Alexandria Journal of Medicine (2014) xxx, xxx–xxx

Alexandria University Faculty of Medicine

Alexandria Journal of Medicine

www.sciencedirect.com

Efficacy of glucosamine sulfate in lowering serum level

of interleukin-1b in symptomatic primary knee

osteoarthritis: Clinical and laboratory study

Enas M. Shahine a,*, Abeer S. Elhadidi b

a Physical Medicine, Rheumatology and Rehabilitation Department, Faculty of Medicine, Alexandria University, Egyptb Clinical Pathology Department, Faculty of Medicine, Alexandria University, Egypt

Received 27 July 2013; accepted 29 January 2014

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KEYWORDS

Glucosamine sulfate;

Interleukin-1b;Primary knee osteoarthritis

Corresponding author. Tel.-mail address: enas2002@ya

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edicine.

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tp://dx.doi.org/10.1016/j.ajm

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steoarthritis: Clinical and laborat

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hahine EM

ory study,

Abstract Objective: To identify the effect of a-D glucosamine sulfate (GS) on serum level of inter-

leukin-1b (IL-1b) in patients with symptomatic primary knee OA.

Methods: Sixty patients (mean age = 52.2 ± 8.6 years), fulfilling the American College of Rheu-

matology criteria of idiopathic knee OA, were randomized to receive either 1500 mg a-D GS and

1200 mg Ibuprofen (group I), or only 1200 mg Ibuprofen (group II) daily for 12 weeks. Patients

were followed up by the Visual Analogue knee pain Scale (VAS), Western Ontario and McMaster

Universities Osteoarthritis Index (WOMAC) functional index and quantitative detection of IL-1bserum levels. Reference serum level of IL-1b was determined in 20 matched healthy volunteers.

Results: Group I showed significant progressive improvement in pain VAS and total WOMAC

scale, pain, stiffness and function subscales during the follow up visits compared to group II. At

baseline, both groups had significantly higher IL-1b serum level than the control group. On follow

up group I showed significant progressive reduction in IL-1b serum level with a final level that was

significantly lower than group II and was not significantly higher than the control group. In group

II the reductions in IL-1b serum level did not reach the level of statistical significance and the final

level persisted significantly higher than that of the control group.

Conclusion: Adding a-D GS to treatment of primary symptomatic knee OA could relieve symp-

toms, improve function and affect some of the disease mechanisms.ª 2014 Alexandria University Faculty of Medicine. Production and hosting by Elsevier B.V. All rights

reserved.

8117577; fax: +20 3 5437245.(E.M. Shahine).

xandria University Faculty of

g by Elsevier

y Faculty of Medicine. Production

.006

, Elhadidi AS Efficacy of glucosamine

Alex J Med (2014), http://dx.doi.org/1

1. Introduction

Osteoarthritis (OA) is the most common chronic synovial joint

disorder and is generally characterized by articular cartilagedeterioration. Although traditionally considered a non-inflam-matory joint disease; it is now well appreciated that inflamma-

tory mediators are produced by articular tissues in OA and

and hosting by Elsevier B.V. All rights reserved.

sulfate in lowering serum level of interleukin-1b in symptomatic primary knee

0.1016/j.ajme.2014.01.006

2 E.M. Shahine, A.S. Elhadidi

have been implicated in disease pathogenesis.1 There is convinc-ing evidence that interleukin-1b (IL-1b) and other inflammatorymediators, synthesized locally by synovial cells and chondro-

cytes, play a pivotal role in driving the pathways associated withOA pathogenesis and promote cartilage degradation.2 IL-1b ismarkedly increased in chondrocytes, synovial membrane and

synovial fluid of osteoarthritic patients with a positive correla-tion between its expression and the severity of chondral damageand increasing grades of OA.3–5 IL-1b initiates the number of

events leading to cartilage damage. This process involves inhibi-tion of biosynthesis of proteoglycans in addition to promotionof their degradation. IL-1b has a repressive effect on glucurono-syltransferase involved in a key step in glycosaminoglycans bio-

synthesis,6 while it upregulates metalloproteinases (MMP),inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2), Prostaglandin E2 and IL-6.7 Thus, IL-1b may be a

therapeutic target in OA. The conservative treatment of OA isstill limited to a few classes of medications, such as analgesicdrugs, nonsteroidal anti-inflammatory drugs (NSAIDs), COX-

