Effect of an Oral Supplement Containing Curcumin Extract (Longvida™) on Lameness due to Osteoarthritis or Degenerative Disease and Gastric Ulcer Scores
 
 
W.F. Young Study Report: 001 January 29, 2019
 
Sponsored by:
W.F. Young, Inc.
302 Benton Drive
East Longmeadow, MA 01028
 
 
Phone: (413) 486-1486
Fax: (413) 526-8990
Email : acairy@wfyoung.com
 
 
 
Principal             _______________________________        ________________
Investigator        Frank M. Andrews, DVM, MS, DACVIM                    Date:
 
 
 
 
Study Monitor   _______________________________        ________________
                                    Amy Cairy                                                                Date:
EXECUTIVE SUMMARY
With the current emphasis on natural products “Clean Sport” for easing pain and improving lameness, an oral herbal supplement, Absorbine® Bute-Less® Performance (BLP, W.F. Young, Inc. East Longmeadow, MA) was fed to horses with lameness due to osteoarthritis. This supplement contains essential vitamins, herbs and extracts, including Curcumin Extract (Longvida™, 1.060 gm/2 oz.), a plant derivative.  A two-period prospective randomized crossover study utilized 10 Thoroughbreds 3 to 20 years of age (mean age: 8.6 years) with diagnosed lameness (AAEP Scale 2-4) due to chronic osteoarthritis of one forelimb (fetlock, pastern, carpus). Lameness was confirmed with a lameness examination, radiographs of the affected limb and when indicated the affected joint was injected with lidocaine to confirm improvement in lameness. A complete blood count (CBC) and biochemical panel were performed before (Day -1) and after the trial (Day 31) to determine health and safety status of treatment. Treatment consisted of 2 ounces (56.7 gm) of BLP top-dressed on grain (1.0 Kg, Omelene 100®, Purina Animal Nutrition, West Shoreview, MN) once daily in the AM for 30 days. Control horses received only grain without supplement added. On Day -1 (before treatment), Day 15 and Day 30, subjective lameness data (lameness examination, joint range of motion, pain-response to flexion), lameness locator, and force plate (peak vertical force [PVF], impulse force [IMP]) data were collected on each horse by a masked (to treatment) clinician (LMR). The clinician was board-certified (Diplomate American College of Veterinary Surgeons) specialist. In addition, on Days 1 and 14 of the study, Blood samples were drawn before the morning BLP dose and then 15, 30, 45, 60, 75, 90, 105, 120, 150, 180 minutes, then hourly for the next 6 hours (15 total blood samples) following dosing. Plasma was collected and frozen at -80°C for pharmacokinetics analysis. Gastroscopy examination was performed on each horse before treatment and on Day 31 to record ulcers scores and stomach health. Daily clinical observations were performed on each horse.
Statistical Analysis: Mean ± SEM was used to summarize data when appropriate. Clinical pathology data (CBC and Biochemical Panel) data was summarized and included in the raw data. Data analyses were performed using SAS 9.4 software (SAS Institute Inc., Cary, NC). A repeated measure analysis of variance (ANOVA) with a mixed effect model was used on the variables measured. Treatment (curcumin or control), day and their interactions were entered as the fixed effects. Each animal was entered as the random effect. Treatment successful (TS) rate was computed with 95% confidence interval. A one-sided binomial test of statistical significance was used to exam if the rate is greater than 50%. A chi square test was used to check the association between the success rate (TS and Treatment Failure). In addition, a logistic regression was used to study the association between treatment success rates (TS), period and the treatment. Significance was considered when P<0.05.
Results: All horses readily ate the supplement and there were no adverse responses observed. The CBC and serum chemistry analysis on whole blood and plasma, respectively, showed no clinically significant values. Subjective lameness examinations, lameness locator values and force plate analysis values showed no treatment by day effect. However, in the first cohort of horses (n=6) treated vs. untreated, overall, peak vertical force (PVF, indicator of weight bearing) was significantly (P=0.0024) higher in the lame limb in curcumin-fed horses curcumin-fed horses and that trend continued in the overall study (P=0.0563). In addition, pain response upon palpation was significantly (P=0.025) lower in the BLP-treated horses by Day 30. When all horses (n=10) were evaluated, 6/10 curcumin-treated horses showed a significant (≥ 5% increase, range: 5-33%) in PVF in the lame limb as measured by the force plate, indicating lameness improvement. In all but 1 of those horses, lameness improved by at ≥ 1 AAEP lameness grade by 30 days of BLP-treatment. Whereas, in the untreated control horses, only 3/10 horses significantly improved by 30 days and 2/10 improved by ≥ 1 AAEP grade.  In addition, all of those horses improved during period 2 of the study, suggesting that BLP might have contributed to a carry-over effect. When improvement in PVF in curcumin-treated horses was compared to untreated controls, there was a significant association (P=.0053) in period 1 of the study and a strong association overall (P=0.1031) in lameness improvement and a logistic regression showed that treatment success (TS) was 2.19X more likely in the BLP-treated horses when compared to the untreated controls.  On gastroscopy examination, nonglandular gastric ulcer scores (EGUS, NGN and NGS) were significantly (P=0.0195; P=0.0100; P=0.0025, respectively) lower in both groups by day 30 of the trial. Glandular ulcer scores were not changed, however glandular ulcers were observed in only a few horses. Plasma free curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and their phase-2metabolites (curcumin-O-sulfate, curcumin-O-glucuronide) were measured and validated using FDA criteria in equine plasma using LC-ESI–MS/MS method. Plasma samples in two horses showed the presence of curcumin-O-sulfate concentrations after the 14th dose. Plasma from horses in the study was frozen at -80 C and will be analysis later for pharmacokinetics.
Discussion: Absorbine® Bute-Less® Performance supplement top-dressed on feed was safe and showed no clinically significant changes clinical observations or CBC or biochemical values after 30 days of treatment. There was an overall treatment effect and less pain on palpation of the affected joint in the first cohort of horses (n=6) receiving BLP.  Although, there was no statistically significant different was noted in other subjective parameters of lameness, peak vertical force (PVF), an indicator of weight-bearing, in the lame limb significantly improved in 6/10 (60%) curcumin-treated horses, compared to 3/10 (30%) same horses when untreated. In addition there was a strong association (P=0.0632; OR=2.19) in lameness improvement in BLP-treated horses.  In other words, lameness in the BLP-treated horses was 2.19X more likely to improve than in the untreated horses by day 30 of treatment. In addition, pain on palpation of the affected limb was improved in BLP-treated horses. Furthermore, fewer ulcers were seen in both groups after 30 days of feeding. Furthermore, curcumin and its metabolites were detected in plasma of the two horses measured by day 14 of treatment showing assay validation and bioavailability of the curcumin.
Conclusions: Absorbine® Bute-Less® Performance containing a proprietary blend of herbs, vitamins, and curcumin (Longvida™), fed to horses, achieved blood levels, reduced pain and improved weight bearing in horses with lameness due to osteoarthritis after 30 days of feeding, while maintaining stomach health. 
TABLE OF CONTENTS
1.0 STUDY TITLE.. 7
Effect of an Oral Supplement Containing Curcumin Extract (Longvida) on Lameness due to Osteoarthritis or Degenerative Disease and Gastric Ulcer Scores. 7
2.0 PROTOCOL. 7
2.1 Protocol number 7
2.2 Type of protocol 7
3.0 STUDY CONTACTS.. 7
3.1 Sponsor 7
3.2 Sponsor’s Representative. 7
3.3 Investigators. 7
3.3.1 Principal Investigator 7
3.3.2 Co- Investigator 8
3.3.3 Co- Investigator 8
3.4 Study Coordinator 8
3.5 Study Monitor 8
3.6 Quality Assurance Unit 8
3.7 Statistician.. 9
4.0 STUDY LOCATION.. 9
5.0 STUDY OBJECTIVES.. 9
5.1 Clinical efficacy and safety. 9
6.0 REGULATORY COMPLIANCE.. 9
7.0 STUDY RATIONALE.. 9
7.1 Test Article Justification.. 10
7.2 Dose Justification. 10
7.3 Clinical Efficacy. 10
8.0 STUDY SCHEDULE.. 10
8.1 Duration of in-life experimental phase. 10
8.2 Schedule of events for each period. 10
8.3 Clinical efficacy, PK and safety study design summary. 12
9.0 STUDY DESIGN.. 12
9.1 Study Design.. 13
9.2 Treatment group. 13
9.3 Blocking factors. 13
9.4 Randomization Procedures. 13
10.0 STUDY PROCEDURES.. 13
10.1 Test Animals. 13
10.1.1 Description.. 13
10.1.2 Number of animals. 14
10.1.3 Source of animals. 14
10.1.4 Identification method. 14
10.2 Test Animal Criteria. 14
10.2.1 Screening procedures. 14
10.2.2 Inclusion criteria. 14
10.2.3 Exclusion criteria. 15
10.2.4 Post – inclusion removal criteria. 15
10.3 Acclimation of Test Animals. 15
10.3.1 Duration.. 15
10.3.2 Medication and/or vaccination during acclimation period. 15
10.3.3 Baseline data collected prior to initiating study. 15
10.4 Masking of study. 16
10.5 Measures to ensure compliance with study protocol 16
10.6 Measures to ensure compliance with Good Clinical Practices. 16
11.0 ANIMAL MANAGEMENT and HOUSING.. 16
11.1 Housing. 17
11.2 Concomitant therapy and/or medications. 17
11.3 Feeding Regimen.. 17
11.4 Water 17
12.0 TEST ARTICLE & CONTROL. 17
12.1 Test article identification. 17
12.1.1 Chemical name. 17
12.1.2 Trade name or supplement 17
12.1.3 Active/inactive ingredients. 17
12.1.4 Dosage form.. 17
12.1.5 Dose to be tested. 17
12.1.6 Manufacturing site. 17
12.1.7 Packaging. 18
12.1.8 Lot number 18
12.1.9 Certificate of analysis. 18
12.1.10 Test article storage during study. 18
12.2 Control product 18
13.0 TREATMENTS & PROCEDURES.. 18
13.1 Dose. 18
13.1.1 Dose administered. 18
13.1.2 Dose preparation.. 18
13.1.3 Method of Administration: 18
13.1.4 Timing. 18
13.2 Daily observations. 19
13.3 Clinical safety. 19
14.0 ASSESSMENT of TREATMENT and CLINICAL SUCCESS.. 19
14.1 Effects to be achieved. 19
14.2 Study procedures. 19
14.2.1 Physical examination procedure. 19
14.2.2 Venipuncture for CBC and serum chemistries. 19
14.2.3 Body weights. 19
14.2.4 Lameness Evaluation Protocol 19
14.2.5 Evaluation of improvement 21
14.2.6 Clinical safety. 21
15.0 DISPOSTION OF STUDY ANIMALS, TEST ARTICLE & CONTROL. 21
15.1 Care to be administered to animals removed from the study. 21
15.2 Disposition of remaining test article and control 21
15.3 Disposition of study animals. 21
16.0  STATISTICAL METHODS.. 21
16.1 Sample size. 21
16.2 Statistical analysis. 21
17.0  COLLECTION AND RETENTION OF SOURCE DOCUMENTATION.. 22
18.0  ADVERSE EVENTS.. 22
18.1 Monitoring for adverse events. 22
18.2 Recording adverse events. 22
18.3 Reporting adverse events. 22
18.4 Necropsy. 22
19.0 ANIMAL WELFARE.. 22
21.0                  RESULTS.. 23
RESULTS (Figures and Tables) 25
20.0 REFERENCES.. 34
21.0 APPENDICES.. 34
APPENDIX 1:  Sample Certificate of Analysis. 36
 

