Real world evidence of effectiveness and safety of an oral formulation containing un-denatured type-II collagen 40 mg and aflapin 100 mg (HAPID^®) in the management of osteoarthritis of knee: findings of a prospective, multi-center, observational study

INTRODUCTION

Osteoarthritis (OA) of knee is a common progressive multifactorial joint disorder and is characterized by chronic pain, stiffness, and functional disability.¹ It accounts for almost four fifths of the burden of OA worldwide and increases with obesity and age.² The pooled global prevalence of knee OA was 16% (95% CI, 14.3-17.8%) and the prevalence increases with age.³ However, the prevalence of the OA of knee is higher in the Asian continent (19.2%; 95% CI, 15.7%-23%) compared to Europe (13.4%; 95% CI, 10.1%-17%) and North America (4⋅1%; 95% CI, 2.1%-6.9%).³ Also, literature shows a higher prevalence and incidence of knee OA in women (21.7%; 95% CI, 19%-24.5%) as compared to men (11.9%; 95% CI, 10.2%-13.8%).^3,4 The prevalence of OA of the knee in India is found to be 28.7%.⁵ In an Indian hospital-based study, 58% of females had onset of symptoms of OA of knees before 50 years of age as compared to only 20% in males (p<0.05).⁶ Also, 64% females with OA of knees had the onset of symptoms either during perimenopausal period or within five years of natural menopause or hysterectomy. The pathological changes in later stage of OA which includes softening, ulceration, and disintegration of the articular cartilage.⁷ Based on the radiology, OA of knee is classified using the Kellgren and Lawrence classification system, which grades the OA severity from grade-0 (no OA) to grade 4 (severe OA).⁸ OA of the hips and knees tends to cause the greatest burden to the population as pain and stiffness in these large weight bearing joints often leads to significant disability requiring surgical intervention.⁹ OA of knee is incurable and the only cure is surgical repair which is considered only at an advanced stage of the disease; however, it involves substantial healthcare costs.¹⁰

The management of OA focuses on multi-modal treatment including patient education and self-management, non-pharmacological management, and pharmacological treatments. Non-pharmacologic therapy includes patient education, weight loss (5% over 20 weeks), moderate exercises, physical therapy, and occupational therapy.¹¹ Pharmacological management of OA includes paracetamol, non-steroidal anti-inflammatory drugs (NSAID), opioid analgesics, intra-articular drugs, and disease modifying OA drugs (DMOAD). However, there are no DMOADs yet approved for OA, and some are in clinical development (targeted against Bone morphogenetic protein-7 (BMP-7), fibroblast growth factor-18 (FGF-18), platelet rich plasma (PRP), matrix metalloproteinases (MMPs), and mesenchymal stem cells (MSCs). NSAIDs and corticosteroids are associated with high prevalence of gastrointestinal/ cardiovascular adverse effects. All efforts to develop safe NSAIDs that spare the gastrointestinal tract and the cardiovascular effects are still far from achieving a breakthrough. Hence, there is a need for a safe and effective alternative for management of OA.

Many nutraceutical products like Boswellia, Aflapin, chondroitin sulphate, glucosamine sulphate, collagen peptide, curcumin, fish oil, ginger, green tea, and rosehip extract have been used for treatment of OA of knee but with varied results.¹² Gum resin extracts of Boswellia serrata have shown anti-inflammatory properties and promising potential as therapeutic interventions against inflammatory diseases like OA. Aflapin is synergistic composition of Boswellia serrata extract enriched in AKBA (3-O-Acetyl-11-keto-beta-boswellic acid) and non-volatile oil portion of B. serrata gum resin.¹³ Undenatured form of type II collagen (UC-II) is nutritional supplement derived from chicken sternum cartilage which plays important role in building of joint cartilage and may also have anti-inflammatory and antioxidant effects.¹⁴ Study published in science in 1993 reported reduced joint swelling with oral type II collagen supplementation in RA.¹⁵ It is expected that compared to NSAIDs, B. serrata extract and UC-II may associated with better tolerability.

The present real-world study is the first in India and probably worldwide which evaluates the efficacy and safety of an oral formulation containing UC-II and aflapin for treatment of OA of knee.

