阅读说明：本技术 用于关节小病的生物标志物和其用途 (Biomarkers for small joint disease and uses thereof ) 是由 陈巨星 K·韦德金德 M·巴斯克斯-阿农 于 2020-03-12 设计创作，主要内容包括：本发明涉及与关节病症相关的生物标志物,以及使用所述生物标志物来诊断关节小病、监测关节小病的进展、确定何时需要治疗并监测治疗效率的方法。还提供了用于治疗关节小病的方法,所述方法包括将螯合微量矿物质施用于被诊断患有或倾向于患有关节小病的动物。(The present invention relates to biomarkers associated with joint disorders and methods of using the biomarkers to diagnose arthropathy, monitor the progression of arthropathy, determine when treatment is needed, and monitor the efficiency of treatment. Also provided are methods for treating a small joint disease comprising administering the chelated trace minerals to an animal diagnosed with or predisposed to having the small joint disease.)

1. A method for treating or preventing a facet joint disorder in an animal, the method comprising:

(a) collecting a blood, urine or synovial fluid sample from the animal;

(b) determining the level of at least one biomarker present in said blood sample, said biomarker selected from osteocalcin, type I collagen C-terminal telopeptide (CTX-1), type II procollagen C-terminal propeptide (P2CP), type II collagen C-terminal telopeptide (CTX-2), type II collagen (C2C), or a combination thereof;

(c) analyzing the level of the at least one biomarker to determine whether the animal has or is predisposed to having arthropathy, wherein the analyzing comprises comparing the level of the at least one biomarker in the blood sample to an arthropathy-positive and/or arthropathy-negative reference level for the at least one biomarker in order to determine whether the animal has or is predisposed to having arthropathy; and

(d) administering to the animal an effective amount of a metal chelate if the animal is determined to have or is predisposed to having a joint ailment.

2. The method of claim 1, wherein step (b) comprises an antibody-based detection method.

3. The method according to claim 1 or 2, wherein the method comprises determining the level of at least two biomarkers and comparing the level to an arthrosis positive and/or arthrosis negative reference level for the at least two biomarkers.

4. The method according to any one of claims 1 to 3, wherein the method comprises determining the levels of at least three biomarkers and comparing the levels to arthropathy positive and/or arthropathy negative reference levels for the at least three biomarkers.

5. The method of any one of claims 1 to 4, wherein the method comprises determining the levels of at least four biomarkers and comparing the levels to arthropathy positive and/or arthropathy negative reference levels for the at least four biomarkers.

6. The method according to any one of claims 1 to 5, wherein the method comprises determining the levels of five biomarkers and comparing the levels to arthropathy positive and/or arthropathy negative reference levels for the five biomarkers.

7. The method of any one of claims 1-6, wherein the metal chelate comprises at least one metal ion and at least one ligand, wherein the at least one ligand is an amino acid, a hydroxy acid, an organic acid, a sugar alcohol, a protein hydrolysate, a polysaccharide, or a polynucleic acid.

8. The method of claim 7, wherein the at least one metal ion is calcium, chromium, cobalt, copper, iron, magnesium, manganese, nickel, potassium, sodium, zinc, or a combination thereof.

9. The method of claim 7, wherein the at least one ligand is a compound of formula (I):

wherein:

R¹is methyl or ethyl; and is

n is 1 or 2.

10. The method of claim 9, wherein R¹Is methyl and n is 2.

11. The method of claim 9 or 10, wherein the at least one metal ion is copper, manganese, zinc, or a combination thereof.

12. The method of any one of claims 1-11, wherein the animal is a livestock animal.

13. The method of claim 12, wherein the livestock animal is a pig or a cow.

14. The method of any one of claims 1-11 wherein the animal is a companion animal.

15. The method of claim 14, whichWherein said companion animal is a cat, dog or horse.ⁱ

Technical Field

The present disclosure relates to biomarkers associated with joint disorders and uses thereof.

