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\begin{document}


\def\RunningAuthor{Venkatesan Palanivel et al.}
\firstPage{8160}
\articleType{Original Article}
\receivedDate{15 Aug 2020}
\acceptedDate{19 Sep 2020}
\revisedDate{18 Sep 2020}
\journalVolume{2020, 11}
\journalIssue{4}
\journalDoi{ijrps.v11i4.709}
\copyrightYear{2020}
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\title{\textbf{Drug-Drug Compatibility Studies of Novel Combination of Drugs for the Treatment of Irritable Bowel Syndrome}}
\author{Sivasakthivel~Muthu\textsuperscript{1},  
            Christakis~Sergides\textsuperscript{2}, 
            Venkatesan~Palanivel\textsuperscript{*}\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Pharmacy, FEAT, Annamalai University, Tamilnadu-608 002, India}~\\{\textsuperscript{2}Research and Development Department, Medochemie Ltd., 3505 Limassol, Cyprus}}

\begin{abstract}
Irritable bowel syndrome (IBS) has become a disorder of concern which affects the large intestine. Symptoms observed includes abdominal pain, cramping, bloating gas, and diarrhoea or constipation, or both. IBS is chronic and needs to manage for long term. Infection caused by bacteria or a virus needs to treat with an antibiotic and other relief of symptoms including intestinal discomfort, bowel disturbances and abdominal pain; are to be treated with antispasmodic. This, urged to design a new drug delivery system that would efficiently release Rifaximin and Pinaverium bromide, in a target specific site to lessen the adverse effects and dosing frequency, when administered in combination. However, certain combinations of drugs may pose safety concerns due to interactions. Thus, the current study was designed to evaluate the interaction of Rifaximin and Pinaverium bromide to develop a combo nano drug delivery system. Thermal stressing and characterization performed to evaluate the interactions of drugs in combination. Rifaximin and Pinaverium bromide combination shown to be stable without any significant physico-chemical interactions. This study proved that Rifaximin and Pinaverium bromide is compatible with each other to develop nanoparticle formulations with combination of both drugs. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Compatibility,\newline Rifaximin,\newline Pinaverium Bromide,\newline Nanoparticles
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Venkatesan~Palanivel~\\ Phone:\ ~\\  Email:\ venkatesan1978@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Rifaximin, 2S-Acetyloxy-5,6,21,23-tetrahydroxy-27-methoxy-2,4,11, 16,20,22,24,26-octamethyl-2,7 (epoxypentoeleca (1,11, 13) trienimino) benzofuro[4,5-e] pyride [1,2-a] benzimidazole-1,15(2H)-dione, is a non-systemic rifampin analogue and a site-specific antibiotic which targets gastrointestinal system \unskip~\citep{1435161:25119561,1435161:25119577}. It showed several indications and used to treat travellers' diarrhoea caused by E. coli; hepatic encephalopathy recurrence; as well as diarrhoea-influenced irritable bowel syndrome (IBS-D) in adults of both sexes. Dosage for Travellers' diarrhoea: One 200mg tablet taken three times a day by oral route \unskip~\citep{1435161:25119565,1435161:25119579}. Dosage for Hepatic encephalopathy: by oral route one 550 mg tablet taken two times a day. The adverse effects associated with Rifaximin for treatment in travellers' diarrhoea (\ensuremath{\geq } 5\%): Flatulence, nausea, headache, abdominal pain, rectal tenesmus and defecation urgency. Most common adverse reactions in hepatic encephalopathy (\ensuremath{\geq } 10\%): Dizziness, flatulence, nausea, fatigue, ascites, headache and peripheral edema. Additionally, Rifaximin reported to have approximately 6 hours of mean elimination half-life, demanding administered in multiple doses daily \unskip~\citep{1435161:25119573,1435161:25119562}. Pinaverium bromide, 4-[(2-bromo-4,5-dimethoxyphenyl)methyl]-4-[2-(2-\{6,6-dimethylbicyclo[3.1.1] heptan-2-yl\}ethoxy)ethyl]morpholin-4-ium, atypical calcium antagonist and a quaternary ammonium compound that acts to restore normal bowel function. Therapeutically administered to get relief of symptoms associated \unskip~\citep{1435161:25119564}  with irritable bowel syndrome (IBS) including abdominal pain \unskip~\citep{1435161:25119574,1435161:25119571,1435161:25119582}, intestinal discomfort and bowel disturbances; and to treat symptoms related to functional illnesses of biliary tract. Dosage is 50mg tablet taken orally three times a day. The dosage maybe increased to maximal total daily dose of 300mg administered 100mg thrice a day. The adverse effects associated with Pinaverium treatment involve nausea, heartburn, distension, epigastric pain and/or fullness, constipation, and diarrhoea. Additionally, Pinaverium bromide reported to have approximately 1.5 hours of mean elimination half-life, demanding administered in multiple doses daily \unskip~\citep{1435161:25119560,1435161:25119563}. This, urged to design and compound a new drug delivery system to effectively modify the release of Rifaximin and Pinaverium bromide, by reducing the dosing frequency and have lesser adverse effects. 

