U.S. patent application number 17/831821 was filed with the patent office on 2022-09-22 for biomarkers for joint ailments and uses thereof.
The applicant listed for this patent is Novus International, Inc.. Invention is credited to Juxing Chen, Mercedes Vazquez-Anon, Karen Wedekind.
Application Number | 20220296640 17/831821 |
Document ID | / |
Family ID | 1000006390999 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220296640 |
Kind Code |
A1 |
Chen; Juxing ; et
al. |
September 22, 2022 |
BIOMARKERS FOR JOINT AILMENTS AND USES THEREOF
Abstract
The present invention relates to biomarkers that are associated
with joint disorders, and methods of using the biomarkers diagnose
joint ailments, monitor the progression of joint ailments,
determine when treatments is indicated, and monitor the efficiency
of treatment. Also provided are methods for treating joint
ailments, which comprise administering chelated trace minerals to
animals diagnosed with or predisposed to having joint ailments.
Inventors: |
Chen; Juxing; (Chesterfield,
MO) ; Wedekind; Karen; (St. Peters, MO) ;
Vazquez-Anon; Mercedes; (Chesterfield, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novus International, Inc. |
St. Charles |
MO |
US |
|
|
Family ID: |
1000006390999 |
Appl. No.: |
17/831821 |
Filed: |
June 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16816766 |
Mar 12, 2020 |
11376279 |
|
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17831821 |
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62817121 |
Mar 12, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/02 20180101;
G01N 2333/575 20130101; G01N 2333/78 20130101; G01N 2800/10
20130101; A61K 33/34 20130101; G01N 33/6893 20130101; A61K 33/32
20130101; A61K 31/198 20130101; A61K 33/30 20130101 |
International
Class: |
A61K 33/30 20060101
A61K033/30; G01N 33/68 20060101 G01N033/68; A61K 31/198 20060101
A61K031/198; A61K 33/34 20060101 A61K033/34; A61P 19/02 20060101
A61P019/02; A61K 33/32 20060101 A61K033/32 |
Claims
1. A method for treating or reducing the severity of lameness due
to inflammation in a pig, the method comprising: (a) determining
the level of a ratio of two biomarkers in a blood sample from the
pig, wherein the ratio of two biomarkers is selected from OC/CTX1,
P2CP/C2C, and P2CP/CTX2; (b) comparing the level of the ratio of
the pig to a non-lame pig having a gait score of about 0 on a 5
point scale to determine whether the pig is lame, wherein the pig
is lame if the level of one or more of the ratios is decreased
compared to the non-lame pig; (c) administering an effective amount
of metal chelate to the pig if the pig is determined to be lame,
wherein the metal chelate results in the at least one ratio
increasing and a reduction of the lameness.
2. The method of claim 1, wherein in step (b) the level of a single
biomarker selected from C2C, CTX1, CTX2, OC and P2CP may be
determined.
3. The method of claim 1, wherein step (b) comprises an
antibody-based detection method.
4. The method of claim 1, 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, hydroxy acid, organic acid, sugar
alcohol, protein, protein hydrolysate, polysaccharide, or
polynucleic acid.
5. The method of claim 4, wherein the at least one metal ion is
calcium, chromium, cobalt, copper, iron, magnesium, manganese,
nickel, potassium, sodium, zinc, or combination thereof.
6. The method of claim 4, wherein the at least one ligand is a
compound of Formula (I): ##STR00002## wherein: R.sup.1 is methyl or
ethyl; and n is 1 or 2.
7. The method of claim 6, wherein R.sup.1 is methyl and n is 2.
8. The method of claim 7, wherein the at least one metal ion is
copper, manganese, zinc, or combination thereof.
9. The method of claim 1, wherein the pig is at risk for developing
lameness due to inflammation.
10. The method of claim 1, wherein the pig is lame and has a gait
score of about 2 or more on a 5 point scale.
11. The method of claim 1, wherein the metal chelate reduces
symptoms of lameness compared to administering an inorganic mineral
to a comparable pig afflicted with lameness due to
inflammation.
12. A method for treating or preventing lameness due to
inflammation in a pig, the method comprising administering a metal
chelate to the pig, wherein the metal chelate reduces symptoms of
lameness compared to administering an inorganic mineral to a
comparable pig afflicted with lameness due to inflammation.
13. The method of claim 12, 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, hydroxy acid, organic acid, sugar
alcohol, protein, protein hydrolysate, polysaccharide, or
polynucleic acid.