2 selective inhibitors and viscosupplementation, which provideprimarily symptomatic relief but have not yet been demonstratedto interfere with the progression of the disease.8 D-Glucosamine,

the biologically active formof glucosamine, serves as ametabolicprecursor in the synthesis of the proteoglycans and glycosamino-glycans of cartilage matrix and synovial fluid. It possesses anti-inflammatory efficacies against OA in animals and humans

and is suggested to delay damage of cartilage and long-term pro-gression of OA as a disease modifying anti-OA drug through itschondroprotective effect.9 It is believed that oral consumption of

large quantities of glucosamine elevates its intra-articular con-centrations and thereby enhances synthesis of the articular carti-lage matrix. Despite the increased use of glucosamine sulfate

(GS) in the treatment of OA, the mechanisms accounting forits in vivo antiarthritic activity are still unclear. The aim of thepresent study was to identify the suppressive effect of a-D gluco-

samine sulfate on serum level of IL-1b in patients with symptom-atic primary knee OA as one of mechanisms which may affectthe disease progression.

Study design: Ibuprofen-controlled, a-D glucosamine sul-

fate study to evaluate its efficacy as a treatment for symptom-atic primary knee OA.

2. Methods

Sixty patients with age range of 46–62 years (52.2 ± 8.6), ful-filling the American College of Rheumatology criteria for idi-

opathic OA of the knee,10 were included in the study. Knee OAwas documented by standardized weight bearing posteroante-rior plain radiographic examination. Severity of OA was

graded according to the Kellgren and Lawrence grading scale(KL).11 Evaluation of each joint compartment was performedwith regard to the presence of joint space narrowing,osteophyte and subchondral sclerosis. Scoring of OA severity

impact on patient’s functional status was determined byLequesne’s algofunctional index (LFI).12 Patients were eligibleto be included if they had knee pain on most days of the pre-

ceding month and in need for daily NSAIDs, had KL grade 2or 3 and score >4 to 611 on LFI. By full history taking, thor-ough clinical examination and specified laboratory investiga-

tions; secondary knee OA, polyarticular or suspectedinflammatory joint diseases, intra-articular or systemiccorticosteroid use within 3 months prior to study, diabetes

Please cite this article in press as: Shahine EM, Elhadidi AS Efficacy of glucosamine

osteoarthritis: Clinical and laboratory study, Alex J Med (2014), http://dx.doi.org/1

mellitus and administration of anticoagulants or antihyperten-sive were excluded. An informed written consent was obtainedfrom all participants prior to enrollment in the study which

was approved by the Local Medical Ethics Committee. Afterenrollment, demographic data were recorded for each patient.All patients underwent a complete history taking and clinical

examination and were randomized to either study group by acomputer generated randomization code. Thirty patients(group I) were allocated to receive 3 capsules of 500 mg a-Dglucosamine sulfate, an active isomer of GS in addition to1200 mg Ibuprofen daily, while 30 patients (group II) receiveda daily dose of 1200 mg Ibuprofen only. Patients were followedup until completion of a 12-week treatment course. In initial

and 3 follow up visits (V1, V2, V3 and V4: baseline, weeks 4,8 and 12, respectively) all the following were conducted: (1) Pa-tients were asked to rate the severity of knee pain on a 0–

100 mm Visual Analogue Scale (VAS) (0; no pain-100; mostsevere pain). (2) Western Ontario and McMaster UniversitiesOsteoarthritis Index (WOMAC) functional index was

assessed.13 Assessment was carried for the total index andfor the joint pain (5 questions), stiffness (2 questions), andlimitation of physical function subscales (17 questions) each

question scored on a scale from 0 to 4, with 0 indicating noneand 4 indicating extreme; therefore 20, 8, 68 and 96 points, arethe worst possible severity scores for pain, stiffness, limitation ofphysical function and total index, respectively. (3) Quantitative

detection of IL-1b serum levels using commercially availableenzyme linked immunosorbent assay (ELISA) quality-vali-dated kit for human (ebioscience. North America, Europe/

International).14 Additionally, IL-1b serum level was deter-mined in a control group of 20 age and sex matched healthyvolunteers who had no clinical signs or symptoms of any

arthritis and had KL radiographic score <1. Authors werenot blind to group assignment.

