Elimination

Rapid elimination due to poor in vivo stability of peptides is the most significant pharmacokinetic barrier limiting the clinical use of peptides. The renal filtration of peptides is also efficient but its role is often less significant (Tang et al. 2004; Werle and Bernkop-Schnürch 2006). The short elimination half-lives, from a few minutes to a few hours, of many peptides can be explained by their physiological role as hormones (Tang et al. 2004).

Rapid degradation allows the strict regulation of endogenous concentrations of peptides, and consequently prompt adjustment of their functions.

The broad spectrum of proteolytic enzyme activities is main reason for the rapid elimination of peptides and these enzymes are distributed ubiquitously throughout the body, most importantly in blood, liver and kidneys (Tang et al. 2004; Werle and Bernkop-Schnürch 2006; Lin 2009). Furthermore, since peptides are often hydrophilic compounds, they are degraded by the soluble enzymes present in blood or enzymes bound at the membrane, rather than enzymes in the cytoplasm (Werle and Bernkop-Schnürch 2006). The proteolytic enzymes responsible for cleaving peptides can be divided into two main categories, namely exo- and endopeptidases (Werle and Bernkop-Schnürch 2006; Lin 2009).

Exopeptidases remove one or two amino acids either from the N- (aminopeptidase) or from the C-terminals of the peptide chain (carboxypeptidase), whereas endopeptidases cleave the peptide bonds within the peptide chain (Werle and Bernkop-Schnürch 2006; Lin 2009).

Each peptidase has its own cleavage specificity for certain peptides and the localization of enzymes between tissues varies (Tang et al. 2004). For example, brush border enzymes are

situated on the luminal membrane of kidneys and they specifically hydrolyze small linear peptides, such as angiotensin I and II, bradykinin (Carone and Peterson 1980) or peptide YY3-36 (Addison et al. 2011). In contrast, larger peptides, such as insulin, are endocytosed at the luminal membrane of the kidney and subsequently degraded in lysozymes (Carone et al. 1982). Another specific feature for peptide elimination is receptor-mediated elimination via cellular uptake at the target sites of peptides (Tang et al. 2004). Receptors can be saturated at therapeutic concentrations, and thus, this elimination route can represent a source of non-linear pharmacokinetics.

Peptides are smaller than 10 kDa in size, and thus they are freely filtered by the glomerus in the kidneys. However, the significance of renal clearance via glomerural filtration is negligible in the elimination of many peptides unless the enzymatic degradation pathway is blocked (Lin 2009). This can be illustrated by the fact that the complete removal of the peptide via kidneys would require approximately 60 minutes for even the peptide strictly confined within blood circulation. However, many peptides are cleared in less than 60 minutes (Lin 2009). This conclusion is further supported by the studies showing that renal impairment does not affect insulin pharmacokinetics (Holmes et al. 2005) and that urine excretion of N-acetyl-seryl-aspartyl-lysyl-proline peptide increased significantly after the enzyme inhibition (Azizi et al. 1999).

Different chemical modifications of peptides have been introduced in order to prolong the elimination half-lives of peptidesi.e. by making the modified peptide less susceptible to enzymatic degradation or renal excretion. The main methods of modification are alteration in the peptide structure, or the conjugation of peptide with protein, polysialic acid or PEG (Werle and Bernkop-Schnürch 2006; Nestor Jr. 2009; Gentilucci et al. 2010). For example, the acetylation of N-terminal or amidation of C-terminal, improved the plasma stability of MART-I^27-35so that the activity of the modified peptide was 5- to 6-fold higher than the activity of the unmodified counterpart after 20 h incubation in plasma (Brinckerhoff et al.

1999). Another example of structure modification is the replacement of labile, natural L-amino acids with more stable unnatural D-L-amino acids since few human enzymes are able to hydrolyze D-amino acids (Powell et al. 1993; Gentilucci et al. 2010). The conjugation of the peptide with a large molecule can increase peptide circulation time through two mechanisms; making the peptide more stable towards enzymes and increasing the molecular size to reduce the extent of renal filtration (Caliceti and Veronese 2003; Werle and Bernkop-Schnürch 2006; Nestor Jr. 2009). The latter approach requires the use of a PEG chain over 38 kDa or alternatively a protein the size of serum albumin. Examples of these approaches reducing elimination and retaining the biological activity of the peptide include the conjugation of GLP-1 via linker to serum albumin (Li et al. 2010) or PEGylation of salmon calcitonin (Ryan et al. 2009) (Table 2.1).

Table 2.1. Recent examples of in vivo studies of peptide delivery systems for different administration routes.

Technology Peptide (Mw) Result Reference

Intravenous

7- to 15-fold increase in elimination half-life after i.v. administration but

Terminal half-life prolonged from 2 to 8 h, and Cmax –values decreased 6- to compared with s.c. injection of solution

(Maggio and 29% in a comparison with s.c. injection

(Xu et al. buccal mucosa for the duration of the experiment (6 h)

(Langoth et al. 2006)

(Table continues on the next page)

Table 2.1. (continues from the previous page)

Technology Peptide (Mw) Result Reference

Intranasal

1.4- to 5-fold increase in the absorption in a comparison with plain solution depending on enhancer in brains in a comparison with i.v.

administration despite lower plasma induced hypoglycemia for 36 h and over 5 days, respectively s.c. insulin by using 80-fold higher dose 160-fold lower s.c. insulin dose after removal of epidermis, no

Traditionally, peptides have been administered by i.v. or s.c. injection and typically with a short duration of action, but non-invasive routes have attracted considerable interest in order to have pain-free administration. Regardless of the administration route, controlled delivery systems are needed if one wishes to achieve sustained release and a prolonged pharmacological effect. Various delivery systems such as polymeric implants, micro- and nanoparticles and liposomes have been developed for peptide administration via different routes (Table 2.1) (Sanders 1990; Dass and Choong 2006; Al-Tahami and Singh 2007).