METHODS

Setting

This study was a prospective, post-marketing, real-world, observational study conducted for assessment of effectiveness and efficacy of an oral formulation containing UC-II and aflapin for treatment of OA of knee. Data was collected from 20 participating sites across India. study duration-Jul-21 to Jul-22.

Informed consent and ethics

In this post-marketing study, data of patients who were prescribed treatment with oral tablet containing un-denatured type-II collagen 40mg and aflapin 100 mg (HAPID^®, Wockhardt, India) for treatment of knee OA was collected prospectively from the participating sites. The study documents were reviewed and approved by the institutional ethics committee (IEC) of D. Y. Patil university school of medicine, Navi Mumbai (DYP/IEC/12/2021, June 10, 2021). The study was conducted in accordance with the principles of declaration of Helsinki (World medical association) and good clinical practice (GCP) guidelines issued by the ICMR and CDSCO, government of India, and was registered with the clinical trials registry of India (CTRI/2021/07/034559, July 02, 2021). This being a prospective study, informed consent was obtained from all the patients, and the strict confidentiality was maintained for the patient’s identity.

Study participants

Data of 505 patients of either gender above 18 years of age who received study treatment were included for the study. Ambulatory adult patients with clinically and/or radiologically confirmed diagnosis of OA of knee and prescribed capsule HAPID^® (Un-denatured type II collagen and Aflapin) for first time, willing to provide written informed consent and comply to the study as per protocol were included in the study. Those with a history or evidence of hypersensitivity to any components of study medication, or those who received visco-supplementation (hyaluronic acid injection in joints) within 9 months prior to date of screening were excluded. Pregnant or lactating women, and patients with known allergy to eggs/ chicken were not included.

Study procedures

Data was collected and recorded in a study specific electronic data capture tool from the participating sites. Patient information about their clinical condition on admission, comorbidities, complications, and details of other treatment (including analgesic and anti-inflammatory drugs) received was collected. This being an observational study, there were no study specific treatments and all treatments for the patients were based at the discretion of the treating clinician. All patients received capsule containing Aflapin 100 mg and un-denatured type II collagen 40 mg orally as per investigator’s discretion for a period of 90 days.

The study outcomes were based on the Western Ontario and McMaster Universities OA Index (WOMAC), which is valid and reliable for defining function in lower extremity disorders.¹⁶ WOMAC consists of a total of 24 questions and three subscales (5 questions related to pain, two questions related to stiffness, and 17 questions related to difficulties in performing activities of daily living in relation to physical function). Pain was assessed using a 0-10 visual analogue scale (VAS). Assessments were done at baseline and then on days 5, 30, 60 and 90 days.

Rescue analgesic

Use of any rescue analgesic (paracetamol 500 mg as per need) for breakthrough pain during study period recorded.

Study outcomes

The primary outcomes for effectiveness were mean change in total WOMAC score from baseline through day 90, mean change in VAS score for pain from baseline through day 90, and mean change in scores for WOMAC subscales (joint pain, joint stiffness, and physical function) from baseline through day 90. Secondary endpoints were for safety and tolerability assessed based on the adverse events reported with the study treatment.

Statistical methods

This being an observational study, there was no study hypothesis. Due to lack of published literature on efficacy of combined treatment with UC-II and aflapin in OA knee, the sample size was not based on any assumptions and calculations, and it was planned to include data of 500 patients on empirical basis. Data were entered in Microsoft office excel worksheet and checked for discrepancies and errors. Descriptive are presented for demography and study outcomes (WOMAC score and VAS score). Measurement data are presented as means and standard deviation (SD), whereas categorical data is presented as numbers with percentages. Within group analysis of baseline data with follow-up visits data (Day 5, 30, 60 and 90) is done for WOMAC scores (total and subscale) and VAS scores using Friedman test (repeat measures non-parametric ANOVA). Individual pair-wise comparisons with baseline are done using Wilcoxon test for paired data. All analyses were done using two-sided tests with alpha 0.05 (95% confidence levels). Statistical analysis was done using Stata 13.1 for Windows (StataCorp LLC, TX, USA).