1.0 STUDY TITLE

Effect of an Oral Supplement Containing Curcumin Extract (Longvida®) on Lameness due to Osteoarthritis or Degenerative Disease and Gastric Ulcer Scores

 

2.0 PROTOCOL

 

2.1 Protocol number

Sponsor’s protocol # :  001
LSU IACUC #: 16-017

           

2.2 Type of protocol

Site specific non-pivotal pilot efficacy, safety and pharmacokinetic study
 

3.0 STUDY CONTACTS

 

3.1 Sponsor

1. W.F. Young Company

302 Benton Drive
East Longmeadow, MA 01028
 

3.2 Sponsor’s Representative

Amy Cairy

1. W. F. Young Company

302 Benton Drive
East Longmeadow, MA 01028
Office: 413-486-1486
Fax: 413-526-8990
acairy@wfyoung.com
              

3.3 Investigators

3.3.1 Principal Investigator

Frank M. Andrews, DVM, MS, DACVIM (LAIM)
Department of Veterinary Clinical Sciences
Louisiana State University School of Veterinary Medicine
Skip Bertman Drive
Baton Rouge, LA  70803
Office: 225-578-9580
Cell: 225 303-8121
fandrews@lsu.edu
 
 
 
 

3.3.2 Co- Investigator

Laura M. Riggs, DVM, PhD, DACVS
Department of Veterinary Clinical Sciences
Louisiana State University School of Veterinary Medicine
Skip Bertman Drive
Baton Rouge, LA  70803
Office: 225-578-9519
lriggs@vetmed.lsu.edu; lriggs@lsu.edu

3.3.3 Co- Investigator

Mandi J. Lopez, DVM, MS, PhD, DACVS
Department of Veterinary Clinical Sciences
Louisiana State University School of Veterinary Medicine
Skip Bertman Drive
Baton Rouge, LA  70803
Office: 225-578-9918
mlopez@vetmed.lsu.edu; mlopez@lsu.edu
 

3.4 Study Coordinator

Michael L. Keowen
Equine Health Studies Program
Louisiana State University School of Veterinary Medicine
Skip Bertman Drive
Baton Rouge, LA  70803
Office: 225-578-9500
mkeowen@lsu.edu
 

3.5 Study Monitor

Amy Cairy

1. F. Young Company

302 Benton Drive
East Longmeadow, MA 01028
Office: 413-486-1486
Fax: 413-526-8990
acairy@wfyoung.com

 

3.6 Quality Assurance Unit

An external quality assurance audit of the data will be conducted at the discretion of the Sponsor after submission of the final report by the principal investigator.
 

3.7 Statistician

Chin-Chi Lui, PhD
Research Associate
Department of Veterinary Clinical Sciences
School of Veterinary Medicine
Louisiana State University
124 Modular Building
Baton Rouge, LA 70803
Office: 225-578-9299
cliu@lsu.edu
 
 

4.0 STUDY LOCATION

Louisiana State University
Equine Health Studies Program
School of Veterinary Medicine
Skip Bertman Drive
Baton Rouge, LA  70803
 

5.0 STUDY OBJECTIVES

 

5.1 Clinical efficacy and safety

Assess the clinical efficacy, safety and pharmacokinetics of oral supplement (Absorbine® Bute-Less® Performance) for the improving pain and lameness due to osteoarthritis in horses.

 

6.0 REGULATORY COMPLIANCE

Study was conducted according to Good Clinical Practice (GCP) guidelines as outlined on FDA Guidance Document # 85, VICH GL9 “Good Clinical Practice (May 2001)”.  All investigators and their personnel involved with the conduct of the study read the guidance document and were thoroughly familiar with the principles of GCPs and their own particular responsibilities to adhered to them during the conduct of the study. All personnel signed a statement indicating they have read and understood the guidance documents and their responsibilities there under.
 

7.0 STUDY RATIONALE

Lameness caused by osteoarthritis (OA) or degenerative joint disease (DJD) is common in horses. Non-steroidal anti-inflammatory agents (NSAIDs) are routinely administered to treat this condition, but when administered systemically these drugs increase the risk of gastrointestinal (GI) and renal toxicity, and when administered topically, penetration through the skin is incomplete. Agents, such as BLP, containing Curcumin Extract (Longvida™, a botanical extract, offers an alternative to NSAIDs for treatment of lameness in horses due to OA or DJD. 
 

