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\def\RunningAuthor{Chitra Khanwelkar et al.}
\firstPage{7160}
\articleType{Original Article}
\receivedDate{16 Aug 2020}
\acceptedDate{23 Sep 2020}
\revisedDate{16 Sep 2020}
\journalVolume{2020, 11}
\journalIssue{4}
\journalDoi{ijrps.v11i4.3840}
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\title{\textbf{The Comparison of Anti-inflammatory and Analgesic Effect of Diclofenac Alone with Combined Treatment with Diclofenac and Serratiopeptidase in Patients having Osteoarthritis of Knee Joint}}
\author{Chitra~Khanwelkar\textsuperscript{*},  
            Kartik~Peethambaran, 
            Sujata~Jadhav~\\[5pt]{Department of Pharmacology, Krishna Institute of Medical Sciences, Karad-415339, Maharashtra, India}}

\begin{abstract}
Oral proteolytic enzymes like serratiopeptidase are very commonly used by clinicians either alone or in combination with non-steroidal anti-inflammatory drugs for analgesia and anti-inflammatory purpose. As the activity of these drugs is not proved in trials, and they are not listed in any country's official pharmacopoeia, it was planned to study their effect in osteoarthritis patients. Two groups (n= 30 each) of diagnosed knee osteoarthritis patients, were treated with diclofenac 50 mg twice a day (BID) and serratiopeptidase 10mg three times a day (TID) + Diclofenac 50 mg BID for two weeks. The pain and difficulty in daily activities were assessed by Visual Analogue Scale (VAS) and Western Ontario and Mc Masters Universities Osteoarthritis (WOMAC OA) index scale before and after the treatment.\textbf{\space }Highly significant improvement in both scales was seen in both groups. There was no statistically significant difference in the improvements found in both groups. Addition of serratiopeptidase has not potentiated analgesic and anti-inflammatory effects of diclofenac. Thus, the analgesic and anti-inflammatory efficacy of serratiopeptidase are not proved.  
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Analgesia,\newline Anti-inflammatory,\newline Diclofenac,\newline Osteoarthritis,\newline Serratiopeptidase
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Chitra~Khanwelkar~\\ Phone:\ +91-9822031043~\\  Email:\ chitrakhanwelkar@gmail.com  
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Inflammation is the local response of the living tissues to injury caused by any agent. It is the natural defence reaction to eliminate the injury-causing agent. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) and glucocorticoids are used in therapy for their potent anti-inflammatory effect for many decades. Because of the potential adverse effects of glucocorticoids, NSAIDs are preferred for controlling inflammation and suppress associated symptoms in cases of joint pains, sprains, dental pain. NSAIDs are available without prescription in chemist shops and maybe overused by patients as self-medication also.

Besides, there is a group of drugs called proteolytic enzymes (chymotrypsin, serratiopeptidase, hyaluronidase) which are widely used alone or in combination with NSAIDs. Oral preparations of serratiopeptidase are prescribed by many clinicians for its proposed anti-inflammatory action, for trauma, arthritis, carpal tunnel syndrome, respiratory tract congestion, parotitis etc. 

Serratiopeptidase is a proteolytic enzyme having a high molecular weight \unskip~\citep{887067:20340532,887067:20340538}. In the gastrointestinal tract, complex proteins are degenerated to simple proteins and then only they can be absorbed. Therefore, it is doubtful that serratiopeptidase reaches the blood circulation intact after oral administration. Therefore, it is also doubtful that it produces an anti-inflammatory effect at the site of action when it is given orally. Serratiopeptidase fixed-dose combinations have very poor rationality score also \unskip~\citep{887067:20340533}. Besides, serratiopeptidase is neither listed in any official pharmacopoeia nor any essential drug list. Considering all these facts, the present study was planned to evaluate whether the anti-inflammatory effect of diclofenac is potentiated by its combination with serratiopeptidase given orally, in patients having osteoarthritis of the knee joint.
    
