Available online http://arthritis-research.com/content/10/4/R85

Research article

Open AccessVol 10 No 4

A double blind, randomized, placebo controlled study of theefficacy and safety of 5-Loxin® for treatment of osteoarthritis of
the knee
Krishanu Sengupta1, Krishnaraju V Alluri2, Andey Rama Satish3, Simanchala Mishra4,
Trimurtulu Golakoti5, Kadainti VS Sarma6, Dipak Dey7 and Siba P Raychaudhuri8
1Cellular and Molecular Biology Division, Laila Impex R&D Center, Jawahar Autonagar, Vijayawada, 520 007 India
2Pharmacology Division, Laila Impex R&D Center, Jawahar Autonagar, Vijayawada, 520 007 India
3Department of Orthopedics, Alluri Sitarama Raju Academy of Medical Sciences (ASRAM), National Highway 5, Eluru, 534 002 India
4Department of Internal Medicine, Alluri Sitarama Raju Academy of Medical Sciences (ASRAM), National High way 5, Eluru, 534 002 India
5Drug Discovery and Development Division, Laila Impex R&D Center, Jawahar Autonagar, Vijayawada, 520 007 India
6Department of Statistics, Prakasam Road, SV University, Tirupati, 517 592 India
7Department of Statistics, 215 Glenbrook Road, University of Connecticut, Storrs, Connecticut 06269, USA
8Department of Medicine, Division of Rheumatology, Allergy and Immunology, School of Medicine, U C Davis and VA Medical Center Sacramento,

Hospital Way, Mather, California 95655, USA
Corresponding author: Siba P Raychaudhuri, sraychaudhuri@ucdavis.edu
Received: 24 Nov 2007 Revisions requested: 21 Dec 2007 Accepted: 30 Jul 2008 Published: 30 Jul 2008
Arthritis Research & Therapy 2008, 10:R85 (doi:10.1186/ar2461)
This article is online at: http://arthritis-research.com/content/10/4/R85© 2008 Sengupta et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Introduction 5-Loxin® is a novel Boswellia serrata extractenriched with 30% 3-O-acetyl-11-keto-beta-boswellic acid
(AKBA), which exhibits potential anti-inflammatory properties by
inhibiting the 5-lipoxygenase enzyme. A 90-day, double-blind,randomized, placebo-controlled study was conducted toevaluate the efficacy and safety of 5-Loxin® in the treatment of
osteoarthritis (OA) of the knee.
Methods Seventy-five OA patients were included in the study.
The patients received either 100 mg (n = 25) or 250 mg (n =25) of 5-Loxin® daily or a placebo (n = 25) for 90 days. Eachpatient was evaluated for pain and physical functions by using
the standard tools (visual analog scale, Lequesne's Functional
Index, and Western Ontario and McMaster Universities
Osteoarthritis Index) at the baseline (day 0), and at days 7, 30,
60 and 90. Additionally, the cartilage degrading enzyme matrix
metalloproteinase-3 was also evaluated in synovial fluid from OA
patients. Measurement of a battery of biochemical parameters inserum and haematological parameters, and urine analysis were
performed to evaluate the safety of 5-Loxin® in OA patients.

Results Seventy patients completed the study. At the end of thestudy, both doses of 5-Loxin® conferred clinically andstatistically significant improvements in pain scores and physicalfunction scores in OA patients. Interestingly, significant
improvements in pain score and functional ability were recordedin the treatment group supplemented with 250 mg 5-Loxin® asearly as 7 days after the start of treatment. Corroborating theimprovements in pain scores in treatment groups, we also notedsignificant reduction in synovial fluid matrix metalloproteinase-3.
In comparison with placebo, the safety parameters were almost
unchanged in the treatment groups.
Conclusion 5-Loxin® reduces pain and improves physicalfunctioning significantly in OA patients; and it is safe for humanconsumption. 5-Loxin® may exert its beneficial effects bycontrolling inflammatory responses through reducing
proinflammatory modulators, and it may improve joint health by
reducing the enzymatic degradation of cartilage in OA patients.
Trail Registration
ISRCTN05212803.)(Clinical

trial

registration

number:

AKBA = 3-O-acetyl-11-keto-beta-boswellic acid; ANOVA = analysis of variance; ASRAM = Alluri Sitarama Raju Academy of Medical Sciences; BMI= Body Mass Index; ELISA = enzyme-linked immunosorbent assay; LFI = Lequesne's Functional Index; MMP = matrix metalloproteinase; NSAID =nonsteroidal anti-inflammatory drug; NU = normalized units; OA = osteoarthritis; VAS = visual analog scale; WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index.
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Introduction
Osteoarthritis (OA) is the commonest form of inflammatory
joint disease, characterized by articular cartilage degradation
with an accompanying peri-articular bone response [1,2]. OA
affects nearly 21 million people in the USA, accounting for
25% of visits to primary care physicians. It is estimated that
80% of the population will have radiographic evidence of OA
by age 65 years, although only 60% of those will be symptomatic [3]. Clinical manifestations of OA of the knee include
pain in and around the joint, stiffness of the joint after rest,
crepitating on motion and limited joint motion, among others[4]. Current recommendations for managing OA focus onrelieving pain and stiffness and improving physical function as
important goals of therapy [5,6]. Currently available medication regimens for most cases include nonopioid analgesics
such as acetaminophen and nonsteroidal anti-inflammatory
drugs (NSAIDs), including cyclo-oxygenase II inhibitors. These
pharmaceutical agents can reduce both pain and inflammation
quite effectively, but long-term use of NSAIDs has been foundto be associated with enhanced risk for gastrointestinal bleeding, hypertension, congestive heart failure and renal insufficiency, among other adverse effects [7-9]. Because of thehigh incidence of adverse events associated with both nonselective and cyclo-oxygenase II selective NSAID therapy, effective and safer alternative treatments for OA are urgently
needed.
In recent years, the gum resin extracted from the ancient herb
Boswellia serrata has gained much attention as a potent antiinflammatory, anti-arthritic and analgesic agent [10,11]. 3-Oacetyl-11-keto-beta-boswellic acid (AKBA) is the most active
component of Boswellia extract and has been demonstrated
to be a potent inhibitor of 5-lipoxygenase (5-LOX), which is a
key enzyme in the biosynthesis of leukotrienes from arachidonic acid in the cellular inflammatory cascade [12,13].
5-Loxin® is a novel B. serrata extract enriched to 30% AKBA
(US Patent publication no.: 2004/0073060A1). In the carrageenan-induced inflammation model, 5-Loxin® treatment
yielded significant improvement in paw inflammation in albino
Wister rats [14]. Cell based in vitro studies and in vivo experiments conducted in Sprague-Dawley rats suggest that 5Loxin® can inhibit proinflammatory cytokines such as tumour
necrosis factor-α, interleukin-1β (unpublished data, Sengupta
K, Alluri KV, and Golakoti T). Furthermore, Affimatrix gene chipanalysis demonstrates 5-Loxin® can potentially inhibit thetumour necrosis factor-α induced gene expression of matrix
metalloproteinases (MMPs), adhesion molecules such as
intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and mediators of apoptosis in human microvascularendothelial cells [14]. Importantly, extensive acute and dosedependent subchronic safety experiments on rats demonstrate that 5-Loxin® does not exhibit toxic manifestations, evenat a dose 2,000 to 3,000 times higher than the human equivalence dose [15]. In addition, 5-Loxin® does not exhibit geno-

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toxicity in the standard AMES bacterial reverse mutation assay(INTOX, 375, Urawade, Pirangut-Urawade Road, Tal. Mulshi,
Pune – 412108, India; study no. 4477/05).
Although a significant number of clinical study reports support
the anti-inflammatory and anti-arthritic properties of Boswellia
extract [16-19], to the best of our knowledge no reports on theefficacy of AKBA-enriched 5-Loxin® in OA in humans havebeen published. Therefore, in the present double-blind andplacebo-controlled clinical study, we sought to evaluate theefficacy and safety of 5-Loxin® in treatment of OA of the knee.
We assessed the effectiveness of 100 mg/day and 250 mg/
day 5-Loxin® on pain, joint stiffness and mobility in OA
patients. We also explored the effect of 5-Loxin® on the cartilage degrading enzyme MMP-3 in OA patients treated with 5Loxin®.