7.1 Test Article Justification

A study was conducted in horses to evaluate the effect of Absorbine® Bute-Less® Performance (BLP) supplement containing, Curcumin, a botanical extract from the Ginger family, for the treatment of naturally occurring osteoarthritis.  Ten healthy horses were assigned to one of two groups in a 2X2 crossover prospective study where all horses received both treatments for 30 days. All horses were either administered BLP (2 ounces [56.7 gm containing 1.060 gm of curcumin], daily) top-dressed on grain (1 Kg; Omelene 100®, Purina Animal Nutrition, West Shoreview, MN) or no treatment (grain alone). Osteoarthritis was naturally occurring in a single front limb and was diagnosed by a board certified (ACVS) surgeon by lameness examination, radiographs, and lidocaine administered in the joint when appropriate. Horses all received hay (approximately, 1.5% body weight) fed twice daily and 1 Kg of grain twice daily. BLP was fed in the morning top-dressed on the grain ration. Feed quantities were adjusted based in body weights measured on Days 15. Treatment began on Day 1 of each period and continued for 30 days. A period of at least 2 weeks of no treatment followed the 1st period to allow for washout of supplement. Clinical evaluations were performed daily and lameness and radiographic evaluation were performed prior to entering into the study (Day -2 to Day 0) and then on Day 15 and Day 30. 
 

7.2 Dose Justification                  

The dose of BLP with curcumin in this study was justified as the dose as it was previously given to horses with chronic osteoarthritis to control pain and lameness.

 

7.3 Clinical Efficacy

Clinical efficacy was defined as an improvement of at least 1 grade in the American Association of Equine Practitioners (AAEP) lameness scale, an improvement of flexion and pain on palpation of 1 grade on the scale, or an increase in peak vertical force (PVF) by ≥ 5% as measured by the force platform in curcumin-treated horses. 
 

 8.0 STUDY SCHEDULE

 

8.1 Duration of in-life experimental phase

Date of onset: 18 June 2016
Date of completion: 30 August 2018
 

8.2 Schedule of events for each period

 
Day (-10 to -4):   Physical Examination
                              Blood sample for baseline CBC/Chemistry
                              Lameness evaluation for study inclusion criteria
                              Radiographs for study inclusion criteria
                              Joint block to confirm joint cause of lameness
                             
Day (-3 to -2):      Acclimation
                              Feed sweet feed twice daily (no test article)
                              Feed hay 2 hours post grain in AM
                              Daily clinical observations
 
Day-1:                  Lameness evaluation
                              Baseline Force platform
                              Acclimation
                              Feed sweet feed twice daily (no test article)
                              Feed hay two hours post grain feeding
                              Daily clinical observations
                              Body weight
                              Feed withheld at 3:30 PM for gastroscopy on Day 0
 
Day 0:                   Gastroscopy
                              Feed sweet feed twice daily
                              Feed hay 2 hours post grain
                              Daily clinical observations
 
Day 1:                   Place IV catheter in either jugular vein
                              Collect blood for PK before feeding BLP
                              Feed sweet feed top-dressed with BLP or control
                              Collect blood for PK after BLP feeding 15, 30, 45, 60, 75, 90,
                              105, 120, 150, 180 minutes, then hourly for 6 hours
                              Feed hay 2 hours post grain in AM and in PM
                              Daily clinical observations
 
Day 1-13:             Feed sweet Feed with BLP or control AM
                              Feed hay twice daily
                              Daily clinical observations
 
Day 14:                Place IV catheter in either jugular vein
                              Collect blood for PK before feeding BLP
                              Feed sweet feed top-dressed with BLP or control
                              Collect blood for PK after BLP feeding 15, 30, 45, 60, 75, 90,
                              105, 120, 150, 180 minutes, then hourly for 6 hours
                              Feed hay 2 hours post grain in AM and in PM
                              Daily clinical observations
 
Day15:                 Feed sweet feed twice daily (BLP fed AM)
                              Lameness evaluation
                              Force platform
                              Feed hay
                              Daily clinical observations
                              Body weight, adjust feed regarding BW
 
Day 16-29:           Feed sweet feed with BLP or control AM
                              Feed hay twice daily
                              Daily clinical observations
 
Day30:                 Feed sweet feed twice daily (BLP AM)
                              Physical Examination
                              Lameness evaluation
                              Force platform
                              Feed hay
                              Daily clinical observations
                              Feed withheld at 3:30 PM
 
Day 31:                Gastroscopy
                              Daily clinical observations
                              Blood collection for CBC/Chemistry
                              Body weight
                              Horses turned out to pasture.
 
Procedures were repeated for period 2 and treatments were assigned based on the treatment-groups in period 1.
 

8.3 Clinical efficacy, PK and safety study design summary

 

	Study Day
Procedure	-10 to -4	-3 to-2	-1	0	1	1-13	14	15	16-29	30	31
Physical Exam	X							X		X
CBC/Chemistry	X							X		X
Acclimation Period		X	X
Body Weight			X					X		X
Lameness evaluation	X		X		X			X		X
Radiographs	X
Local block (if needed)	X
Force Platform			X		X			X		X
Gastroscopy				X							X
Administer Test Article					X	X	X	X	X	X
Daily observations		X	X	X	X	X	X	X	X	X	X
Sweet Feed (AM) and (PM) and Hay		X	X*	X	X	X	X	X	X	X*
IV Catheter/Blood PK					X		X
Withhold feed			X							X

*No PM feeding (feed withheld at 3:30 PM)

 

9.0 STUDY DESIGN

 

9.1 Study Design

Ten Thoroughbred horses (6 geldings and 4 mares, mean age 8.6 [range: 3-20 years of age]), with a mean body weight of 498.2 Kg (range: 379-611 Kg) or 1096 lbs. (range: 833-1344 lbs.) with front limb lameness due to naturally occurring osteoarthritis in one joint in the front limb, were selected from horses from the LSU Equine Health Studies Program Research and Teaching Herd.  A physical exam, CBC/biochemical panel, lameness evaluation (including use of perineural or intraarticular blocks as required) and radiographs were performed for assessment of inclusion/exclusion criteria on or before Day-10 to Day -4 of initiation of treatment. Once meeting the inclusion criteria horses were randomly assigned to a treatment group (BLP or no treatment [control]). Lameness exams, force platform analysis and in lameness locator analysis were conducted on Day -1 (baselines), at the midpoint of the study (Day 15) and last day of dosing (Day 30). Whole blood samples were obtained from the jugular vein of the horses via preplaced jugular catheter on Days 1 (first day of treatment) and 14 (after the 14th dose) to determine the pharmacokinetics of curcumin and its metabolites.  Blood samples were drawn before the morning BLP dose and then 15, 30, 45, 60, 75, 90, 105, 120, 150, 180 minutes, then hourly for the next 6 hours (15 total blood samples) following dosing.  Whole blood samples were centrifuged at 1,500 RPMs and plasma decanted into two cryogenic vials and immediately frozen at 80°C.
Endoscopy of the stomach (gastroscopy) was performed on Day 0 (before treatment began) and on Day 3, 24 hours after the last treatment, to determine the effects of treatment on gastric ulcer scores. Persons performing the lameness examination (LMR), force plate evaluation (MJL) and assigning ulcer scores (FMA) were masked to the treatment groups. 
 

9.2 Treatment group

BLP-treatment: Horses received Absorbine® Bute-Less® Performance (2 oz., top-dressed on 1 Kg Omelene 100® grain, once daily) for 30 days.
Controls: Horses received grain ration without BLP supplement.
 

9.3 Blocking factors

Not applicable for this study.
 

9.4 Randomization Procedures

Horses entered into period 1 were randomized to treatment groups by a flip of a coin.

 

10.0 STUDY PROCEDURES

 

10.1 Test Animals

Equus caballus, domestic horse

10.1.1 Description

10.1.1.1 Age
Range between 3 and 20 years of age (mean age: 8.6 years of age)
10.1.1.2 Sex
Study included 4 mares and 6 geldings (male castrated horses)   
10.1.1.3 Breed/Class
Thoroughbreds
10.1.1.4 Initial body weight
Mean initial body weight was 498 Kg (1096 lbs.) with a range from 379 Kg (833 lbs.) to 611 Kg. (1344 lbs.).
10.1.1.5 Physiological State
Clinically healthy horses
Non-pregnant or non-lactating

10.1.2 Number of animals

Thoroughbred horses (n=10)

10.1.3 Source of animals

LSU Equine Health Studies Program Research and Teaching Herd

10.1.4 Identification method

LSU unique number tag attached to halter and description of horse
Stall card on each stall

 

10.2 Test Animal Criteria

10.2.1 Screening procedures

All horses were screened to determined health and eligibility to be included into the study. All horses entered into study met the inclusion/exclusion criteria described in sections 10.2.2 and 10.2.3.
 
