**2. Oral route**

## **2.1 Chemical modification**

Chemical modification of proteins and peptides is one of the strategies adopted to make these more clinically applicable [21]. This strategy eliminates some of the undesirable properties of proteins and peptides, such as susceptibility to enzyme hydrolysis, improper solubility, poor membrane permeability, etc. There are several ways of performing the chemical modification, including replacement of a specific amino acid or modification of the structure of amino acids. For example, replacement of an L-amino acid with its D-counterpart may lead to resistance to enzymatic hydrolysis, enhanced cell-membrane permeability, etc. In desmopressin (an analog of vasopressin), the C-terminal L-Arg has been replaced by D-Arg (**Figure 3**). Also, the N-terminal amino group (of cysteine) has been deaminated (**Figure 3**). These chemical modifications resulted in substantially higher oral bioavailability of desmopressin than that of vasopressin [22].

Another method of chemical modification is to incorporate a lipophilic moiety (e.g., fatty acid) in the protein or peptide. The lipophilic moiety will increase the

**Figure 3.** *Structure of vasopressin (left) and desmopressin (right).*

overall hydrophobicity of the molecule, which may lead to an increase in its intestinal absorption. Increased hydrophobicity may also lead to increased stability of the protein or peptide [23]. For example, three amino acids (Gly, Phe, and Lys) in insulin were attached to 1, 3-dipalmitoyl glycerol moiety. This modification resulted in an increased hydrophobicity of insulin as well as increased intestinal absorption. The enzymatic hydrolysis of insulin was also reduced, which resulted in increased bioavailability [24].

#### *2.1.1 PEGylation*

PEGylation involves the covalent attachment of one or more polyethylene glycol (PEG) molecule(s) to a protein or peptide. PEG has some special features that make it suitable for protein and peptide delivery systems [25–28]. One important feature is that it is soluble in both organic and aqueous solvents. Another feature is that PEG can be obtained in a wide range of molecular weights. It can be obtained both as a linear or branched chain. PEG is more hydrophilic than other polymers of comparable sizes. Also, PEG is highly flexible since there is no bulky substituent around the backbone to hinder the rotation. Finally, PEG is non-toxic and non-immunogenic.

PEG improves the pharmacokinetic properties of proteins and peptides by protecting these from enzymatic hydrolysis (**Figure 4**) and by increasing solubility. PEG acts as a shield that prevents the hydrolysis of the protein or peptide by blocking its access to the proteolytic enzyme (**Figure 5**) [29]. The attachment of PEG with a protein or peptide increases its size, which increases the circulation half-life of the protein or peptide by decreasing the clearance by the renal filtration process. The attachment of PEG to a protein or peptide also reduces immunogenicity by reducing the recognition by the β-cell or antibodies. Finally, the attachment of PEG to proteins or peptides prevents their possible aggregation [30].

PEGylation process can interfere with the molecular recognition between the protein or peptide and the receptor due to the modified structure of the PEGylated protein or peptide. For example, PEGylation of a protein or peptide may cause steric hindrance, which may prevent proper binding of the protein or peptide to the target. However, this drawback is balanced by the improvements in pharmacokinetic properties, such as enhanced absorption and circulation half-life [31]. The development of Nobex Corporation's HIM2 was based on the PEGylation approach. HIM2 is hexyl insulin monoconjugate 2 that is attached to a PEG. The attachment of PEG improves the stability of the molecule and enhances bioavailability that allows oral administration [32].

#### *2.1.2 Peptidomimetics*

Peptidomimetics mimic the structure of a protein or peptide and show acceptable pharmacokinetic properties while retaining biological activity. Different modifications (N-alkylation, isosteric replacement of the amide bond, etc.) can be performed on a peptide to improve its pharmacokinetic properties [33]. Structural

**Figure 4.** *Enzymatic hydrolysis of the peptide bond.*

**Figure 5.** *PEGylaiton blocks enzymatic hydrolysis of peptides.*

modifications are mainly performed to make the peptide more stable in the GIT by making it less susceptible to enzymatic degradation. Solubility, lipophilicity, and the flexibility of the peptide can also be altered to enhance stability as well as absorption. Modification of a peptide may occur at the backbone of the peptide or the side chains of amino acids, or both. The alkylation of the backbone N-atom has been shown to improve the bioavailability of peptide molecules [34].

N-alkylation, in general, leads to an increase in lipophilicity of the peptide as well as a steric hindrance. N-alkylation also leads to a reduction in H-bonding since the H-atom of the backbone amide has been replaced by an alkyl group. This decrease in H-bonding may lead to destabilization of α-helix and β-sheets, which may alter the conformation of the peptide. N-alkylation method has been used to make cyclosporine (**Figure 6**) where the N-atoms of the backbone have been alkylated by methyl groups (green spheres) [35].

Isosteric replacement of amide bond is another strategy for peptidomimetics design. This process can lead to alteration in H-bonding as well as peptide-folding. Thus, this process may affect the conformation of the peptide. For example, the replacement of a carbon-atom by an N-atom gives azapeptide class. Azapeptides can be of two types – azatides and peptoids. In azatides, all the α-carbon in the peptide backbone has been replaced by nitrogen atom. In peptoids, the α-carbon is replaced by N-atom, and the N-atom of the backbone is replaced by C-atoms. Ritonavir (**Figure 7**) is an example of a drug developed by the peptidomimetics approach [36, 37].

#### **2.2 Enzyme inhibition**

Inhibition of enzymes like proteases is a good way of improving the stability of proteins and peptides in the GIT since these enzymes hydrolyze peptide bonds in proteins and peptides administered via the oral route. Proteases can be classified into different categories depending on the catalytic amino acid residue

**Figure 6.** *Backbone N-alkylation in cyclosporine.*

**Figure 7.** *Ritonavir.* (Ser, Thr, Asp, Glu, etc.) in the active site of the enzyme [38]. For example, serine proteases contain a serine residue in the active that acts as a nucleophile during the hydrolysis of the peptide bond. Another class of proteases is metalloproteases, where the water molecule used for hydrolysis is ligated to a metal ion (Zn2+, Co2+, Mn2+, etc.) in the active site of the enzyme. Thus, one way of preventing hydrolysis of the protein or peptide drug is to co-administer protease inhibitors [39]. Insulin, for example, undergoes degradation in the GIT by different enzymes like trypsin, chymotrypsin, etc. Therefore insulin is co-administered with various synthetic (e.g., camostat mesylate) and naturally occurring inhibitors (soybean trypsin inhibitor) of trypsin and chymotrypsin. This coadministration leads to enhanced bioavailability of insulin [40]. However, the administered protein other than the therapeutic protein will not be degraded, leading to toxic side effects. Besides, the non-degraded proteins may also cause metabolic changes in the GIT [41].

Another way of protease inhibition is to alter the pH of the medium in which these enzymes work. This pH change may lead to the inactivation of the proteases. It has been reported that lowering the pH of the intestine to 4.5 or below leads to the inhibition of trypsin and chymotrypsin [23].