Materials and methods
Recruitment of patients
This trial was performed at Alluri Sitarama Raju Academy ofMedical Sciences (ASRAM), Eluru, Andhra Pradesh, India
from July 2006 to October 2006 (clinical trial registration
number: ISRCTN05212803). The study protocol was evaluated and approved by the ASRAM Institutional Review Board.
An overview of the clinical study is provided in Figure 1. Briefly,
236 patients out of 823 attending the orthopaedic Outpatients Department of the ASRAM Hospital were selected,
based on the signs, symptoms and radiological changes consistent with OA in the first phase of the screening procedure.
A total of 75 patients suffering for more than 3 months withmedial tibiofemoral OA were selected using inclusion/exclusion criteria summarized in Table 1. All patients signed theInstitutional Review Board approved consent form. Patientswere otherwise healthy, were aged 40 years or older, and had
a diagnosis of OA, fulfilling the American College of Rheumatology classification criteria [4]. After recruitment, the patientswere randomly distributed into three groups; demographic
data and baseline characteristics are summarized in Table 2.

Before study enrollment, patients were required to be taking
an NSAID at prescription strength for at least 30 days or
acetaminophen 1,200 to 4,000 mg/day on a regular basis (atleast 25 of the preceding 30 days) with a history of therapeutic
benefit. Eligibility required patients to meet specific flare criteria upon medication washout. At screening, patients had todemonstrate a visual analog scale (VAS) score between 40
and 70 mm during the most painful knee movement, andLequesne's Functional Index (LFI) score greater than 7 points
after 7-day withdrawal of usual medication.
Study design
A total of 75 selected patients with symptoms of moderate tomild OA were recruited into the study. Each patient was randomly assigned to a treatment group using a randomizationtable generated using validated computer software (RAN-Available online http://arthritis-research.com/content/10/4/R85

Table 1
Inclusion/exclusion criteria
Criteria

Details

Inclusion

Patients must understand risks and benefits of the protocol and be able to give informed consent
Male and female patients aged 40 to 80 years
Females of child-bearing potential must agree to use an approved form of birth control and to have a negative pregnancy test result.
Unilateral or bilateral osteoarthritis of the knee for more than 3 months
Visual analogue scale score during the most painful knee movement between 40 and 70 mm after 7 days of withdrawal of usual
medication
Lequesne's Functional Index score greater than 7 points after 7 days of withdrawal of usual medication
Ability to walk
Availability for the duration of the entire study period

Exclusion

History of underlying inflammatory arthropathy or severe rheumatoid arthritis
Hyperuricaemia (>440 μmol/l) and/or past history of gout
Recent injury in the area affected by osteoarthritis of the knee (past 4 months) and expectation of surgery in the next 4 months
Intra-articular corticosteroid injections within the preceding 3 months
Hypersensitivity to nonsteroidal anti-inflammatory drugs, abnormal liver or kidney function tests, history of peptic ulceration and uppergastrointestinal haemorrhage, congestive heart failure, hypertension, hyperkalaemia
Major abnormal findings on complete blood count, history of coagulopathies, haematological or neurological disorders
High alcohol intake (>2 standard drinks per day)Pregnant, breastfeeding, or planning to become pregnant during the study
Use of concomitant prohibited medication other than ibuprofen
Obesity (body mass index > 30 kg/m2)

CODE; IDV, Gauting, Germany). Treatment allocation
depended only on the time sequence in which patientsentered the study, thus minimizing selection bias. The clinical

trial pharmacist and statistician ensured that treatment codes
remained confidential. The patients were distributed into three

Table 2
Demographic data and baseline characteristics of the patients
Characteristics

Placebo (n = 23)

100 mg/day 5-Loxin® (n = 24)

250 mg/day 5-Loxin® (n = 23)

Sex (male/female; n)

5/18

7/17

8/15

Age (years)

52.43 ± 9.65

52.37 ± 8.37

53.22 ± 8.73

Body weight (kg)61.48 ± 10.69

61.08 ± 10.67

54.84 ± 10.19

Body mass index (kg/m2)

26.05 ± 4.29

25.91 ± 4.94

22.64 ± 4.07

Visual analog score (mm)56.88 ± 12.04

57.05 ± 8.71

55.62 ± 9.26

Lequesne's Functional Index

12.76 ± 2.6

12.1 ± 2.76

12.04 ± 3.03

Pain subscale

38.04 ± 9.7

48.08 ± 14.05

37.17 ± 13.8

Stiffness subscale

33.15 ± 13.3

31.8 ± 17.6

27.7 ± 16.8

Function subscale

41.3 ± 9.6

41.5 ± 11.1

38.6 ± 11.1

WOMAC score

Values are expressed as mean ± standard deviation. WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index.