Background

Lameness has been identified as a welfare problem for all livestock species, which leads to a reduction in productivity and profitability of the farm. Dyskinesias occur in connection with hoof and limb injuries, nervous system disorders, metabolic and infectious disorders, and mechanical and structural problems. The incidence of sow and cow lameness can vary from 5% to 40% and is associated with lower reproductive performance, food intake, longevity and increased mortality. Lameness is the most common cause of sows and cows leaving the herd prematurely after reproductive problems. The cause of lameness is mainly associated with osteochondrosis in the bone joints, which is difficult to identify in living animals, however. Visual gait lameness scores are commonly used to identify lameness in production animals (pigs, cattle and chickens). However, such subjective scoring systems lack sensitivity and consistency among scorers and result in delayed lameness detection. Earlier detection of lameness may improve the chances of solving the problem through a nutrition program. Therefore, there is a need for an accurate, objective method that will enable earlier identification of lameness. Furthermore, solutions for reducing the severity and incidence of lameness, such as nutritional interventions, are needed.

Disclosure of Invention

One aspect of the present disclosure provides a method for treating or preventing a facet joint disease in an animal, the method comprising: (a) collecting a blood sample from the animal; (b) determining the level of at least one biomarker present in said blood sample, said biomarker selected from osteocalcin, type I collagen C-terminal telopeptide (CTX-1), type II procollagen C-terminal propeptide (P2CP), type II collagen C-terminal telopeptide (CTX-2), type II collagen (C2C), or a combination thereof; (c) analyzing the level of the at least one biomarker to determine whether the animal has or is predisposed to having arthropathy, wherein the analyzing comprises comparing the level of the at least one biomarker in the blood sample to an arthropathy-positive and/or arthropathy-negative reference level for the at least one biomarker in order to determine whether the animal has or is predisposed to having arthropathy; and (d) administering to the animal an effective amount of a metal chelate if the animal is determined to have or is predisposed to having facet joint disease.

Other aspects and iterations of the present disclosure are described in more detail below.

Drawings

Figure 1 presents P2CP concentrations for four treatments (healthy ITM, healthy MTX, lameness ITM, lameness MTX) in a naturally occurring lameness model.

Figure 2 presents the time course of visual gait scores following urate injection to induce acute lameness. Gait scores are presented for animals injected with urate that were fed MTX or ITM two months prior to the injection.

Fig. 3A presents the time course of serum levels of C2C in animals injected with urate. Fig. 3B plots the time course of serum levels of CTX2 in animals injected with urate. FIG. 3C presents the time course of the serum levels of osteocalcin in urate-injected animals previously fed MTX or ITM.

Figure 4A presents the average weight borne by the front-to-side legs of MTX and ITM animals over time. Figure 4B shows the average weight borne by the anterior ipsilateral leg of the MTX and ITM animals over time. Figure 4C presents the average weight borne by the hind-contralateral legs of MTX and ITM animals over time. Figure 4D shows the average weight borne by urate-injected legs of MTX and ITM animals over time.

Detailed Description

The present disclosure provides biomarkers that can be used as objective indicators of arthrosis in animals (e.g., livestock animals). The biomarkers can be used to diagnose arthropathy, monitor the progression of arthropathy, determine when treatment is needed, and monitor the efficiency of treatment.

(I) Biomarkers

One aspect of the present disclosure encompasses a set of serum biomarkers associated with arthropathy. The biomarkers are associated with the synthesis and degradation of bone and cartilage. The biomarkers can be used to distinguish healthy animals from animals with arthropathy (e.g., lameness), monitor the progression of arthropathy or joint disease, or monitor the efficacy of treatment.

One biomarker is osteocalcin. Osteocalcin, also known as bone gamma-carboxyglutamic acid containing protein (BGLAP), is a non-collagenous protein hormone present in bone and dentin. Osteocalcin is a marker of bone synthesis.

