METHODS FOR ENHANCING PEPTIDE AND PROTEIN DELIVERY

METHODS FOR ENHANCING PEPTIDE AND PROTEIN DELIVERY - Ocular Delivery and Therapeutics of Proteins and Peptides

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III. METHODS FOR ENHANCING PEPTIDE AND PROTEIN DELIVERY

Irrespective of the noninvasive route employed to deliver the peptide and protein drugs, some inherent delivery problems must be overcome. Table 3 lists some of the general methods that can be employed to negotiate such formulation difficulties.

Physical methods for increasing absorption such as iontophoresis and phonophoresis have been examined extensively (25–28). Such methods of enhancement are quite complex and may lead to chemical and physical instability of the permeating protein (29–31). Erratic results may also be obtained due to different degrees of surface absorption.

Prolongation of the biological half-life of proteins and enhancement of their systemic bioavailability are also necessary. An attempt has been made by coadministering proteins with known enzyme inhibitors (32). The cova-

498 Dey et al. Table 3 General Methods for Enhancing Protein Delivery 1. By increasing absorption through:

(a) Application of physical methods like iontophoresis or phonophoresis (b) Coadministration with permeation enhancers (c) Incorporation into liposomes or other carriers (d) Chemical modification of primary structure and development of

prodrugs 2. By minimizing metabolism through: (a) Covalent attachment to a polymer (b) Chemical modification of the primary structure (c) Targeting to specific tissues (d) Coadministration with an enzyme inhibitor 3. By prolonging blood levels through: (a) Use of bioadhesives (b) Protection using liposomes, polymers, or other carrier

lent attachment of polymers has also been shown to protect a number of proteins from enzymatic hydrolysis (33–37). Thus, azopolymers may be used to deliver proteins orally whereby the system could bypass the digestive enzymes of the small intestine; however, in the flora of the colon the polymer will release the protein. In any event, the problem of low bioavailability still exists, and permeation enhancers may have to be used to overcome this obstacle.

The use of chemical permeation enhancers has been reviewed exten- sively relative to nasal, oral, and rectal absorption of proteins and peptides (38–41). Three major mechanisms of action are possible for these enhancers: perturbation of membrane integrity, expansion of the paracellular pathway, and increase in the thermodynamic activity of the permeating species.

The following section will briefly discuss the studies performed in other noninvasive routes of protein and peptide delivery, the findings of which may be useful in designing ocular peptide delivery systems.

A. Oral Route The oral route, though the most convenient, is the least likely to be success-

ful because of extensive degradation of proteins and peptides in the gut. Based on the concentration-dependent absorption of 1-desamino-8-d-argi- nine-vasopressin (DDAVP), Lundin and Artursson (42) suggested the pro- cess to be mediated by passive transport. Other investigators have shown that the permeability of peptides can be enhanced through prodrug mod- ification designed to utilize the peptide transport system of the digestive tract (43–46). Coadministration of enzyme inhibitors may offer some pro-

Peptides and Proteins as Therapeutic Agents 499 tection in conjunction with a delivery system that can target the protein to

the site of optimal absorption (38,47). The incorporation of absorption enhancers to improve the oral bioavailability of proteins has been well documented (48). Lundin et al. (49) showed that sodium taurodihydrofusi- date (STDHF) enhanced both the in vitro and in vivo absorption of DDAVP. Schilling and Mitra (50) used the everted gas sac technique to evaluate the optimal site of insulin absorption. The addition of sodium glycocholate and linoleic acid enhanced insulin absorption in the duodenum and jejunum by eight- and threefold, respectively.

B. Nasal Route The extensive network of blood capillaries underneath the nasal mucosa

could provide effective systemic absorption of drugs. The nasal route is capable of providing a rapid absorption with a bioavailability relatively similar to that following subcutaneous injection. Nasal delivery of peptides and proteins has been reviewed (51,52). The polypeptides intended for deliv- ery by this route should be readily soluble in a low mucosal irritant vehicle. It must also be absorbed in effective amounts to make this mode of admin- istration both economical and acceptable (53). This route has been shown to

be acceptable for peptides with 10 residues or less (54,55). When the number of amino acids in the peptide approaches 20 or more, satisfactory bioavail- ability is obtained only with a permeation enhancer (56). The nasal mucosa contains enzymes capable of hydrolyzing peptides such as leucine enkepha- lin. It appears that the human nasal passage contains a variety of peptidases with wide specificities. The enhancing effect of two enzyme inhibitors, amas- tatin and bestatin, a mucolytic agent, N-acetyl-l-cysteine, and the permea-