However, certain combinations of drugs may pose interactions, leading to physico-chemical degradations. Physical interactions are those changes in the drug characteristics that do not form any chemical bond or break the drug structure, this can be identified by alterations in the organoleptic parameters. Chemical interactions are those changes in the chemical structure of the molecule which results in reduced drug content by formation of other molecule such as degradation products \unskip~\citep{1435161:25119570,1435161:25119559,1435161:25119581}. Physico-chemical interactions may cause safety concerns. Hence, mandatory thorough evaluation of drug-drug compatibility study is necessary. This study was focused to evaluate the interaction of Rifaximin \unskip~\citep{1435161:25119572}  and Pinaverium bromide to develop a combo nano drug delivery system \unskip~\citep{1435161:25119558,1435161:25119566,1435161:25119568}.
    
\section{MATERIALS AND METHODS}
Rifaximin and Pinaverium bromide were procured respectively from Optimus Drugs, India and Jubilant Generics, India. 

The HPLC grade water was prepared by using Evoqua water technologies, UK. All materials were of high purity grade and used without further purification.

\textbf{Description}

Visual observation of the test samples for any change in colour and texture and recorded.


\begin{table*}[!htbp]
\caption{\boldmath {Thermal stress conditions, testing interval and Test parameters} }
\label{tw-4c750382f184}
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Tests  & \multicolumn{4}{p{\dimexpr(.7834\linewidth-2\tabcolsep)}}{\cAlignHack Condition}\\
\rowcolor{kwdboxcolor} & \cAlignHack 50\ensuremath{^\circ}C/75\%RH & \cAlignHack 40\ensuremath{^\circ}C/75\%RH & \cAlignHack 30\ensuremath{^\circ}C/75\%RH & \cAlignHack 25\ensuremath{^\circ}C/60\%RH\\
\rowcolor{kwdboxcolor} & \cAlignHack Open & \cAlignHack Open & \cAlignHack Open & \cAlignHack Open\\
\tblmidrule 
Description  &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51}\\
FTIR  &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51}\\
DSC  &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51}\\
Water content  &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51}\\
Assay  &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51}\\
Relates substances  &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51} &
  \cAlignHack \ding{51}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Description observations in Thermal stress conditions} }
\label{tw-f8a4f9f73523}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
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\tbltoprule \rowcolor{kwdboxcolor}Sample & Code &  & \multicolumn{4}{p{\dimexpr(.5847\linewidth-2\tabcolsep)}}{\cAlignHack Description }\\
\rowcolor{kwdboxcolor} &  & Initial & \cAlignHack 50\ensuremath{^\circ}C/75\%RH  & \cAlignHack 40\ensuremath{^\circ}C/75\%RH & \cAlignHack 30\ensuremath{^\circ}C/75\%RH & \cAlignHack 25\ensuremath{^\circ}C/60\%RH\\
\rowcolor{kwdboxcolor} &  &  & \cAlignHack Open & \cAlignHack Open & \cAlignHack Open & \cAlignHack Open\\
\tblmidrule 
Pinaverium bromide &
  PIN-AD0297  &
  White to off-white powder &
  \cAlignHack NVC  &
  \cAlignHack NVC &
  \cAlignHack NVC &
  \cAlignHack NVC\\
Rifaximin &
  RIF-A22142  &
  Red-orange powder &
  \cAlignHack NVC  &
  \cAlignHack NVC &
  \cAlignHack NVC &
  \cAlignHack NVC\\
Pinaverium bromide: Rifaximin (1:1) &
  PRCS0519  &
  Orange powder &
  \cAlignHack NVC  &
  \cAlignHack NVC &
  \cAlignHack NVC &
  \cAlignHack NVC\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{NVC: No Visual Change}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {FTIR Samples} }
\label{tw-405a5cb4d084}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.28630000000000006\linewidth-2\tabcolsep}p{\dimexpr.7137\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Sample No. & Sample Name\\