RESULTS

Patient characteristics and pre-treatment data

Of the 505 patients included for the final analysis, there were 45% male and 55% females. Table 1 shows demography and patient profile of all patients included in study. About 56.4% patients newly diagnosed knee OA. Most of patients (96%) received 90 days of study treatment and only 20 (4%) patients received 60 days treatment.

Table 2 shows prev. medication received by patients, and concomitant medication received along with study treatment. Other therapies received by patients in past were oral NSAID’s (19.2%), topical NSAID’s (1.4%), exercise therapy (5.1%), weight loss (2.6%), and other therapies (0.6%). Other therapies received by the patients were antihypertensives (1.6%), oral hypoglycaemics (0.6%) and thyroxine (0.4%).

WOMAC score

Table 3 presents the WOMAC scores for the three sub-scales and the total score at baseline and change from baseline on days 5, 30, 60 and 90. Improvements are seen from baseline for all 3 sub-scale scores and total scores on WOMAC scale immediately on day 5 with study treatment. There is progressive improvement up to day 90 with gross reductions in mean scores on day 90. Figure 1 presents mean scores for WOMAC physical function sub-scale and total scores from baseline through day 90. Figure 2 shows mean scores for WOMAC pain and stiffness sub-scale scores from baseline through day 90. Statistically significant reduction (p<0.0001) in all sub-scale scores and total scores for WOMAC seen over 90 days.

Pain (VAS) score

Table 4 presents the pain scores (0-10 VAS) at baseline and change from baseline on days 5, 30, 60 and 90. Figure 3 presents the mean scores for pain (0-10 VAS) from baseline through day 90. Great reduction in the pain scores is seen over 90 days (p<0.0001).

* p<0.05; ** p<0.001 (Versus baseline, Wilcoxon test).

* p<0.05; ** p<0.001 (Versus baseline, Wilcoxon test).

*P<0.001 (Versus baseline, Wilcoxon test).

# Friedman test (repeat measures); * Wilcoxon test.

# Friedman test (repeat measures); * Wilcoxon test.

Rescue analgesic

Of the total 505 patients rescue analgesic was required for breakthrough pain only for 138 (27.3%) patients. The mean (SD) duration of rescue analgesic use was 15.02 (16.83) days. The need for rescue was similar in males (26.4%) and females (28.1%). However, the need for rescue was lesser in normal weight individuals (17.7%), as compared to overweight and obese patients (30.1%).

Safety

There were no adverse events reported by any of the patients during the study period.

DISCUSSION

OA is the commonest form of arthritis which is associated with poor quality of life due to disability and hampered physical function. Prevalence of OA increases with age and is about 50% in people above 60 years of age.¹⁷ Several systemic factors have been identified as risk factors for OA, and these may act by increasing the susceptibility of joints to injury, by direct damage to joint tissues, or by impairing the process of repair in damaged joint tissue.¹⁸ Local factors are most commonly biomechanical in nature and adversely affect the forces applied to the joint. Certain specific risk factors have been identified including obesity and metabolic disease, age, sex, ethnicity and race, genetics, nutrition, smoking, bone density and muscle function.¹⁸ A systematic review and meta-analysis of the risk factors for the onset of OA of the knee, has reported that there is a consistent strong association between increased BMD (bone mineral density) and the onset of knee OA in women.¹⁹

Despite several drugs available for pharmacological therapy, there are no curative therapies currently available for OA, and patients in advanced disease often require surgical management. There is a desperate need for effective therapies to decrease disease progression, and achieve symptom relief for pain and stiffness, to improve the quality of life in patients with OA.

