanized in one such survey, which may reflect the severity and nature of the cases evaluated. A large study in the UK found a prevalence of 7.1% and estimated that around 8,000 horses suffered from laminitis each 5 year, about 600 of which were euthanized because of it and 1,300 animals were left permanently unsound and so contributed to the pool of over 16,500 chronically laminitic animals (EQnckley K, Henderson L. 35th Congress of the British Equine Veterinary Association; 1996; Warwick, UK; 1996. p. 62)

10 Laminitis literally means inflammation of the laminae which connect the pedal bone to the hoof wall. The dermal laminae interdigitate with the epidermal laminae to suspend the foot in the hoof. The force created by the animal's weight is passed down through the bones of the leg to the pedal bone. The laminae help transfer and dissipate the force from the bone to the hoof wall. In severe cases, the condition may weaken the bond between

15 the hoof wall and the pedal bone. This may cause the pedal bone to sink ('sinker') or rotate and drop ('founder') due to the upward pull of the ligaments and tendons.

A horse with laminitis is likely to be in pain and discomfort. When severe, horses may need to be euthanased. Once significant changes to the laminae have occurred, they are 20 often irreversible. Even after successful treatment a horse may become more likely to suffer from recurring attacks.

The actual pathological process of laminitis is thought to be one of inflammation followed by degeneration and separation of the laminae. Currently there are 3 main 25 theories regarding the pathogenic/etiologic mechanisms involved, as follows. Within each of these theories, multiple factors might be implicated and aspects of all of these may be inter-related and occur simultaneously.

1. Primarily related to a disturbance in the blood flow to the feet, perhaps resulting 30 from increased venous resistance - leading to a period of ischaemia of the sensitive dermal lamellae and reperfusion injury, or compartment syndrome.

5519Hv1 2. Related to inflammatory, toxic, metabolic, and or enzymatic mechanisms: hematogenous delivery of laminitis trigger factors to the epidermal laminae leads to the activation of metabolic or enzymatic events which in turn result in disruption to the lamellar ultrastructure. In particular, activation of matrix metalloproteinases (MMPs)

5 break down the basement membrane bonding the dermal to the epidermal lamellae.

3. Related to traumatic/mechanical factors. These may include damage to the vascular endothelium and/or perivascular nerves due to concussion or stagnant hypoxia, due to blood pooling in the digits with excessive or prolonged weight bearing.

10 Laminitis is believed to occur secondarily to a number of conditions including certain gastrointestinal diseases, for example, those which result in an increase in the permeability of the gastrointestinal tract, such as colic. Certain infectious and toxic conditions (for example endometritis) and certain endocrine disorders (for example hyperadrenocorticism) are also associated with laminitis. In addition, laminitis may occur

15 following adverse mechanical influences on the foot including excess weight bearing or excess foot trimming. The most common predisposing factors for laminitis are, however, believed to be hyperadrenocorticism and excessive carbohydrate reaching the hindgut and being rapidly fermented. Rapid fermentation within the stomach and small intestine may also be involved.

20

Horses are non-ruminant herbivores that have evolved to subsist principally on a diet of fibrous vegetation, much of which cannot be broken down by mammalian enzymes. Digestion of this fibrous material is principally carried out in the caecum and colon by a large population of microorganisms, primarily bacteria but also including protozoa, yeasts

25 and fungi. These microorganisms are responsible for the digestion of fibrous material allowing the horse to use this material as a source of energy.

Wild and feral equids are thought to suffer far less from dietary induced metabolic disorders because they subsist on a diet that their digestive system has evolved to cope

30 with, although they may suffer if they get access to inappropriate feeds and pastures.

Metabolic disorders are much more frequently seen in domestic animals due to the

551914V1 addition of less fibrous foods to their diet, most noticeably cereals and access to starch/sugar/fructan rich pastures.

Access to large amounts of cereals or to lush grass or to other pasture species (such as 5 legumes or clover or herbacious weeds or stemmy grass) or forage with a high sugar/starch or fructan content are the most commonly recognised feed related predisposing causes of laminitis especially in ponies. Sugar is produced as an energy source in plants, including cereals and grasses, via photosynthesis. Depending upon the plant's energy requirements, this sugar is either metabolised directly or stored as storage

10 carbohydrates for use at a later time. The main storage carbohydrate for cereals is starch (chains of glucose units linked together). Some pasture species may also contain starch as the main storage carbohydrate, such as clover which can have a starch content of up to 50%. However for some grass species, starch may account for as little as 10-15% of the total storage carbohydrate with the remainder being present as simple sugars, such as

15 sucrose, or more complex molecules such as fructans.

Fructans (oligo- and polyfructosyl sucrose) are polymers of fructose and can form between 5 and 50% of the dry matter of grass. Unlike starch and other simple sugars, fructans are believed to be effectively non-hydolysable (i.e. not broken down to any

20 appreciable or significant extent by mammalian enzymes) and pass to a greater extent unmodified to the hindgut, where they act in a similar manner to a starch overload, as both can be rapidly fermented by the microbes in the gastrointestinal tract, in particular the hindgut. Some fermentation may occur in the stomach and small intestine which may also be involved in the pathophysiology. A typical managed pasture may contain several

25 types of grasses, clover (or other legume) and various herby weed species, which may therefore present a risk to ponies predisposed to laminitis from an excess of starch, sugars and/or fructan. It should be appreciated that in addition to the amount of fructans ingested it may also be necessary to determine the type of fructan ingested. The effect of the fructan may be related to for example the speed and degree of breakdown. Thus, a

30 long chain branched chain fructan may pose less of a risk than a short straight chain fructan. Furthermore there may be large numbers of performance horses that suffer from subclinical laminitis as a result of their high grain low forage diets.

551914V1 The upper part of the equine gastrointestinal tract has a relatively small capacity and the horse has digestive and metabolic limitations to high grain, starch and sugar based diets. Similar to the intake of fructans, large grain meals or any overload of starch or sugar may

5 overwhelm the digestive capacity of the stomach and small intestine, leading to much of the material passing through to the hindgut where it may be rapidly fermented. This fermentation leads to a production of excess lactic acid and a drop in hindgut pH. The main lactate producing bacterial genus are Streptococcus and Lactobacilli, such as the species S. bovis and L. mucosae, S. ruminatum and L. reuteri. Production of lactic acid

10 causes a rapid drop in the pH of the hindgut from about 7 to 6 or less. At this pH, certain more acid resistant bacteria thrive, potentially altering the balance of the flora, which in turn, potentially affects normal fermentation. At extremes of pH, predominantly only acid resistant bacteria and other microflora will persist. The increasingly acid environment affects the intestinal mucosa in particular, increasing permeability. This

15 process can result in diarrhoea, colic and absorption of substances such as endotoxin from the dead bacteria and other compounds such as vasoactive amines and various exotoxins including matrix metalloprotease activators, believed to be involved in the pathogenesis of laminitis, see Figure 1.

0 There is now an increasing awareness of the role of glucose and insulin resistance in laminitis.

Glucose is important in maintaining lamellar integrity and has been shown to be essential for the viability of hoof explants in culture. Culture without glucose or inhibition of

25 glycolysis causes basement membrane zone separation under tension and high circulating corticosteroid levels and/or inhibition of insulin activity could result in such an effect. It is possible that in the insulin resistant state, glucose transporters are down regulated in the lamellar tissues and therefore glucose entry into the epithelial cells may be impaired, as has been demonstrated in chronic laminitis.

30

It has been well recognized that obese animals, especially ponies, are more prone to laminitis and this may in part be linked with mechanical trauma due to the increased load.

551914V1 However, it is likely that the risk for laminitis in obese animals is more appropriately attributed to the development of insulin resistance. The "syndrome" of obesity, insulin resistance and laminitis in mature horses has been referred to as either "peripheral Gushing' s syndrome" or an equine "metabolic syndrome". It has been suggested that

5 there may be a progression of insulin resistance in laminitic prone ponies from compensated insulin resistance (i.e. insulin sensitivity greatly decreased but compensated for by increased insulin secretion), being a predisposing factor in healthy but genetically predisposed ponies, through to a decompensated insulin resistance later in the course of the disease. Insulin resistance may also be linked to an increased risk of laminitis in other

10 ways including - increased oxidative stress, increased cytokine and inflammatory market release and increased susceptibility to vasoconstrictor agents etc. Some of these potential pathways are illustrated in Figure 1.