14. The method of claim 13, wherein the at least one metal ion is
calcium, chromium, cobalt, copper, iron, magnesium, manganese,
nickel, potassium, sodium, zinc, or combination thereof.
15. The method of claim 13, wherein the at least one ligand is a
compound of Formula (I): ##STR00003## wherein: R.sup.1 is methyl or
ethyl; and n is 1 or 2.
16. The method of claim 15, wherein R.sup.1 is methyl and n is
2.
17. The method of claim 15, wherein the at least one metal ion is
copper, manganese, zinc, or combination thereof.
18. The method of claim 12, wherein the pig is at risk for
developing lameness due to inflammation.
19. The method of claim 12, wherein the pig has been diagnosed as
lame.
20. The method of claim 19, wherein the pig has a gait score of
about 2 or more on a 5 point scale.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 16/816,766, filed Mar. 12, 2020, which claims
the benefit of U.S. Provisional Application No. 62/817,121, filed
Mar. 12, 2019, which is hereby incorporated by reference in its
entirety.
FIELD
[0002] The present disclosure relates to biomarkers that are
associated with joint disorders and uses thereof.
BACKGROUND
[0003] Lameness has been identified as a welfare issue in all
livestock species that leads to reduction in productivity and
profitability of the farm. The incidence of locomotion disorders
has been associated with hoof and limb lesions, neurological
disorders, metabolic and infectious disorders and mechanical and
structural problems. The incidence of lameness can vary from 5 to
40% in sows and dairy cows, and has been associated with lower
reproduction performance, intake, longevity, and increased
mortality. After reproductive problems, lameness is the most common
reason for sows and dairy cows resulting in premature removal from
the herd. Causes of lameness have been mostly associated with
osteochondrosis in bone joints, however this is difficult to
identify in live animals. Visual gait lameness scores are commonly
used to identify lameness in production animals (pigs, cows and
chickens). However, this subjective scoring system lacks
sensitivity and consistency among scorers and leads to delayed
detection of lameness. Earlier detection of lameness improves the
opportunity for resolution with nutritional programs. Thus, there
is a need for accurate, objective methodologies that will enable
earlier identification of lameness. Furthermore, solutions are
needed for reducing severity and incidence of lameness such as
nutritional intervention.
SUMMARY
[0004] One aspect of the present disclosure provides a method for
treating or preventing a joint ailment 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 the
blood sample, the biomarker being chosen from osteocalcin,
C-terminal telopeptide of type I collagen (CTX-1), procollagen type
II C-terminal propeptide (P2CP), C-terminal telopeptide of type II
collagen (CTX-2), type II collagen (C2C), or combination thereof;
(c) performing an analysis of the level of the at least one
biomarker to determine whether the animal has or is predisposed to
having a joint ailment, wherein the analysis includes comparing the
level of the at least one biomarker in the blood sample to joint
ailment-positive and/or joint ailment-negative reference levels of
the at least one biomarker in order to determine if the animal has
or is predisposed to having a joint ailment; and (d) administering
an effective amount of a metal chelate to the animal if the animal
is determined to have or to be predisposed to having a joint
ailment.
[0005] Other aspects and iterations of the disclosure are described
in more detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 presents the P2CP concentrations from the four
treatments (Healthy ITM, Healthy MTX, Lame ITM, Lame MTX) in the
naturally occurring lameness model.
[0007] FIG. 2 presents a time-course of visual gait scores
following urate injection to induce acute lameness. Presented are
the gait scores for urate-injected animals fed MTX or ITM for two
months prior to the injection.
[0008] FIG. 3A presents a time-course of serum levels of C2C in
urate-injected animals. FIG. 3B plots a time-course of serum levels
of CTX2 in urate-injected animals. FIG. 3C presents a time-course
of serum levels of osteocalcin in urate-injected animals previously
fed MTX or ITM.
[0009] FIG. 4A presents the average weight borne by the front
contralateral leg of MTX and ITM animals over time. FIG. 4B shows
the average weight borne by the front ipsilateral leg of MTX and
ITM animals over time. FIG. 4C presents the average weight borne by
the rear contralateral leg of MTX and ITM animals over time. FIG.
4D shows the average weight borne by the urate-injected leg of MTX
and ITM animals over time.