\tblmidrule 
\cAlignHack 1 &
  Pinaverium bromide 25\ensuremath{^\circ}C/60\%RH\\
\cAlignHack 2 &
  Rifaximin 25\ensuremath{^\circ}C/60\%RH\\
\cAlignHack 3 &
  Pinaverium bromide + Rifaximin 25\ensuremath{^\circ}C/60\%RH\\
\cAlignHack 4 &
  Pinaverium bromide 30\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 5 &
  Rifaximin 30\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 6 &
  Pinaverium bromide + Rifaximin 30\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 7 &
  Pinaverium bromide 40\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 8 &
  Rifaximin 40\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 9 &
  Pinaverium bromide + Rifaximin 40\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 10 &
  Pinaverium bromide 50\ensuremath{^\circ}C/75\%RH \\
\cAlignHack 11 &
  Rifaximin 50\ensuremath{^\circ}C/75\%RH\\
\cAlignHack 12 &
  Pinaverium bromide + Rifaximin 50\ensuremath{^\circ}C/75\%RH\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {DSC thermogram samples with endothermal peak temperature} }
\label{tw-7b00067cbbee}
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\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1661\linewidth-2\tabcolsep}p{\dimexpr.5021999999999999\linewidth-2\tabcolsep}p{\dimexpr.3317\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sample No.  & Sample Name  & \cAlignHack Endothermic peak temperature (\ensuremath{^\circ}C)  \\
\tblmidrule 
\cAlignHack 1 &
  Pinaverium bromide 25\ensuremath{^\circ}C/60\%RH  &
  \cAlignHack 166.19\\
\cAlignHack 2 &
  Rifaximin 25\ensuremath{^\circ}C/60\%RH  &
  \cAlignHack 62.00\\
\cAlignHack 3 &
  Pinaverium bromide + Rifaximin 25\ensuremath{^\circ}C/60\%RH  &
  \cAlignHack 61.94, 167.98\\
\cAlignHack 4 &
  Pinaverium bromide 30\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 166.13\\
\cAlignHack 5 &
  Rifaximin 30\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 61.46\\
\cAlignHack 6 &
  Pinaverium bromide + Rifaximin 30\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 54.00, 167.74\\
\cAlignHack 7 &
  Pinaverium bromide 40\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 166.43\\
\cAlignHack 8 &
  Rifaximin 40\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 67.93\\
\cAlignHack 9 &
  Pinaverium bromide + Rifaximin 40\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 64.69, 167.68\\
\cAlignHack 10 &
  Pinaverium bromide 50\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 165.95\\
\cAlignHack 11 &
  Rifaximin 50\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 76.22\\
\cAlignHack 12 &
  Pinaverium bromide + Rifaximin 50\ensuremath{^\circ}C/75\%RH  &
  \cAlignHack 64.03, 167.49\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Water content (\%) values in different storage conditions} }
\label{tw-fdf6377f1517}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.23979999999999997\linewidth-2\tabcolsep}p{\dimexpr.19669999999999995\linewidth-2\tabcolsep}p{\dimexpr.2083\linewidth-2\tabcolsep}p{\dimexpr.18000000000000004\linewidth-2\tabcolsep}p{\dimexpr.17520000000000003\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Tests  & \multicolumn{4}{p{\dimexpr(.7602000000000001\linewidth-2\tabcolsep)}}{\cAlignHack Condition }\\
\rowcolor{kwdboxcolor} & \cAlignHack 25\ensuremath{^\circ}C/60\%RH Open & \cAlignHack 30\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 40\ensuremath{^\circ}C/75\%RH Open  & \cAlignHack 50\ensuremath{^\circ}C/75\%RH \\
\tblmidrule 
Pinaverium bromide  &
  \cAlignHack 0.34 &
  \cAlignHack 0.64 &
  \cAlignHack 0.54 &
  \cAlignHack 0.43\\
Rifaximin  &
  \cAlignHack 5.99 &
  \cAlignHack 4.35 &
  \cAlignHack 6.38 &
  \cAlignHack 6.39\\
Pinaverium bromide + Rifaximin  &
  \cAlignHack 3.95 &
  \cAlignHack 3.04 &
  \cAlignHack 2.71 &
  \cAlignHack 4.37\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Assay (\%) values in different storage conditions} }