However, it is not possible currently to diagnose insulin resistance by means of a simple

15 test. Baseline evaluations of glucose and insulin are known to be unreliable as indicators and more complex procedures such as the euglycaemic hyperinsulinaemic clamp or the 'minimal model' are required which are very time consuming, technically demanding, costly and complicated and cannot be easily undertaken in practice or on multiple occasions. 0

Conventional methods for the prevention and/or treatment of laminitis include changes in the feeding and/or keeping of the animals to reduce their access to carbohydrates (such as cereals, high sugar grasses, high fructan grasses). The influx of rapidly fermentable carbohydrate (starch, water soluble carbohydrates: sugars and/or fructan) primarily into 5 the large intestine results in overgrowth of gram-positive bacteria and a decrease in gram negative bacteria. The streptogrammin antibiotic Virginiamycin, for example, has been used and marketed to prevent pasture-induced laminitis by preventing the overgrowth of gram positive caecal bacteria based on work done in Australia which suggested that pre- dosing with virginiamycin prevented the development of laminitis following

30 administration of a wheat slurry. However, virginiamycin is only available in Europe under special licence and certain conditions as it has been banned for use as a growth promoter in pigs and poultry in the European Union due to concerns over antibiotic

551914v1 resistance. Anecdotally, it is not effective in all cases. Obviously the use of other antibiotics which are less selective or are bactericidal in nature could be counterproductive and lead to other problems.

5 Since laminitis occurs all around the world in horses and ponies and has major welfare implications it is obviously important to be able to recognise and treat the condition in its early stages so that the pain and suffering is kept to a minimum. However, ideally it would be preferred to be able to recommend certain interventions/countermeasures that avoid or prevent the condition from occurring in the first place. Turning certain ponies or

10 horses out onto 'lush' or 'stressed' pastures especially in the spring and autumn is thought to be a common predisposing factor. Currently it is believed that the high levels of water- soluble carbohydrates (which include the simple sugars as well as the more complex storage carbohydrates such as fructans) and /or starch may be involved in this process. One way to avoid pasture associated laminitis is therefore to prevent access to pasture and

15 to feed forage alternatives that are known to be low in rapidly fermentable material. For the majority of ungulate animals, total restriction is not always a viable or desired option for financial, welfare and health reasons. It also may not be necessary for those animals that are not predisposed to laminitis. So ideally, the first stage in any countermeasures would be to determine whether an individual animal has a predisposition to laminitis.

20

There is therefore a need for a simple, fast and reliable test for a predisposition to laminitis in ungulate animals, such as horses.

The inventors have surprisingly found that the administration of an effectively non- 25 hydrolysable non-structural carbohydrate (NSC) such as a fructan causes an unexpectedly high insulin response in ungulate animals having a predisposition to laminitis. This effect may also be seen following administration of fructose.

The first aspect of the present invention therefore provides a method of diagnosis of a 30 predisposition to laminitis in an ungulate animal comprising the steps of: a) measuring the level of insulin in one or more basal samples obtained from the animal when fed a basal diet;

551914V1 b) measuring the level of insulin in one or more subsequent samples obtained from the animal when fed said basal diet and additionally a source of a non-hydrolysable nonstructural carbohydrate or fructose; and c) comparing the levels of insulin in step a) and step b). 5

In one feature of the first aspect of the invention, the ungulate animal is selected from the group consisting of horses, ponies, equids, cows, goats or sheep. More preferably, the ungulate animal is a horse, pony or equid. The method of diagnosis is, however, appropriate for use in relation to any animals which are known to be at risk of developing 10 laminitis.

The sample obtained from the animal may be a biological sample which may be blood, blood plasma or urine. The levels of insulin in the samples may be measured using any appropriate standard techniques known in the art.

15

Non-structural carbohydrates (NSCs) represent the sum of starch, sugars, fructan, pectins, cellulose, hemi-cellulose, mucilages and gums. According to the first aspect of the invention, the subsequent samples are obtained after administration of a source of non- hydrolysable NSCs. Non-hydrolysable NSCs are those NSCs which are not broken down

20 by mammalian enzymes to any appreciable or significant effect. It will therefore be appreciated that starch and sugars such as glucose are excluded from the definition of non-hydrolysable non-structural carbohydrates because they are hydrolysable and therefore can be broken down by mammalian enzymes.

25 Fructans, pectins, cellulose, hemi-cellulose, mucilages and gums therefore do fall within the definition of non-hydrolysable non-structural carbohydrate and are effectively not broken down by mammalian enzymes. In a preferred feature of the first aspect of the invention, the non-hydrolysable non-structural carbohydrate is a fructan. It should however be appreciated that such carbohydrates are only effectively non-hydrolysable,

30 i.e. a small portion of these carbohydrates may be subject to some breakdown including some hydrolytic breakdown for example in the stomach.

551914V1 According to the first aspect of the invention, the subsequent samples may alternatively be obtained after administration of a source of fructose.

In a further feature, the dose of non-hydrolysable non-structural carbohydrate 5 administered is in the range of 0.1 to 9g/kg body weight per day for up to 10 days and preferably within the range of 0.1 to 5g/kg, more preferably within the range 0.5 to 4g/kg bodyweight per day. The dose of fructose administered is in the range of 0.001g/kg bodyweight to 10g/kg bodyweight per day for up to 10 days and preferably within the range of 0.1 - lg/kg bodyweight per day. 10

These doses may or may not be given at once or in divided doses throughout the day so that any individual dose does not increase the risk of laminitis occurring in the animal to which it is administered. It has been found by other researchers that administration of a bolus dose of 7.5g/kg bodyweight of fructan has initiated laminitis in animals not prone to 15 this condition. It is therefore important to avoid administering such high doses as a single bolus.

It should be appreciated that the non-hydrolysable non-structural carbohydrate or fructose may be administered as one bolus dose or be fed to the animal during one day, after 20 which the insulin (and other metabolite) levels may be measured. Alternatively, several doses may be administered over a course of several days and testing for insulin levels is therefore carried out while still feeding/administering the non-hydrolysable non-structural carbohydrate or fructose.

25 It should also be appreciated that the basal diet may also contain non-hydrolysable nonstructural carbohydrates or fructose, although preferably at a low or restricted level. The source of non-hydrolysable non-structural carbohydrate or fructose is administered in addition to the basal diet and therefore provides an increase in the overall level of non- hydrolysable non-structural carbohydrate or fructose.

30

In another feature of the first aspect of the invention, the one or more subsequent samples are obtained from the animal during the time period of from 1 hour to 7 days after single

551914V1 administration or continued administration of the non-hydrolysable non-structural carbohydrate source or fructose. Preferably, the subsequent sample is obtained from the animal during the time period of from lhr to 60hrs, more preferably during the time period of from 20 hr to 50hrs after administration or the start of intermittent daily 5 administration of the non-hydrolysable non-structural carbohydrate source or fructose source. The subsequent samples may be obtained after 3, 6, 9, 12, 15, 18, 21 or 24 hours or 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 days after administration or the start of intermittent daily administration of the non-hydrolysable non-structural carbohydrate source or fructose source. 10

In another feature of the first aspect of the invention the levels of insulin in step a) and step b) are compared with the levels from normal ungulate animals i.e. those animals which have had no history of laminitis in a defined period of more than 2 years.

15 Typically, the insulin levels are increased following administration of the non- hydrolysable non-structural carbohydrate or fructose. Animals having a predisposition may display a significantly higher increase in insulin levels and/or a level of increase which is maintained for longer than in normal animals following administration of the non-hydrolysable non-structural carbohydrate or fructose. Additionally, the insulin levels 0 may move to a level outside the normal (reference) range or normal response range provided by the testing laboratory following administration of the non-hydrolysable nonstructural carbohydrate or fructose. Therefore, a putative diagnosis of a predisposition to laminitis can be made.

25 In one feature of the first aspect of the invention, the method of diagnosis may additionally comprise measuring the levels of glucose. In a further feature, other parameters including metabolites, hormones etc involved in the maintenance of energy metabolism and in the homeostasis of glucose such as triglycerides, thyroxine, triiodothyronine and Cortisol may be monitored. The levels of glucose and these other

30 parameters may be measured using standard laboratory techniques including RIA, ELISA, HPLC, GC, MS. The difference in the levels of these parameters obtained before

551914v1 and after carbohydrate administration can then be compared to data obtained for animals known not to be predisposed to laminitis.