Compliance to medications was determined by asking the

patients about missed doses. Safety was investigated by record-ing the occurrence of adverse events.

2.1. Statistical analysis

Data were analyzed using SPSS version 17. Data were summa-rized using descriptive statistics: mean ± standard deviation(±SD). The statistical analysis was performed using paired

sample t-tests for intra-group comparisons. While independentsample t-tests was used for inter-groups comparisons. A prob-ability value (P value) 6 0.05 was considered statistically

significant.

3. Results

Of the 153 patients who underwent screening, 60 patients wererandomized into the study groups. All patients in the two stud-ied groups completed the 12-week treatment course without

withdrawals or drop outs. Knee OA was moderate to severeat enrollment according to KL grading and LFI scores. Mostpatients in the 2 groups (63.3% of group I and 66.6% of groupII) had bilateral knee OA. Table 1 demonstrates the compara-

ble baseline characteristics of the two studied groups. Patientsin both groups were comparable regarding KL grading, LFIscores, pain VAS as well as total WOMAC OA scale and its

3 subscales (P > 0.05). The mean age of the control group

sulfate in lowering serum level of interleukin-1b in symptomatic primary knee

0.1016/j.ajme.2014.01.006

Table 1 Patients’ demographic and clinical characteristics in the two studied groups at baseline.

Group I (n= 30) Group II (n= 30) p

Age of patients (years) 52.7 ± 7.9 51.6 ± 9.4 .528

Gender (man/woman) 8/22 (men = 26.6%) 10/20 (men = 33.3%) .703

BMI (kg/m2) 35.0 ± 6.2 35.9 ± 5.6 .706

Disease duration (months) 6.1 ± 5.1 5.0 ± 6.8 .258

Number of symptomatic knees 5 right, 6 left, 19 bilateral 6 right, 4 left, 20 bilateral .263

KL score 2.7 ± 0.7 2.5 ± 0.8 .980

LFI 9.0 ± 1.2 8.8 ± 2.1 .534

Pain VAS (mm) 60.5 ± 15.7 69.0 ± 13.3 .073

WOMAC OA index total scale 54.5 ± 10.5 53.3 ± 10.3 .719

Pain subscale 11.3 ± 4.1 10.9 ± 3.7 .753

Stiffness subscale 7.0 ± 2.3 6.9 ± 1.6 .818

Function subscale 36.2 ± 6.8 35.5 ± 8.2 .772

IL-1b (pg/ml) 6.2 ± 0.6 6.0 ± 0.6 .482

Except when indicated otherwise, values are the mean ± SD.

P is significant if <0.05.

Efficacy of glucosamine sulfate in lowering serum level of interleukin-1b in symptomatic primary 3

was 49.4 ± 9.2 years. They were 7 men (35%) and 13 women.Their mean BMI was 33.9 ± 5.2 kg/m2. The mean IL-1b ser-

um level in the control group was 5.4 ± 0.5 pg/ml.Group I showed significant progressive and constant

improvement in pain VAS and total WOMAC scale, pain,

stiffness and function subscales during the 3 follow up visitscompared to group II. Additionally, the final change in thepain VAS and total WOMAC scale, pain, stiffness and func-

tion subscales scores showed significant differences in favorof group I where the mean pain VAS decreased from60.5 ± 15.7 mm at V1 to 18.2 ± 13.1 mm at V4 (70% reduc-tion) in group I and from 69.0 ± 13.3 mm at V1 to

42.5 ± 14.7 mm at V4 (38.5% reduction) in group II(P < 0.001) and the mean total WOMAC score decreasedfrom 54.5 ± 10.5 at V1 to 29.9 ± 8.6 at V4 (45.4% reduction)

in group I and from 53.3 ± 10.3 at V1 to 45.2 ± 9.8 at V4(15% reduction) in group II (P < 0.001) (Table 2).