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Figure 1

Flow chart of the patients who participated in the clinical trial
trial. Evaluations of physical activity and pain scores, serum biochemistry, haematology,
urine biochemistry and proinflammatory cytokines were done at baseline (day 0) and on days 7, 30, 60 and 90 during follow up. Assessments ofmatrix metalloproteinase-3 were done on days 0 and 90 only.

groups: placebo (n = 25); 30% AKBA enriched B. serrata
extract (5-Loxin®) low-dose group (100 mg/day), in which
patients received 50 mg encapsulated 5-Loxin® twice daily (n= 25); and 5-Loxin® high-dose group (250 mg/day), in which
patients received 125 mg encapsulated 5-Loxin® twice daily (n= 25). Patients in the placebo group received two capsules ofsimilar color, taste and appearance but filled only with rice
bran.
Each patient completed a questionnaire, providing details
regarding demographics, medical history and nutritional status, at the baseline evaluation and during the follow-up evaluations on days 7, 30, 60 and 90. At the baseline evaluation,
and at each visit during the 90-day follow up period, allpatients were assessed for pain scores and physical ability.
Various parameters of serum biochemistry, haematology andurine analysis were carried out on each evaluation day. Serumsamples were collected at all evaluation days for proinflammatory modulators. Knee joint synovial fluid was aseptically collected at baseline and at day 90 for evaluation of MMP-3
concentration. Safety was monitored by clinical and laboratory
assessments conducted at study visits and patient-reported
adverse experiences.
Functional disability and pain score evaluation
The investigators assessed the functional disability reportedby the patients at baseline and on each follow-up visit (days 7,
30, 60 and 90). Questionnaire-based assessment of pain,
stiffness and physical function were done using the Western
Ontario and McMaster Universities Osteoarthritis Index
Page 4 of 11(page number not for citation purposes)(WOMAC) index [20], LFI [21] and VAS [22]. The WOMAC
index produces scores for three subscales: pain, stiffness andphysical function. The pain, stiffness and function subscales ofthe WOMAC were converted to a 0 to 100 normalized units(NU) scale [23]. The pain subscale was the average of the first
five questions of WOMAC and measured using the NU scalefrom 0 mm ('no pain') to 100 mm ('extreme pain') for eachquestion. The stiffness subscale was the average of questions
6 and 7, measured using the NU scale from 0 mm ('no stiffness') to 100 mm ('extreme stiffness') for each question. The
physical function subscale was the average of questions 8
through 24 of the WOMAC and measured by NU scale from0 mm ('no difficulty') to 100 mm ('extreme difficulty') for eachquestion. Analyses of these end-points were based upon thetime-weighted average change from baseline over 90 days.
Haematological and biochemical evaluations
For assessment of safety of 5-Loxin®, several parameters were
evaluated in serum, urine and whole blood of all patients ateach visit of the study duration (Table 3). Serum biochemicalparameters and haematological parameters were measured
using the automated analyzer HumaStar 300 (Human, Wiesbaden, Germany) and the haematological counter Humacount(Human), respectively. The urine analysis was carried out by
microscopy and by using UroColor™10 Dip Sticks (Standard
Diagnostics, Kyonggi-do, Korea).

Available online http://arthritis-research.com/content/10/4/R85

Table 3
Parameters tested in serum biochemistry, haematology andurine analysis
Analysis

Details

Serum biochemistry

Albumin
Alkaline phosphataseTotal bilirubin
Cholesterol
Creatinine
Creatine kinase-N-acetyl cysteine

reader (Bio-Rad, Hercules, CA, USA). A standard curve was
generated by plotting the optical density at respective known
concentration of MMP3. The sensitivity of MMP-3 detection
ELISA kit is 9 pg/ml.
Rescue medication
Patients were prescribed ibuprophen 400 mg tablets (maximum 400 mg thrice daily; total 1,200 mg) as rescue analgesia
on days 7, 30 and 60, based on pain intensity reported to thestudy physician by the patient. However, the patients were
instructed not to take medicine at least 3 days before each
evaluation. No other OA interventions were allowed during thestudy period.