Another biomarker is type I collagen C-terminal telopeptide or CTX-1, which is a marker of bone degradation or turnover (Turnover). Type I collagen makes up about 90% of the bone organic matrix. CTX-1 is involved in bone turnover because it is the portion of the molecule that is cleaved by osteoclasts during bone resorption. CTX1 is a marker of bone degradation.

Yet another biomarker is type II procollagen C-terminal propeptide or P2CP, which is a biomarker for cartilage synthesis. Type II collagen is the major organic component of cartilage. P2CP is a marker of cartilage synthesis.

An additional biomarker is type II collagen C-terminal telopeptide or CTX-2, which is a biomarker of cartilage degradation. After cartilage degradation, fragments of CTX-2 are released into the circulation. CTX2 is a marker of cartilage degradation.

Yet another biomarker is type II collagen or C2C, which is a biomarker of cartilage degradation.

After determining the levels of the biomarkers of arthropathy in healthy animals and animals with arthropathy (naturally occurring arthropathy or chemically induced arthropathy), these levels can be correlated with standard indicators of lameness or arthropathy to establish biomarker reference levels for healthy animals and animals with arthropathy/lameness. The ratio of synthesis/degradation (P2CP/C2C, P2CP/CTX2, osteocalcin/CTX 1) may be associated with lameness.

Standard indicators of lameness or arthropathy include visual gait scores and force plate tests. The gait or movement of the animals was observed and scored. Gait can be scored on a 5 point scale, ranging from "0" for animals that are walking normally to "4" for animals that are unwilling to walk and bear weight with one or more legs. A force plate can be used to measure the weight of each forelimb, where differences indicate that the animal is biased towards one limb. By correlating biomarker levels with visual lameness/arthropathy criteria, reference levels for healthy and lameness animals can be established.

(II) methods for diagnosing and/or monitoring progression of arthropathy

Another aspect of the disclosure provides methods for diagnosing and/or monitoring progression of arthropathy in an animal. For example, the method can be used to distinguish between a healthy animal and an animal with a small joint disease, and if the animal has a small joint disease, the progression of the small disease can be monitored. The method comprises the following steps: (a) collecting a blood sample from the animal; (b) determining the level of at least one of the biomarkers disclosed herein present in the blood sample; and (c) analyzing the level of the at least one biomarker to determine whether the animal has or is predisposed to having arthropathy, wherein the analyzing comprises comparing the level of the at least one biomarker in the blood sample to an arthropathy positive and/or arthropathy negative reference level for the at least one biomarker, in order to determine whether the animal has or is predisposed to having arthropathy, and/or monitoring the progression of arthropathy in animals known to have joint problems.

Small joint disease

As used herein, "arthropathy" refers to a disease or disorder of a joint or joint tissue. Joint tissue comprises bone and connective tissue, i.e. cartilage, tendons and ligaments. Arthropathy encompasses arthritis or osteoarthritis, which is a degenerative joint disease or condition resulting from the progressive degeneration of articular cartilage within one or more joints. Arthritis is a general description of any condition that causes inflammation of the joints. Rheumatoid arthritis is a chronic inflammatory disorder of the joints. Other arthropathies include osteochondrosis, gouty arthritis, bursitis, tenosynovitis, epicondylitis, synovitis, ankylosing spondylitis, Sjogren's syndrome, psoriatic arthritis, and Lyme disease. Some joint disorders may be due to hoof or footpad diseases or disorders.

Step (a)

The first step of the method comprises collecting a blood sample from the animal. Various methods of collecting blood, urine or synovial fluid are known in the art. Generally, methods of collecting blood include accessing the blood using a skin piercing element and collecting the blood therein into some type of collection device. Entering blood may also involve the use of a fluid pathway, a capillary channel (e.g., a capillary tube), a fluid transfer medium (e.g., a hydrophilic porous material), or some mechanical or vacuum means in combination with a skin piercing element. In general, the sample collection method preferably maintains the integrity of the sample so that the abundance value of each molecular feature can be accurately measured. The blood sample may be a whole blood sample, a plasma sample or a serum sample.

