**2.3 Absorption enhancers**

Absorption enhancers are substances co-administered with protein or peptide drugs to enhance their absorption. In general, these absorption enhancers reversibly damage the physical barrier in the cell wall, which prevents a protein or peptide from crossing the intestinal wall. Thus, absorption enhancers provide a temporal path for the proteins or peptides to cross the intestinal wall and to be absorbed [42]. There are two main ways by which absorption enhancers cause the temporal opening – transcellular pathway and paracellular pathway. The transcellular mechanism involves the structural change in the cell membrane of the epithelial cells. This structural alteration leads to enhanced passive diffusion of proteins and peptides through the cell. In the paracellular pathway, the absorption enhancers facilitate the opening of tight junctions between the epithelial cells, which allows the protein and peptide to diffuse through the intercellular space present between the epithelial cells. Another absorption enhancing mechanism involves the reduction of viscosity of the mucus in the intestinal wall, which enhances the diffusion of proteins and peptides [25].

There are different types of absorption enhancers based on their molecular structure and mechanism of action. For example, ethylene diamine tetraacetic acid (EDTA) works by chelating Ca-ions, which are important in maintaining the tight junction between the cells. Therefore, when Ca-ions are complexed by EDTA, the tight junction between the cells will be opened, allowing the proteins and peptides to cross the intercellular space. However, surfactants (sodium lauryl sulfate, Tween 40, etc.) work by disrupting the intestinal membrane allowing the protein and peptide to cross the cells via the transcellular pathway [43].

It is worth mentioning that the absorption enhancers are potentially toxic since some of them disrupt the integrity of the intestinal membrane. Damage of the intestinal membrane can cause proteins or peptides other than the protein or peptide of interest to be absorbed, leading to toxicity. Besides, pathogens (virus, bacteria, etc.) may also get absorbed, which may lead to various pathological conditions. Severe damage of the intestinal membrane may also lead to inflammatory conditions and ulceration of the epithelium. Therefore, the toxicology of the absorption enhancers needs to be fully understood before their long-term applications [44].

#### **2.4 Site specific delivery in GIT**

Different regions of the GIT show differences in the absorption of proteins and peptides. These differences are due to different pH values and different distribution of proteolytic enzymes at different regions of the GIT. The pH affects both the solubility and the stability of the protein or peptide, while the proteolytic enzymes are responsible for the degradation of the protein or peptide. There is also variability in the distribution of active transporters involved in peptide transport and the efflux pumps that can lower the absorption of proteins or peptides along the GIT [45, 46].

Extensive research has been performed to locate the optimum absorption site in the GIT for proteins or peptides [47]. These results indicate that the colon region of the GIT is one of the optimum sites for protein or peptide absorption mainly due to the lower protease activity in the colon area compared to other areas in the GIT, such as the stomach and small intestine. Therefore, several strategies have been employed to deliver the therapeutic protein or peptide intact to the colon. One approach is to design a prodrug (**Figure 8**) with adequate stability in the other regions of the GIT [48]. However, it should be converted to the parent therapeutic protein or peptide in the colon. Since the microflora in the colon produces reductive enzymes, the prodrug should be designed by linking the therapeutic protein or peptide via a bond (e.g., azo bond) that can be cleaved by reductive enzymes in the colon [49]. These reducing enzymes can also be utilized to attach a polymeric carrier to the protein or peptide (**Figure 9**). This polymer will protect the therapeutic

**Figure 9.** *A prodrug with a polymeric carrier.*

**Figure 8.**

peptide molecule along the GIT and will release the protein or peptide at the colon site, enhancing its absorption.
