Efficacy of glucosamine sulfate in lowering serum level of interleukin-1β in symptomatic primary knee osteoarthritis: Clinical and laboratory study

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 22 February 2014

Abstract

Objective

To identify the effect of α-d glucosamine sulfate (GS) on serum level of interleukin-1β (IL-1β) in patients with symptomatic primary knee OA.

Methods

Sixty patients (mean age = 52.2 ± 8.6 years), fulfilling the American College of Rheumatology criteria of idiopathic knee OA, were randomized to receive either 1500 mg α-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-1β serum levels. Reference serum level of IL-1β 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-1β serum level than the control group. On follow up group I showed significant progressive reduction in IL-1β 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-1β 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 α-d GS to treatment of primary symptomatic knee OA could relieve symptoms, improve function and affect some of the disease mechanisms.

KEYWORDS:

• Glucosamine sulfate
• Interleukin-1β
• Primary knee osteoarthritis

1 Introduction

Osteoarthritis (OA) is the most common chronic synovial joint disorder and is generally characterized by articular cartilage deterioration. Although traditionally considered a non-inflammatory joint disease; it is now well appreciated that inflammatory mediators are produced by articular tissues in OA and have been implicated in disease pathogenesis.¹ There is convincing evidence that interleukin-1β (IL-1β) and other inflammatory mediators, synthesized locally by synovial cells and chondrocytes, play a pivotal role in driving the pathways associated with OA pathogenesis and promote cartilage degradation.² IL-1β is markedly increased in chondrocytes, synovial membrane and synovial fluid of osteoarthritic patients with a positive correlation between its expression and the severity of chondral damage and increasing grades of OA.^3–5 IL-1β initiates the number of events leading to cartilage damage. This process involves inhibition of biosynthesis of proteoglycans in addition to promotion of their degradation. IL-1β has a repressive effect on glucuronosyltransferase involved in a key step in glycosaminoglycans biosynthesis,⁶ while it upregulates metalloproteinases (MMP), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), Prostaglandin E2 and IL-6.⁷ Thus, IL-1β may be a therapeutic target in OA. The conservative treatment of OA is still limited to a few classes of medications, such as analgesic drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 selective inhibitors and viscosupplementation, which provide primarily symptomatic relief but have not yet been demonstrated to interfere with the progression of the disease.⁸ d-Glucosamine, the biologically active form of glucosamine, serves as a metabolic precursor in the synthesis of the proteoglycans and glycosaminoglycans 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 progression of OA as a disease modifying anti-OA drug through its chondroprotective effect.⁹ It is believed that oral consumption of large quantities of glucosamine elevates its intra-articular concentrations and thereby enhances synthesis of the articular cartilage matrix. Despite the increased use of glucosamine sulfate (GS) in the treatment of OA, the mechanisms accounting for its in vivo antiarthritic activity are still unclear. The aim of the present study was to identify the suppressive effect of α-d glucosamine sulfate on serum level of IL-1β in patients with symptomatic primary knee OA as one of mechanisms which may affect the disease progression.

Study design: Ibuprofen-controlled, α-d glucosamine sulfate study to evaluate its efficacy as a treatment for symptomatic primary knee OA.

2 Methods

Sixty patients with age range of 46–62 years (52.2 ± 8.6), fulfilling the American College of Rheumatology criteria for idiopathic OA of the knee,¹⁰ were included in the study. Knee OA was documented by standardized weight bearing posteroanterior plain radiographic examination. Severity of OA was graded according to the Kellgren and Lawrence grading scale (KL).¹¹ Evaluation of each joint compartment was performed with regard to the presence of joint space narrowing, osteophyte and subchondral sclerosis. Scoring of OA severity impact on patient’s functional status was determined by Lequesne’s algofunctional index (LFI).¹² Patients were eligible to be included if they had knee pain on most days of the preceding month and in need for daily NSAIDs, had KL grade 2 or 3 and score >4 to ⩽11 on LFI. By full history taking, thorough clinical examination and specified laboratory investigations; secondary knee OA, polyarticular or suspected inflammatory joint diseases, intra-articular or systemic corticosteroid use within 3 months prior to study, diabetes mellitus and administration of anticoagulants or antihypertensive were excluded. An informed written consent was obtained from all participants prior to enrollment in the study which was approved by the Local Medical Ethics Committee. After enrollment, 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 a computer generated randomization code. Thirty patients (group I) were allocated to receive 3 capsules of 500 mg α-d glucosamine sulfate, an active isomer of GS in addition to 1200 mg Ibuprofen daily, while 30 patients (group II) received a daily dose of 1200 mg Ibuprofen only. Patients were followed up 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) Patients were asked to rate the severity of knee pain on a 0–100 mm Visual Analogue Scale (VAS) (0; no pain-100; most severe pain). (2) Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) functional index was assessed.¹³ Assessment was carried for the total index and for the joint pain (5 questions), stiffness (2 questions), and limitation of physical function subscales (17 questions) each question scored on a scale from 0 to 4, with 0 indicating none and 4 indicating extreme; therefore 20, 8, 68 and 96 points, are the worst possible severity scores for pain, stiffness, limitation of physical function and total index, respectively. (3) Quantitative detection of IL-1β serum levels using commercially available enzyme linked immunosorbent assay (ELISA) quality-validated kit for human (ebioscience. North America, Europe/International).¹⁴ Additionally, IL-1β serum level was determined in a control group of 20 age and sex matched healthy volunteers who had no clinical signs or symptoms of any arthritis and had KL radiographic score <1. Authors were not blind to group assignment.