Step (b)

The second step of the method comprises determining the level of at least one biomarker present in the blood sample. Various methods may be used to determine the level or concentration of one or more biomarkers. Biomarkers can be detected and quantified using antibody-based detection methods. For example, enzyme-linked immunosorbent assays (ELISAs) can be used to determine the levels of biomarkers. The ELISA may be a direct ELISA, a sandwich ELISA, a competition ELISA or a reverse ELISA. The detection method may be optical (i.e., colorimetric or fluorescent) or electrochemical. In particular embodiments, one or more biomarkers can be detected using a sandwich ELISA and a colorimetric assay.

In other embodiments, antibody-based detection methods may include protein immunoprecipitation, immunoelectrophoresis, western blotting, or protein immunostaining. In still other embodiments, the level of one or more biomarkers can be quantified using High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS).

Step (c)

The next step of the method comprises analyzing the level of at least one biomarker to determine whether the animal is healthy, predisposed to or likely to suffer from arthrosis, or whether the animal suffers from arthrosis. The analysis comprises comparing the level of the at least one biomarker in the blood sample with an arthropathy positive and/or arthropathy negative reference level for the at least one biomarker. If the level of the at least one biomarker falls within the range of the negative reference level for arthropathy, the animal is healthy and does not suffer from arthropathy. The animal has an arthropathy if the level of the at least one biomarker falls within the range of a positive reference level for the arthropathy. The severity of the arthropathy can be estimated based on the level of at least one biomarker. The progression of arthropathy can be monitored by comparing the levels of at least one biomarker over time.

In some embodiments, the levels of both biomarkers may be determined. For example, the levels of osteocalcin and CTX1 can be determined and/or the osteocalcin/CTX 1 ratio can be determined. Alternatively, the levels of P2CP and C2C may be determined and/or the ratio of P2CP/C2C may be determined. In other embodiments, the levels of three biomarkers may be determined. In further embodiments, the levels of four biomarkers may be determined. In still other embodiments, the levels of all five biomarkers can be determined.

Animal(s) production

Suitable animals include, but are not limited to, livestock animals, companion animals, laboratory animals, and zoological animals. In particular embodiments, the animal may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cattle, poultry, goats, sheep, llamas, alpacas, aquatic animals, and the like. In an exemplary embodiment, the animal may be a pig, such as a sow. In other embodiments, the animal may be a cow.

In other embodiments, the animal may be a companion animal. Non-limiting examples of companion animals can include pets such as dogs, cats, horses, rabbits, birds, or rodents (e.g., mice, rats, hamsters, guinea pigs). In yet other embodiments, the animal may be an zoological animal. As used herein, "zoological animal" refers to an animal that can be found in a zoo. Such animals may include non-human primates, large felines, wolves, bears, river horses, kangaroos, and the like. In still other embodiments, the animal may be a laboratory animal. Non-limiting examples of laboratory animals may include rodents, canines, felines, and non-human primates.

(III) methods for treating or preventing arthrosis

Another aspect of the disclosure provides a method for treating or preventing a facet joint disease in an animal. The method may also be used to monitor the efficacy of a treatment method, wherein the treatment method may be modified accordingly. The method comprises the following steps: (a) collecting a blood sample from the animal; (b) determining the level of at least one of the biomarkers disclosed herein present in the blood sample; (c) analyzing the level of the at least one biomarker to determine whether the animal has or is predisposed to having arthropathy, wherein the analyzing comprises comparing the level of the at least one biomarker in the blood sample to an arthropathy-positive and/or arthropathy-negative reference level for the at least one biomarker in order to determine whether the animal has or is predisposed to having arthropathy; and (d) administering to the animal an effective amount of a metal chelate if the animal is determined to have or is predisposed to having facet joint disease.

Steps (a), (b) and (c) of the method are as described in part (II) above, as are suitable arthropathies and animals.