DETAILED DESCRIPTION
[0010] The present disclosure provides biomarkers that may be used
as objective indicators of joint ailments in animals, e.g.,
livestock animals. The biomarkers may be used to diagnose joint
ailments, monitor the progression of joint ailments, determine when
treatment is indicated, and monitor the efficiency of
treatment.
(I) Biomarkers
[0011] One aspect of the present disclosure encompasses a panel of
serum biomarkers associated with joint ailments. The biomarkers are
involved with bone and cartilage synthesis and degradation. The
biomarkers may be used to distinguish healthy animals from animals
with joint ailments (e.g., lame animals), to monitor the
progression of joint ailments or joint diseases, or monitor the
efficacy of treatment.
[0012] One biomarker is osteocalcin. Also known as bone
gamma-carboxyglutamic acid-containing protein (BGLAP), osteocalcin
is a noncollagenous protein hormone found in bone and dentin.
Osteocalcin is a marker for bone synthesis.
[0013] Another biomarker is C-terminal telopeptide of type I
collagen or CTX-1, which is a marker for bone degradation or
turnover. Type I collagen accounts for about 90% of the organic
matrix of bone. CTX-1 relates to bone turnover because it is the
portion of the molecule that is cleaved by osteoclasts during bone
resorption. CTX1 is a marker for bone degradation.
[0014] Still another biomarker is procollagen type II C-terminal
propeptide or P2CP, which is a biomarker for cartilage synthesis.
Type II collagen is the major organic constituent of cartilage.
P2CP is a marker for cartilage synthesis.
[0015] A further biomarker is C-terminal telopeptide of type II
collagen or CTX-2, which is a biomarker for cartilage degradation.
Following the degradation of cartilage, fragments of CTX-2 are
released into circulation. CTX2 is a marker for cartilage
degradation.
[0016] Yet another biomarker is type II collagen or C2C, which is a
biomarker for cartilage degradation.
[0017] Upon the determination of levels of the joint ailment
biomarkers in healthy animals and animals with joint ailments
(either naturally occurring joint ailments or chemically induced
joint ailments), these levels may be correlated with standard
indicators of lameness or joint ailments to establish biomarker
reference levels in healthy animals and animal with joint
ailments/lameness. The ratio of synthesis/degradation (P2CP/C2C,
P2CP/CTX2, osteocalcin/CTX1) may be associated with lameness.
[0018] Standard indicators of lameness or joint ailments include
visual gait scores and force plate tests. Gait or locomotion of
animals is observed and scored. Gait may be scored on a 5-point
scale, ranging from "0" for animals with a normal gait to "4" for
animals that are reluctant to walk and bear weight on one or more
legs. Force plates may be used to measure the weight of each
forelimb, wherein differences suggest that the animal is favoring
one limb. By correlating biomarker levels with visual lames/joint
ailments standards, reference levels for healthy and lame animals
may be established.
(II) Methods for Diagnosing Joint Ailments and/or Monitoring
Progression of Joint Ailments
[0019] Another aspect of the present disclosure provides methods
for diagnosing joint ailments in animals and/or monitoring the
progression of joint ailments in animals. For example, the methods
may be used to distinguish between healthy animals and animals with
joint ailments, and if an animal has a joint ailment, the
progression of the ailment may be monitored. The methods comprise
(a) collecting a blood sample from the animal, (b) determining the
level of at least one of the biomarkers disclosed herein that is
present in the blood sample; and (c) performing an analysis of the
level of the at least one biomarker to determine whether the animal
has or is predisposed to having a joint ailment, wherein the
analysis includes comparing the levels of the at least one
biomarker in the blood sample to joint ailment-positive and/or
joint ailment-negative reference levels of the at least one
biomarker in order to determine if the animal has or is predisposed
to having a joint ailment and/or monitoring the progression of
joint ailments in animals known to have joint problems.
Joint Ailments
[0020] As used herein, "joint ailments" refer to diseases or
disorders of joints or joint tissues. Joint tissues include bone
and connective tissue, i.e., cartilage, tendons, and ligaments.
Joint ailments include arthritis or osteoarthritis, which are
degenerative joint diseases or disorders due to the gradual
deterioration of the articular cartilage within one or more the
joints. Arthritis is a general description for any condition that
causes inflammation in the joints. Rheumatoid arthritis is a
chronic inflammatory disorder of the joints. Other joint ailments
include osteochondrosis, gouty arthritis, bursitis, tenosynovitis,
epicondylitis, synovitis, ankylosing spondylitis, Sjogren's
syndrome, psoriatic arthritis, and Lyme disease. Some joint
disorders may arise due to hoof or foot pad diseases or
disorders.