10.2.1.1 Physical exam  
Physical exam was performed by a veterinarian, either the Principal Investigator or designee.  Physical examination results were recorded on a standardized form and found in the raw data.
10.2.1.2 Complete blood count and serum chemistry profile
Whole blood samples were obtained from the jugular vein of the horses and immediately placed in vacutainer tubes, one containing EDTA and another containing lithium heparin anticoagulant. Samples were submitted immediately to the LSU Clinical Pathology laboratory and a complete blood count (CBC) and biochemical panel was performed.  Flagged whole blood and plasma values outside the reference range were evaluated by the PI to determine if they were clinically significant. Data from the blood work were summarized and included in the raw data within the study books.

10.2.2 Inclusion criteria

10.2.2.1 Age and body condition
All horses were between 3 and 20 years of age (mean age: 8.6 yrs.) and not grossly obese or underweight in the opinion of the PI or designee.
10.2.2.2 Vaccination status
Horses were vaccinated during the routine schedule for the EHSP Research and Teaching herd; however, horses were not vaccinated during the study periods or washout periods.
10.2.2.3 Health status
Horse were judged to be in good general health based on results of a medical history, physical exam, and laboratory profile.  Horses are part of a stable herd and health records are routinely maintained per facilities SOP.
10.2.2.4 Previous medication and treatment
Horses received no drugs or supplements for the treatment of lameness or pain for at least 14 days prior to Day 0.
10.2.2.5 Lameness evaluation
Lameness evaluations were performed by a co-investigator (LMR), an ACVS board certified surgeon, on or before Day-10 to Day-4, prior to entering the study. Front limb lameness scores varied from 2 to 4 on the AAEP lameness scale and was located to one joint (fetlock, carpus) in the front limb.
10.2.2.6 Radiographic evidence
Radiographs of the affected joint was evaluated by a qualified veterinarian and/or veterinary radiologist on or before Day-10 to -4. All horses were diagnosed with chronic osteoarthritis (degenerative joint disease) based on the radiographic findings.

10.2.3 Exclusion criteria

10.2.3.1 Does not meet Inclusion criteria
All horses enrolled in the study met the inclusion criteria per Section 10.2.

10.2.4 Post – inclusion removal criteria

10.2.4.1 Event that requires discontinuation of test article
No horses were removed due to events unrelated to the test article.
 

10.3 Acclimation of Test Animals

10.3.1 Duration

Animals were acclimated in stalls starting on Day -3. 

10.3.2 Medication and/or vaccination during acclimation period

No medication or vaccinations were given during the acclimation period. 

10.3.3 Baseline data collected prior to initiating study

10.3.3.1 Body Weight
Body weight in kilograms and pounds (calculated) were obtained on Days -1, 15 and 30.
10.3.3.2 Physical Exam
Physical exam was performed by a licensed veterinarian on Day -1 and was recorded on a standard form.  No health issues were noted in the horses examined. Data on physical examination was included in the raw data.
10.3.3.3 Baseline blood plasma samples
Blood samples were drawn on Day -1 for CBC/Chemistry analysis.
10.3.3.4 Daily clinical observations
Daily clinical observations for baseline comparisons began on Day -3.
10.3.3.5 Lameness evaluation
Lameness evaluations were performed by the LMR on Day-1 for baseline.  Results were recorded on a standardized form and included in the raw data.
10.3.3.6 Range of Motion and Palpation
Baseline evaluation for range of motion and reaction to palpation of the affected joint was conducted on Day -1.  Results were individually recorded on a standardized form and contained in the raw data.
10.3.3.7 Force Plate Analysis
Baseline force plate analysis was conducted on Day -1 per procedure outlined in section 14.2.4.3 and all data collected was found in the raw data.

 

10.4 Masking of study

Investigators performing the lameness examination (LMR), force plate analysis (MJL) and assigning gastroscopy ulcer scores (FMA) were mask to the treatment groups.
 

10.5 Measures to ensure compliance with study protocol

All investigators and their personnel involved with conduct of the study read the protocol and were thoroughly familiar with the study design and their own particular responsibilities. All personnel signed a statement indicating they had read and understood the protocol and the responsibilities assigned to them therein.
 

10.6 Measures to ensure compliance with Good Clinical Practices

A copy of FDA – CVM Guidance # 85, Good Clinical Practices, was provided to the Principal Investigator prior to initiation of the study. All investigators and their personnel involved with conduct of the study read the guidance document and were thoroughly familiar with the principles of GCPs and their own particular responsibilities and adhered to them during the conduct of the study. All personnel signed a statement indicating they have read and understood the guidance document and their responsibilities there under.
 

11.0 ANIMAL MANAGEMENT and HOUSING

 

11.1 Housing

The horses were housed at the EHSP research barn (School of Veterinary Medicine Campus, Baton Rouge, LA) in 12’ x 12’ box stalls during the study to facilitate treatment.
 

11.2 Concomitant therapy and/or medications

No concomitant therapy with any drug or vaccine were administered during the study or washout periods. 
 

11.3 Feeding Regimen

Horses were fed grass hay at approximately 1.5% body weight and 2 Kg grain (Omelene® 100) were fed daily divided into two feedings.  The hay was not weighted. Horses were weighed on Day -1, Day 15 and Day 30 during the study. All horses maintained their weight during the study.
 

11.4 Water

Animals were allowed access to potable water.  Water consumption was not measured but was observed for normalcy. There were no known contaminants believed to be present at levels that interfered with the study. Analysis of water samples are done throughout the year and available upon request. 

 

12.0 TEST ARTICLE & CONTROL 

 

12.1 Test article identification

12.1.1 Chemical name

Not applicable 

12.1.2 Trade name or supplement

Absorbine® Bute-Less® Performance (BLP)

12.1.3 Active/inactive ingredients

A proprietary blend of essential vitamins, herbs and extracts, including Curcumin Extract (Longvida™, 1.060 gm/2 oz.), a plant extract.

12.1.4 Dosage form

Oral top-dress on grain

12.1.5 Dose to be tested

BLP: 2 oz. (56.7 gm) containing 1.060 gm Longvida® form of curcumin) (See label below)
Absorbine® Bute-Less® Performance Label Bucket Label.
 

12.1.6 Manufacturing site

Uckele Health & Nutrition, Blissfield, MI

12.1.7 Packaging

BLP was supplied in 10 lb. resealable bags with a 2 oz. scoop included in the bag.

12.1.8 Lot number

                              TAP-1617274

12.1.9 Certificate of analysis

A certificate of analysis was included in this report (Figure X).

12.1.10 Test article storage during study

All test articles were stored securely in the test facility’s research building at room temperature and according to the facility’s standard practice.
 

12.2 Control product 

Omelene 100™ (Purina Animal Nutrition, West Shoreview, MN), without top-dressed supplement was stored in the feed room until use in the barn for feeding.

 

13.0 TREATMENTS & PROCEDURES

 

13.1 Dose

13.1.1 Dose administered

All horses readily ate the 2 oz. doses of the BLP supplement.  Horses were observed while eating and all the supplement doses were consumed.

13.1.2 Dose preparation

The BLP supplement was supplied in a powder form.  Doses (2 oz.) were weighed initially in the scoop provided and found to be 2 oz by the use of the calibrated scale in the laboratory. After the initial measurement, the scoop was used to administer the dose of BLP.

13.1.3 Method of Administration:

The BLP supplement was place in the scoop to level portion and immediately top-dressed on Omelene™ 100 (1 Kg; Purina Animal Nutrition, West Shoreview, MN). Horses were observed while eating the dose and it was confirmed and recorded in the dosing form that all horses consumed the BLP.

13.1.4 Timing

The BLP-dose, top-dressed on the grain portion of the diet (Omelene™ 100, Purina Animal Nutrition, West Shoreview, MN) was fed to all horses in the morning for the duration of the study. Exact time of each treatment was recorded and included in the raw data. Lameness evaluations were done 2 hours after dosing.
 

13.2 Daily observations

Daily observations were conducted by study personnel once daily. General behavior, appearance, attitude, neurologic status and appetite were observed and findings recorded on a standardized Daily Observation form and included in the raw data. All horses appeared clinically normal during the study period.