GlucoseHigh-density lipoprotein
Low-density lipoprotein
Potassium
Serum glutamic oxaloacetate transaminase
Serum glutamate pyruvate transaminase
Triglycerides
Urea
Haematology

Total count and differential count
Erythrocyte sedimentation rate
Haemoglobin
Platelet count
Mean corpuscular volume
Mean corpuscular hemoglobin

Urine analysis

Specific gravity
pH
Albumin
Bile salt
Bile pigment
GlucoseRed blood cell count
Ketone bodies

Assessment of matrix metalloproteinase-3 in synovial
fluids
MMP-3 (R&D Systems, Minneapolis, USA) were quantitatively
measured by ultrasensitive ELISA method. Assay procedures
adhered to the protocol supplied by the manufacturers. Briefly,
synovial fluid samples were incubated on capture antibody
coated 96-well microplates. Specifically bound antigen was
detected by appropriate biotinylated detection antibody andwas probed with horseradish peroxidase enzyme. The specificimmune reaction was detected by substrate solution and thecolour development was read with the help of micro-plate

Statistical analysis
We performed detailed statistical analyses using SAS software to evaluate the efficacy of two doses of 5-Loxin® in comparison with the placebo group in terms of improvement in
pain and physical ability scores, and to assess biomolecularmarkers at baseline and days 7, 30, 60 and 90 of treatment.
Pair-wise changes were examined by carrying out a least significant difference test for all possible pairs. The significanceof the effects of the treatment groups was compared by using
one-way analysis of variance (ANOVA) followed by Tukey's
multiple comparison tests. Results with P < 0.05 are considered statistically significant.

This is a three-arm (two doses of 5-Loxin® and placebo), randomized, double-blind, placebo-controlled, single-centre trial
conducted over 90 days. The trial's primary objective was todetermine the effects of 5-Loxin® on pain, physical function
and joint stiffness. For power calculations, the estimates forvariability and assumed mean changes for each treatmentgroup were based on results from previous placebo-controlled
studies of celecoxib, etoricoxib and rofecoxib conducted inpatients with OA [24-27]. We believe that an intervention thatgives an average improvement of mean change + 1 standard
deviation, rather than mean change only, will provide results ofgreater significance [28]. Our trial is designed to have morethan 80% power to detect a situation in which either active
drug dosage yields an improvement to at least mean change+ 0.9 standard deviation, under a conservative assumption,
and we tested differences between groups in mean improvement using ANOVA (α = 0.05, two-sided). With 25 patients
per group, we would have a 93% chance of observing at least
one example of any side effect occurring in 10% or more of thepatient population at a specific dosage.

Results
Baseline characteristics
Descriptive statistics comparing demographic variables, baseline disease characteristics and baseline outcome measures(that is, WOMAC pain, function and stiffness subscores) areprovided in Table 2. Overall, the treatment groups receiving 5Loxin® low dose (100 mg/day, n = 25), 5-Loxin® high dose

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(250 mg/day, n = 25) and placebo (n = 25), were similar withrespect to sex, age, Body Mass Index and pain severity (Table2). The patients were randomly distributed into three groups.
Although there are some differences in baseline characteristics of gender, body mass index and WOMAC scores, thoseare statistically not significant.
Clinical efficacy
We compared the scores between the treatment groups
obtained at day 90. Both doses of 5-Loxin® conferred clinicallyand statistically significant improvements in pain scores andphysical ability scores in OA patients between baseline andday 90 (Table 4).

Tukey's multiple comparison test revealed statistically significant improvements by 48.83% (P < 0.001), 23.79% (P <0.036) and 39.61% (P = 0.009) in VAS, LFI and WOMAC
pain scores, respectively, in the low-dose (100 mg 5-Loxin®)group versus the placebo group (Table 4). Improvements by
42.5% (P = 0.120) and 28.62% (P = 0.100) score in
WOMAC stiffness and WOMAC functional ability, respectively were also achieved in the low-dose group (Table 4).
In comparison with the placebo group, the high-dose (250 mg
5-Loxin®) group also exhibited statistically significant improvements in all parameters (Table 4). The high-dose group
showed improvements by 65.94% (P < 0.001), 31.34% (P <0.017), 52.05% (P < 0.001), 62.22% (P = 0.014) and
49.34% (P = 0.002) in VAS, LFI, WOMAC pain, WOMAC

Table 4Student's t-test (paired) analyses for comparison of the scores obtained from the low-dose and high-dose 5-Loxin® groups at day 90nBaseline
Mean