It is important to also test for unexpected increases or changes in the levels of such

5 metabolites, in addition to testing for changes in insulin levels, such that the presence of other diseases which may be responsible for increased insulin levels may be excluded.

For example, the present inventors have found that while a significant increase in insulin levels is found in horses having a predisposition to laminitis following administration of the non-hydrolysable non-structural carbohydrate or fructose, the glucose levels did not

10 change significantly. If, however, a significant increase in glucose levels is found in a horse, in combination with a significant increase in insulin levels following administration of the non-hydrolysable non-structural carbohydrate or fructose then the increased insulin levels could be indicative of other conditions (such as hyper adrenocorticism) in addition to or instead of a predisposition to laminitis, e.g. pancreatic

15 disease.

The non-hydrolysable non-structural carbohydrate or fructose of the first aspect can be provided as a solid, liquid or semi-solid form. In particular, the carbohydrate can be provided as a powder, an aerosol, a bolus, a paste, a gel, drops, a solution, a syrup, a

20 suspension, an emulsion, a tablet, or a capsule. The carbohydrate could be provided as a solid for addition to water or as an oil based liquid. The carbohydrate can be mixed with any suitable carrier routinely used in the art such as magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose, water, ethanol, glycerine, polyethylene glycol, an oil, an aqueous gum, cellulose, silicate, tragacanth, gelatin, glycerine, etc. The carbohydrate

25 may additionally comprise a suspending agent, a preservative, a flavouring or a colouring agent.

The non-hydrolysable non-structural carbohydrate or fructose of the first aspect can be provided as a food supplement or a feedstuff. 30

The food supplement or feedstuff of the first aspect of the invention can be solid, semisolid, or liquid. For example, the food supplement or feedstuff could be in the form of a

551914V1 powder, a paste, a pellet or a drink. The food supplement or feedstuff can additionally contain ingredients which enable the feedstuff to be formulated in a particular form. For example, the feedstuff can contain molasses or a molasses/oil mixture, for example, cane molasses with approximately 6% or above oil such as Molglo (e.g. to bind the ingredients 5 together or as a palatability agent) or oat feed, wheat feed or another suitable filler ingredient. The feedstuff may also contain fibre sources such as grasses, straw (chopped or ground), sugar beet, soya hulls and oats, and fat sources such as corn oil, soya oil, processed canola oil, coconut oil, palm oil or sunflower oil.

10 The food supplement can be added to a food or administered prior to or after feeding. It may be fed with other feed ingredients as part of the ration or on its own. The supplement could be administered together with a standard feedstuff used or with the feedstuff of the invention. The mixing may occur when the feedstuff is prepared or packaged or may occur when the feedstuff is provided to the animals. The supplement

15 may alternatively be supplied as a topping to a feedstuff. The food supplement can be in the form of a food snack or a drink (for example snack bars, biscuits and sweet products). The drink may be aqueous or oil based.

The non-hydrolysable non-structural carbohydrate or fructose may be administered by 0 any conventional method for example by oral, parenteral, mucosal (i.e. buccol, sublingual), and the compositions adapted accordingly. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. In a preferred aspect, the non-hydrolysable non-structural carbohydrate or fructose is administered as a bolus

25 dose in an injectable form or as a bolus dose in a meal.

The non-hydrolysable non-structural carbohydrate or fructose may be prepared by standard techniques known in the art depending upon the mode of administration. The non-hydrolysable non-structural carbohydrate or fructose may be supplied as part of a 30 sterile, composition which will normally include an acceptable carrier. This composition may be in any suitable form, (depending upon the desired method of administering it to the animal). It may be provided in unit dosage form, will generally be provided in a sealed

551914v1 container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

5 Non-hydrolysable non-structural carbohydrate compositions or fructose compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, as a paste, syrups or suspensions (in aqueous or nonaqueous liquids; or as edible foams or whips; or as emulsions). Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic

10 acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For the preparation of solutions and syrups, excipients which may be used include, for example, water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) may be used to provide oil-in- water or water in oil suspensions. Pastes may include a flavouring, other feedstuff s

15 suitable for horses, carrier agents etc.

Compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and

20 aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the

25 sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The non-hydrolysable non-structural carbohydrate or fructose compositions may contain 30 preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants. They

551914v1 may also contain therapeutically active agents in addition to the substance of the present invention.

The non-hydrolysable non-structural carbohydrate or fructose may be provided by a 5 natural or a synthetic source or a combination thereof. The non-hydrolysable nonstructural carbohydrate or fructose may be provided as a purified, semi-purified or a crude extract. The non-hydrolysable non-structural carbohydrate or fructose may be provided by the addition of a natural source such as a feedstuff or a mixture of feedstuff s.

10 The non-hydrolysable non-structural carbohydrate or fructose of the composition for the purposes of this invention may be isolated from a natural source. The required component may be removed from the other components of the natural source and purified until only the required component is present. Alternatively, the required component may be partially purified from the other components of the natural source (and provided for

15 example as a crude or partially purified extract of the natural source) or provided by the addition of the natural source. When the component is isolated from or provided by a natural source, the natural source may be chemically and/or biologically modified, for example by genetic modification.

20 Examples of feedstuffs naturally rich in non-hydrolysable non-structural carbohydrate for the present invention include temperate grasses, especially the high fructan storing varieties grown under the environmental conditions likely to promote high fructan accumulation. Typically, warm season grasses (C4) have lower NSC contents than cool season species (C3). Not only does the type of plant affect NSC status, but environmental

25 conditions can also cause large and significant changes e.g. the NSC content of a given plant species can range from 95 to 560 g/kg dry matter (DM) with corresponding ranges in fructan of 32 to 439 g/kg DM depending upon the temperature at which it is grown, with higher and lower values being associated with cooler (5-10° C) and warmer (15-25° C) temperatures, respectively. The NSC content of a particular grass can therefore be

30 increased by subjecting the grass to stress.

551914V1 Other fructan rich materials such as vegetables (e.g. onions), flowers (e.g. dahlia tubers) and within the Asteraceae Group e.g artichoke (Cynara scolymus), globe thistle (Echinops ritro), chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus) provide further examples of feedstuffs naturally rich in non-hydrolysable non- 5 structural carbohydrate. In addition, in the future it may be possible to use genetically engineered crops which produce high levels of specific fructan molecules as the specificity of fructosyltransferases determines the type of glycosidic bond formed and the donor and acceptor substrates used, which enables the synthesis of many structurally diverse fructans in crops such as sugar beet and potato

10

A synthetic source for the purposes of this invention may provide a component substantially free of any other materials which may then be mixed with other components. The synthetic source of the component may be more than 70% pure, preferably more than 85% pure, more preferably more than 95% pure prior to mixing with any other

15 components. The synthetic source may be any material which has been prepared from the available starting materials by a series of biochemical or chemical reactions. The product of these reactions may then be partially or fully purified. The structure of the component from the synthetic source may correspond exactly to the structure of the component in nature or it can be analogue of that structure. The synthetic component may be provided

20 in a form for example a masked form such as an ester or amide which can be modified in the body to produce one or more active components

Preferably, the synthetic source is provided by a commercially sourced fructan of any type including the phlein or levan -type ( β2, 6- linked) or the inulin (β2, 1 -linked) type 25 e.g. Raftilose extracted from the roots of chicory, or mixtures thereof or a commercially sourced fructose.

In a second aspect of the invention there is provided a method of determining the level of insulin resistance in a mammal comprising the steps of

30 a) measuring the level of insulin in one or more basal samples obtained from the mammal when fed a basal diet;

551914v1 b) measuring the levels of insulin in subsequent samples obtained from the mammal when fed said basal diet and additionally a source of non-hydrolysable non-structural carbohydrate or fructose; and c) comparing the levels of insulin levels in step a) and step b)

In one feature of the second aspect of the invention the levels of insulin in step a) and step b) are compared with the levels from normal mammals, i.e. those with an expected level of insulin resistance for their age,

10 Typically, the insulin levels are increased following administration of the non- hydrolysable non-structural carbohydrate. Animals having an increased level of insulin resistance may display a significantly higher increase in insulin levels and/or a level of increase which is maintained for longer than in normal mammals following administration of the non-hydrolysable non-structural carbohydrate or fructose.