In the last few decades, many nutraceutical products like Boswellia, Aflapin, chondroitin sulphate, glucosamine sulphate, collagen peptide, curcumin, fish oil, ginger, green tea, and rosehip extract have been used for treatment of OA of knee but with varied results.¹² These supplements have been found to be effective in knee OA in various studies, and no serious side effects have been reported for any of these supplements.¹² Due to there are significant anti-inflammatory properties, gum resin extracts of Boswellia serrata containing 3-O-acetyl-11-keto-β-boswellic acid (AKBA) are a promising potential as therapeutic interventions for inflammatory diseases such as OA.¹³ However, the AKBA has poor bioavailability following oral administration. Aflapin is a synergistic composition of Boswellia serrata extract enriched in AKBA (3-O-Acetyl-11-keto-beta-boswellic acid) and non-volatile oil portion of B. serrata gum resin.¹³ Due to this formulation, the availability of AKBA in systemic circulation is increased by 51.78%.²⁰ In the last two decades, preparations of the gum resin of Boswellia serrata (a traditional ayurvedic medicine) and of other Boswellia species have experienced increasing popularity in Western countries.²³ Animal studies and pilot clinical trials support the potential of Boswellia serrata gum resin extract (BSE) for the treatment of a variety of inflammatory diseases like inflammatory bowel disease, rheumatoid arthritis, OA, and asthma. The pharmacological effects of BSE were mainly attributed to suppression of leukotriene formation via inhibition of 5-lipoxygenase (5-LO) by two boswellic acids, 11-keto-β-boswellic acid (KBA) and acetyl-11-keto-β-boswellic acid (AKBA).²¹ These two boswellic acids have also been chosen in the monograph of Indian frankincense in European -pharmacopoeia 6.0. Compared to NSAIDs, it is expected that B. serrata may be associated with better tolerability.

UC-II is derived from chicken sternum cartilage and is glycosylated to improve stability.²² Collagen supplements are rich in amino acids such as glycine, proline, and hydroxyproline; all of which play important roles in the building of joint cartilage and may also have anti-inflammatory and antioxidant effects and have been speculated to act as signalling molecules.¹⁴ Studies have shown that undenatured type II collagen is effective in the treatment of RA and OA.^23,24 A White Paper on collagen hydrolyzates and ultrahydrolyzates in OA reports evidence of efficacy in a few small-scale clinical trials.²⁵

In an early study with 40 mg UC-II for 42 days in patients with OA, there was an average reduction of 26% in the pain score.³⁰ This was observed in 80% patients with OA (4/5), and there were no adverse effects reported by patients.³⁰ We observed a 15.2% reduction in pain score on day 5 which increased progressively to 48.3% reduction by day 90. Thus, we observed a greater reduction in pain score as compared to Bagchi et al. This greater reduction in pain scores in our study could be probably due to use of aflapin along with UC-II. In a multicenter randomized, double-blind, three-arm, placebo-controlled study, efficacy and tolerability of UC-II was compared with placebo and glucosamine hydrochloride plus chondroitin sulfate (GC) in patients with OA of knee over 180 days.²⁶ On day 180, the UC-II therapy demonstrated a significant reduction in overall WOMAC score compared to placebo (p=0.002) and GC (p=0.04). Also, there were significant changes for all three WOMAC subscales: pain (p=0.0003 vs. placebo; p=0.016 vs. GC); stiffness (p=0.004 vs. placebo; p=0.044 vs. GC); physical function (p=0.007 vs. placebo). These observations are coherent with our study findings. Safety of UC-II was similar to placebo and GC.³¹ Similarly, a randomized comparative clinical trial (Crowley et al) of 90-day therapy with UC-II versus combination therapy with GC (glucosamine and chondroitin sulphate) in OA of knee showed significantly greater reduction in WOMAC score and pain scores with UC-II as compared to GC therapy.²⁷ We observed a significant improvement in WOMAC scores for all domains of pain, stiffness and physical function by treatment with combination therapy of orally administered Aflapin and UC-II over a 90-day period. Also, in the study by Crowley et al there were significant reduction in pain scores with UC-II therapy. They reported significant enhancement in daily activities suggesting an improvement in their quality of life with UC-II therapy.³² In our study we observed a significant reduction of pain scores from 7.08 (2.27) to 6.00 (1.71) on day 5 (p<0.05) and 3.66 (2.21) on day 90 (p<0.0001). Also, improvement in the mean scores for physical function domain of WOMAC suggests that the quality of life improved with combination therapy of Aflapin and UC-II.