Day 90SD

Mean

95% CI (versus placebo)P

SD

Visual analogue scale score
Placebo

23

56.88

12.04

41.76

15.98

100 mg 5-Loxin®

24

57.05

8.71

21.37

7.13

-27.67, -13.11<0.0001<0.05

250 mg 5-Loxin®

23

55.62

9.26

14.22

6.8

-34.94, -20.19<0.0001

Placebo

23

12.76

2.6

10.19

3.24

100 mg 5-Loxin®

24

12.1

2.76

7.78

4.61

-4.74, -0.07<0.0001

250 mg 5-Loxin®

23

12.04

3.03

7

3.5

-5.19, -1.18<0.0001

Placebo

23

38.04

2.03

31.74

2.58

100 mg 5-Loxin®

24

42.08

2.93

19.17

3.55

-21.33, to -3.83<0.0001

250 mg 5-Loxin®

23

37.17

2.88

15.22

2.50

-23.78 to -9.28<0.0001

Placebo

23

33.15

2.73

24.45

2.37

100 mg 5-Loxin®

24

31.77

3.61

14.06

3.71

-38.87, -6.85<0.0001

250 mg 5-Loxin®

23

27.72

3.44

9.24

2.07

-43.35, -17.45<0.0001

Lequesne's Functional Index
0.031

WOMAC pain subscale
0.1212WOMAC stiffness subscale
0.2983

WOMAC function subscale
Placebo

23

41.30

2.02

34.07

1.09

100 mg 5-Loxin®

24

41.48

2.31

24.32

4.28

-18.64, -0.82<0.0001

0.1048250 mg 5-Loxin®

23

38.56

2.32

17.267

1.98-21.39, -12.23<0.0001

Placebo

15

902.1

275.6

928.5

216.020.4886

100 mg 5-Loxin®

16

893.6

270.1

637.2

224.5

<0.0001

250 mg 5-Loxin®

14

926.9

270.5

497.5

167.5

<0.0001

MMP-3 (ng/ml)

CI, confidecne interval; MMP, matrix metalloproteinase; WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index.

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stiffness and WOMAC functional ability scores, respectively.
Student's t-test analyses revealed that MMP-3 concentration(P < 0.0001) in synovial fluids and VAS pain scores (P =0.001) were significantly lower in the high-dose group than in
the low-dose group. It is worth noting that both low-dose andhigh-dose treatment groups exhibited improvement in painscores and physical ability scores as early as 7 days after thestart of treatment, and these indices continued to improvethroughout the 90 days of treatment (Figure 2). After 7 days,
the low-dose and high-dose treatment groups exhibited
10.09% (P = 0.05) and 12.18% (P = 0.02) reductions in VAS,
respectively, compared with the placebo group. In addition,
WOMAC physical ability also improved by 14.38% (P < 0.01)
after 7 days of treatment with high-dose 5-Loxin® (Figure 2).

Figure 2

Function, pain and stiffness scores.
scores Presented are the mean scores for (a) visual analog scale, (b) Lequesne's Functional Index, (c) Western Ontario
and McMaster Universities Osteoarthritis Index (WOMAC)-pain, (d) WOMAC-stiffness, and (e) WOMAC-functional ability in the low-dose (100 mg/
day 5-Loxin®) and high-dose (250 mg/day 5-Loxin®) groups and placebo group at different time points, as indicated. Each bar represents mean concentration ± standard deviation. In comparison with placebo, the change in scores in the treatment groups was tested for significance using Tukey's
multiple comparison test; asterisk indicates statistical significance.

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Assessment of MMP-3 concentration
OA is a degenerative joint disorder; in molecular pathogenesis
of OA, proteolytic enzymes such as MMPs are highly elevatedin body fluids such as serum and synovial fluids, which cause
potential damage in cartilage tissues [29]. Therefore, in orderto determine whether 5-Loxin® treatment can normalize the
MMP level, we evaluated the concentration of MMP-3 in synovial fluids collected from the patients. Figure 3 illustrates
changes in MMP-3 concentration in synovial fluid samples collected from patients of all groups included in the study. Pairwise comparisons indicated that at the end of the study both
treatment groups exhibited highly significant reductions in
MMP-3 in synovial fluid. Compared with the placebo group,
the low-dose (100 mg) and high-dose (250 mg) 5-Loxin®
groups showed 31.37% (P = 0.002) and 46.4% (P < 0.001)
reductions in MMP-3 concentration, respectively. Regular ttests revealed that high-dose 5-Loxin® treatment significantly
reduced (P < 0.0001) synovial MMP-3 concentration whencompared with the low-dose group (Table 4). Compared withbaseline, the Wilcoxon sign-rank-sum test revealed that the
low-dose and high-dose groups conferred 28.69% (P =0.0013) and 46.33% (P < 0.0001) reductions in synovial fluid
MMP-3 concentration at day 90. The MMP-3 level in the placebo group remained virtually unchanged at day 90 comparedwith baseline.
Biochemical evaluations
As a part of the safety evaluation, laboratory tests were performed to evaluate different biochemical parameters in serumand urine, and haematological parameters. The tested paramFigure 3