Step (d)

If it is determined in step (c) that the animal has or is predisposed to having arthrosis, the next step comprises administering to the animal an effective amount of a metal chelate.

The metal chelate includes at least one ligand and at least one metal ion. The ligand may be an amino acid, a hydroxy acid (e.g., an alpha hydroxy acid), an organic acid, a sugar alcohol, a protein hydrolysate (e.g., soy protein hydrolysate), a polysaccharide, or a polynucleic acid.

In some embodiments, the ligand may be an amino acid. Suitable amino acid derivatives include alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, homocysteine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, caseinate and valine.

In other embodiments, the ligand may be an organic acid. Non-limiting examples of suitable organic acid moieties include adipates, ascorbates, caprylates, citrates, xanthohumates, fumarates, glucoheptonates, gluconates, glutarates, glycerophosphates, humates, lactates, ketoglutarates, malates, malonates, orotates, oxalates, pantothenate, picolinates, pyridonates, sebacates, succinates, and tartrates.

In still other embodiments, the ligand may be a sugar alcohol. Suitable sugar alcohols include, but are not limited to, sorbitol, mannitol, xylitol, lactitol, isomalt, maltitol, erythritol, and Hydrogenated Starch Hydrolysates (HSH).

In a particular embodiment, the ligand is a compound of formula (I).

Wherein R is¹Is methyl or ethyl and n is 1 or 2. In exemplary embodiments, R¹Is methyl and n is 2, and the compound of formula (I) is the methionine hydroxy analogue (or 2-hydroxy-4- (thiomethyl) butanoic acid, HMTBA).

The at least one metal ion may be calcium, chromium, cobalt, copper, germanium, iron, lithium, magnesium, manganese, molybdenum, nickel, potassium, sodium, rubidium, tin, vanadium, zinc, or a combination thereof. In certain embodiments, the at least one metal ion may be calcium, chromium, cobalt, copper, iron, magnesium, manganese, nickel, potassium, sodium, zinc, or combinations thereof. In particular embodiments, the at least one metal ion may be copper, manganese, zinc, or a combination thereof.

In the metal chelate, the ratio of the at least one ligand and the at least one metal ion may vary. For example, the ligand to metal ratio can be in the range of 1:1 to about 3:1 or higher. In embodiments where the metal ion is divalent, the ligand to metal ratio may be 2: 1.

In particular embodiments, the metal chelate may include the methionine hydroxy analog copper (i.e., MHA-Cu to (HMTBA)₂Cu), the methionine hydroxy analog manganese (i.e., MHA-Mn or (HMTBA)₂Mn), the methionine hydroxy analog zinc (i.e., MHA-Zn or (HMTBA)₂-Zn) or a combination of any or all of the foregoing (which may be available under the trade name novius International, Inc.) from nomavis InternationalObtained).

The effective amount of the metal chelate administered to the animal can and will vary depending on, for example, the type and age of the animal and/or the severity of the arthropathy. Suitable amounts can be readily determined by one skilled in the art.

Typically, the metal chelate is included in the feed ration of the animal. Feed rations are typically formulated to meet the nutrient and energy requirements of a particular animal. The nutrient and energy content of many common animal feed ingredients has been measured and made available to the public. The National Research Council (National Research Council) published books containing tables of common feed ingredients and their corresponding measured nutrient and energy contents. Additionally, estimates of nutrient and maintenance energy requirements are provided for animals of different life stages, ages, sexes or uses. One skilled in the art can use this information to estimate the nutritional and maintenance energy requirements of the animal and to determine the nutrient and energy content of the animal feed ingredients.

Examples of the invention

The following examples illustrate various embodiments of the disclosure.