15 Additionally, the insulin levels may move to a level outside the normal (reference) range or normal response range provided by the testing laboratory following administration of the non-hydrolysable non-structural carbohydrate or fructose. Therefore, a putative diagnosis of increased level of insulin resistance can be made and/or a comparative degree of insulin resistance/insulin sensitivity can be derived.

20

In one feature of the second aspect of the invention, the mammal is a human In another feature of the second aspect of the invention, the mammal is an ungulate animal.

Obesity in man increases the likelihood of developing type 2 diabetes, hypertension and

25 coronary heart disease. In addition, obesity, and in particular visceral adipose tissue accumulation is associated with a variety of diabetogenic, atherogenic, thrombotic and inflammatory metabolic abnormalities often referred to as insulin resistance or metabolic syndrome Insulin resistance has been found to be a risk factor for several human diseases including coronary heart disease, hypertension, polycystic ovarian syndrome, as

30 well as diabetes mellitus type 2.

551914v1 Insulin resistance has also been implicated in the pathogenesis of a number of equine diseases, such as laminitis, pituitary adenoma, hyperlipidaemia and certain developmental orthopaedic diseases. It also influences reproductive efficiency and exercise ability. There is increasing evidence that insulin-resistant muscle is less able to oxidise and utilise 5 fatty acids, and this may be an important factor in obesity.

Equine obesity has been associated with an increased risk of laminitis, hyperlipaemia, heat intolerance, insulin insensitivity, increased bone and joint injuries, placental restriction and lowered birth weights, increased risk of development orthopaedic disease 10 etc.

In a third aspect of the invention there is provided a method of diagnosis of a predisposition to laminitis in an ungulate animal comprising the steps of: a) measuring the level of insulin in one or more basal samples from said animal fed a 15 high non-hydrolysable non-structural carbohydrate containing diet or feedstuff. b) measuring the level of insulin in subsequent samples from an animal whilst being fed a lower non-hydrolysable non-structural carbohydrate or fructose containing diet or feedstuff. c) comparing the change in levels of insulin in step a) and step b)

20 wherein step a) may be performed either before or after step b) and the animal is allowed to adapt to a diet or feedstuff before said diet or feedstuff is changed.

In a fourth aspect of the invention there is provided a method for determining the level of insulin resistance in a mammal comprising the steps of: 25 a) measuring the level of insulin in one or more basal samples from said mammal fed a high non-hydrolysable non-structural carbohydrate containing or high fructose containing diet or feedstuff. b) measuring the level of insulin in subsequent samples from a mammal whilst being fed a lower non-hydrolysable non-structural carbohydrate containing or lower

30 fructose containing diet or feedstuff. c) comparing the change in levels of insulin in step a) and step b),

551914V1 wherein step a) may be performed either before or after step b) and the mammal is allowed to adapt to a diet or feedstuff before said diet or feedstuff is changed.

In one feature of the third and fourth aspects of the invention, the mammal or ungulate 5 animal is therefore first fed a high non-hydrolysable non-structural carbohydrate containing or high fructose containing diet or feedstuff and then fed a lower non- hydrolysable non-structural carbohydrate containing or fructose containing diet or feedstuff. In an alternative feature of the third aspect of the invention, the mammal or ungulate animal is first fed a low non-hydrolysable non-structural carbohydrate 10 containing or low fructose containing diet or feedstuff and then fed a higher non- hydrolysable non-structural carbohydrate containing or higher fructose containing diet or feedstuff.

The high non-hydrolysable non-structural carbohydrate containing or high fructose 15 containing feedstuff may be grass or preserved forage with or without added non- hydrolysable non-structural carbohydrate containing or fructose containing feedstuffs. The low non-hydrolysable non-structural carbohydrate feedstuff or low fructose feedstuff may be grass or preserved forage or other feedstuffs with a lower non-hydrolysable nonstructural carbohydrate content or lower fructose content than the high non-hydrolysable 20 non-structural carbohydrate or high fructose material. Each type of feedstuff may be fed for a period of 1 to 21 days, preferably 1 to 7 days and samples may be obtained from the animal during this time.

It should be appreciated that a 'high' non-hydrolysable non-structural carbohydrate

25 containing or 'high' fructose containing diet comprises a diet or feedstuff having a level of carbohydrate which is at least 2.5% higher (i.e. it has at least 2.5% more of the relevant carbohydrate) than the level of the 'lower' non-hydrolysable non-structural carbohydrate or 'lower' fructose. Thus, a "high" non-hydrolysable non-structural carbohydrate containing or "high" fructose containing diet would for example have at least 12.5% of

30 the relevant carbohydrate compared to a "lower" non-hydrolysable non-structural carbohydrate or "lower" fructose diet which contained 10% of the relevant carbohydrate.

In a preferred feature, the concentration of the non-hydrolysable non-structural

551914v1 carbohydrate or fructose in the high non-hydrolysable non-structural carbohydrate containing or fructose containing diet or feedstuff is at least 5% or at least 10% higher than that of the lower non-hydrolysable non-structural carbohydrate containing or fructose containing diet or feedstuff, more preferably at least 20% higher. 5

The change in diet or feedstuff from a high non-hydrolysable non-structural carbohydrate or high fructose to a lower non-hydrolysable non-structural carbohydrate or low fructose vice versa may be effected by either the replacement or partial replacement of the original diet with one or more alternative feedstuffs. For example, a lower non-hydrolysable non- 10 structural carbohydrate diet may be initially fed to a horse. The diet may then be completely replaced with a higher non-hydrolysable non-structural carbohydrate feedstuff, or alternatively the level of non-hydrolysable non-structural carbohydrate may be increased by replacing a portion (for example, a third) of the original diet with a higher non-hydrolysable non-structural carbohydrate containing feedstuff or a mixture of such 15 feedstuffs.

A dexamethasone suppression test which includes measurement of insulin levels in addition to Cortisol levels can also be used in the diagnosis of a predisposition to laminitis. The dexamethasone suppression test may be used alone in the diagnosis of a 20 predisposition to laminitis. Alternatively, the dexamethasone suppression test may be used in addition to the methods of the first, second, third or fourth aspects of the invention to form a double diagnostic test for a predisposition to laminitis.

The dexamethasone suppression test is usually employed to diagnose and differentiate 25 between the various types of Cushing's syndrome and other hypercortisol states.

The 19 hour overnight dexamethasone suppression test may suitably be used. This test is described in Donaldson et al, JVIM 2005; 19:217-22. This test involves measurement of blood Cortisol levels before and after administration of a dose of dexamethasone. 30

Dexamethasone is an exogenous steroid that provides negative feedback to the pituitary to suppress the secretion of ACTH. However, in animals with Cushing's syndrome, the

551914v1 Cortisol levels following administration of dexamethasone are not suppressed. The presence of Gushing' s syndrome in animals can therefore be identified.

However, it has now found that measurement of insulin levels in addition to Cortisol 5 levels before and following dexamethasone administration can be used in the diagnosis of a predisposition to laminitis.

In animals which have a predisposition to laminitis, a rise in insulin levels is seen following dexamethasone administration in comparison to animals which are not 10 predisposed to laminitis. Comparison of insulin levels before and after dexamethasone administration can therefore additionally be used to indicate the presence of prelaminitic metabolic syndrome, and therefore a predisposition to laminitis.

Accordingly, the methods of the first, second, third or fourth aspects of the invention for 15 the diagnosis of a predisposition to laminitis may additionally comprise the steps of: a) measuring the levels of insulin in one or more basal samples obtained from the animal b) measuring the levels of insulin in one or more subsequent samples obtained from the animal following administration of a dose of dexamethasone; and 0 c) comparing the levels of insulin in step a) and step b).

The dose of dexamethasone may be administered at night and the subsequent samples obtained the following day, typically after 19 hours.

25 The dexamethasone may be administered in a dose of 0.01 to 0.1 mg/kg bodyweight, preferably 0.02 to 0.06mg/kg.

The measurement of insulin levels prior to and following the administration of a source of a non-hydrolysable non-structural carbohydrate or fructose in the method of the first, 30 second, third or fourth aspect of the invention and the measurement of insulin levels prior to and following administration of dexamethasone are carried out separately, allowing sufficient time for recovery of insulin levels after each test.