 

13.3 Clinical safety

Additionally, safety was evaluated by examining changes in daily observations, body weight and laboratory parameters during the study. No changes in clinical parameters were seen during the study period.
 

14.0 ASSESSMENT of TREATMENT and CLINICAL TREATMENT SUCCESS

 

14.1 Effects to be achieved

It was hypothesized that BLP will improve lameness sufficient to result in improvement in lameness evaluation parameters in treated animals. Lameness in these horses was evaluated by subjective lameness exams, force plate analysis and Lameness Locator.
           

14.2 Study procedures

All study procedures took place from 18 June 2016 to 30 August 2018.

14.2.1 Physical examination procedure

Physical examination was performed on Day -1 and then again on Day 30. No clinical abnormalities were noted on any of the physical examinations in the horses enrolled in the study.

14.2.2 Venipuncture for CBC and serum chemistries

The CBC and plasma biochemical panels on Days -1 and 31 showed no clinical significant values throughout the study period and values are included in the raw data.

14.2.3 Body weights

Body weights for each horse were obtained on Day -1 and Days 15 and 30.

14.2.4 Lameness Evaluation Protocol

 
14.2.4.1 Lameness Examinations
Lameness examinations were performed on each horse on Day -1, 15 and 30.  Except on Day -1, lameness examinations were conducted between 2 and 6 hours following the morning treatment and were performed on a hard, flat surface. Each horse was observed in hand at a walk and a trot in a straight line and lunging in both clockwise and counterclockwise directions. All lameness exams were conducted by the Co-I (LMR), who was masked to treatment. The AAEP lameness scoring system was used.
 
14.2.4.2 Range of Motion and Palpation
Range of motion (flexion) and response to palpation of the designated affected joint were performed.  Assessment of the range of motion for the affected joint and pain reaction to palpation of the affected joint were performed by the LMR.
 
14.2.4.3 Force Plate Analysis
Kinetic gait analysis was performed on each horse on Days -1, 15 and 30 of the study, according to established criteria (5,6,9) following the lameness evaluation.  A 900 x 900 mm force platform embedded in the center of a 40 m concrete runway (Model #BP900900, Advanced Mechanical Technology) was used for all gait trials. The force platform surface is the same color and texture as the runway.  An experienced handler trotted horses for all trials. All trials were considered successful when the forelimb contacted the force platform followed by contact of the ipsilateral hind limb at a velocity of 2.00-3.80 m/s and acceleration of 0.9 to -0.9 m/s2. All recordings were entered based on these criteria. A series of three reflective photocell sensors (Mek92-PAD, Doslyn Clark Controls, Inc.) were used to determine velocity and acceleration in each trial. The middle sensor was positioned so that it bisects the center of the force platform.  Data logging was triggered by a force of 50 N.  Trials were rejected if the hoof was not straight on the force platform, was not completely on the force platform or was within 5 cm of the force platform edge. The velocity and acceleration ranges selected include a comfortable trot for all of the subjects included in the study. There was little difference in velocity between sound and lame horses, and horses selected a preferred stride length and frequency to obtain an energetically optimal velocity (9). Based on this information, a particular speed was not imposed on the horses, but individual limb stance times were recorded.  Five successful trials were recorded for each limb (5). Ground reaction forces were recorded at a rate of 100 Hz and processed with commercially available software (Acquire V7.3, Sharon Software).  Recorded forces included peak vertical force (PVF) and impulse, both normalized to body weight. Mean vertical loading and unloading rates as well as stance time were evaluated according to described methods.7 Data was recorded in Appendix X.  
 
14.2.4.4 Gastroscopy
All horses’ stomachs and the proximal duodenum were evaluated using a 3-m endoscope (Karl Storz, El Segundo, CA) on Days 0 and 31. Feed was withheld for 12-24 hours and water was withheld prior to gastroscopy to improve the visualization of the stomach. Horses were sedated using xylazine (0.4mg/kg, IV) 5 minutes prior to the gastroscopy.  The gastroscope was introduced into the stomach via the nostril, nasopharynx and esophagus. The stomach was insufflated with air and feed material was washed off using tap water introduce using a 60 cc syringe attached to the biopsy channel. Ulcer scores were assigned and scores are found in study raw data.
 

14.2.5 Evaluation of improvement

Each horse evaluated according to section 16.2.3 had its clinical response summarized as follows:
 
Treatment Failure (TF)                  No response
Treatment Success (TS)              Horse improved ≥ 1 grade of lameness
                                                                                    and/or
                                                            ≥ 5% improvement in PVF in the lame
limb as recorded on the force platform

14.2.6 Clinical safety

Clinical safety was evaluated by examining changes in daily observations, body weight and laboratory parameters during the study. No adverse responses or clinically abnormalities were noted throughout the trial. Daily observations for each horse are found in the raw data in the study book.

 

15.0 DISPOSTION OF STUDY ANIMALS, TEST ARTICLE & CONTROL

 

15.1 Care to be administered to animals removed from the study

No horses were removed from the study.
 

15.2 Disposition of remaining test article and control

All unused test material was retained at the test facility. Test article inventory records were maintained on the appropriate form that accompanies initial shipment and records are found in the study book.
 

15.3 Disposition of study animals

All study animals were returned to the EHSP Research and Teaching herd at the test facility at the completion of the study.
 

16.0  STATISTICAL METHODS

 

16.1 Sample size

10 horses

 

16.2 Statistical analysis

All hypothesis testing was evaluated at P < 0.05.
 
Data analyses were performed using SAS 9.4 software (SAS Institute Inc., Cary, NC). A repeated measure analysis of variance (ANOVA) with a mixed effect model was used to the variables measured. Treatment (curcumin or control), day and their interactions were entered as the fixed effects. Each animal was entered as the random effect. Treatment successful rate was computed with 95% confidence interval. A one-sided binomial test of statistical significance was used to exam if the rate is greater than 50%. A chi square test was used to check the association between the success rate and treatment. In addition, a logistic regression was used to study the association between the success rate, period and the treatment. Significance was considered when P<0.05.
Clinical pathology values were summarized.
 

17.0  COLLECTION AND RETENTION OF SOURCE DOCUMENTATION

 
All data were gathered and recorded according to standard clinical practices at the facility and recorded on the standardized forms in use at the study facility and on forms designed specifically for this study. Completed forms were placed in the study folder as raw data and held at the study facility. All raw data was retained in the study book and summarized in pertinent data were placed in tables and figures and attached to the study report.
 
All original raw data was retained by the Principal Investigator at the Louisiana State University School of Veterinary Medicine, Equine Health Studies Research building Rm. 101.  A copy of all raw data will be sent to the sponsor with a copy of the final study report.

 

18.0  ADVERSE EVENTS

 

18.1 Monitoring for adverse events

There were no adverse events that occurred during the study.  However, one horse had abdominal pain during the study, but was a control horse.  Rectal examination showed that the horse had an impaction colic and the episode resolved with medical treatment.
 

18.2 Recording adverse events

No adverse events were recorded during the study.  The colic event was recorded in the raw data.
 

18.3 Reporting adverse events

No adverse events were seen in this study. The colic event was reported to the PI and it was determined to not be study supplement related as the horse was a control horse.
 

18.4 Necropsy

No horses were euthanatized during the study.

 

19.0 ANIMAL WELFARE

This protocol was reviewed and approved by the LSU Institutional Animal Care and Use Committee (IACUC) on 19 May 2016 and was assigned IACUC Protocol #16-017.
 
 

21.0    RESULTS

All horses readily ate the supplement and there were no adverse responses observed. The CBC and serum chemistry analysis on whole blood and plasma, respectively, showed no clinically significant values. Horse numbers, gender, age, body weights and date horses entered into the study are found in Table 1.
 
Table 1. Horse number, gender, age, body weights and date horses entered the treatment phase of the study and whether they were treated or untreated controls and period treated. All horses were Thoroughbreds.
 