Example 1: description of the protocol

Two different lameness models (naturally occurring and chemically induced) were evaluated in this study. In both models, objective measures of lameness (serum biomarkers, force plate, thermal imaging) were compared to visual gait scores. The study design was a 2 x 2-factor array consisting of two dietary treatments (chelated trace minerals and inorganic trace minerals) and two herds (lameness and healthy non-lameness). Chelated trace Minerals (MTX) include Methionine Hydroxy Analogue (MHA) chelates (i.e., MHA-Cu, MHA-Mn, and/or MHA-Zn), and Inorganic Trace Minerals (ITM) include inorganic sulfates.

Four groups of pigs (8 lames per group/8 non-lames, total 32 lames/32 non-lames/total 64) were fed with a diet treatment for a period of two months. For the naturally occurring portion of the test (example 2), lameness measurements were taken at baseline (d0), month 1 (d28), and month 2 (d 53). At the end of the 2 month period, healthy animals (8 per group; 4 for MTX, and 4 for ITM) were then injected with sodium urate crystals (10mg/mL, and injection volume 0.2mL) only in the right posterior distal interphalangeal joints (example 3).

Most methods for gait scoring are based on uneven or asymmetric weight bearing. As shown in Table 1, a score of 5 (0-4) was used, with score 4 being the most severe.

Force plate analysis quantifies the amount of force applied to four separate loading units per limb. Two data points per second were captured over a duration of 2 minutes. Lameness animals typically bear less weight on a painful or structurally sound limb.

Thermography measures the amount of heat emitted from a body surface in the form of infrared radiation. Clinical disease studies have shown that a difference between two identical anatomical regions of greater than 1 ℃ indicates the presence of an abnormality, such as inflammation. The high degree of symmetry between the left and right sides of the animal is an important element in diagnosing unilateral problems associated with lameness. Therefore, these objective measures of lameness should be correlated with a visual gait score that is also based on asymmetric weight bearing.

All statistical analyses were performed using SAS, using pigs as experimental unit. All lameness measures were analyzed using two-way analysis of variance (ANOVA) to test the main impact of dietary treatment and health and lameness and their interaction; forelimb and hindlimb differences were also compared. In addition, data were also assessed using a hybrid model, including days, lameness, diet treatment, weight, group, and gender. Days were included as replicate measures using the autoregressive covariance structure, and data collected the day before the study began was included as covariates (for force plate data only).

Pearson's (Pearson) correlation coefficients and regression analysis are used to explore the relationship between objective measures and gait scores.

Example 2 biomarker levels in healthy animals and naturally occurring lameness animals

Serum biomarkers were measured in healthy (H) and lame (L) animals (n-48) on days 0, 25 and 53 (2 months). The levels of serum CTX1, Osteocalcin (OC), C2C, P2CP and CTX2 were measured by enzyme-linked immunosorbent assay (ELISA) according to the procedure described in the commercial kit (e.g., MyBiosource). The results are presented in table 2.

^1.Treatment (ITM or MTX).

These results reveal that biomarkers are able to distinguish between health and lameness and demonstrate the beneficial effects of MTX. In lameness animals, the cartilage degradation markers CTX2(P ═ 0.0002) and C2C (P ═ 0.0176) were elevated and the cartilage synthesis/degradation ratio (P2CP/CTX 2; P ═ 0.0908) was decreased (table 2). Differences in dietary treatment were also observed: cartilage synthesis biomarkers (P2CP, P ═ 0.0114) and the ratio of cartilage synthesis/degradation (P2CP/CTX2, P ═ 0267) increased with MTX (table 2). As shown in fig. 1, there was no diet (ITM and MTX) interaction of health and lameness (P-0.9727). This suggests that MTX treatment increased cartilage synthesis in both healthy and lameness pigs and may have a prophylactic joint health effect.

Example 3: urate-induced lameness

At the end of two months, sodium urate crystals (10mg/mL, and injection volume 0.2mL) were injected into the right posterior distal interphalangeal joint of healthy pigs from each of the four groups. Thus, during the urate induction portion of the experiment, a total of 16 pigs were evaluated for MTX and 16 for ITM. Data collection related to urate injection time occurred at baseline (d-1/d53), 6 and 12 hours post-injection (d0/d54), 24 hours (d1/55), 48 hours (d2/56), 72 hours (d3/57) and 144 hours (d 6/60).