\section{Materials and Methods}
It was an open-labelled comparative, prospective, interventional study involving 60 patients of either sex having age more than 30 years attending orthopaedic Outpatient Department (OPD) suffering from mild to moderate osteoarthritis of the knee joint. These patients were enrolled after confirmation of diagnosis by an orthopaedic specialist and after taking their written informed consent. The study was conducted in a tertiary care hospital of Krishna Institute of Medical Sciences, Karad, Maharashtra. The Institutional Ethics Committee of Krishna Institute of Medical Sciences, Karad approved the study protocol.

Patients fulfilling inclusion criteria (Table~\ref{tw-4730984bf781}) were randomly assigned in 1:1 fashion to either of two treatment groups. Each group consisted of 30 patients. Group A received tablet diclofenac sodium 50 mg twice a day (BID) for two week. Group B received tablet diclofenac sodium 50 mg BID and tablet serratiopeptidase 10 mg three times a day (TID) for two weeks. Efficacy of treatment was assessed by measuring the severity of pain in the affected joint by using Visual Analogue Scale (VAS) (0-10 scale) \unskip~\citep{887067:20340534} before starting treatment and at the end of 2 weeks treatment. Pain, stiffness and level of difficulty during different daily activities was assessed before starting treatment and at the end of 2 weeks of treatment, by using Western Ontario and Mc Masters Universities Osteoarthritis (WOMAC OA) index \unskip~\citep{887067:20340547}.

Each sub-dimension in WOMAC OA index was graded according to the Likert scale (0-4 points). Total pain score, total stiffness score, the total level of difficulty score was calculated by adding scores of individual sub-dimensions of each patient. Means of total scores of pain, stiffness and level of difficulty were calculated for all 60 patients at day 0 and end of 2 weeks. Safety of treatments was assessed by the recording of adverse effects complained by patients after telephonic calls every week and by questions asked at the end of the two weeks.

\textbf{Statistical Analysis}

Student's paired t-test was used to compare results obtained in group A and group B at day 0 and the end of 2 weeks of treatment. The difference in efficacy was assessed by comparing the mean difference in symptom scores between baseline and 2-week score in both groups, in both VAS and WOMAC scale tests. The statistical analysis of the difference in the efficacy of two treatments was done by using student's unpaired t-test. P-value {\textless}0.05 was considered significant, and {\textless}0.01 was considered highly significant.
    
\section{Results}
All 60 enrolled patients of senile osteoarthritis of knee joint completed the study. Out of the total, 60 patients, 32 were female, and 28 were males. Maximum patients were having age between 41 to 70 years (Table~\ref{tw-d521c113bdec}).