 

Horse	Sex	Age (Yrs)	Wt. (lbs)	Wt. (Kg)	Date	Day	Period	Treat
467	G	8	1292.3	587.4	28-Jun-16	-1	1	Curcumin
			1277.3	580.6	13-Jul-16	15	1	Curcumin
			1280.4	582.0	28-Jul-16	30	1	Curcumin
			1242.5	564.8	27-Sep-16	-1	2	Control
			1232.4	560.2	12-Oct-16	15	2	Control
			1236.0	561.8	28-Oct-16	30	2	Control
384	M	14	1182.5	537.5	28-Jun-16	-1	1	Control
			1172.6	533.0	13-Jul-16	15	1	Control
			1159.6	527.1	29-Jul-16	30	1	Control
			1148.4	522.0	27-Sep-16	-1	2	Curcumin
			1141.8	519.0	12-Oct-16	15	2	Curcumin
			1159.6	527.1	28-Oct-16	30	2	Curcumin
140	G	15	1278.0	580.9	10-Apr-17	-1	1	Control
			1302.0	591.8	26-Apr-17	15	1	Control
			1298.0	590.0	11-May-17	30	1	Control
			1256.0	570.9	20-Jun-17	-1	2	Curcumin
			1317.8	599.0	6-Jul-17	15	2	Curcumin
			1344.2	611.0	21-Jul-17	30	2	Curcumin
628	G	3	1053.8	479.0	10-Apr-17	-1	1	Curcumin
			1058.2	481.0	26-Apr-17	15	1	Curcumin
			1058.2	481.0	11-May-17	30	1	Curcumin
			1057.1	480.5	20-Jun-17	-1	2	Control
			1080.0	490.9	6-Jul-17	15	2	Control
			1078.0	490.0	21-Jul-17	30	2	Control
633	G	5	1036.0	470.9	30-May-17	-1	1	Control
			1017.1	462.3	14-Jun-17	15	1	Control
			935.7	425.3	29-Jun-17	30	1	Control
			1016.0	461.8	7-Aug-17	-1	2	Curcumin
			999.9	454.5	24-Aug-17	15	2	Curcumin
			1005.0	456.8	7-Sep-17	30	2	Curcumin
625	M	4	832.9	378.6	30-May-17	-1	1	Curcumin
			905.1	411.4	14-Jun-17	15	1	Curcumin
			906.0	411.8	30-Jun-17	30	1	Curcumin
			953.0	433.2	7-Aug-17	-1	2	Control
			968.0	440.0	24-Aug-17	15	2	Control
			1009.8	459.0	7-Sep-17	30	2	Control
635	G	3	1070.0	486.4	8-Jan-18	-1	1	Curcumin
			1084.6	493.0	24-Jan-18	15	1	Curcumin
			1031.8	469.0	8-Feb-18	30	1	Curcumin
			1061.9	482.7	16-Mar-18	-1	2	Control
			1064.8	484.0	4-Apr-18	15	2	Control
			1081.0	491.4	19-Apr-18	30	2	Control
48	M	20	939.0	426.8	8-Feb-18	-1	1	Curcumin
			942.0	428.2	28-Feb-18	15	1	Curcumin
			965.8	439.0	15-Mar-18	30	1	Curcumin
			1059.9	481.8	19-Apr-18	-1	2	Control
			998.8	454.0	9-May-18	15	2	Control
			1000.7	454.9	24-May-18	30	2	Control
652	M	8	1061.0	482.3	19-Apr-18	-1	1	Curcumin
			1058.2	481.0	9-May-18	15	1	Curcumin
			1054.5	479.3	24-May-18	30	1	Curcumin
			1108.6	503.9	26-Jul-18	-1	2	Control
			1084.7	493.1	15-Aug-18	15	2	Control
			1047.6	476.2	30-Aug-18	30	2	Control
657	G	6	1137.4	517.0	24-May-18	-1	1	Curcumin
			1137.4	517.0	13-Jun-18	15	1	Curcumin
			1106.7	503.0	28-Jun-18	30	1	Curcumin
			1138.5	517.5	26-Jul-18	-1	2	Control
			1084.7	493.1	15-Aug-18	15	2	Control
			1147.3	521.5	30-Aug-18	30	2	Control

 
 
Lameness
All horses had moderate to severe radiographic evidence of OA. Lameness scores according to the AAEP Lameness scale ranged between from 2 and 4 throughout the trial and were variable during the study periods (Table 2). Lameness scores showed no significant treatment*day effects (Figure 1). Six of 10 (60%) horses in both groups improved at least one lameness grade during the study (Table 2). Only 1 horse (#140) showed no improvement in lameness score in either group by day 30 of the study. Five horses improved at least 1 lameness grade regardless of treatment, however 4/5 were control horses that improved were in the control group during period 2 of the study, suggesting a carry-over effect of BLP treatment during period 1. In addition, in the initial 6-horse cohort, pain response upon palpation was significantly (P=0.025) lower in BLP-treated horses by Day 30 of treatment (Figure 5).
 
Table 2. Horse numbers, Day, period, treatment groups, lame limb, lameness grade, range of motion, and pain response in horses. (* denotes horses that improved 1 lameness grade during the study)
 

Horse	Date	Day	Period	Treat	Lame Limb	L. Score	Motion	Response
467	28-Jun-16	-1	1	Curcumin	LF	3	0	1
	13-Jul-16	15	1	Curcumin	LF	2	0	1
	28-Jul-16	30	1	Curcumin	LF	2*	0	0
	27-Sep-16	-1	2	Control	LF	2	0	1
	12-Oct-16	15	2	Control	LF	0	0	0
	28-Oct-16	30	2	Control	LF	1*	0	1
384	28-Jun-16	-1	1	Control	RF	3	3	3
	13-Jul-16	15	1	Control	RF	3	3	2
	29-Jul-16	30	1	Control	RF	2*	2	3
	27-Sep-16	-1	2	Curcumin	RF	2	2	3
	12-Oct-16	15	2	Curcumin	RF	3	3	2
	28-Oct-16	30	2	Curcumin	RF	2*	2	2
140	10-Apr-17	-1	1	Control	LF	3	1	1
	26-Apr-17	15	1	Control	LF	2	0	1
	11-May-17	30	1	Control	LF	4	0	1
	20-Jun-17	-1	2	Curcumin	LF	3	1	0
	6-Jul-17	15	2	Curcumin	LF	2	0	0
	21-Jul-17	30	2	Curcumin	LF	3	0	0
628	10-Apr-17	-1	1	Curcumin	LF	3	0	0
	26-Apr-17	15	1	Curcumin	LF	3	0	1
	11-May-17	30	1	Curcumin	LF	3	0	0
	20-Jun-17	-1	2	Control	LF	3	0	0
	6-Jul-17	15	2	Control	LF	3	1	0
	21-Jul-17	30	2	Control	LF	2*	0	0
633	30-May-17	-1	1	Control	LF	4	3	2
	14-Jun-17	15	1	Control	LF	4	3	2
	29-Jun-17	30	1	Control	LF	3*	2	1
	7-Aug-17	-1	2	Curcumin	LF	3	1	1
	24-Aug-17	15	2	Curcumin	LF	4	3	2
	7-Sep-17	30	2	Curcumin	LF	4	3	1
625	30-May-17	-1	1	Curcumin	RF	3	1	1
	14-Jun-17	15	1	Curcumin	RF	2	2	2
	30-Jun-17	30	1	Curcumin	RF	2*	0	0
	7-Aug-17	-1	2	Control	RF	3	0	0
	24-Aug-17	15	2	Control	RF	1	0	1
	7-Sep-17	30	2	Control	RF	2*	0	1
635	8-Jan-18	-1	1	Curcumin	RF	3	2	2
	24-Jan-18	15	1	Curcumin	RF	2	2	1
	8-Feb-18	30	1	Curcumin	RF	2*	1	1
	16-Mar-18	-1	2	Control	RF	1	0	0
	4-Apr-18	15	2	Control	RF	1	0	0
	19-Apr-18	30	2	Control	RF	2	0	0
48	8-Feb-18	-1	1	Curcumin	RF	4	1	0
	28-Feb-18	15	1	Curcumin	RF	3	1	1
	15-Mar-18	30	1	Curcumin	RF	3*	1	0
	19-Apr-18	-1	2	Control	RF	1	1	0
	9-May-18	15	2	Control	RF	1	0	0
	24-May-18	30	2	Control	RF	0*	0	0
652	19-Apr-18	-1	1	Curcumin	RF	2	0	1
	9-May-18	15	1	Curcumin	RF	2	0	0
	24-May-18	30	1	Curcumin	RF	1*	2	1
	26-Jul-18	-1	2	Control	RF	2	1	1
	15-Aug-18	15	2	Control	RF	2	0	1
	30-Aug-18	30	2	Control	RF	0*	0	1
657	24-May-18	-1	1	Curcumin	LF	2	1	1
	13-Jun-18	15	1	Curcumin	LF	2	1	1
	28-Jun-18	30	1	Curcumin	LF	3	0	0
	26-Jul-18	-1	2	Control	LF	3	1	1
	15-Aug-18	15	2	Control	LF	3	1	1
	30-Aug-18	30	2	Control	LF	2*	0	1