\label{tw-c93b23dc3922}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.263\linewidth-2\tabcolsep}p{\dimexpr.1818\linewidth-2\tabcolsep}p{\dimexpr.2049\linewidth-2\tabcolsep}p{\dimexpr.1752\linewidth-2\tabcolsep}p{\dimexpr.1751\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Tests & \multicolumn{4}{p{\dimexpr(.737\linewidth-2\tabcolsep)}}{\cAlignHack Condition}\\
\rowcolor{kwdboxcolor} & \cAlignHack 25\ensuremath{^\circ}C/60\%RH Open & \cAlignHack 30\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 40\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 50\ensuremath{^\circ}C/75\%RH Open\\
\tblmidrule 
Pinaverium bromide &
  \cAlignHack 100.2 &
  \cAlignHack 102.2 &
  \cAlignHack 103.5 &
  \cAlignHack 99.9\\
Rifaximin &
  \cAlignHack 98.2 &
  \cAlignHack 97.3 &
  \cAlignHack 93.2 &
  \cAlignHack 92.7\\
Pinaverium bromide from Pinaverium bromide + Rifaximin mixture &
  \cAlignHack 102.2 &
  \cAlignHack 100.5 &
  \cAlignHack 99.4 &
  \cAlignHack 97.5\\
Rifaximin from Pinaverium bromide + Rifaximin mixture &
  \cAlignHack 96.0 &
  \cAlignHack 93.1 &
  \cAlignHack 91.2 &
  \cAlignHack 89.9\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Related substances (\%) values of Pinaverium bromide and Pinaverium bromide in Pinaverium bromide + Rifaximin mixture in different storage conditions} }
\label{tw-01fd623a92e6}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.21159999999999997\linewidth-2\tabcolsep}p{\dimexpr.18840000000000003\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Main impurities  & \multicolumn{4}{p{\dimexpr(.7884\linewidth-2\tabcolsep)}}{\cAlignHack Pinaverium bromide }\\
\rowcolor{kwdboxcolor}(RRT) & \cAlignHack 25\ensuremath{^\circ}C/60\%RH Open & \cAlignHack 30\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 40\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 50\ensuremath{^\circ}C/75\%RH\\
\tblmidrule 
Unknown (\%) (RRT 1.255) &
  \cAlignHack ND &
  \cAlignHack 0.05 &
  \cAlignHack 0.06 &
  \cAlignHack 0.05\\
Unknown (\%) \mbox{}\protect\newline (RRT 1.320) &
  \cAlignHack ND &
  \cAlignHack ND &
  \cAlignHack ND &
  \cAlignHack 0.05\\
Total (\%) &
  \cAlignHack 0.00 &
  \cAlignHack 0.05 &
  \cAlignHack 0.06 &
  \cAlignHack 0.10\\
\cellcolor[HTML]{E5E5E5}{Main impurities } &
  \multicolumn{4}{p{\dimexpr(.7884\linewidth-2\tabcolsep)}}{\cellcolor[HTML]{E5E5E5}\cAlignHack {Pinaverium bromide in Pinaverium bromide + Rifaximin mixture }}\\
\cellcolor[HTML]{E5E5E5}{(RRT)} &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{25\ensuremath{^\circ}C/60\%RH Open } &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{30\ensuremath{^\circ}C/75\%RH Open } &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{40\ensuremath{^\circ}C/75\%RH Open } &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{50\ensuremath{^\circ}C/75\%RH }\\
Unknown (\%) (RRT 1.255) &
  \cAlignHack - &
  \cAlignHack - &
  \cAlignHack - &
  \cAlignHack -\\
Unknown (\%) \mbox{}\protect\newline (RRT 1.320) &
  \cAlignHack - &
  \cAlignHack - &
  \cAlignHack - &
  \cAlignHack -\\
Total (\%) &
  \cAlignHack 0.00 &
  \cAlignHack 0.00 &
  \cAlignHack 0.00 &
  \cAlignHack 0.00\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{-: Not Detected}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Related substances (\%) values of Rifaximin and Rifaximin in Pinaverium bromide + Rifaximin mixture in different storage conditions} }
\label{tw-d9904b61a9ef}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2796\linewidth-2\tabcolsep}p{\dimexpr.19509999999999998\linewidth-2\tabcolsep}p{\dimexpr.19330000000000002\linewidth-2\tabcolsep}p{\dimexpr.1685\linewidth-2\tabcolsep}p{\dimexpr.16349999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Main impurities (RRT) & \multicolumn{4}{p{\dimexpr(.7204\linewidth-2\tabcolsep)}}{\cAlignHack Rifaximin}\\