Step (a)
[0021] 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, a method of
collecting blood comprises accessing the blood using a
skin-piercing element and collecting the blood therein into some
type of a collection device. Accessing the 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 kind of mechanical or vacuum means in
conjunction with the skin-piercing element. Generally speaking, the
sample collection method preferably maintains the integrity of the
sample such that abundance values for each molecular feature can be
accurately measured. A blood sample may be a whole blood sample, a
plasma sample, or a serum sample.
Step (b)
[0022] The second step of the method comprises determining the
level of at least one biomarker present in the blood sample. A
variety of method may be used to determine the level or
concentration of the biomarker(s). The biomarker may be detected
and quantified using an antibody-based detection method. For
example, the level of the biomarker may be determined using an
enzyme-linked immunosorbent assay (ELISA). The ELISA may be a
direct ELISA, a sandwich ELISA, a competitive ELISA, or a reverse
ELISA. The detection method may be optical (i.e., colorimetric or
fluorometric) or electrochemical. In specific embodiments, the
biomarker(s) may be detected using a sandwich ELISA with
colorimetric detection.
[0023] On other embodiments, the antibody-based detection method
may comprise protein immunoprecipitation, immunoelectrophoresis,
Western blotting, or protein immunostaining. In still other
embodiments, the biomarker(s) levels may be quantitated using high
performance liquid chromatography (HPLC) or liquid
chromatography--mass spectrometry (LC/MS).
Step (c)
[0024] The next step of the method comprises performing an analysis
of the level of the at least one biomarker to determine whether the
animal is healthy, is predisposed or likely to develop a joint
ailment, or has a joint ailment. The analysis comprises comparing
the level of the at least one biomarker in the blood sample to
joint ailment-positive and/or joint ailment-negative reference
levels of the at least one biomarker. If the level of the at least
one biomarker falls within the range of joint ailment-negative
reference levels, then the animal is healthy and does not have a
joint ailment. If the level of the at least one biomarker falls
within the range of joint ailment-positive reference levels, then
the animal has a joint ailment. The severity of the joint ailment
may be estimated based upon the level of the at least one
biomarker. The progression of the joint ailment may be monitored by
comparing the level of the at least one biomarker over time.
[0025] In some embodiments, the levels of two biomarkers may be
determined. For example, the levels of osteocalcin and CTX1 may be
determined and/or the ratio of osteocalcin/CTX1 may 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 additional
embodiments, the levels of four biomarkers may be determined. In
still other embodiments, the levels of all five biomarkers may be
determined.
Animals
[0026] Suitable animals include, but are not limited to, livestock
animals, companion animals, lab animals, and zoological animals. In
specific embodiments, the animal may be a livestock animal.
Non-limiting examples of suitable livestock animals may include
pigs, cows, poultry, goats, sheep, llamas, alpacas, aquatic
animals, etc. In exemplary embodiments, the animal may be a pig,
e.g., a sow. In other embodiments, the animal may be a dairy
cow.
[0027] In other embodiments, the animal may be a companion animal.
Non-limiting examples of companion animals may 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
a zoological animal. As used herein, a "zoological animal" refers
to an animal that may be found in a zoo. Such animals may include
non-human primates, large cats, wolves, bears, hippos, kangaroos,
etc. In still other embodiments, the animal may be a laboratory
animal. Non-limiting examples of a laboratory animal may include
rodents, canines, felines, and non-human primates.
(III) Methods for Treating or Preventing Joint Ailments
[0028] Another aspect of the present disclosure provides methods
for treating or preventing joint aliments in animals. The methods
may also be used to monitor the efficacy of the treatment method,
wherein the treatment method may be modified accordingly. The
methods comprise (a) collecting a blood sample from the animal, (b)
determining the level of at least one of the biomarkers disclosed
herein that is present in the blood sample; (c) performing an
analysis of the level of the at least one biomarker to determine
whether the animal has or is predisposed to having a joint ailment,
wherein the analysis includes comparing the levels of the at least
one biomarker in the blood sample to joint ailment-positive and/or
joint ailment-negative reference levels of the at least one
biomarker in order to determine if the animal has or is predisposed
to having a joint ailment; and (d) administering an effective
amount of a metal chelate to the animal if the animal is determined
to have or to be predisposed to having a joint ailment.