At V1, group I and group II had significantly higher mean

IL-1b serum level than the control group (P = .001 and .003;respectively). On subsequent follow up (V2, V3 and V4), groupI showed significant progressive reduction in mean IL-1b ser-um level (P = .002, .033 and .002; respectively). The final

change in IL-1b serum level at V4 was significantly lower thanV1 level in group I (P = .001) and was significantly lower thanV4 level in group II (P = .001) (Tables 2 and 3). Additionally,

it was not significantly higher than the control group’s level(P = .073). In group II there were reductions in mean IL-1b

Table 2 Change in the VAS, WOMAC OA index scores and seru

baseline (V1) values.

Measure Group I (n = 30)

Difference mean ± SD

VAS 42.2 ± 15.6

WOMAC total scale 24.6 ± 7.3

WOMAC pain subscale 7.6 ± 3.2

WOMAC stiffness subscale 4.1 ± 2.1

WOMAC function subscale 12.9 ± 6.9

IL-1b 1.0 ± 0.7

* P is significant if <0.05.

Please cite this article in press as: Shahine EM, Elhadidi AS Efficacy of glucosamine

osteoarthritis: Clinical and laboratory study, Alex J Med (2014), http://dx.doi.org/1

serum level during V2, V3 and V4 but did not reach the levelof statistical significance (P = .382, .360 and .358; respec-

tively) and its final V4 level persisted significantly higher thanthe level of the control group (P = .017).

Compliance to the study medication and safety was good

without statistically significant differences in the proportionor pattern of adverse events between both groups. The mostfrequently reported complaints consisted predominantly of

transient episodes of abdominal pain (5%), dyspeptic symp-toms (12%), nausea and diarrhea.

4. Discussion

Data of the present study showed that 12-week oral adminis-tration of a-D GS and NSAID could significantly improve pri-mary knee OA pain, stiffness, limitation of function and lower

the elevated serum level of IL-1b throughout the study com-pared to oral administration of NSAID solely for the sametreatment period. Such results were obtained by assessment

using the pain VAS and WOMAC index that is, the mostwidely used algo-functional index for knee OA. Therefore, a-D GS could be classified as a symptom-modifying drug in

OA. The main finding of this study is that a-D GS could reducethe increase of IL-1b serum level in patients with primary kneeOA. Consistent with this data, an experimental study15 showed

that reduction of IL-1b level occurred not only in the joints,but also in the serum of rats with adjuvant-induced arthritis

m level of IL1-b in each group at week 12 (V4) compared with

Group II (n= 30)

P Difference mean ± SD P

.000* 26.5 ± 17.3 .000*

.000* 8.1 ± 7.5 .000*

.000* 3.4 ± 2.8 .000*

.000* 2.1 ± 2.2 .000*

.000* 2.5 ± 7.4 .144

.000* .18±.3 .149

sulfate in lowering serum level of interleukin-1b in symptomatic primary knee

0.1016/j.ajme.2014.01.006

Table 3 Comparison between the two studied groups regarding serum level of IL-1b at weeks 4, 8 and 12 follow up visits.

Measure Group I (n= 30) mean ± SD Group II (n= 30) mean ± SD P

V2-IL1-b (pg/ml) 5.7 ± 0.6 5.9 ± 0.6 .300

V3-IL1-b (pg/ml) 5.4 ± 0.9 5.9 ± 0.5 .032*

V4-IL1-b (pg/ml) 5.4 ± 0.6 5.9 ± 0.3 .001*

Values are the mean ± SD.* P is significant if <0.05.