\rowcolor{kwdboxcolor} & \cAlignHack 25\ensuremath{^\circ}C/60\%RH Open & \cAlignHack 30\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 40\ensuremath{^\circ}C/75\%RH Open & \cAlignHack 50\ensuremath{^\circ}C/75\%RH\\
\tblmidrule 
Unknown (\%) (RRT 0.494) &
  \cAlignHack 0.05 &
  \cAlignHack 0.05 &
  \cAlignHack - &
  \cAlignHack -\\
Unknown (\%) \mbox{}\protect\newline (RRT 0.621) &
  \cAlignHack 0.06 &
  \cAlignHack - &
  \cAlignHack 0.10 &
  \cAlignHack 0.16\\
Unknown (\%) \mbox{}\protect\newline (RRT 0.642) &
  \cAlignHack 0.12 &
  \cAlignHack 0.13 &
  \cAlignHack 0.14 &
  \cAlignHack 0.15\\
Impurity D+H (\%) \mbox{}\protect\newline (RRT 0.761) &
  \cAlignHack - &
  \cAlignHack - &
  \cAlignHack - &
  \cAlignHack 0.06\\
Total (\%) &
  \cAlignHack 0.23 &
  \cAlignHack 0.18 &
  \cAlignHack 0.24 &
  \cAlignHack 0.37\\
\cellcolor[HTML]{E5E5E5}{Main impurities (RRT) } &
  \multicolumn{4}{p{\dimexpr(.7204\linewidth-2\tabcolsep)}}{\cellcolor[HTML]{E5E5E5}\cAlignHack {Rifaximin in Pinaverium bromide + Rifaximin mixture }}\\
\cellcolor[HTML]{E5E5E5}{} &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{25\ensuremath{^\circ}C/60\%RH Open } &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{30\ensuremath{^\circ}C/75\%RH Open } &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{40\ensuremath{^\circ}C/75\%RH Open } &
  \cAlignHack \cellcolor[HTML]{E5E5E5}{50\ensuremath{^\circ}C/75\%RH }\\
Unknown (\%) \mbox{}\protect\newline (RRT 0.416) &
  \cAlignHack -  &
  \cAlignHack -  &
  \cAlignHack -  &
  \cAlignHack 0.07\\
Unknown (\%) \mbox{}\protect\newline (RRT 0.429) &
  \cAlignHack -  &
  \cAlignHack -  &
  \cAlignHack -  &
  \cAlignHack 0.10\\
Unknown (\%) \mbox{}\protect\newline (RRT 0.495) &
  \cAlignHack 0.05 &
  \cAlignHack -  &
  \cAlignHack -  &
  \cAlignHack - \\
Unknown (\%) \mbox{}\protect\newline (RRT 0.621) &
  \cAlignHack 0.07 &
  \cAlignHack 0.06 &
  \cAlignHack 0.13 &
  \cAlignHack 0.32\\
Unknown (\%) \mbox{}\protect\newline (RRT 0.643) &
  \cAlignHack 0.12 &
  \cAlignHack 0.12 &
  \cAlignHack 0.15 &
  \cAlignHack 0.26\\
Impurity D+H (\%) \mbox{}\protect\newline (RRT 0.761) &
  \cAlignHack -  &
  \cAlignHack -  &
  \cAlignHack 0.05 &
  \cAlignHack 0.10\\
Total (\%) &
  \cAlignHack 0.00 &
  \cAlignHack 0.00 &
  \cAlignHack 0.00 &
  \cAlignHack 0.00\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{-: Not Detected}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\bgroup
\fixFloatSize{images/8d47b0d5-1116-4f37-82e9-c0465ace1df9-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/8d47b0d5-1116-4f37-82e9-c0465ace1df9-upicture1.png}{\includegraphics{images/8d47b0d5-1116-4f37-82e9-c0465ace1df9-upicture1.png}}{}
\makeatother 
\caption{FTIR Chromatograms of Samples 1-6}
\label{f-2bed3c3a83a6}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/9cf73c9d-c40f-4cc2-b370-8832282fbfd6-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/9cf73c9d-c40f-4cc2-b370-8832282fbfd6-upicture2.png}{\includegraphics{images/9cf73c9d-c40f-4cc2-b370-8832282fbfd6-upicture2.png}}{}
\makeatother 
\caption{\boldmath {FTIR Chromatograms of Samples 7-12}}
\label{f-535738ca6225}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/7390f287-bc87-4b8f-bec8-aef704344708-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/7390f287-bc87-4b8f-bec8-aef704344708-upicture3.png}{\includegraphics{images/7390f287-bc87-4b8f-bec8-aef704344708-upicture3.png}}{}
\makeatother 
\caption{\boldmath {DSC Thermograms of Samples 1-6}}
\label{f-9f7cbddcc933}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/f159022e-80b8-4bbd-8cd2-1461707f1edd-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/f159022e-80b8-4bbd-8cd2-1461707f1edd-upicture4.png}{\includegraphics{images/f159022e-80b8-4bbd-8cd2-1461707f1edd-upicture4.png}}{}
\makeatother 
\caption{\boldmath {DSC Thermograms of Samples 7-12}}
\label{f-63146c4b427d}
\end{figure*}
\egroup
\textbf{Fourier Transform Infrared Spectroscopy (FTIR)}