[0029] Steps (a), (b), and (c) of the method are as described above
in section (II), as are suitable joint ailments and animals.
Step (d)
[0030] If, at step (c), the animal is determined to have or to be
predisposed to having a joint ailment, the next step comprises
administering an effective amount of a metal chelate to the
animal.
[0031] The metal chelate comprises at least one ligand and at least
one metal ion. The ligand may be an amino acid, a hydroxy acid
(e.g., alpha hydroxy acid), an organic acid, a sugar alcohol,
protein, protein hydrolysate (e.g., soy protein hydrolysate),
polysaccharide, or polynucleic acid.
[0032] In some embodiments, the ligand may be an amino acid.
Suitable amino acid derivatives include alanate, arginate,
asparaginate, aspartate, cysteinate, glutaminate, glutamate,
histidinate, homocysteinate, isoleucinate, lysinate, methionate,
phenylalinate, prolinate, serinate, threonate, typtophanate,
tyrosinate, and valinate.
[0033] In other embodiments, the ligand may be an organic acid.
Non-limiting examples of suitable organic acid moieties include
adipate, ascorbate, caprylate, citrate, fulvate, furmarate,
glucoheptonate, gluconate, glutarate, glycerophosphate, humate,
lactate, ketoglutarate, malate, malonate, orotate, oxlate,
pantothenate, picolinate, pidolate, sebacate, succinate, and
tartrate.
[0034] In still other embodiments, the ligand may be a sugar
alcohol. Suitable sugar alcohols include, without limit, sorbitol,
mannitol, xylitol, lactitol, isomalt, maltitol, erythritol, and
hydrogenated starch hydrolysates (HSH).
[0035] In specific embodiments, the ligand is a compound of Formula
(I).
##STR00001##
wherein R.sup.1 is methyl or ethyl and n is 1 or 2. In exemplary
embodiments, R.sup.1 is methyl and n is 2 and the compound of
Formula (I) is methionine hydroxy analog (or
2-hydroxy-4-(thiomethyl)butanoic acid, HMTBA).
[0036] 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
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 combination thereof.
In specific embodiments, the at least one metal ion may be copper,
manganese, zinc, or combination thereof.
[0037] The ratio of the at least one ligand and the at least one
metal ion may vary in the metal chelate. For example, the ratio of
ligand to metal may range from 1:1 to about 3:1 or higher. In
embodiments in which the metal ion is divalent, the ratio of ligand
to metal may be 2:1.
[0038] In particular embodiments, the metal chelate may comprise
methionine hydroxy analog copper (i.e., MHA-Cu to
(HMTBA).sub.2-Cu), methionine hydroxy analog manganese (i.e.,
MHA-Mn or (HMTBA).sub.2-Mn), methionine hydroxy analog zinc (i.e.,
MHA-Zn or (HMTBA).sub.2-Zn), or a combination of any or all of the
foregoing (which are available from Novus International, Inc.,
under the tradename MINTREX.RTM.).
[0039] The effective amount of the metal chelate that is
administered to the animal can and will vary, depending for example
upon the type and age of the animal and/or the severity of the
joint ailment. Persons skilled in the art can readily determine the
appropriate amount.
[0040] In general, the metal chelate is included in the feed
rations of the animal. Feed rations typically are formulated to
meet the nutrient and energy demands of a particular animal. The
nutrient and energy content of many common animal feed ingredients
have been measured and are available to the public. The National
Research Council has published books that contain tables of common
feed ingredients and their respective measured nutrient and energy
content. Additionally, estimates of nutrient and maintenance energy
requirements are provided for animals of different life stages,
age, sex, or use. This information can be utilized by one skilled
in the art to estimate the nutritional and maintenance energy
requirements of animal and determine the nutrient and energy
content of animal feed ingredients.
EXAMPLES
[0041] The following examples illustrate various embodiments of the
present disclosure.
Example 1: Protocol Description
[0042] Two different models of lameness (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 scoring. The study
design was a 2.times.2 factorial arrangement consisting of two
dietary treatments (chelated trace minerals vs inorganic trace
minerals) and two populations of pigs (lame vs healthy non-lame).
The chelated trace minerals (MTX) comprised methionine hydroxy
analog (MHA) chelate (i.e., MHA-Cu, MHA-Mn, and/or MHA-Zn) and the
inorganic trace minerals (ITM) comprised mineral sulfates.