4 E.M. Shahine, A.S. Elhadidi

fed on combination of GS and chondroitin sulfate. Cytokine

analysis revealed increased inflammatory proteins in sera ofOA patients compared to the control, including IL-1b, IL-1receptors antagonist and IL-6. The levels of these cytokines

were higher in OA synovial fluid than sera, consistent withtheir origin from joint tissue, indicating that local inflamma-tion within OA joint tissues can be reflected in serum. Theabnormal production and leakage of inflammatory cytokines

into serum are analogous to that observed for biomarkers ofjoint tissue synthesis and degradation.16 On the contrary, ser-um concentration of IL-1b was found to be below detection

limit in OA patients in another study.17 This contradictionmight be explained by the fact that inflammatory cytokinesin OA joints traffic through synovium and out into circulation,

so they may or may not be present at higher than normal levelsin sera of OA patients according to the magnitude of joint tis-sue inflammation. Participants in the present study had moder-

ate to severe knee OA at enrollment. It was hypothesized thatthe effects of GS are better realized in patients with moderateto severe OA, which have greater involvement of IL-1b.18 It isnot clear whether GS can modulate the intra-articular synthe-

sis of IL-1b so it can lower its serum level or has another sys-temic mechanism in this regard. Future studies are needed toidentify the mechanism responsible for systemic effect of GS

in lowering serum level of IL-1b in patients with primary kneeOA.

Short-term studies designed to describe the pattern of the

symptomatic effect of GS, showed a significantly better effectin decreasing pain in patients with primary knee OA comparedto placebo therapy and conventional NSAID use in the first4 weeks of treatment, with improvement in pain and function

up to 40–50% relative to basal conditions within 12 weeks.19,20

The rapid effects on symptoms observed for short treatmentcourses are better explained by the anti-inflammatory effect

of GS. In human osteoarthritic chondrocytes, it was foundthat GS blocks inflammatory signaling and inhibits the synthe-sis of proinflammatory mediators stimulated by IL-1b through

a nuclear factor jB-dependent mechanism.21 Glucosamine sul-fate suppresses IL-1b induced phospholipase A222,23 andCOX-2 expression and activation,24 iNO22–24 and reactive oxy-

gen species synthesis.23 It also reduces the intra-articular con-centration of IL-1b and tumor necrosis factor-a.15,25

The long-term effects of GS in primary knee OA arethought to be related to its effect on cartilage. Pharmacoki-

netic studies have shown that, crystalline GS is bioavailableboth systemically and in articular cartilage after oral adminis-tration.26,27 Glucosamine is incorporated into the components

of the glycosaminoglycan chains by the chondrocytes,28 stimu-lates the synthesis of physiological proteoglycans,26 reversessome of the negative effects of IL-1b on cartilage metabolism.

There are many reports suggesting that GS reverses the de-

Please cite this article in press as: Shahine EM, Elhadidi AS Efficacy of glucosamine

osteoarthritis: Clinical and laboratory study, Alex J Med (2014), http://dx.doi.org/1

crease in proteoglycan synthesis and in UDP-glucuronosyl-

transferase I mRNA expression induced by IL-1b.6 In theporcine cartilage explant model, GS showed a chondroprotec-tive effects through inhibition of IL-1b effect on hyaluronic

acid and sulfated glycosaminoglycan degradation.29 In articu-lar chondrocytes culture, exogenous glucosamine effectivelyrendered the chondrocytes unresponsive to IL-1b stimula-tion.30 In the human articular chondrocytes model, GS inhib-

ited the IL-1b-mediated increase of MMP activity.26,31

Additionally, GS suppresses the expression of the catabolicgenes in human osteoarthritic explants.32 Thus it is suggested

that the potential therapeutic effects of GS on pathologicalOA cartilage is more probably due to its ability to inhibitthe deleterious effects of IL-1b signaling (anti-catabolic activ-

ities), rather than due to cartilage matrix biosynthesis (ana-bolic activities). There are reports showing that there wasless joint space narrowing in knee OA patients who were on

glycosaminoglycan over a period of 3 years, compared toplacebo.33

Safety of GS during short-term treatment was significantlybetter than conventional NSAIDs.19,20 The present study con-

firmed good treatment tolerance over 12-week administrationwithout significant toxic effect patterns being identified.

Conclusion: incorporation of a-D GS to NSAID for pri-

mary symptomatic knee OA could relieve symptoms, improvefunction and affect some of the disease mechanisms. Monitor-ing of IL-1b is useful in monitoring treatment response in pri-

mary knee OA.

Conflict of interest

The authors declare no conflicts of interest.

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sulfate in lowering serum level of interleukin-1b in symptomatic primary knee

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