As shown in fig. 2, the gait score of MTX fed pigs was low (major effect of diet: P ═ 0.0057). Additionally, MTX-fed pigs had lower gait scores at the peak of lameness (P0.0244 at 12 hours after urate injection and 0.0155 at 48 hours after urate injection). Since pigs had received 2 months of dietary treatment (MTX and ITM) prior to injection of urate, these findings suggest that MTX supplementation reduces the severity of urate-induced lameness.

The time course of serum biomarkers after urate administration is shown in figures 3A-3C. Changes in biomarkers are closely related to changes in gait scores. For example, similar to gait scores, cartilage degradation biomarkers (C2C and CTX2) peaked 12 hours after urate injection (fig. 3A, 3B), and bone synthesis biomarkers (osteocalcin) peaked between 12 and 48 hours after urate injection (fig. 3C). Similar to gait scores, after 72 hours (if not earlier), the three biomarkers (C2C, CTX2, osteocalcin) returned to baseline or tended to stabilize relative to the initial concentration.

Tables 3-5 present biomarker levels (no covariates) after urate administration. As indicated in fig. 3A-3C, there was a clear or significant effect of time (hours) for C2C (P ═ 0.0853), CTX2(P ═ 0.1014), and osteocalcin (OC, P ═ 0.0011). Beneficial effects of MTX were also observed; both the bone synthesis biomarker (OC, P ═ 0.0700) and the ratio of bone synthesis/degradation (OC/CTX 1; P ═ 0.0470) increased, and the ratio of cartilage synthesis/degradation (P2 CP/C2C; P ═ 0.0990) decreased with MTX.

Although both bone and cartilage synthesis/degradation are predicted to increase with MTX, bone and cartilage synthesis are negatively correlated based on correlation analysis (opposite slopes for osteocalcin (+) and P2CP (-) in table 6 and table 7). Similarly, Billinghurst et al (Am J Vet Res, 2004,65(2):143-50) reported a negative direction between bone and cartilage synthesis (e.g., osteocalcin reduction and P2CP increase) in foals with osteochondrosis.

As shown in table 7, for the urate-induced lameness model, correlation and regression analysis demonstrated that biomarkers were significantly correlated with lameness or gait score. Of the 8 biomarkers evaluated, five biomarkers were significantly correlated with P < 0.05; the two biomarkers are related by P < 0.10; only one biomarker was not relevant. Furthermore, the slope is in the expected direction: positive for cartilage and bone degradation markers, negative for bone and cartilage synthesis/degradation ratios, and mixed for synthetic markers (positive slope for osteocalcin; negative slope for P2 CP).

Force plate analysis is presented in fig. 4A-4D. These analyses also demonstrate that MTX has a beneficial effect on load bearing. For the urate-injected feet (right posterior, RR), higher weight bearing occurred at all time points after injection in MTX animals (fig. 4D; significantly higher at D +3 and D + 6). Higher weight bearing also occurred on the contralateral hind legs of the MTX animals (figure 4C; higher at D +1 and D +2 post injection). These findings indicate that lameness was less severe in MTX fed pigs, consistent with gait score results (figure 2; gait score was less severe for MTX fed pigs).

In summary, serum biomarkers of cartilage degradation (C2C, CTX2) increased and bone synthesis biomarkers (osteocalcin) changed in lameness pigs. These biomarkers can be used objectively to measure lameness in pigs. MTX administration improves bone and cartilage metabolism by increasing bone synthesis (osteocalcin) relative to degradation (osteocalcin/CTX 1 ratio) and cartilage synthesis (P2CP) relative to degradation (P2CP/CTX2 ratio). In summary, lameness increased cartilage degradation biomarkers and altered bone synthesis biomarkers, and administration of MTX increased bone and cartilage synthesis relative to degradation, thereby reducing lameness.