The samples were recorded for FTIR spectra in a Cary 630 Spectrometer (Agilent Technologies) using potassium bromide (KBr) disk technique \unskip~\citep{1435161:25119576,1435161:25119575,1435161:25119580}. Samples equivalent to 2mg of Pinaverium bromide, Rifaximin, and Pinaverium bromide + Rifaximin mixture were diluted with potassium bromide (about 100mg) respectively with glass mortar and pestle to compress disc. The samples were scanned after baseline correction against a blank KBr disc in the range of 4000{\textendash}400 cm\ensuremath{^{-1}} wave number.

\textbf{Differential Scanning Calorimetry (DSC)}

The DSC thermogram of samples were recorded in a Q100 Differential Scanning Calorimeter  (TA Instruments) using DSC aluminium sample pans. Samples equivalent to 50mg of Pinaverium bromide, Rifaximin, and Pinaverium bromide + Rifaximin mixture were placed in the aluminium sample pan individually and were scanned in the temperature range of 20{\textendash}200\ensuremath{^\circ}C \unskip~\citep{1435161:25119578,1435161:25119567,1435161:25119569}.

\textbf{Water Content }

Water content of samples were analysed by Karl Fischer titration method using 870 KF Titrino Plus (Metrohm AG). Samples equivalent to 100mg of Pinaverium bromide, Rifaximin, and Pinaverium bromide + Rifaximin mixture were tested individually for water content using a mixture of 1 volume of formamide and 2 volumes of methanol as the solvent.