551914v1 In a fifth aspect of the invention there is provided a composition comprising a non- hydrolysable non-structural carbohydrate or fructose for use in the diagnosis of a predisposition to laminitis in an ungulate animal. 5

In a sixth aspect of the invention there is provided a composition comprising a non- hydrolysable non-structural carbohydrate or fructose for use in determining the level of insulin resistance in a mammal, in particular an ungulate animal.

10 In a seventh aspect of the invention there is provided the use of a non-hydrolysable nonstructural carbohydrate or fructose in the preparation of a composition for the diagnosis of a predisposition to laminitis in an ungulate animal.

In a preferred feature of the seventh aspect of the invention, the composition is

15 administered to the animal and the blood levels of insulin prior to and after said administration are compared with control results from an animal having no laminitic history for a defined period. The control results may alternatively comprise those falling within the normal (reference) range or the normal response range provided by the testing laboratory or may be from data determined from the use of this dynamic test in the future.

20

In an eighth aspect of the invention there is provided the use of a non-hydrolysable nonstructural carbohydrate or fructose in the preparation of a composition for the diagnosis of insulin resistance in a mammal, in particular an ungulate

25 In a preferred feature of the eighth aspect of the invention the composition is administered to the mammal and the blood levels of insulin prior to and after said administration are compared with control results from a comparable mammal(s) with an expected normal level of insulin resistance. The control results may alternatively comprise those falling within the normal (reference) range or the normal response range provided by the testing

30 laboratory, or may be from data determined from the use of this dynamic test in the future.

5519UV1 In a ninth aspect of the invention there is provided a kit for use in the diagnosis of a predisposition to laminitis in an ungulate animal comprising a non-hydrolysable nonstructural carbohydrate or fructose and instructions for diagnosis. The kit may also include a feedstuff. 5

In a tenth aspect of the invention there is provided a kit for use in determining the level of insulin resistance in a mammal comprising a non-hydrolysable non-structural carbohydrate or fructose and instructions for use. The kit may also include a feedstuff.

10 In an eleventh aspect of the invention there is provided a method for the diagnosis of predisposition to laminitis in an ungulate animal as substantially defined herein by reference to the examples.

In a twelfth aspect of the invention there is provided a method for determining the level of 15 insulin resistance in a mammal, preferably an ungulate animal, as substantially defined herein by reference to the examples.

AU preferred features of each of the aspects of the invention apply to all other aspects mutatis mutandis.

20

The present invention will now be described with reference to the following examples and drawings which are present for the purposes of illustration and are not to be construed as being limiting on the invention. In the examples reference is made to a number of drawings, in which:

25

FIGURE 1 illustrates the possible sequence of events in cereal overload or grass associated laminitis and the potential influence that insulin resistance and raised Cortisol levels may have.

30 FIGURES 2a and 2b show the concentration of triglycerides in blood samples from normal and laminitic ponies prior to and following administration of a 3g/kg bodyweight per day divided dose of inulin.

551914V1 FIGURES 3 a and 3b show the concentration of glucose in blood samples from normal and laminitic ponies prior to and following administration of a 3g/kg body weight per day divided dose of inulin. 5

FIGURES 4a and 4b show the concentration of insulin in blood samples from normal and laminitic ponies prior to and following administration of a 3g/kg bodyweight per day divided dose of inulin.

10 FIGURES 5 a and 5b show the insulin glucose ratio in blood samples from normal and laminitic ponies prior to and following administration of a 3g/kg bodyweight per day divided dose of inulin.

FIGURES 6a and 6b show the concentration of insulin in blood samples from normal and 15 laminitic ponies prior to and following administration of a 3g/kg bodyweight per day divided dose of inulin.

FIGURES 7a and 7b show the concentration of glucose in blood samples from normal and laminitic ponies prior to and following administration of a 3g/kg bodyweight per day 20 divided dose of inulin.

FIGURE 8 shows the concentration of insulin in blood samples from laminitic ponies prior to and following administration of lg/kg body weight per day of inulin.

25 FIGURE 9 shows the concentration of insulin in blood samples from normal and laminitic ponies prior to and following administration of 0.3g/kg bodyweight per day of inulin.

FIGURE 10 shows the concentration of glucose in blood samples from normal and 30 laminitic ponies prior to and following administration of 0.3g/kg bodyweight per day of inulin.

551914v1 FIGURE 11 shows the concentration of insulin in blood samples from normal and laminitic ponies prior to and following administration of lg/kg body weight per day of inulin.

5 FIGURE 12 shows the composition of WSC fraction of whole grass.

FIGURE 13 shows the composition of the fructan components of the Timothy extract after treatment with HCl (0.075M-pH2) and/or foregut digestive enzymes.

10 FIGURES 14a and 14b show the concentration of triglycerides in blood samples from normal and laminitic ponies when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non structural carbohydrate material than the hay diet. The solid lines represent the mean.

15 FIGURES 15a and 15b show the concentration of glucose in blood samples from normal and laminitic ponies when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non structural carbohydrate material than the hay diet. The solid lines represent the mean.

0 FIGURE 16 shows the concentration of insulin in blood samples from normal and laminitic ponies when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non structural carbohydrate material than the hay diet. The mean level of insulin for each group is shown by the solid line and the dotted line represents the normal range.

25

FIGURE 17 shows the concentration of insulin in blood samples from individual laminitic ponies when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non structural carbohydrate material than the hay diet. The mean level of insulin for each diet is shown by the solid

30 line.

551914V1 FIGURE 18 shows the concentration of insulin in blood samples from laminitic ponies when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non structural carbohydrate material than the hay diet. The solid lines show the change in insulin level for each individual animal 5 following the change in diet.

FIGURES 19a and 19b show the concentration of insulin in normal and laminitic ponies over time when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non-structural carbohydrate 10 material than the hay diet.

FIGURES 20a and 20b show the insumr.glucose ratio for normal and laminitic ponies when fed initially a grass based diet and then a hay diet where the grass contained higher levels of effectively non-hydrolysable non structural carbohydrate material than the hay 15 diet.

FIGURES 21a and 21b show the concentration of insulin in blood samples from normal and laminitic ponies prior to and following administration of a 0.04g/kg bodyweight dose of dexamethasone. 20

551914v1 Examples

Example 1: Changes in insulin, glucose and triglycerides in ponies following feeding with 5 inulin.

Method

10 Eleven adult non-obese mixed native breed ponies (4 mares and 7 geldings) were used in a first study. Six of the ponies had one or more episodes of acute laminitis in the previous two years, but none had shown clinical signs in the 3 months leading up to the study (laminitis-prone group); the remaining five had not shown any signs of laminitis in at least the previous 3 years (normals). Following an initial period of 2 weeks on a basal hay

15 diet, inulin was then fed at 3g /kg bodyweight, split into three equal feeds. The basal hay continued to be provided but a high protein dried grass (15% protein plus some NSCs) was fed as one third of the forage ration, to provide an amino acid source which would mimic that present in lush grass.

0 Blood samples were taken when the ponies were equilibrated on the hay diet (2 days before introducing inulin) and then 1, 2 and 5 days after the inulin phase began. Samples were taken at the same time, approximately 3hrs post prandially, and heparinised plasma was prepared immediately by centrifugation and the samples stored at -80 ⁰C until analysis. 5

The concentrations of triglycerides, glucose and insulin in each sample was analysed using standard commercial techniques. Median values were then calculated for both groups on each day.

30 Concentrations of triglycerides, glucose and insulin on each sampling day of the inulin diet phase of the study were compared to the control value on the hay diet using a paired Friedman's multiple comparison test with Dunn's post hoc analysis (non-parametric analysis; data not normally distributed). Values were compared between normal and laminitic animals on each of the sampling days using an unpaired Kruskal-Wallis test,

35 again with Dunn's post hoc analysis.

551914V1 A second study was carried out on a similar group of ponies and using the above method by feeding inulin at a concentration of lg/kg body weight per day.