\begin{table*}[!htbp]
\caption{\boldmath {Inclusion and exclusion criteria} }
\label{tw-4730984bf781}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.4647\linewidth-2\tabcolsep}p{\dimexpr.5353\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Inclusion criteria & Exclusion criteria\\
\tblmidrule 
1. Either sex &
  1. Rheumatoid/ psoriatic arthritis\\
2. Mild/moderate senile osteoarthritis: knee pain and/or knee stiffness &
  2. Pregnant/ lactating mothers\\
3. Age more than 30 years &
  3. Patients with cardiovascular, hepatic, central nervous system disorders\\
 &
  4. Known hypersensitivity to serratiopeptidase or diclofenac\\
 &
  5. Patients receiving any other drug by oral/ injectable or topical route for osteoarthritis\\
 &
  6. Patients participated in any clinical trial in the last 30 days\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Age-wise distribution of osteoarthritis patients} }
\label{tw-d521c113bdec}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.45230000000000004\linewidth-2\tabcolsep}p{\dimexpr.54769999999999996\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Age Group & Number of patients\\
\tblmidrule 
30- 40 years &
  3\\
41-50 years &
  12\\
51-60 years &
  18\\
61-70 years &
  22\\
71-80 years &
  5\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Symptom scores as per WOMAC OA and VAS in group A (Diclofenac) n=30} }
\label{tw-c90f56a34a34}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1319\linewidth-2\tabcolsep}p{\dimexpr.35120000000000005\linewidth-2\tabcolsep}p{\dimexpr.19330000000000005\linewidth-2\tabcolsep}p{\dimexpr.185\linewidth-2\tabcolsep}p{\dimexpr.1386\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Scale & Symptom & Score at 0 week \mbox{}\protect\newline Mean \ensuremath{\pm}SD & Score at 2 weeks Mean \ensuremath{\pm} SD & P-value\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.1319\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{WOMAC}} &
  Pain in knee joint &
  8.33\ensuremath{\pm} 2.74 &
  4.46\ensuremath{\pm}  2.25 &
  \textless\ 0.0001\\
 &
  Stiffness of joint &
  3.73 \ensuremath{\pm}  1.59 &
  1.63 \ensuremath{\pm}  1.47 &
  \textless\ 0.0001\\
 &
  Level of difficulty in daily activities &
  17.86\ensuremath{\pm}  3.83 &
  10.96\ensuremath{\pm}  3.57 &
  \textless\ 0.0001\\
 &
  Total WOMAC OA score &
  29.93\ensuremath{\pm}  6.56 &
  17.06\ensuremath{\pm}  6.08 &
  \textless\ 0.0001\\
VAS \mbox{}\protect\newline  &
  Overall pain intensity &
  5.57\ensuremath{\pm} 1.65 &
  3.033 \ensuremath{\pm}  1.45 &
  \textless\ 0.0001\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Symptom scores as per WOMAC OA and VAS in group B (Diclofenac + Serratiopeptidase) n=30} }
\label{tw-ee3bb1167314}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.11530000000000001\linewidth-2\tabcolsep}p{\dimexpr.3495\linewidth-2\tabcolsep}p{\dimexpr.2082\linewidth-2\tabcolsep}p{\dimexpr.1868\linewidth-2\tabcolsep}p{\dimexpr.1402\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Scale & Symptom & Score at 0 week \mbox{}\protect\newline Mean \ensuremath{\pm} SD & Score at 2 weeks Mean \ensuremath{\pm} SD & P-value\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.11530000000000001\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{WOMAC}} &
  Pain in knee joint &
  8.56 \ensuremath{\pm} 2.90 &
  4.13\ensuremath{\pm} 2.82 &
  \textless\ 0.0001\\
 &
  Stiffness of joint &
  5.03\ensuremath{\pm} 2.34 &
  2.46 \ensuremath{\pm} 1.97 &
  \textless\ 0.0001\\
 &
  Level of difficulty in daily activities &
  21\ensuremath{\pm}5.58 &
  12 \ensuremath{\pm} 6.27 &
  \textless\ 0.0001\\
 &
  Total WOMAC score &
  34.66 \ensuremath{\pm} 9.31 &
  18.8 \ensuremath{\pm} 10.29 &
  \textless\ 0.0001\\
VAS \mbox{}\protect\newline  &
  Overall pain intensity &
  6.50 \ensuremath{\pm} 1.757 &
  3.93 \ensuremath{\pm} 1.721 &
  \textless\ 0.0001\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {The comparison of improvement in scores as per WOMAC and VAS in Groups A and B} }
\label{tw-a0f0d618718e}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1252\linewidth-2\tabcolsep}p{\dimexpr.3878\linewidth-2\tabcolsep}p{\dimexpr.19160000000000004\linewidth-2\tabcolsep}p{\dimexpr.19020000000000003\linewidth-2\tabcolsep}p{\dimexpr.10520000000000001\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Scale & Symptom & Improvement in Group A \mbox{}\protect\newline Mean \ensuremath{\pm} SD & Improvement in Group B \mbox{}\protect\newline Mean \ensuremath{\pm} SD & P-value\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.1252\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{WOMAC}} &
  Pain in knee joint &
  3.86\ensuremath{\pm} 2.25 &
  4.43\ensuremath{\pm} 2.82 &
  0.39\\
 &
  Stiffness of joint &
  2.1\ensuremath{\pm} 1.34 &
  2.56\ensuremath{\pm} 1.81 &
  0.263\\
 &
  Level of difficulty in daily activities &
  6.9\ensuremath{\pm} 3.9 &
  8.93\ensuremath{\pm} 5.17 &
  0.108\\
 &
  Total WOMAC score &
  12.86\ensuremath{\pm} 5.65 &
  15.86 \ensuremath{\pm} 9.04 &
  0.17\\
VAS &
  Overall pain intensity &
  2.53 \ensuremath{\pm} 1.38 &
  2.57\ensuremath{\pm} 1.56 &
  0.93\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{p {\textgreater} 0.05 is considered not significant}
\end{tablenotes}
\end{threeparttable}