Reduction of Synovial MMP-3 levels
levels. Presented are the matrix metalloproteinase (MMP)-3 levels in synovial fluid collected from 5-Loxin®
treated and placebo patients with osteoarthritis. At day 90 there wasno significant change in MMP-3 concentration in the placebo groupcompared with baseline. In comparison with the placebo group, at theend of the study the groups receiving100 mg/day and 250 mg/day 5Loxin® showed 31.37% (P = 0.002) and 46.4% (P < 0.001) reductions in MMP-3 concentration, respectively. Change in MMP-3 concentration between the active treatment groups was not significant (P =0.213). Each bar represents mean concentration of MMP-3 (ng/ml synovial fluid) ± standard deviation.

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eters in serum biochemistry, and haematological and urine
analysis are summarized in Table 3. The significance of the differences between baseline and 90 days was tested by using
repeated measures ANOVA. The F ratio is considered significant if P < 0.05. Although minor changes were observed insome of the parameters, they remained within the normal laboratory range. Statistical analyses of these parameters did not
identify any statstically significant changes. Similarly, haematological and urinary parameters also exhibited no significant
changes in the active treatment groups compared with placebo (data not shown). These findings further demonstrate thesafety of 5-Loxin® in humans.
Adverse events
During the course of the 90-day study period, some minor
adverse events were noted: diarrhoea, nausea, abdominal
pain, mild fever (up to 37.5°C [99.5°F]) and general weakness.
The patients who reported these minor events were distributed evenly throughout the placebo and active treatmentgroups. The numbers of minor adverse events reported by thepatients during the study are summarized in Table 5.
Dropouts
Five patients (one from the low-dose [100 mg 5-Loxin®] group,and two each from placebo and high-dose [250 mg 5-Loxin®]group) were excluded from the study because they were suffering from a nonfatal viral infection during the course of study.

Discussion
To the best of our knowledge, this is the first clinical study toevaluate the efficacy of 5-Loxin® in OA. This study also provides important information regarding the possible molecularmechanisms of action of an anti-inflammatory compound of
herbal origin in the treatment of OA. We demonstrated that 5Loxin® has potential efficacy in terms of reducing pain andimproving the physical ability of OA patients. A novel aspect ofthe present study is its evaluation of the effect of 5-Loxin®
treatment on the cartilage degrading enzyme MMP-3 in synovial fluid from OA patients. In this 90-day clinical study, we also
assessed the safety of 5-Loxin® in OA patients.
Pain, stiffness of joints, reduced joint movement and physicaldisability are the major clinical manifestations of OA [1,30].
Our study demonstrates that 5-Loxin® potentially improvespain, joint stiffness and physical function in OA patients (Figure 2). The statistical analyses revealed that the improvements
in physical parameters and reductions in synovial MMP-3 levels were significantly decreased in the treatment groups ascompared with the placebo group (Figures 2 and 3). In addition, in order to check improvements in the treatment groups,
we compared the data for all parameters between the baselineand day 90. Paired t-test revealed that both treatment groups
had highly statistically significant improvements in all parameters. Comparing the high-dose versus the low-dose groups atday 90, significant differences were observed only for VAS forAvailable online http://arthritis-research.com/content/10/4/R85

Table 5
Incidence of adverse events
Adverse events

Placebo (n = 23)

100 mg/day 5-Loxin® (n = 24)

250 mg/day 5-Loxin® (n = 23)

Diarrhoea

322

Nausea1

3

2

Vomiting

11

1

Abdominal pain

4

2

2

Pedal edema

0

1

0

Itching2

1

4

General weakness

2

4

2

Constipation

0

0

1

Mild fever (up to 37.5°C)

1

2

2

Stomach burn

4

0

3

Allergya

3

0

1

Headache001

Miscellaneousb

9

2

6

Sum of events

30

18

27

aAllergy includes redness of skin and sinus allergy. bMiscellaneous group includes body pain, loss of hair, chest pain and eye infection.