(LPC) on the nasal absorption of human growth hormone (HGH) was studied by O’Hagan et al. (57). The highest bioavailability relative to the subcutaneous injection was found with amastatin, followed by LPC and palmitoyl-d,l-carnitine. Tengammuay and Mitra (58,59) found that mixed micelles of sodium glycocholate and fatty acids were more effective in enhancing the nasal delivery of peptides than the bile salt itself. Vadnere

dextrin were capable of increasing the bioavailability of leuprolide when given intranasally.

C. Buccal Route Delivery of macromolecules through the buccal membrane has also received

considerable attention in recent years (61–63). Both keratinized and non-

500 Dey et al. keratinized mucosae have been used in studying the in vitro rate of penetra-

tion of drugs through the buccal tissue. In vivo absorption of peptides/ proteins from the buccal cavity is likely to be influenced by the presence of mucosal secretions and immunological reactions among other factors. Molecular size may not be the limiting factor in the buccal delivery of peptides (64). Gandhi and Robinson (65) reported that amino acid penetrate the buccal membrane by an active process, whereas peptide drugs permeate passively. The buccal cavity exhibits greater proteolytic enzyme activity than the nasal or vaginal mucosa (64). The metabolic activity is shown to reside primarily in the epithelium (67). Aungst and Rogers (8,68) studied a variety of absorption enhancers to determine their effects on buccal absorption and showed that significant changes in the morphology of this mucosal barrier take place following exposure to the absorption enhancers.

D. Pulmonary Delivery of protein and peptide drugs via the pulmonary route has also

received significant attention in recent years. The walls of the alveoli are thinner than the epithelial/mucosal membrane; the surface area of the lung is much greater and the lungs receive the entire blood supply from the heart, all of which work in favor for the absorption of protein drugs more rapidly and to a greater extent. Of course, the lungs are rich in enzymes, and over- coming this barrier is no easy task. Peptide hydrolases, peptidases, and a wide variety of proteinases are present in the lung cells (69). However, some proteinases inhibitors are also present at concentrations varying with the disease state, which might work to prevent the destruction of administered peptides (70). Liposomal delivery of peptide and protein drugs through the pulmonary route have been attempted (71). Molecular modifications have also been undertaken to explore this route of protein and peptide delivery (72).

E. Ocular Route Lee reviewed the factors affecting corneal drug penetration (73).

Rojanasakul et al. showed that polylysine permeated through epithelial sur- face defects via an intracellular pathway when administered to the eye, whereas insulin predominates in the surface cells of the cornea (23). They noted that there was a significant amount of aminopeptidase activity present in the ocular fluids and tissues. Figure 1 summarizes the results of the metabolism of topically applied enkephalins to the eye (74). Pretreatment with the peptidase inhibitor bestatin had a significant protease inhibitory effect, albeit in the tears only.

Peptides and Proteins as Therapeutic Agents 501

Fig. 1 Concentration of intact (open bar) and degraded (marked bar) leucine enkephalin (===), methionine enkephalin (\ \ \) or [D-Ala 2 ]Met-enkephalinamide (filled bar) recovered in each part of the rabbit eye. (From Ref. 74.)

Studies have been conducted with absorption enhancers to improve the delivery of peptides and proteins into the systemic circulation via the ocular route (75–77). Table 4 lists some penetration enhancers that have been used in the ocular delivery of peptide-like drugs. Ocular delivery of insulin to generate a therapeutic glucose-lowering response requires a pene- tration enhancer (78). Yamamoto et al. (79) reported that the bioavailability

502 Dey et al. Table 4 Penetration Enhancers Used to Improve Ocular Absorption Enhancer

Effect Azone

Threefold increase in cyclosporine absorption Cetrimide, cytochalasin B

Increased absorption of inulin EDTA

Threefold increase in glycerol absorption Taurocholate, taurodeoxycholate

Increased permeation of insulin and FITC-dextran

of insulin could be improved in the following descending order by coadmi- nistration of the permeation enhancers: polyoxyethylene-9-lauryl

late.