 
Peak vertical force (PVF) values (a measure of weight bearing) in the lame limb were significantly (P=0.0024) increased in the BLP-treated horses in the first cohort of horses examined (n=6) (Figure 2). When all horses (n=10) were included, there was a > 5% improvement in mean PVF in the BLP-treated horses by Day 30 when compared to baseline values (Figure 3). No change in overall PVF was seen in the control horses at Day 15 or 30 (Figure 3). When horses were considered individually as either treatment successes (TS) or treatment failures (TF) (≥ 5% improvement in PVF by day 30), 6/10 BLP-treated horses showed a ≥ 5% (P<0.05) increase in PVF in the lame limb, while only 3/10 control horses were considered TS, based on the same criteria (Table 3, Figure 4.1-4.10). One of the control horses improved > 5% in PVF by Day 15, but by Day 30, there was not difference compared to baseline values, so this horse (#657) was considered a TF (Figure 4.10). Also, in the untreated group, all 3 of 10 horses improved during period 2 after a two week washout period.  In addition, data showed a significant period effect on TS favoring the 1st period for BLP-treated horses and the 2nd period for control horses. Thus, BLP-treatment might have contributed to a carry-over effect in control horses during 2nd period in untreated controls leading to TS in the control group. A two-week washout period might not be long enough to clear all of the BLP-tissue effects. In addition, AAEP lameness improved ≥ 1.0 grade in all but one horse that improved ≥ 5% in PVF. In 6/10 BLP-treated horses, PVF increased in the lame limb from 5.0% to 33%. When improvement in PVF in BLP-treated horses was compared to untreated controls, there was a strong association (P=0.0055) during period 1 and a lesser association (P=0.1031) in period 2 for TS. When a data were analyzed using a logistic regression model, BLP-treatment effect was associated with an odds ratio for improvement in lameness was 2.19.
This finding of improvement in objective lameness evaluation was also seen in Q Score as measured by the Lameness Locator.  Q Score is a measure lameness severity, the higher the number the more severe the lameness.  The BLP-treated horses in this study, showed a mean decrease of 33% in Q Score compared to the control horses showing only a 17% reduction in Q Score.  All except 1 horse deemed treatment successes by ≥ 5% improvement in PVF also showed improvement in Q Score, confirming lameness improvement with treatment.
 
Table 3. Treatment success (TS) and treatment failures (TF) in horses as determined by a ≥ 5% improvement in peak vertical force (PVF) in the lame limb as measured by the force platform. All TS horses, except for one horse, showed an improvement in 1 grade on AAEP lameness examination.
 

Horse Number	Curcumin Treated	Control
467	TS	TF**
384	TF*	TF
140	TF	TF
628	TF	TS
633	TF	TF
625	TS	TF
635	TS	TF
48	TS	TS
652	TS	TS
657	TS	TF***
TS vs. TF	6 vs. 4	3 vs. 7

*Force platform was not working on Day 30, so data not collected on that day. So called this horse #384 a treatment failure.
**Force platform was not working on Day 30, so data was not collected. PVF increased ≥ 5% on Day 15, but did not have data for Day 30 so called TF.
***PVF improved ≥ 5% on day 15 in control horse #657, but by day 30 there was no improvement, so was considered a TF.
 
Gastric Ulcers and Kidney Parameters
On gastroscopy examination, nonglandular gastric ulcer scores (EGUS scoring system) significantly (P=0.0195) lower in both groups by day 30 of the trial (Figure 6). No treatment*day effect was noted regarding gastric ulcer scores. In addition, glandular ulcer scores were not changed, but were observed in only a few horses.
Serum biochemical parameters, especially for the kidneys (BUN and creatinine) showed no clinical significant increases in plasma in treated horses.  Use of non-steroidal anti-inflammatory drugs, such as phenylbutazone, can lead to increases in these kidney parameters (azotemia), however azotemia was not noted in any horses in this study after 30 days of feeding BLP.
 
Plasma Concentrations
Plasma curcumin and its metabolite curcumin-O-sulfate were measured using LC-ESI–MS/MS method. The linearity of the aforementioned curcuminoids and curcumin-O-sulfate was in the range of 0.5–1000 ng/mL and 1–1000 ng/mL for curcumin-O-glucuronide with 85–115% accuracy and <15% precision in equine plasma. The method was validated based on US FDA criteria and applied to characterize the pharmacokinetics of curcumin-O-sulfate in equine plasma. No plasma concentrations of curcumin or its metabolites were found after the first BLP dose.  However, Plasma from the 2 horses measured contained curcumin-O-sulfate concentrations (Figure 7). The time to maximum plasma concentration (Tmax) was approximately 1.5 hours (after the 15th dose) and the maximum concentration (Cmax) ranged from 1.25 to 2.5 ng/ml in the two horses measured. Further analysis of plasma samples will take place in the future. Pharmacokinetic analysis will be done after all plasma samples are analyzed and will be added as a supplement to this report.
 
22.0 DISCUSSION
The BLP supplement top-dressed on feed and fed to horses was readily eaten, safe and showed no clinically significant changes in clinical examination parameters or CBC and biochemical values. Palatability of any supplement is important especially when top-dressed on the grain portion of the feed. The consumption of the BLP supplement was confirmed by observation of the horse eating the supplement and later in plasma samples, which showed that a metabolite of curcumin (curcumin-O-sulfate) present.
Lameness due to naturally occurring osteoarthritis (OA) can vary within and among horses and vary by the day examined. In this study, we sought to perform a comprehensive lameness evaluation using the AAEP subjective lameness examination, Lameness Locator® Technology (Equinosis®Q with Lameness Locator®, Equinosis, LLC, 104 E Broadway Columbia, MO.) and an equine force platform analysis as done in an earlier study (6). In this study, subjective AAEP lameness grades assigned by a masked (to treatment) expert showed variability in the horses by day and period sampled. Lameness grades improved ≥ 1 AAEP lameness grade in 6/10 (60%) of the BLP-treated and untreated control horses, which resulted in no statistically significant treatment by day effects. Subjective lameness examinations are limited by observer error (7,8) and there might be overestimation of changes in lameness after treatment (9).  Therefore, subjective measures of lameness might not be the best way to determine improvement in lameness in horses. Therefore, a comprehensive lameness examination might decrease the variability of gait analysis.
The comprehensive lameness examination in this study also included range of motion and pain on palpation of the affected front limb, as well as objective evaluation of lameness using the force platform and lameness locator. There was no treatment by day effect on range of motion in this group of horses, however in the first cohort of horses (n=6) pain was significantly decreased on palpation in the BLP-treated horses when compared to the untreated controls. The active ingredient of BLP is curcumin and curcumin extracts have been shown to be anti-inflammatory and inhibit interleukin-6 levels in people and horses leading to decreased joint inflammation and pain (10,11,12). This statistical difference was not seen when all the horses (n=10) were evaluated, however there was a strong period effect. This was likely due to horse in the second cohort (n=3) showing less initial pain on palpation of the affected limb, which might be a result of carry-over of treatment.
Objective measures of lameness provide quantitative values that can be used to determine improvement and takes away bias associated with subjective measures.  Subjective lameness scores in this study were not statistically different when evaluated by an expert clinician.  However, objective measurement of weight bearing, as measured by peak vertical force (PVF) of the limbs on the force platform can be helpful in determining improvement of lameness that might be missed on subjective evaluation.  An improvement of ≥ 5% in weight bearing is considered to be equivalent to a 1 grade improvement in lameness score.  In the study reported here, BLP-treatment resulted in 5% to 33% improvement in PVF in 6/10 (60%), whereas only 3/10 of the untreated control horses improved ≥ 5%.  Also, all horses that improved by ≥ 5% in PVF on the force platform showed at least a ≥1 grade improvement on the AAEP lameness scale. In support of BLP-treatment, there was a significant association (P=0.0055) with treatment success (TS) in the BLP-treated horses in period 1 of the study and an overall association (P=0.1031) for the whole study, when compared to untreated controls.  Also, BLP-treated horses were 2.19X more likely to improve their lameness than control horses. Kinetic gait analysis is an established mechanism to overcome many of the limitations faced with subjectively evaluating lameness (13).  This finding of improvement in objective lameness evaluation was also seen in the Q Score as measured by the Lameness Locator.  Q Score is a measure lameness severity.  The BLP-treated horses showed a mean decrease (33%) in Q Score compared to the control horses showing a decrease of only 17%.  All except 1 of the horses deemed a treatment success also showed improvement in Q Score, confirming lameness improvement.
Since treatment of lameness in horses involves the use phenylbutazone or other nonsteroidal anti-inflammatories (NSAIDs), it is important to show that any supplement used to maintain musculoskeletal health in horses is safe and maintains stomach, gastrointestinal and renal health.  NSAIDs toxicity is mostly targeted at the stomach and kidneys. No evidence of changes in kidney values (BUN, Creatinine) were seen within biochemical panels. Gastroscopy examination showed gastric ulcers in a majority of horses prior to treatment (Day 0), however by 30 days of treatment (Day 31) ulcer scores decreased in most of the horses in the non-glandular region of the stomach. There was a significantly (P < 0.05) decrease in non-glandular ulcer scores in both groups by the end of the trial. Therefore, from the data presented here, it can be concluded that BLP-treatment does not lead to an increase in gastric ulcers or increase in kidney parameters in stall-confined horses suffering from lameness due to osteoarthritis.
Curcumin and its metabolites were detected in plasma by day 14 of treatment and the technique was validated for equine plasma (14). It is important to determine if the primary active ingredient, curcumin, in the BLP supplement is absorbed and bioavailable to reach tissue concentrations. In this study, detectable plasma concentrations were found after the 14th dose. Initial plasma samples taken on Day 1, after the first dose, no curcumin or it’s metabolites were detected. It is likely that the ingredients of BLP will not reach plasma levels until at least 14 days after feeding. Only 3/7 horses considered TS showed a ≥ 5% improvement in lameness after 15 days of treatment, whereas all 6 horses showed improvement by 30 days.  Thus, supplementation for at least 30 days will be needed to improvement in musculoskeletal health.  In addition, there was variable improvement in lameness throughout the study and more horses improved in the control group during period 2 of the study, which might suggest that there was a carry-over effect from initial treatment.
 