Compliance to medications was determined by asking the patients about missed doses. Safety was investigated by recording the occurrence of adverse events.

2.1 Statistical analysis

Data were analyzed using SPSS version 17. Data were summarized using descriptive statistics: mean ± standard deviation (±SD). The statistical analysis was performed using paired sample t-tests for intra-group comparisons. While independent sample t-tests was used for inter-groups comparisons. A probability value (P value) ⩽ 0.05 was considered statistically significant.

3 Results

Of the 153 patients who underwent screening, 60 patients were randomized into the study groups. All patients in the two studied groups completed the 12-week treatment course without withdrawals or drop outs. Knee OA was moderate to severe at enrollment according to KL grading and LFI scores. Most patients in the 2 groups (63.3% of group I and 66.6% of group II) had bilateral knee OA. Table 1 demonstrates the comparable baseline characteristics of the two studied groups. Patients in both groups were comparable regarding KL grading, LFI scores, pain VAS as well as total WOMAC OA scale and its 3 subscales (P > 0.05). The mean age of the control group 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-1β serum level in the control group was 5.4 ± 0.5 pg/ml.

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/m^2)	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-1β (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.

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 visits compared to group II. Additionally, the final change in the pain VAS and total WOMAC scale, pain, stiffness and function subscales scores showed significant differences in favor of group I where the mean pain VAS decreased from 60.5 ± 15.7 mm at V1 to 18.2 ± 13.1 mm at V4 (70% reduction) 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 decreased from 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).

Table 2 Change in the VAS, WOMAC OA index scores and serum level of IL1-β in each group at week 12 (V4) compared with baseline (V1) values.

Measure	Group I (n = 30)		Group II (n = 30)
	Difference mean ± SD	P	Difference mean ± SD	P
VAS	42.2 ± 15.6	.000^*	26.5 ± 17.3	.000^*
WOMAC total scale	24.6 ± 7.3	.000^*	8.1 ± 7.5	.000^*
WOMAC pain subscale	7.6 ± 3.2	.000^*	3.4 ± 2.8	.000^*
WOMAC stiffness subscale	4.1 ± 2.1	.000^*	2.1 ± 2.2	.000^*
WOMAC function subscale	12.9 ± 6.9	.000^*	2.5 ± 7.4	.144
IL-1β	1.0 ± 0.7	.000^*	.18±.3	.149

* P is significant if <0.05.

At V1, group I and group II had significantly higher mean IL-1β serum level than the control group (P = .001 and .003; respectively). On subsequent follow up (V2, V3 and V4), group I showed significant progressive reduction in mean IL-1β serum level (P = .002, .033 and .002; respectively). The final change in IL-1β serum level at V4 was significantly lower than V1 level in group I (P = .001) and was significantly lower than V4 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-1β serum level during V2, V3 and V4 but did not reach the level of statistical significance (P = .382, .360 and .358; respectively) and its final V4 level persisted significantly higher than the level of the control group (P = .017).

Table 3 Comparison between the two studied groups regarding serum level of IL-1β 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-β (pg/ml)	5.7 ± 0.6	5.9 ± 0.6	.300
V3-IL1-β (pg/ml)	5.4 ± 0.9	5.9 ± 0.5	.032^*
V4-IL1-β (pg/ml)	5.4 ± 0.6	5.9 ± 0.3	.001^*

Values are the mean ± SD.

* P is significant if <0.05.

Compliance to the study medication and safety was good without statistically significant differences in the proportion or pattern of adverse events between both groups. The most frequently reported complaints consisted predominantly of transient episodes of abdominal pain (5%), dyspeptic symptoms (12%), nausea and diarrhea.