[0043] Four groups of pigs (8 lame/8 non-lame per group) for a
total of 32 lame/32 non-lame/64 total were fed the dietary
treatments for a period of two months. For the naturally-occurring
portion of the trial (Example 2), lameness measurements were taken
at baseline (d0), month 1 (d28) and month 2 (d53). At the end of
the 2-month period, only the healthy animals (n=8 per group; 4 on
MTX and 4 on ITM) were then injected with sodium urate crystals (10
mg/mL and 0.2 mL injection volume) into the right rear distal
interphalangeal joint (Example 3).
[0044] Most methods used for gait scoring are based on uneven or
asymmetrical weight-bearing. As shown in Table 1, a 5-point scale
(0-4) was used, with 4 being most severe.
TABLE-US-00001 TABLE 1 Scoring System for Subjective Lameness
Evaluation Lameness Score Description 0 Animal moves freely and
used all 4 limbs and feet evenly 1 Animal shows weight-shifting
activities away from affected limb upon standing but show no
lameness of limping when walking 2 Animal obviously shifts weight
away from affected limb when standing and shows limping or adaptive
behavior when walking (e.g., head bob, quickened step on affected
limb) 3 Animal is reluctant to stand and/or walk, obvious limp and
adaptive behaviors when walking 4 Animal is non-weight bearing on
the affected limb when either standing or walking
[0045] The force-plate analysis quantifies the amount of force each
limb applies to four separate loading cells. Two data points per
second were captured over a 2-minute time duration. A lame animal
typically bears less weight on the limb that is painful or
structurally unsound.
[0046] Thermal imaging measures the heat emitted from a body
surface as infrared radiation. Studies of clinical disease have
shown that a difference greater than 1.degree. C. between two of
the same anatomical regions indicate an abnormality such as
inflammation. The high degree of symmetry between the left and
right side of an animal is a valuable asset in the diagnosis of a
unilateral problem associated with lameness. Thus, these objective
measures of lameness should correlate to the visual gait score,
which is also based on asymmetrical weight-bearing.
[0047] All statistical analyses were performed with SAS using the
pig as the experimental unit. All lameness measurements were
analyzed using a two-way analysis of variance (ANOVA) to test the
main effects of dietary treatment and healthy vs lame plus their
interactions; differences in front vs rear limbs were also
compared. In addition, data were also evaluated using a mixed model
including day, lameness, dietary treatment, bodyweight, group and
gender. Day was included as a repeated measure using an
autoregressive covariance structure, and data collected the day
before the start of the study were included as covariates (for
force-plate data only).
[0048] Pearson correlation coefficients and regression analyses
were used to explore the relationship between objective
measurements vs the gait score.
Example 2. Biomarker Levels in Healthy and Naturally-Occurring Lame
Animals
[0049] Serum biomarkers were measured at day 0, day 25 and day 53
(2 months) in healthy (H) and lame (L) animals (n=48). The levels
of serum CTX1, osteocalcin (OC), C2C, P2CP, and CTX2 were measured
by enzyme-linked immunosorbent assay (ELISA) according to the
procedures described in the commercial kits (e.g., MyBiosource).
The results are presented in Table 2.
TABLE-US-00002 TABLE 2 Biomarker Levels in Healthy and
Naturally-Occurring Lame Animals P2CP/C2C OC/CTX1 P2CP/CTX2 ratio
ratio ratio CTX1 OC P2CP CTX2 C2C ITM 1.32 15.2 1.08 15.45 161.5
7.28 7.41 14.09 MTX 1.04 15.2 1.27 16.58 163.2 8.28 6.95 14.63 SEM
0.14 0.98 0.06 0.54 3.70 0.28 0.24 0.47 Group <.0001 <.0001
<.0001 <.0001 <.0001 <.0001 <.0001 <.0001 Gender
0.5349 0.7119 0.0007 0.1197 0.9446 0.0047 0.0020 0.0330 H or L
0.0890 0.8477 0.0908 0.0073 0.8430 0.2020 0.0002 0.0176 Trt.sup.1
0.1686 0.9498 0.0267 0.1398 0.7454 0.0114 0.1694 0.4124 H or L*Trt
0.2736 0.4433 0.1480 0.1134 0.1397 0.9727 0.7631 0.5731 Day 0.3520
0.0773 0.7891 <.0001 0.2912 0.9771 0.4635 0.4111 Trt*day 0.4409
0.5765 0.4150 0.3543 0.9514 0.8792 0.3368 0.2474 P2CP/CTX2 OC/CTX1
H or L ratio CTX2 C2C Day ratio CTX1 Healthy (H) 1.25 6.53 13.57 0
13.0 18.3 Lame (L) 1.11 7.82 15.15 28 16.3 14.1 SEM 0.06 0.24 0.47
56 16.3 15.5 % delta 11.2 16.5 10.4 SEM 1.2 0.66 .sup.1Treatment
(ITM or MTX).