5 A third study was carried out on two normal ponies and two laminitis-prone ponies by feeding inulin at a concentration of 0.3g/kg bodyweight per day. The inulin was added to a mixture of alfalfa and hay. A fourth study was carried out by feeding four normal ponies and three laminitis-prone ponies with lg/kg bodyweight inulin per day. The inulin was added to a mixture of basal hay and high protein dried grass. Blood samples for the

10 third and fourth studies were taken every two hours, two days prior to adding inulin to the diet (i.e. when on a hay only diet) and then every two hours after the administering inulin on the second day of inulin feeding.

15 Results and Discussion First study:

The plasma triglyceride levels were not significantly different between the normals and laminitic ponies, (medians of 3.6 and 5.0 mmol/1, respectively on the hay diet) although 2 of the normals and 4 of the laminitics were outside of the normal range (0.17-0.46

20 mmol/1). Most remarkable was the spread of the values, with the range of values for the normals on hay being 0.37mmol/l and a range for the laminitics of 1.07 mmol/1 (Figure 2a and b). The inclusion of inulin had no apparent effect on plasma triglycerides in either group.

25 Plasma glucose levels were within or just below the normal range of 5-7 mmol/1 in all of the ponies throughout the study (Figure 3 a and b). Glucose concentrations in the normal ponies did not change significantly following introduction of the inulin in the diet (median 5.3 (range 5.1-5.4) on hay vs 4.9 (4.8-5.5) on hay + inulin, day 2). In the laminitic ponies, there was a trend towards glucose increasing on the inulin diet vs the

30 hay diet, although this did not reach statistical significance (median 5.15 (range 4.9-5.4) on hay vs 5.9 (4.8-6.5) on hay + inulin, day 2).

551914v1 These results raise an important point. When researchers induced laminitis by feeding a fructan they found glucose levels peaked at 24 - 28hrs but the plasma insulin concentrations did not show significant effects of time. However, in the above first study, there was no real difference in the glucose levels but real massive differences were found 5 with the insulin levels and these remained high throughout the feeding period.

Plasma insulin concentrations were within the normal range (5.5-36 iu/ml) in all but one (Bella; 127 iu/ml) of the normal ponies on the hay diet. In the laminitic group, 3 of the 6 ponies were above the normal range (median 44.7 iu/ml (range 8.9-152.0). However, 10 there was no significant difference between normals and laminitics on the hay diet, confirming the difficulty in using basal blood samples as an indicator. The concern here is that Bella may in fact have an increased risk of laminitis and therefore additional precautions have now been put in place to reduce the risk in this individual.

15 In the normal group of ponies, insulin concentrations increased by 2.0+0.3 fold between diets (median on hay diet 14.4 μlU/ml (range 9.5-31.2, with one outlier (Bella) at 127.0) vs. 37.9 (23.1-38.4 plus one individual (Bella) at 174.0) μlU/ml on hay plus inulin, 48hrs (no significant difference). (Figure 4a). Normal range 5.5-36.0 μlU/ml). However, in the ponies predisposed to laminitis, their insulin levels were not significantly different from

20 the normal ponies while on the basal hay diet (44.7 (8.9-152) μlU/ml; 3 within normal range), insulin concentrations increased dramatically and significantly by the 24hr sample with a peak at 48hrs (5.5±2.2 fold increase) following the addition of fructan carbohydrates (peak of 137.0 (52.1-576.0) μlU/ml at 48 hrs; p<0.03) with two individuals exceeding 500 iu/ml (Figure 4b), in comparison with the normal animals where there was

25 no significant increase in insulin concentrations at this 48hr point.

On all three days of sampling while on the hay + inulin diet, all of the ponies predisposed to laminitis had pm plasma insulin levels, above the normal range. This was a significant increase compared with the hay diet (P<0.0057). Insulin levels were significantly greater 30 in the laminitics compared with the normals at 48h after starting on inulin (P<0.05).

551914V1 As a very rough estimate of insulin sensitivity, an insulin: glucose ratio was calculated for each time point (Figures 5a and b). Four out of the 5 normal ponies (except Bella) consistently had insulin: glucose ratios below 10, throughout both diet periods.

5 In the laminitic group, 3 ponies had insulin: glucose ratios below 10 on the hay diet (median 8.35 (range 1.8-28.7)). On the inulin diet, however, all of the laminitic ponies had increased insulin:glucose ratios over 10 (median value 20 or more throughout the 5 day sampling period).

10 Fig 5 highlights the difference in the insulin response in the normals and laminitics to the feeding of 3g/kg BW/day of a commercially sourced fructan .

Fig 6 shows the changes in glucose with the feeding of this diet in a group of laminitics and those not known to be prone to laminitis and shows that the combination of 15 monitoring both glucose and insulin levels is advantageous.

Similar findings were seen for the second study when lg/kg body weight inulin was fed once a day to ponies in the second study as illustrated by the insulin response in the laminitis prone group (Figure 8). 0

Figures 9 and 10 show the results for the third study. It can be seen that the insulin levels are very similar at the start (time=0hrs). However, by two hours it can be seen that the levels in the laminitic ponies are higher than the levels for the same ponies when fed hay only and for normal ponies when fed the inulin or hay diets. By four hours post inulin 5 administration, the difference between the laminitic ponies and the other ponies becomes very marked. It can be seen from Figure 10 that the response is not due to glucose since the levels do not vary between the groups.

Figure 11 shows the results for the fourth study. The results show a different pattern to 30 that of the fourth study, although again there is a very marked difference in the response by the laminitis prone animals. However on this occasion the insulin levels are high at the start.

551914v1 However, it seems that the normal ponies also start to respond to the administration of inulin, but they then compensate and the levels return to baseline levels. The levels in the laminitics do not return to baseline and are very high (above 500μiU/ml in comparison to normal levels of less than 50μiU/ml).

5

The data obtained in the above studies provides evidence that the majority of ponies in the group tested that were considered to be predisposed to laminitis showed signs of abnormalities in their control of plasma insulin concentrations. It is interesting that half of this group have normal plasma insulin concentrations when sampled on a hay diet. 10 When inulin was supplemented, however, plasma insulin concentrations rose to above the normal range in all cases, in some to a massive degree.

Administration of inulin and subsequent analysis of the level of rise in plasma insulin concentrations following such inulin administration can therefore be used as a test or the

15 base of a test for identifying animals having a predisposition to laminitis. Appropriate action can then be taken to avoid subjecting any animals identified to further conditions which are likely to put them at risk of developing laminitis. For example, changes may be made in the diet of such animals so that access to carbohydrates is restricted or suitable preventative treatments (e.g. antibiotics) may be administered.

20

Although glucose was not elevated in the normal ponies in the first study, a slight increase was noted in the laminitics on the inulin diet, but this was not significant. Therefore if glucose levels were the stimulus for the rise in insulin concentrations, the insulin response was hugely disproportionate to the stimulus in many of these individuals.

25 However, the results of the third study indicate that the response is not occurring due to glucose.

It is possible that the fructose released from the inulin in the stomach (by acid hydrolysis) causes the very marked inappropriate increase in insulin levels seen in the fourth study. 30 This suggests that oral administration of fructose might result in similar responses - even though in humans the increases in blood glucose and insulin after fructose ingestion are only 10 to 20% of those measured after an equivalent amount of glucose. However one

551914v1 previous study in normal horses showed no difference in the insulin response to glucose or fructose. The effect of fructose was investigated further in Example 2.

Example 2: Carbohydrate breakdown 5

Nilsson et al., (1988 J Nutr 1325 - 1330) showed that fructans act as dietary fibre in humans and rats, and are not degraded by mammalian enzymes but readily fermented by bacteria. It has been suggested that it also acts as a dietary fibre in horses and passes relatively undigested through the stomach and small intestine (foregut) of the horse to the

10 caecum and colon (hindgut) where it is very rapidly fermented by the bacterial population. This rapid fermentation results in the increased production of acidic fermentation products (primarily the volatile fatty acids (VFA) acetate, propionate, butyrate and lactate) and thus a decrease in the pH of the hindgut, a condition known as acidosis. It has been thought that fructans act in a similar way in the horse i.e. they are not

15 broken down by mammalian enzymes and therefore are digested by the bacteria within the gastro-intestinal tract of the horse, in particular the hind gut.