\end{table*}
In control group A treated with tablet diclofenac 50 mg BID, it was observed that individual symptom scores for each parameter like pain in the knee joint, stiffness and level of difficulty during various daily activities were very highly significantly reduced at the end of 2 weeks of treatment as compared to day 0 scores, as assessed by WOMAC OA Index (p\textless\ 0.0001). The intensity of pain in the arthritic knee joint was also found to be very highly significantly reduced at the end of 2 weeks treatment with diclofenac in this group (p\textless\ 0.0001) (Table~\ref{tw-c90f56a34a34}). 

The group B received diclofenac 50 mg BID and serratiopeptidase 10 mg TID for two weeks. In this group also there was a great reduction(p{\textless}0.0001) in the mean individual scores for pain, stiffness and difficulty during daily activities at the end of 2 weeks of treatment as assessed by WOMAC OA scale. The overall intensity of pain in this group was also very highly significantly decreased (p\textless\ 0.0001) at the end of 2 weeks of treatment, as assessed by using the VAS scale (Table~\ref{tw-ee3bb1167314}).

The differences in all scores between 0 week and 2 weeks, for all parameters of WOMAC and VAS, were calculated and means of group A and B were compared using student's unpaired t-test. There was no significant statistical difference seen in the improvements in group A and B (p{\textgreater} 0.05). Thus, both treatments were equally effective, and no treatment was superior as compared to the other treatment. (Table~\ref{tw-a0f0d618718e})

During the entire treatment course in both the groups, no serious adverse effects were observed/ reported. All the patients completed the treatments, and no treatment was stopped due to any adverse effect. Only 2 patients in the diclofenac group, while one patient in diclofenac + serratiopeptidase group has reported nausea and epigastric pain. The difference was not statistically significant.
    
\section{Discussion}
The present study was aimed to evaluate the efficacy of serratiopeptidase, a proteolytic enzyme obtained from a nonpathogenic bacterium Serratia E15 \unskip~\citep{887067:20340532}, in controlling inflammation and pain associated with osteoarthritis of knee joint in human. In this study, a comparison was done between anti-inflammatory and analgesic effects of diclofenac 100 mg/day and combination treatment of diclofenac 100 mg/day with serratiopeptidase 30 mg/day, administered orally for two weeks. The efficacy of treatments was assessed by measuring the improvement in symptom scores in pain, stiffness and level of difficulty in various daily activities by using WOMAC OA index \unskip~\citep{887067:20340547}. The overall intensity of pain was assessed by using a visual analogue scale.

Our study results show that there was a very highly significant reduction in inflammation and pain at the end of 2 weeks of both treatments. Further, it was also observed that there was no significant difference in the efficacy of both treatments in producing relief in parameters like joint pain, joint stiffness and difficulty in performing daily activities. Thus, it can be concluded from these observations that the addition of oral serratiopeptidase to diclofenac did not potentiate the anti-inflammatory and analgesic effect of diclofenac in osteoarthritis. This result creates doubt about the anti-inflammatory effect of oral serratiopeptidase. This result also raises a question about the bioavailability of oral serratiopeptidase. Serratiopeptidase is a polypeptide having large molecular weight.