[0050] These results reveal that the biomarkers were able to
distinguish between healthy vs lame, as well as demonstrate
beneficial effects of MTX. Cartilage degradative markers, CTX2
(P=0.0002) and C2C (P=0.0176) were elevated and the ratio of
cartilage synthesis/degradation (P2CP/CTX2; P=0.0908) was decreased
in lame animals (Table 2). Dietary treatment differences were also
observed: cartilage synthesis biomarker (P2CP, P=0.0114) and the
ratio of cartilage synthesis/degradation (P2CP/CTX2, P=.0267) were
increased with MTX (Table 2). As shown in FIG. 1, there was not a
diet (ITM vs MTX) * healthy vs lame interaction (P=0.9727). This
indicates that MTX treatment is increasing cartilage synthesis in
both healthy and lame pigs and may have preventative joint health
effects.
Example 3: Urate-Induced Lameness
[0051] At the end of two months, sodium urate crystals (10 mg/mL
and 0.2 mL injection volume) were injected into the right rear
distal interphalangeal joint of healthy pigs from each of the four
groups. Thus, a total of 16 pigs for MTX, and 16 for ITM were
evaluated during the urate-induced portion of the trial. Data
collection, relative to time of urate injection, occurred at
baseline (d -1/d 53), 6 and 12 hr (d0/d54), 24 hr (d1/55), 48 hr
(d2/56), 72 hr (d3/57) and 144 hr (d6/60) post-injection.
[0052] As shown in FIG. 2, pigs fed MTX had lower gait scores (Main
effect of diet: P=0.0057). In addition, gait scores at peak
lameness (12hr post-urate injection P=0.0244, 48hr post-urate
injection P=0.0155) were lower in pigs fed MTX. Since the pigs had
been on the dietary treatments (MTX vs ITM) for 2 months prior to
urate-injections, these findings suggest that MTX supplementation
lowers the severity of urate-induced lameness.
[0053] The time-course for the serum biomarkers after urate
administration is shown in FIG. 3A-3C. Biomarker changes closely
paralleled the changes in gait score. For example, cartilage
degradative biomarkers (C2C and CTX2), similar to gait score,
peaked 12 hr post-urate injection (FIG. 3A, 3B), bone synthesis
biomarker (osteocalcin) peaked between 12 and 48 hr post-urate
injection (FIG. 3C). Similar to gait scores, after 72 hr (if not
sooner) these three biomarkers (C2C, CTX2, osteocalcin) returned to
baseline or plateaued relative to initial concentrations.
[0054] Tables 3-5 present the biomarker levels after urate
administration (without covariate). There were numerical or
significant effects of time (Hr) for C2C (P=0.0853), CTX2
(P=0.1014), and osteocalcin (OC, P=0.0011) as indicated in FIG.
3A-3C. Beneficial effects of MTX were also observed; bone synthesis
biomarker (OC, P=0.0700) and the ratio of bone
synthesis/degradation (OC/CTX1; P=0.0470) were increased and the
ratio of cartilage synthesis/degradation (P2CP/C2C; P=0.0990) was
decreased with MTX.