\textbf{Assay}

Assay of Pinaverium bromide was measured by HPLC with WATERS Alliance e2695 equipped with Photodiode Array Detector 996. The separation was achieved with Symmetry C18, 150mm x 4.0mm, 3.5\ensuremath{\mu }m, No. 310. The elution was performed with mobile phase 1\% triethylamine in water and acetonitrile (40:60), adjusted the pH to 3.5\ensuremath{\pm}0.05 using formic acid. Diluent is same as mobile phase. 1mL/min was the flow rate with controlled temperature at 28\ensuremath{\pm}2\ensuremath{^\circ}C. PDA detector was set at the wavelength of 254nm and injection volume was 10\ensuremath{\mu }L for every samples and standard. Standard solution is prepared by weighing 50mg of Pinaverium bromide standard in a 100mL flask, dissolve with 30mL of diluent and make up the volume with the diluent. Test solution is prepared by weighing the sample equivalent to 50mg of Pinaverium bromide in a 100mL flask, dissolve with 60mL of diluent with shaking for 20min and make up the volume with the diluent. Assay of Rifaximin was measured by HPLC with WATERS Alliance e2695 equipped with Photodiode Array Detector 996. The separation was achieved with InerSustain C18, 250mm x 4.6mm, 5\ensuremath{\mu }m. The elution was performed with mobile phase buffer+solvent mixture (37:63), buffer is prepared with 3.16\% ammonium formate in water, adjusted the pH to 7.2\ensuremath{\pm}0.05 using ammonia solution and solvent mixture is methanol: acetonitrile (50:50). Diluent is Acetonitrile-water mixture (40:60). 1.4mL/min was the flow rate with controlled temperature at 40\ensuremath{\pm}2\ensuremath{^\circ}C. PDA detector was set at the wavelength of 276nm and injection volume was 20\ensuremath{\mu }L for every samples and standard. Standard solution is prepared by weighing 40mg of Rifaximin working standard in a 100mL flask, dissolve with 50mL of diluent and make up the volume with the diluent, dilute 5mL of this solution to 50mL with diluent. Test solution is prepared by weighing the sample equivalent to 40mg of Rifaximin in a 100mL flask, dissolve with 50mL of diluent with shaking for 20min and make up the volume with the diluent, dilute 5mL of this solution to 50mL with diluent.

\textbf{Related Substances}

Related substances of Pinaverium bromide were measured by HPLC with WATERS Alliance e2695 equipped with Photodiode Array Detector 996. The separation was achieved with Symmetry C18, 150mm x 4.0mm, 3.5\ensuremath{\mu }m, No. 310. The elution was performed by Gradient with mobile phase A [0.5\% triethylamine in water and acetonitrile (40:60), adjusted the pH to 3.5\ensuremath{\pm}0.05 using formic acid] and mobile phase B [0.5\% triethylamine in water and acetonitrile (60:40), adjusted the pH to 3.5\ensuremath{\pm}0.05 using formic acid]. Diluent is same as mobile phase A. 1mL/min was the flow rate with controlled temperature at 28\ensuremath{\pm}2\ensuremath{^\circ}C. PDA detector was set at the wavelength of 254nm and injection volume was 20\ensuremath{\mu }L for every samples and reference. Reference solution is prepared by weighing 5.0mg of each impurity A, impurity B and Pinaverium Br standard in a 100mL flask, and dissolve with mobile phase; dilute 5mL of this solution to 25mL with mobile phase. Test solution is prepared by weighing the sample equivalent to 100mg of Pinaverium bromide in a 50mL flask, dissolve with 20mL of mobile phase with shaking for 20min and make up the volume with the mobile phase. Related substances of Rifaximin was measured by HPLC with WATERS Alliance e2695 equipped with Photodiode Array Detector 996. The separation was achieved with InertSustain C18, 250mm x 4.6mm, 5\ensuremath{\mu }m. The elution was performed with mobile phase buffer+solvent mixture (37:63), buffer is prepared with 3.16\% ammonium formate in water, adjusted the pH to 7.2\ensuremath{\pm}0.05 using ammonia solution and solvent mixture is methanol: acetonitrile (50:50), 1.4mL/min was the flow rate with controlled temperature at 40\ensuremath{\pm}2\ensuremath{^\circ}C. Diluent is Acetonitrile-water mixture (40:60). PDA detector was set at the wavelength of 276nm and injection volume was 20\ensuremath{\mu }L for every samples and reference. Reference solution is prepared by weighing 100 mg of rifaximin in a 20mL flask, add 1mL of 30\% hydrogen peroxide solution, shaken and kept aside for 30min, and make up the volume with the diluent, 1mL of this solution was diluted to 50mL with diluent and then diluted 1mL of this solution to 10mL with the diluent. Test solution is prepared by weighing the sample equivalent to 40mg of Rifaximin in a 20mL flask, add 8mL of acetonitrile shaking for 20min and make up the volume with the water.