The water soluble carbohydrate (WSC) present in six temperate grass species that are commonly used in UK pastures (Timothy (T) , Meadow fescue (M) , Cocksfoot (C), 20 Perennial ryegrass (PR), Italian ryegrass (IR) and Hybrid ryegrass (HR)), was characterised and the fructan content partially purified.

The total WSC content of the grasses was extracted by boiling dried, ground grass in water, and was then analysed using quantitative and qualitative high performance liquid 25 chromatography (HPLC) and thin layer chromatography (TLC). The fructan fraction was extracted by boiling fresh grass in 80% ethanol then precipitated out of the resulting solution using 100% ethanol. The composition of the WSC fraction of whole grass can be seen in Figure 12.

30 The WSC and fructan extracted were then used to investigate the capacity of the stomach and small intestine to digest grass WSC in vitro. First, the partially purified fructan was incubated at 39⁰C with synthetic enzymes, pepsin mixed with hydrochloric acid (HCl, pH 2.5) for 0.5, 1, 2 or 3 hours, and porcine pancreatin overnight, to mimic conditions in the

551914V1 foregut. It was then subjected to similar treatment but using gastric and intestinal fluids collected from humanely slaughtered horses, either filtered to remove the microbes present or unfiltered. Whole grass (dried and ground) was also subjected to this treatment. Finally, both whole grass and partially purified fructan were incubated with 5 HCl of differing pH levels, 3, 4 and 5, to look at the effect of acid hydrolysis.

Results and Discussion

Results from the above study can be seen in Figure 13, which shows very surprisingly 10 significant degradation with the pepsin/HCL mix of the inulin fructan components with an increasing conversion of the inulin series to raffinose series with increasing exposure to the pepsin/HCL.

In other species it has been suggested that during their passage through the

15 gastrointestinal tract, inulin-type fructans never produce fructose (Roberfroid & Delzenne

Ann Rev Nutr 1988; 18 117 - 43). However, the table below shows a shift in the proportion of Timothy extract classified as high molecular weight (HMW) i.e. long chain polymers of fructose (fructans) which are presumed to be resistant to mammalian enzymes or low molecular weight (i.e. the sum of fructose, glucose and sucrose) which 0 are presumed digestible and absorbable in mammalian foregut.

Treatment HMW (long chain LMW (sum of fructose, polymers of fructose i.e. sucrose and glucose) fructans)

1. Control - water 0 h 98 2

2. water - 3 h 98 2

3. HCL- 3 h 81 19

4. pepsin/ HCl- Vi h 85 15

5. pepsin/HCL -I h 84 16

6. pepsin /HCL -2h 81 19

551914V1 This showed that around 20% of the HMW material can be broken down to LMW fractions with HCL at pH 2. Similar amounts of conversion occurred with Pepsin/HCL for up to 2hrs with around 25% conversion seen actually by 3hrs of incubation.

5 In conclusion, since fructans are polymers of fructose, this acid hydrolysis will result in the cleavage of fructose molecules sufficient to result in an increase in the amount of fructose that is available. This was also the case when the whole grass or fructan were incubated with the digestive fluids; some degradation occurred but only low levels.

10 There was also increased conversion of the HMW to LMW when the extract was incubated overnight with pancreatin (to 47 & 53% respectively) suggesting that this may have an additional effect.

Whilst it is known that fructan linkages may be broken down under mild acidic 15 conditions, it has previously been shown that no significant breakdown of fructan occurs in the stomach of man or rats (which have similar pre-caecal digestive systems to the horse). Nilsson et al 1988 concluded that this acid hydolysis was of limited importance. In fact only 12 - 14% loss of ingested fructans has been shown for the whole of the gastric and ileal tract in other species and most of the loss was thought to be due to fermentation 20 by the microbial population in the ileum (Roberfroid & Delzenne 1988 Ann Rev Nutr 18 117 - 43). It was also concluded that fructans 'proceed undigested through the upper part of the gastrointestinal trace into the colon ' (Roberfroid & Delzenne 1988 Ann Rev Nutr 18 117 - 43) and are classified as 'nondigestible' oligosccharides.

25 As horses are continual gastric acid producers it is possible that the pH in the stomach may reach at certain times of day (and especially in the morning after having eaten most if not all of their feed the evening before) sufficiently low levels of pH to be able to promote sufficient acid hydolysis of any ingested fructan to result in a release of fructose molecules. Therefore, if fructan is administered in the morning to a horse then the gastric

30 pH is likely to be very low, leading to breakdown of fructan to fructose. However, if the fructan is administered after a meal then there may be limited breakdown of fructan to fructose.

551914V1 These in themselves were very unexpected findings, but did suggest that under certain conditions within the time period that fructan is present within the stomach (and therefore when exposed to low pH there could be some conversion and release of fructose). 5

Discussion of the conclusions that may be drawn from the results of Examples 1 and 2

It has been shown in the results of Example 1, that dramatic increases in insulin levels are seen following administration of inulin to a laminitis-prone horse. These effects are seen 10 without the occurrence of a concurrent increase in glucose. The results of Example 2 indicate that the reason for the absence of a rise in the glucose levels is because the effect seen is due to the breakdown of fructan to fructose.

Whilst work in man has suggested that fructose does not stimulate insulin release it has

15 been previously shown that administration of fructose in normal healthy horses may result in similar insulin increases to that seen following glucose administration. This means that only a small response would have been expected in line with the limited amount of fructose that could have been released from the 0.3g/kg bodyweight fructan administration. However, as discussed above, a significant response was seen. Even if

20 all the inulin administered was broken down to fructose (or fructose itself administered initially), a similar amount of glucose would not have lead to such a high level of insulin present. This suggests that animals prone to laminitis are unusually sensitive to fructose. It is possible that fructose itself may promote insulin resistance together with endothelial dysfunction which may increase the risk of laminitis.

25

In conclusion, the results of Examples 1 and 2 suggest that the administration of even small amounts of fructose to ponies may result in unexpectedly high insulin responses in those prone to laminitis/ with insulin resistance. The administration of fructose to ponies may therefore be used as an alternative to inulin in the diagnosis of a predisposition to

30 laminitis.

551914V1 Example 3: Changes in insulin sensitivity in ponies predisposed to laminitis, when changing from a higher non-hvdrolysable non-structural carbohydrate and fructan containing material (ie grass) to a lower non-hydrolysable non-structural carbohydrate 5 and fructan containing diet (mature timothy hay) diet.

Method

10 ponies which had had one or more episodes of acute laminitis in the previous two 10 years, but had shown not clinical signs in the 3 months leading up to the study (laminitis- prone group); and 9 which had not shown any signs of laminitis in at least the previous 3 years (normals) ponies were used. The ponies were maintained out on grass. Blood and urine samples were taken. The ponies were then housed and were fed a lower nonstructural carbohydrate diet and in particular a lower fructan containing diet: i.e. mature 15 timothy hay. The blood samples, urine samples and blood pressure were resampled after a time period of 1, 3, 5 and 7 days following the start of feeding of the hay.

Concentrations of triglycerides, glucose and insulin on each sampling day of the inulin diet phase of the study were compared to the control value on the higher fructan hay diet 20 using similar methods to the previous example.

Results and Discussion

One normal pony with respect to laminitis history in the past 3 yrs - aubergine - had significantly high triglyceride concentrations (Fig 14a). However apart from this 25 individual no significant differences were seen between those prone to laminitis and those not believed to be prone (Figures 14a and 14b).

No major differences were seen between the two groups for glucose as shown in Figures 15a and 15b . 30

On the higher fructan containing diet the insulin concentrations were significantly higher in the laminitis prone group than the normals when taken as a group (Figure 16). However the majority of the individuals in both groups had basal levels within the

551914V1 accepted normal range. The normal/reference range is represented by levels which fall at or below the dotted line shown in Figure 16 and is provided by the testing laboratory. In this instance the reference/normal range for the laboratory used was 5.5-36.0 μlU/ml.

5 In the apparently normal animals, 2 out of the 9 had insulin levels above the normal range and 3 out of 10 laminitis also had insulin levels above the normal range (Figure 16). The two apparently normal animals would be considered to be obese on body condition scoring. Obesity has been associated with insulin resistance. Following the change in diet the mean (as illustrated by the solid line) insulin levels of the laminitis significantly 10 decreased after 7 days of feeding the hay (see Figure 17 and Figure 18) resulting in more of the individuals having values with in the accepted normal range - again illustrating the power of such a dynamic test.