pain and synovial fluid MMP3 concentration (Table 4). Thisfinding suggests that the higher dose of 5-Loxin® has better
therapeutic efficacy against OA. We observed that, in comparison with baseline, there were downward trends in VAS score,
LFI and WOMAC scores in the placebo group. We believethat this might be partly attributable to the placebo effect[31,32] manifesting while patients completed thequestionnaires, and partly due to the consumption of ibuprophen as rescue medication by more patients in the placebogroup during the study. Interestingly, at the end of the study
we found that the total number of participants requesting rescue medication was 16.7% and 72.2% higher in the placebogroup than in the groups receiving 100 mg and 250 mg 5Loxin®, respectively.
An important observation in the present study is that 250 mg/
day 5-Loxin® had a significant effect in lowering VAS score by
12.18% (P = 0.02) and WOMAC function score by 14.38%(P < 0.01) in OA patients as early as 7 days after the start oftreatment. These findings therefore indicate that 5-Loxin® confers prompt and significant pain relief and improvement inphysical ability in OA patients. Existing information reveals that
glucosamine usually takes 6 weeks to achieve significant beneficial effect in terms of pain relief in OA [33]. In addition, a randomized, double-blind, placebo-controlled trial [34] showedthat 4,000 mg milk protein concentrate per day (Microlactin™;
Stolle Milk Biologics Inc., Cincinnati, OH, USA) yields significant improvement in WOMAC score after 2 weeks of treatment on OA patients.

In OA patients, MMPs such as MMP-3 are over-expressed andabundant in fluids of the synovial cavity, and cause degeneration of cartilage tissue [35,36]. 5-Loxin® was able to reduce
the elevated MMP-3 level in synovial fluid. This finding indicates that reduction in synovial fluid MMP-3 level by 5-Loxin®
is consistent with improvements in abnormal joint physiology
in OA. Therefore, these data together demonstrate that 5Loxin® potentially has effects in terms of reducing the pain andimproving physical ability and joint health; it is most likely thatthese improvements occur through downregulation of cartilage degrading enzymes such as MMP-3.
Earlier, in acute and subchronic toxicity studies we demonstrated that 5-Loxin® (a 30% enriched product of AKBA, whichis the active component of Boswellia extract) is safe and nontoxic in rats [15]. Additionally, 5-Loxin® did not exhibit mutagenicity in the standard AMES test (INTOX; study no. 4477/
05). In the present study biochemical parameters in serum,
haematological parameters and urine analysis (Table 3) also
did not reveal any major adverse effect of the test compound
in OA patients. Taken together, these observations further
demonstrate that 5-Loxin® is potentially safe in the treatment
of OA in humans.

Conclusion
In summary, the present study provides the evidence in support of the potential efficacy and safety of 5-Loxin® in patientswith OA: 5-Loxin® significantly improved joint function andexhibited better therapeutic efficacy at 250 mg/day than at
100 mg/day; it reduces pain rapidly, as early as after 1 weekof treatment; it reduces levels of the cartilage degrading
Page 9 of 11(page number not for citation purposes)

Arthritis Research & Therapy

Vol 10 No 4

Sengupta et al.

enzyme MMP-3 in synovial fluid; and, most importantly, 5Loxin® is safe for human consumption, even long term. This
study provides important information about the efficacy andsafety of 5-Loxin® in the treatment of OA, which may be usefulin promoting 5-Loxin® as a promising alternative therapeuticstrategy that may be used as a nutritional supplement against
OA.

8.

Competing interests

12.

This study is funded by Laila Impex R&D Center, India. KS, TG
and KVA are employee of Laila Impex Research Centre, Vijayawada, India. ARS and SM are employee of ASRAM, Eluru,
India. KVSS (SV University, Tirupati, India), DD (University ofConnecticut, Storrs, CT, USA) and SPR (University of California Davis Medical Center, Davis, CA and VA Medical Center
Sacramento, CA, USA) are consultants for the Laila Impex
Research Center.

9.
10.
11.

13.

14.

15.

Authors' contributions
KS contributed to the design of the project and data analysis,
and was primarily responsible for writing the manuscript. KVA
contributed to the design of the project, patient recruitment
and management, and data collection. ARS and AM worked
with patients to obtain informed consent, conducted clinicalevaluations, took samples and evaluated therapeutic responseof 5-Loxin®. TG contributed as the study coordinator andhelped to review the manuscript. KVSS and DD helped in clinical data analysis. SPR helped in designing the study, conducting data analysis and writing the manuscript.

Acknowledgements
This study is funded by Laila Impex R&D Center, India.

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