\textbf{Experimental Studies}

\textbf{Thermal Stress Conditions and Sample Preparation}

Samples for thermal stress testing were prepared in the ratio of Pinaverium bromide: Rifaximin (1:1). Individual drugs and the combined drugs samples were placed in the Espec constant climate chamber and followed In-house stress conditions as shown in Table~\ref{tw-4c750382f184}.
    
\section{RESULTS AND DISCUSSION}
Samples were removed from the ovens after the stressing period and analysed for Description, FTIR, DSC, Water content, Assay and Related Substances.

\textbf{Description}

Description (appearance) of samples remained same without any visual changes during the storage period (Table~\ref{tw-f8a4f9f73523}). Hence, it is proved that the drug-drug combinations were physically stable.

\textbf{FTIR Spectrometry}

IR chromatograms of samples as shown in Table~\ref{tw-405a5cb4d084}  are presented in Figure~\ref{f-2bed3c3a83a6}  and Figure~\ref{f-535738ca6225}. FT-IR spectrum of samples (1 to 12) with absorption bands which were characteristic and comparable with the absorption bands of an individual sample. Hence, it is proved that the drug-drug combinations were chemically stable.

\textbf{Differential Scanning Calorimetry}

DSC thermograms of samples as shown in Table~\ref{tw-7b00067cbbee}  are displayed in Figure~\ref{f-9f7cbddcc933}  and Figure~\ref{f-63146c4b427d}. Pinaverium bromide illustrate an endothermal peak at around 165\ensuremath{^\circ}C, while Rifaximin shows a broad endothermal peak between 40 {\textendash} 100\ensuremath{^\circ}C. It is obvious that Pinaverium Bromide melting point is stable under four different storage conditions. In contrast, in the case of Rifaximin, it looks that the temperature of the endothermic peak increases with increase in the storage temperature. In particular, Rifaximin stored at 25\ensuremath{^\circ}C/60\%RH exhibited endothermic peak at 60\ensuremath{^\circ}C while Rifaximin stored at 50\ensuremath{^\circ}C/75\%RH endothermic peak was at 76\ensuremath{^\circ}C. In the case of combination of Pinaverium Bromide and Rifaximin, both endothermic peaks were observed with Pinaverium peak being more intense. Hence, it is proved that the drug-drug combinations were chemically stable.

\textbf{Water Content}

Water content varies in different storage conditions which were comparable with individual sample and presented in Table~\ref{tw-fdf6377f1517}. Hence, it is proved that the drug-drug combinations were physically stable.

\textbf{Assay}

Assay of individual drugs and drug mixtures are presented in Table~\ref{tw-c93b23dc3922}. Assay results of drugs from drug mixtures were comparable with individual drugs. Hence, it is proved that the drug-drug combinations were chemically stable.

\textbf{Related Substances}

Related substances of individual drugs and drug mixtures are presented in Table~\ref{tw-01fd623a92e6}  and Table~\ref{tw-d9904b61a9ef}. Related substances results of drugs from drug mixtures shows there is no any significant degradation in comparison with the individual drugs. Hence, it is proved that the drug-drug combinations were chemically stable.
    
\section{CONCLUSION }
The novel combination of Pinaverium bromide and Rifaximin proven to be stable without any significant physico-chemical interactions in the various tests performed in drug-drug compatibility studies at various stress conditions. Hence, this research proved that the Pinaverium bromide and Rifaximin are compatible with each other and this knowledge can be useful in developing modified-release nanoparticle formulations containing a combination of Pinaverium bromide and Rifaximin for the better treatment of irritable bowel syndrome (IBS).

\textbf{Funding Support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that there is no conflict of interest.
    

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\end{document}
