(W1230-01-02) Novel Use of Excised Bovine Stomach to Study Mucoadhesive and Rheological Synergism Properties of Gastro-Retentive Antacid Formulations

Purpose: The current study is aimed to study mucoadhesive strength of antacid formulations intended to act as gastro-retentive delivery systems by novel utilization of bovine stomach portions, followed by rheological measurements to understand correlation of rheological synergism with mucoadhesiveness. It is known that polysaccharide-based hydrocolloid systems tend to form “entangled systems” with mucin, a high molecular weight, heavily glycosylated protein found in mucus, thus providing the “adhesive force” of any mucoadhesive formulation. Hence, it was postulated that an indication of synergistic amplification of resistance to rheological deformation would indicate a heightened mucoadhesion response in freshly excised mucus containing tissue due to increased mucin binding. Our experiments may act as a direction to understand mucoadhesive nature of formulations based on combination of rheology & histological investigations utilizing a bovine stomach portion.

Methods: Gastro-retentive oral formulations were prepared using various types & levels (0.8 to 5.0% w/w) of mucoadhesive polymers comprising of natural hydrocolloids & celluloses. They included formulations singly or in binary combinations of hydrocolloids (HC) such as alginate (HC#1), gellan gum (HC#2), agar (HC#3), modified starch (HC#4), pectin (HC#5), chitosan (HC#6), guar gum (HC#7), xanthan gum (HC#8), locust bean gum (HC#9), carrageenan (HC#10), methylcellulose (MC) & hydroxypropyl methylcellulose (HPMC). Freshly excised bovine stomach, particularly, the reticulum portion, was obtained from slaughterhouses & tissue samples containing mucus membrane were prepared in phosphate buffer (pH 5.8). A fixed amount of formulation was applied to the tissue of fixed area & was subjected to consecutive washings for 4 hours with artificial saliva (pH 6.7), at every 30 minutes interval. Hydrochloric acid could not be used to simulate stomach acidity due to tissue degradation at low pH. At time zero & after every wash, the samples were subjected to optical microscopy & images were analyzed by contrast area measurement against control (Software: Keyence Vision Terminal V2.4.0000), thereby calculating the area of the tissue which retained the formulation from being washed away at each time point (Figure 1). Formulations were screened based on their response to bioadhesive tests & few were chosen as shown in Figure 2. The order of formulations in the decreasing order of adhesiveness were as: F006 > F021 > F019 > F007 > F018 > F017 > F022 > F156 (as control). For a simplified rheology study, 3 formulas F006 (Ratio of HC#10/HC#5=6.25) with highest, F007 (Ratio of HC#10/HC#5=0.16) with intermediate adhesiveness were chosen, along with F156 as control & mixed with pure mucin obtained from bovine submaxillary gland (CAS # 84195-52-8, Sigma Aldrich) in a fixed concentration & subjected to dynamic oscillatory rheology. The changes in storage modulus (G’) & loss modulus (G”) were calculated to understand any “rheological synergism” with mucoadhesion results obtained from bovine mucus tissue (F. Madsen et al.1998).

Results: Significant increases in mucoadhesion force as an indication of rheological synergism were observed in certain binary combinations whereas no or negative interaction was seen for the rest. The mucin interaction (∆G’/G’) for combination of hydrocolloid is established & correlated with results from bovine tissue. Formulation F006 singly and with mucin showed increase in viscoelastic moduli G’ & G” & correlated with mucoadhesive results obtained with bovine tissues (Figure 3). In some cases, effect of one hydrocolloid was augmented by the presence of another, but only up to a certain concentration (results not shown). In addition, the total calculated charge of the hydrocolloid & degree of hydration played a major role in obtaining a strong bioadhesive force. A correlation with rheological effects & bovine tissue, was only observed where the ∆G’/G’ was greater than 5. Formulation F006 along had ∆G’/G’ value of 14 and with mucin, it was 41. The major limitation of this study was to maintain integrity & variability of mucin in mucus membrane in the excised bovine tissue & mimicking the low pH of stomach due to degradation of the excised tissue at low pH. This can be overcome by developing an ex vivo model with in-line modifications which was beyond the scope of this study. A previous study (F. Madsen et al.1998) indicated that rheology could be used as a powerful tool in delineating mucin binding interactions, but it was not correlated with results obtained from actual tissues. However, our study may act as a rapid tool for predicting muco-adhesion by mucin interaction with actual stomach mucin present in mucus membrane.

Conclusion: Although precise combination of hydrocolloids could not be disclosed here due to confidential nature of the project, this approach utilizes a novel method towards understanding and correlating mucoadhesive studies of various hydrocolloid systems using rheology and mucoadhesive histology in tandem. This methodology might contribute towards developing rapid screening and predictive mucoadhesive dosage forms using bovine stomach which has large surface area, easy to handle and source. However, more studies with other animal models are warranted to perfect this model for greater accuracy.

References: Madsen F et al., Journal of Controlled Release 50 (1998) 167–178


Mucoadhesive studies utilizing bovine stomach followed by image analysis


Mucoadhesive capacity measured by percentage retained by relevant formulations over time


Calculation of rheological synergism of relevant formulations