In a double-blind, randomized, placebo-controlled clinical study by Sengupta et al which evaluated the early efficacy of aflapin in 15 subjects with OA of knee, aflapin caused clinically and statistically significant improvements in pain scores and physical function scores.²⁸ These improvements were recorded as early as 7 days after starting therapy. In another 30-day, double-blind, randomized, placebo-controlled study of aflapin in the management of clinical symptoms of OA of the knee, 60 subjects received either 100 mg (n=30) of Aflapin or placebo (n=30) daily for 30 days.²⁹ Aflapin provided clinically and statistically significant improvements in pain scores and physical function scores with effects starting as early as 5 days of treatment. We also observed improvement (p>0.05) in pain and WOMAC scores on day 5 after starting the combination therapy with aflapin and UC-II. In our study we also observed significant improvements in pain scores and WOMAC scores on day 5 after starting the combination therapy with aflapin and UC-II. The combination of aflapin and UC-II could be additive for the therapeutic effects due to different mechanisms of each ingredient. Aflapin, can provide powerful anti-inflammatory efficacy and anti-arthritic actions, whereas UC-II can provide basic elements required for building of joint cartilage. UC-II also has antioxidant and anti-inflammatory actions which could potentiate the effects of aflapin.

The common adverse effects reported by Crowley et al were gastrointestinal (5/26) and musculoskeletal (7/26) observed in 16/26 (61.5%) patients on UC-II therapy.³² Sengupta et al reported no change in safety parameters including laboratory parameters with aflapin therapy. However, in our study none of the patients reported any adverse effects after starting therapy with aflapin and UC-II.

This post-marketing clinic-based study captured the real-world safety and efficacy data of use of capsules containing Un-denatured type II collagen 40 mg and Aflapin 100 mg administered for 90 days for treatment of OA of knee. Being devoid of potential adverse effects, such as gastric ulceration and cardiovascular toxicity, UC-II and aflapin combination can offer a valuable therapeutic option for the management of knee OA. The well-established safety profile of both ingredients can show their potential for clinical use.

Our study has some limitations in terms of being a post-marketing study. Due to the observational study design, strict control of confounding factors could not be obtained. Hence, this limits the extrapolation of study results to large scale population.

CONCLUSION

The excellent safety and efficacy profile of UC-II and aflapin combination makes it a desirable pharmacological treatment modality for management of patients of knee OA.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Ethics Committee