4 Discussion

Data of the present study showed that 12-week oral administration of α-d GS and NSAID could significantly improve primary knee OA pain, stiffness, limitation of function and lower the elevated serum level of IL-1β throughout the study compared to oral administration of NSAID solely for the same treatment period. Such results were obtained by assessment using the pain VAS and WOMAC index that is, the most widely used algo-functional index for knee OA. Therefore, α-d GS could be classified as a symptom-modifying drug in OA. The main finding of this study is that α-d GS could reduce the increase of IL-1β serum level in patients with primary knee OA. Consistent with this data, an experimental study¹⁵ showed that reduction of IL-1β level occurred not only in the joints, but also in the serum of rats with adjuvant-induced arthritis fed on combination of GS and chondroitin sulfate. Cytokine analysis revealed increased inflammatory proteins in sera of OA patients compared to the control, including IL-1β, IL-1 receptors antagonist and IL-6. The levels of these cytokines were higher in OA synovial fluid than sera, consistent with their origin from joint tissue, indicating that local inflammation within OA joint tissues can be reflected in serum. The abnormal production and leakage of inflammatory cytokines into serum are analogous to that observed for biomarkers of joint tissue synthesis and degradation.¹⁶ On the contrary, serum concentration of IL-1β was found to be below detection limit in OA patients in another study.¹⁷ This contradiction might be explained by the fact that inflammatory cytokines in OA joints traffic through synovium and out into circulation, so they may or may not be present at higher than normal levels in sera of OA patients according to the magnitude of joint tissue inflammation. Participants in the present study had moderate to severe knee OA at enrollment. It was hypothesized that the effects of GS are better realized in patients with moderate to severe OA, which have greater involvement of IL-1β.¹⁸ It is not clear whether GS can modulate the intra-articular synthesis of IL-1β so it can lower its serum level or has another systemic mechanism in this regard. Future studies are needed to identify the mechanism responsible for systemic effect of GS in lowering serum level of IL-1β in patients with primary knee OA.

Short-term studies designed to describe the pattern of the symptomatic effect of GS, showed a significantly better effect in decreasing pain in patients with primary knee OA compared to placebo therapy and conventional NSAID use in the first 4 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 treatment courses are better explained by the anti-inflammatory effect of GS. In human osteoarthritic chondrocytes, it was found that GS blocks inflammatory signaling and inhibits the synthesis of proinflammatory mediators stimulated by IL-1β through a nuclear factor κB-dependent mechanism.²¹ Glucosamine sulfate suppresses IL-1β induced phospholipase A2^22,23 and COX-2 expression and activation,²⁴ iNO^22–24 and reactive oxygen species synthesis.²³ It also reduces the intra-articular concentration of IL-1β and tumor necrosis factor-α.^15,25

The long-term effects of GS in primary knee OA are thought to be related to its effect on cartilage. Pharmacokinetic studies have shown that, crystalline GS is bioavailable both systemically and in articular cartilage after oral administration.^26,27 Glucosamine is incorporated into the components of the glycosaminoglycan chains by the chondrocytes,²⁸ stimulates the synthesis of physiological proteoglycans,²⁶ reverses some of the negative effects of IL-1β on cartilage metabolism. There are many reports suggesting that GS reverses the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by IL-1β.⁶ In the porcine cartilage explant model, GS showed a chondroprotective effects through inhibition of IL-1β effect on hyaluronic acid and sulfated glycosaminoglycan degradation.²⁹ In articular chondrocytes culture, exogenous glucosamine effectively rendered the chondrocytes unresponsive to IL-1β stimulation.³⁰ In the human articular chondrocytes model, GS inhibited the IL-1β-mediated increase of MMP activity.^26,31 Additionally, GS suppresses the expression of the catabolic genes in human osteoarthritic explants.³² Thus it is suggested that the potential therapeutic effects of GS on pathological OA cartilage is more probably due to its ability to inhibit the deleterious effects of IL-1β signaling (anti-catabolic activities), rather than due to cartilage matrix biosynthesis (anabolic activities). There are reports showing that there was less joint space narrowing in knee OA patients who were on glycosaminoglycan over a period of 3 years, compared to placebo.³³

Safety of GS during short-term treatment was significantly better than conventional NSAIDs.^19,20 The present study confirmed good treatment tolerance over 12-week administration without significant toxic effect patterns being identified.

Conclusion: incorporation of α-d GS to NSAID for primary symptomatic knee OA could relieve symptoms, improve function and affect some of the disease mechanisms. Monitoring of IL-1β is useful in monitoring treatment response in primary knee OA.

Conflict of interest

The authors declare no conflicts of interest.

Notes

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 22 February 2014