23.0 CONCLUSIONS
Absorbine® Bute-Less® Performance containing curcumin (Longvida™), fed to horses with lameness (AAEP grade 2-4) due to osteoarthritis, achieved blood levels, reduced pain and improved weight-bearing after 30 days of feeding, while maintaining stomach health.  It also appears that effects of the BLP supplement might last for several weeks after treatment has been discontinued.
 
 

FIGURES

Figure 1. Mean ± SEM subjective (AAEP) lameness scores by Day -1 (before treatment) and Days 15 and 30 of treatment. A significant treatment*day effect was not observed.

 
Figure 2. Graph showing a significantly (P=0.0024) higher peak vertical force (PVF) in Absorbine® Bute-less Performance®-treated horses compared to controls by day 30 of feeding in the first cohort of horses (n=6).

 

Figure 3. Mean ± SEM peak vertical force (Force) in lame and sound front limbs on Day -1 before treatment and on Day 15 and 30 of treatment. *A significant (>5%) increase in PVF on Day 30 in the lame limb of BLP-treated horses, but not in the control horses.

 

Figures 4.1-4.10: Peak vertical force (Ng/Kg), corrected for body weight, as measured by the force platform in the control and BLP-treated horses in lame limb treated (tx Lame), sound limb treated (tx Sound), lame limb controls (cont Lame) and sound limb control (cont Sound). (†) and (*) denote significant improvement (≥ 5% increase) compared to baseline of the same control and BLP-treated limbs. TS=Treatment Success; TF=Treatment Failure.

 

 

Figure 5. Graph showing a significantly (P=0.025) lower response to “Pain on Palpation” score in the lame joint in the BLP-supplemented horses compared to controls by day 30. First 6 horses in the cohort.

 

Figure 6. Mean ± SEM EGUS (gastric ulcer) scores (0-3) by Day -1 (before treatment) and Days 15 and 30 of treatment. A treatment*day effect was not observed, however EGUS scores were significantly (P<0.05) lower in both groups after 30 days of treatment.

 Figure 7. The plasma concentration-time profiles of curcumin-O-sulfate after two weeks of BLP supplement containing curcumin (Longvida®) top-dressed on feed in two horses.6
 
 

20.0 REFERENCES 

1) Johnson, P., 2002. The equine metabolic syndrome peripheral Cushing's syndrome. Veterinary Clinics of North America-Equine Practice 18, 271-293.
2) Khumsap, S., Clayton, H., Lanovaz, J., 2001. Effect of walking velocity on ground reaction force variables in the hind limb of clinically normal horses. American Journal of Veterinary Research 62, 901-906.
3) Khumsap, S., Clayton, H., Lanovaz, J., Bouchey, M., 2002. Effect of walking velocity on forelimb kinematics and kinetics. Equine Veterinary Journal. Supplement 34, 325-329.
4) Lopez, M., Quinn, M., Markel, M., 2006a. Evaluation of gait kinetics in puppies with coxofemoral joint laxity. American Journal of Veterinary Research 67, 236-241.
5) Williams, G., Silverman, B., Wilson, A., Goodship, A., 1999. Disease-specific changes in equine ground reaction force data documented by use of principal component analysis. American Journal of Veterinary Research 60, 549-555.
6) Mirza MH, Bommala P, Richbourg HA, Rademacher N, Kearney MT and Lopez MJ (2016) Gait Changes Vary among Horses with Naturally Occurring Osteoarthritis Following Intra-articular Administration of Autologous Platelet-Rich Plasma. Front. Vet. Sci. 3:29. doi: 10.3389/fvets.2016.00029.
7) Keegan KG, Dent EV, Wilson DA, Janicek J, Kramer J, Lacarrubba A, et al. Repeatability of subjective evaluation of lameness in horses. Equine Vet J (2010) 42:92–7. doi:10.2746/042516409X479568.
8) Hammarberg M, Egenvall A, Pfau T, Rhodin M. Rater agreement of visual lameness assessment in horses during lungeing. Equine Vet J (2016) 48:78–82. doi:10.1111/evj.12385.
9) Arkell M, Archer RM, Guitian FJ, May SA. Evidence of bias affecting the interpretation of the results of local anaesthetic nerve blocks when assessing lameness in horses. Vet Rec (2006) 159:346–9. doi:10.1136/vr. 159.11.346.
10) Monteza G, Sahebkar, A. Curcumin: An effective inhibitor of interleukin-6. Current Pharmaceutical Design. (2017) 23 (6), 921-931.
11) Farinacci, M, Gaspardo, B, Colitti, M, Stefanon, B. Dietary administration of curcumin modifies transcriptional profile of genes involved in inflammatory cascade in horse leukocytes. Italian Journal of Animal Science. (2009) 8 (Suppl. 2), 84-86.
12) Funk, JL, Frye, JB, Oyarzo, JN, et al. Efficacy and mechanism of action of turmeric supplements in the treatment of experimental arthritis.  Arthritis & Rheumatism (2006) 54 (11), https://doi.org/10.1002/art.22180.
13) Merkens HW, Schamhardt HC. Evaluation of equine locomotion during different degrees of experimentally induced lameness. I: lameness model and quantification of ground reaction force patterns of the limbs. Equine Vet J Suppl (1988) 20:99–106. doi:10.1111/j.2042-3306.1988.tb04655.x
14) Liu, Y, Siard, M, Adams, A, et al. Simultaneous quantification of free curcuminoids and their metabolites in equine plasma by LC-ESI–MS/MS. J. Pharma and Biomed Analysis. 154 (2018) 31–39.)
 

APPENDIX 1: Sample Certificate of Analysis