REFERENCES

1. 1. Hunter DJ, Bierma-Zeinstra S. Osteoarthritis. Lancet. 2019;393(10182):1745-59.
2. 2. GBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388(10053):1545-602.
3. 3. Cui A, Li H, Wang D, Zhong J, Chen Y, Lu H. Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies? E Clin Med. 2020;29-30:100587.
4. 4. Thati S. Gender Differences in Osteoarthritis of Knee: An Indian Perspective. J Midlife Health. 2021;12(1):16-20.
5. 5. Pal CP, Singh P, Chaturvedi S, Pruthi KK, Vij A. Epidemiology of knee osteoarthritis in India and related factors. Indian J Orthop. 2016;50(5):518-522.
6. 6. Nadkar MY, Samant RS, Vaidya SS, Borges NE. Relationship between osteoarthritis of knee and menopause. J Assoc Physicians India. 1999;47(12):1161-3.
7. 7. Litwic A, Edwards MH, Dennison EM, Cooper C. Epidemiology and burden of osteoarthritis. Br Med Bull. 2013;105:185-99.
8. 8. Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis. 1957;16(4):494-502.
9. 9. Oliveria SA, Felson DT, Reed JI, Cirillo PA, Walker AM. Incidence of symptomatic hand, hip, and knee osteoarthritis among patients in a health maintenance organization. Arthritis Rheum. 1995;38(8):1134-41.
10. 10. Bannuru RR, Osani MC, Vaysbrot EE, Arden NK, Bennell K, Bierma-Zeinstra SMA et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. Osteoarthritis Cartilage. 2019;27(11):1578-89.
11. 11. Oo WM, Yu SP, Daniel MS, Hunter DJ. Disease-modifying drugs in osteoarthritis: current understanding and future therapeutics. Expert Opin Emerg Drugs. 2018;23(4):331-347.
12. 12. Vaishya R, Agarwal AK, Shah A, Vijay V, Vaish A. Current status of top 10 nutraceuticals used for Knee Osteoarthritis in India. J Clin Orthop Trauma. 2018;9(4):338-48.
13. 13. Sengupta K, Kolla JN, Krishnaraju AV, Yalamanchili N, Rao CV, Golakoti T, Raychaudhuri S, Raychaudhuri SP. Cellular and molecular mechanisms of anti-inflammatory effect of Aflapin: a novel Boswellia serrata extract. Mol Cell Biochem. 2011;354(1-2):189-97.
14. 14. Paul C, Leser S, Oesser S. Significant Amounts of Functional Collagen Peptides Can Be Incorporated in the Diet While Maintaining Indispensable Amino Acid Balance. Nutrients. 2019;11(5):1079.
15. 15. Trentham DE, Dynesius-Trentham RA, Orav EJ, Combitchi D, Lorenzo C, Sewell KL et al. Effects of oral administration of type II collagen on rheumatoid arthritis. Science. 1993;261(5129):1727-30.
16. 16. Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW. Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee. J Rheumatol. 1988;15(12):1833-40.
17. 17. Felson DT. The epidemiology of knee osteoarthritis: results from the Framingham Osteoarthritis Study. Semin Arthritis Rheum. 1990;20(3-1):42-50.
18. 18. Litwic A, Edwards MH, Dennison EM, Cooper C. Epidemiology and burden of osteoarthritis. Br Med Bull. 2013;105:185-99.
19. 19. Blagojevic M, Jinks C, Jeffery A, Jordan KP. Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis. Osteoarthritis Cartilage. 2010;18(1):24-33.
20. 20. Abdel-Tawab M, Werz O, Schubert-Zsilavecz M. Boswellia serrata: an overall assessment of in vitro, preclinical, pharmacokinetic and clinical data. Clin Pharmacokinet. 2011;50(6):349-69.
21. 21. Anazodo MI, Subramanian LR, Ammon HP. Boswellic acids: novel, specific, nonredox inhibitors of 5-lipoxygenase. J Pharmacol Exp Ther. 1992;261(3):1143-6.
22. 22. Bagi CM, Berryman ER, Teo S, Lane NE. Oral administration of undenatured native chicken type II collagen (UC-II) diminished deterioration of articular cartilage in a rat model of osteoarthritis (OA). Osteoarthritis Cartilage. 2017;25(12):2080-90.
23. 23. Barnett ML, Kremer JM, St Clair EW, Clegg DO, Furst D, Weisman M et al. Treatment of rheumatoid arthritis with oral type II collagen. Results of a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum. 1998;41(2):290-7.
24. 24. Bagchi D, Misner B, Bagchi M, Kothari SC, Downs BW, Fafard RD et al. Effects of orally administered undenatured type II collagen against arthritic inflammatory diseases: a mechanistic exploration. Int J Clin Pharmacol Res. 2002;22(3-4):101-10.
25. 25. Mobasheri A, Mahmoudian A, Kalvaityte U, Uzieliene I, Larder CE, Iskandar MM et al. A White Paper on Collagen Hydrolyzates and Ultrahydrolyzates: Potential Supplements to Support Joint Health in Osteoarthritis? Curr Rheumatol Rep. 2021;23(11):78.
26. 26. Lugo JP, Saiyed ZM, Lane NE. Efficacy and tolerability of an undenatured type II collagen supplement in modulating knee osteoarthritis symptoms: a multicenter randomized, double-blind, placebo-controlled study. Nutr J. 2016;15:14.
27. 27. Crowley DC, Lau FC, Sharma P, Evans M, Guthrie N, Bagchi M et al. Safety and efficacy of undenatured type II collagen in the treatment of osteoarthritis of the knee: a clinical trial. Int J Med Sci. 2009;6(6):312-21.
28. 28. Sengupta K, Krishnaraju AV, Vishal AA, Mishra A, Trimurtulu G, Sarma KV et al. Comparative efficacy and tolerability of 5-Loxin and Aflapin Against osteoarthritis of the knee: a double blind, randomized, placebo controlled clinical study. Int J Med Sci. 2010;7(6):366-77.
29. 29. Vishal AA, Mishra A, Raychaudhuri SP. A double blind, randomized, placebo controlled clinical study evaluates the early efficacy of aflapin in subjects with osteoarthritis of knee. Int J Med Sci. 2011;8(7):615-22.