TABLE-US-00003 TABLE 3 Biomarker Levels as Affected by
Urate-induced Lameness P2CP/C2C P2CP/CTX2 OC/CTX1 ratio ratio ratio
C2C CTX2 P2CP OC CTX1 ITM 1.67 1.11 11.13 9.72 11.10 6.67 139.1
18.6 MTX 0.89 1.17 13.70 10.33 11.42 6.77 145.3 18.9 SEM 0.34 0.06
0.92 0.42 0.36 0.17 2.5 0.57 Grp <0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Gender
0.5818 0.0835 0.4405 0.0053 <0.0001 0.0011 0.9614 0.6233 Trt
0.0990 0.4955 0.0470 0.2960 0.5168 0.6921 0.0700 0.6233 Hr 0.7260
0.1063 0.3021 0.0853 0.1014 0.3398 0.0011 0.3205 Hr*Trt 0.6260
0.426 0.3950 0.7466 0.9413 0.9822 0.8181 0.8439
TABLE-US-00004 TABLE 4 Time Course of Select Biomarkers Hour C2C
CTX2 OC OC (ITM) OC (MTX) 0 9.73 11.17 139.1 133 145.2 6 10.15
12.27 137.5 133.0 142 12 12.84 13.06 147.2 146.3 148.2 24 9.48
10.93 153.0 152.2 153.8 48 9.23 10.49 151.2 143.2 159.1 72 9.58
10.39 136.5 135.4 137.7 144 9.15 10.52 130.95 130.3 131.6 SEM 0.8
0.59 4.0 4.0 4.0 P value 0.0853 0.1014 0.0011
TABLE-US-00005 TABLE 5 Gender and Biomarker Levels P2CP/CTX2 Gender
C2C CTX2 P2CP ratio Barrow 10.8 12.6 7.13 1.07 Gilt 9.2 9.6 6.31
1.21 SEM 0.42 0.36 0.17 0.06 P value 0.0053 <0.0001 0.0011
0.0835
[0055] Although it was predicted that both bone and cartilage
synthesis/degradation would increase with MTX, based on correlation
analyses, bone and cartilage syntheses were negatively correlated
(Table 6 and opposite slopes for osteocalcin (+) and P2CP (-) in
Table 7). Similarly, a negative direction between bone and
cartilage synthesis was reported by Billinghurst et al. (Am J Vet
Res, 2004,65(2): 143-50) in foals with osteochondrosis (e.g.,
decreased osteocalcin and increased P2CP).
TABLE-US-00006 TABLE 6 P2CP is Negatively Correlated to Osteocalcin
Bone Cartilage synthesis synthesis Correlation P- Model marker
marker coefficient value Urate-induced Osteocalcin P2CP -0.2898
0.0007 Naturally- Osteocalcin P2CP -0.3813 0.0116 occurring
TABLE-US-00007 TABLE 7 Biomarkers are Correlated to Gait Score
Expected Marker Direction Slope P-value Degradative CTX2 .uparw.
+0.9 NS C2C .uparw. +1.4 0.0072 CTXI .uparw. +5.9 <0.0001
Synthesis P2CP .uparw..dwnarw. -0.38 0.0914 Osteocalcin
.uparw..dwnarw. +26.1 0.0070 Ratio of Synthesis/ Degradative
P2CP/CTX2 .dwnarw. -0.27 0.0010 P2CP/C2C .dwnarw. -0.87 0.0716
Osteocalcin/CTXI .dwnarw. -4.8 0.0138
[0056] As shown in Table 7, for the urate-induced lameness model,
correlation and regression analyses demonstrated that biomarkers
were significantly correlated to lameness or gait score. Out of the
8 biomarkers evaluated, five were significantly correlated at
P<0.05; two at P<0.10; only one was not significant.
Furthermore, the slopes were in the expected direction: positive
for cartilage and bone degradative markers, negative for the ratio
of synthesis/degradation in bone and cartilage and mixed for
synthetic markers (positive slope for osteocalcin; negative slope
for P2CP).
[0057] Force-plate analyses are presented in FIG. 4A-4D. These
analyses also demonstrated beneficial effects of MTX on
weight-bearing. For the urate-injected foot (right rear, RR),
higher weight-bearing occurred at all timepoints post-injection in
MTX animals (FIG. 4D; significantly higher at D+3 and D+6). Higher
weight-bearing also occurred on the contralateral rear leg in MTX
animals (FIG. 4C; higher at D+1 and D+2 post-injection). These
findings suggest that the lameness was less severe in pigs fed MTX,
which agreed with the gait score findings (FIG. 2; gait score was
less severe for MTX-fed pigs).
[0058] Taken together, serum biomarkers for cartilage degradation
(C2C, CTX2) were increased and bone synthesis biomarker
(osteocalcin) were altered in lame pigs. Those biomarkers can be
used objectively to measure lameness in pigs. Feeding MTX improved
metabolism of bone and cartilage by increasing both bone synthesis
(osteocalcin) over degradation (osteocalcin/CTX1 ratio) and
cartilage synthesis (P2CP) over degradation (P2CP/CTX2 ratio). In
summary, lameness increased cartilage degradation biomarkers and
altered bone synthesis biomarkers, feeding MTX improved bone and
cartilage synthesis over degradation, therefore reducing
lameness.
* * * * *