Even more unexpectedly when the pattern of response was evaluated in the 2 groups over

15 the 7 day period a significant difference was seen, despite all animals being treated in an identical manner. In the majority of the normals (classified according to their laminitic history) apart from Aubergine and Flicka (the two obese normal individuals) the housing of the animals and the change in diet resulted in very little change in insulins (Figure 19a). However, in the laminitic prone animals and the two obese normals there was an

20 increase in the insulin levels especially after day 3 - which was very considerable in a number of the individuals (Figure 19b). This again may represent an inability to cope with the stress of housing/the effect of increased restriction of movement. In one survey it was found that a significantly higher proportion of acute-laminitis cases than controls were in the no-regular exercise category. The authors questioned 'if regular exercise is

25 protective against laminitis, is it merely because it decreases obesity, - or does it mediate through other physiological effects?' It is certainly well recognized in humans that exercise and exercise training have numerous beneficial effects including those on glucose homeostasis and enhanced insulin sensitivity and it is also believed to have a similar effect in the horse.

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551914V1 This dynamic test may provide an indication of a predisposition to laminitis as well as the degree of insulin resistance present in an individual as insulin resistance is linked with obesity, as previously discussed.

5 Example 4: Changes in insulin levels in ponies following dexamethasone administration.

Method

10 A similar group of ponies to those used in Example 1 were used in a further diagnostic test for a predisposition to laminitis.

A 19 hour overnight dexamethasone suppression test was performed using a standard method, for example the test described in Donaldson et al, JVIM 2005; 19:217-22.

15

Briefly, blood samples were taken from the ponies. A 0.04 mg/kg dose of dexamethasone was then administered. Subsequent blood samples were then taken after 19 hours. Heparinised plasma was prepared immediately by centrifugation and the samples stored at -80 ⁰C until analysis.

20

The concentrations of Cortisol and insulin in each sample was analysed using standard commercial techniques. Median values were then calculated for both groups on each day.

Concentrations of Cortisol and insulin were compared to values obtained prior to 25 dexamethasone administration.

Ponies having a Cortisol concentration of greater than 25nmol/l were diagnosed with possible pituitary pars intermedia dysfunction (PPID), equine Cushing's syndrome.

30 Ponies having a Cortisol concentration which was suppressed to less than 25nmol/l were considered not at risk of PPID.

The insulin levels were measured in the blood samples taken from ponies not at risk of PPID. Ponies with insulin levels after dexamethasone administration of greater than

551914V1 85μIU/ml were considered to have possible prelaminitic metabolic syndrome and therefore have a predisposition to laminitis. Ponies with insulin levels after dexamethasone administration of less than 85μIU/ml were considered not at risk of developing laminitis.

Claims

Claims -

1. A method of diagnosis of a predisposition to laminitis in an ungulate animal 5 comprising the steps of: a) measuring the levels of insulin in one or more basal samples obtained from the animal when fed a basal diet; b) measuring the levels of insulin in one or more subsequent samples obtained from the animal when fed said basal diet and additionally a source of a non-hydrolysable non- 10 structural carbohydrate or fructose; and c) comparing the levels of insulin in step a) and step b).

2. A method of determining the level of insulin resistance in a mammal comprising the steps of

15 a) measuring the levels of insulin in one or more basal samples obtained from the mammal when fed a basal diet; b) measuring the levels of insulin in subsequent samples obtained from the mammal when fed said basal diet and additionally a source of non-hydrolysable non-structural carbohydrate or fructose; and 20 c) comparing the levels of insulin levels in step a) and step b)

3. The method of claim 2 wherein the mammal is an ungulate animal.

4. The method of claims 1 or 3 wherein the ungulate animal is selected from the 25 group consisting of horses, ponies, equids, cows, goats or sheep.

5. The method of claims 1 to 4 wherein the non-hydrolysable non-structural carbohydrate is selected from the group consisting of fructans, pectins, cellulose, hemi- cellulose, mucilages and gums.

30

6. The method of claims 1 to 5 wherein the non-hydrolysable non-structural carbohydrate or fructose are administered by an oral, parenteral or mucosal route.

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7. The method of claims 1 to 6 wherein the non-hydrolysable non-structural carbohydrate or fructose are administered as a bolus dose in an injectable form, as a bolus dose in a meal or in a divided dose.

5

8. The method of claims 1 to 7 wherein the method additionally comprises measuring the levels of glucose prior to and following administration of non-hydrolysable non-structural carbohydrate or fructose.

10 9. The method of claims 1 to 8 wherein the method additionally comprises measuring the levels of metabolites and hormones involved in the maintenance of energy metabolism and in the homeostasis of glucose prior to and following administration of the non-hydrolysable non-structural carbohydrate or fructose.

15 10. The method of claims 1 to 9 wherein the non-hydrolysable non-structural carbohydrate is administered in a dose of 0.1 to 9g/kg body weight per day for up to 10 days.

11. The method of claims 1 to 9 wherein the fructose is administered in a dose of 20 0.001g/kg bodyweight to 10g/kg bodyweight per day for up to 10 days.

12. The method of claims 1 to 10 wherein the non-hydrolysable non-structural carbohydrate or fructose is provided by a natural or synthetic source or a mixture thereof.

25 13. The method of claim 12 wherein the natural source is provided by a source selected from the group consisting of a grass, vegetables and flowers or mixtures thereof.

14. The method of claim 12 wherein the synthetic source is provided by a 30 commercially sourced fructan or fructose.

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15. The method of claims 1 to 14 wherein the levels of insulin in step a) and step b) are compared with the levels from normal animals.

16. A method of diagnosis of a predisposition to laminitis in an ungulate animal 5 comprising the steps of: a) measuring the level of insulin in one or more basal samples from said animal fed a high non-hydrolysable non-structural carbohydrate containing or high fructose containing diet or feedstuff. b) measuring the level of insulin in subsequent samples from an animal whilst being 10 fed a lower non-hydrolysable non-structural carbohydrate containing or lower fructose containing diet or feedstuff. c) comparing the change in levels of insulin in step a) and step b), wherein step a) may be performed either before or after step b) and the animal is allowed to adapt to a diet or feedstuff before said diet or feedstuff is changed . 15

17. A method for determining the level of insulin resistance in a mammal comprising the steps of: a) measuring the level of insulin in one or more basal samples from said mammal fed a high non-hydrolysable non-structural carbohydrate containing or high fructose 0 containing diet or feedstuff. b) measuring the level of insulin in subsequent samples from a mammal whilst being fed a lower non-hydrolysable non-structural carbohydrate containing or lower fructose containing diet or feedstuff. c) comparing the change in levels of insulin in step a) and step b),

25 wherein step a) may be performed either before or after step b) and the mammal is allowed to adapt to a diet or feedstuff before said diet or feedstuff is changed .

18. A composition comprising a non-hydrolysable non-structural carbohydrate or fructose for use in the diagnosis of a predisposition to laminitis in an ungulate animal.

30

19. A composition comprising a non-hydrolysable non-structural carbohydrate or fructose for use in determining the level of insulin resistance in a mammal.

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20. The use of a non-hydrolysable non-structural carbohydrate in the preparation of a composition for the diagnosis of a predisposition to laminitis in an ungulate animal.

5 21. The use of claim 19 wherein the composition is administered to the mammal and the blood levels of insulin prior to and after said administration are compared with control results from a normal mammal.

22. The use of a non-hydrolysable non-structural carbohydrate or fructose in the 10 preparation of a composition for the diagnosis of insulin resistance in a mammal.

23. The use of claim 21 wherein the composition is administered to the animal and the blood levels of insulin prior to and after said administration are compared with control results from a comparable animal with an expected normal level of insulin resistance..

15

24. A kit for use in the diagnosis of predisposition to laminitis in an ungulate animal comprising a non-hydrolysable non-structural carbohydrate and instructions for diagnosis.

25. A kit for use in determining the level of insulin resistance in a mammal 20 comprising a non-hydrolysable non-structural carbohydrate or fructose and instructions for diagnosis.

26. A method for the diagnosis of predisposition to laminitis in an ungulate animal as substantially defined herein by reference to the examples.

25

27. A method for determining the level of insulin resistance in a mammal as substantially defined herein by reference to the examples.

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