Polypeptides given orally are usually degraded by intestinal proteases and then absorbed. There is a meagre number of animal and human studies showing the presence of serratiopeptidase in blood after oral administration \unskip~\citep{887067:20340545,887067:20340536}. For supporting adequate oral bioavailability, more authentic pharmacokinetic data is required. Few studies have shown the anti-inflammatory activity of serratiopeptidase in animal models \unskip~\citep{887067:20340544,887067:20340537}. Antiinflammatory effect of serratiopeptidase in inflammatory venous disease was shown in a clinical study \unskip~\citep{887067:20340546}. Other two clinical studies have also documented anti-inflammatory effect of serratiopeptidase \unskip~\citep{887067:20340543,887067:20340539}. In 6 clinical triasls, the efficacy of oral proteolytic enzymes in osteoarthritis is found to be comparable to diclofenac and tolerability was found better than diclofenac \unskip~\citep{887067:20340529}. In one study, bromelain, another proteolytic enzyme, was found to produce an equal anti-inflammatory effect as diclofenac in osteoarthritis patients \unskip~\citep{887067:20340528}. Two other studies have suggested that oral proteolytic enzyme therapy with bromelain, rutosid and wobenzyme gives either comparable or superior analgesic effect in osteoarthritis patients when compared to oral diclofenac \unskip~\citep{887067:20340541,887067:20340542}. Our results are not matching with all these studies. A study in which the relative efficacy of various analgesic and anti-inflammatory drugs used in practice was compared, failed to show the significant activity of serratiopeptidase \unskip~\citep{887067:20340540}. Nirale and Menon used gel formulations of serratiopeptidase and diclofenac for testing local anti-inflammatory effect in rodents. They claim that both had an anti-inflammatory effect, but there was no statistical difference in their effects \unskip~\citep{887067:20340538}.

 One study concludes that the cost of treatment increases more than five times if serratiopeptidase is combined with NSAIDs. This study also concludes that the trend of prescribing oral proteolytic enzymes along with NSAIDs is very common and is mostly influenced by recommendations of medical representatives \unskip~\citep{887067:20340530}. It is also a worrying fact that this drug is not listed in any official pharmacopoeia, national formulary, and it is not marketed in countries like the USA and UK \unskip~\citep{887067:20340535}.

In a review article, it is suggested that the existing scientific evidence is insufficient to support analgesic anti-inflammatory actions of serratiopeptidase \unskip~\citep{887067:20340531}. 

Therefore, we suggest that more authentic larger clinical trials and pharmacokinetic studies will be required to prove or disprove the efficacy of serratiopeptidase and other proteolytic enzymes as an analgesic and anti-inflammatory drugs. Though few preparations containing serratiopeptidase were banned in India in 2016, many formulations having combinations of serratiopeptidase and NSAIDs and isolated serratiopeptidase are still available in the market. These are being used by clinicians very commonly. Therefore, awareness should be created among clinicians about the lack of proven efficacy of this drug and its absence in any official drug formulary or pharmacopoeia and also essential drug lists. Its use should be discouraged by controlling authorities. This will increase cost-effectiveness and rationality in drug treatment of common ailments like arthritis and other painful and inflammatory conditions.
    
\section{Conclusion}
Addition of serratiopeptidase does not potentiate analgesic and anti-inflammatory effect of diclofenac in osteoarthritis patients. Therefore the combined use of these drugs or use of fixed-dose combination having these drugs as analgesic or anti-inflammatory can not be recommended.

\textbf{Conflict of interest}

The authors declare that they have no conflict of interest for this study.

\textbf{Funding support}

Krishna Institute of Medical Sciences ``Deemed to be University'', Karad, Maharashtra.
    

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