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Industry perspective oligos &

JYOTHI THUNDIMADATHIL*, ARCHANA GANGAKHEDKAR *Corresponding author American Company Inc. Vista, CA, 92081, USA

Jyothi Thundimadathil?????

Improving stability of peptide drugs through chemical modifications

KEYWORDS: peptide, cyclic peptides, pseudo-peptides, pegylation, peptidomimetics, glycopeptides.

Potential for high efficacy and low toxicity coupled with advances in solid phase technology and Abstract purification methods have led to the wide acceptance of peptide based drugs in recent years. However, the development of peptide drugs is often hampered by limitations such as short half-life, rapid , proteolytic cleavage and poor permeation across biological membranes. Pharmacokinetics of peptides can be improved by chemical modifications during or after the . This mini-review will focus on different methods of peptide modifications to increase the metabolic stability, bioavailability and pharmacokinetic properties of peptides. This will provide researchers with a comprehensive overview of methods and approaches that can be considered in drug discovery and development programs.

IN TRODUCTION cyclization, incorporation of non-proteinogenic amino acids, modification of peptide bonds, modification of amino acids side In the last few decades of scientific research, a large number of chains and or terminal residues, conjugation and hydrocarbon biologically active peptides have been discovered and stapling will be discussed (Table 1). knowledge of the structures, properties, and functions of these peptides has grown considerably (1, 2). Peptides have important roles in normal body functions as well as in disease physiology END-PROTECTION METHODS acting as hormones, neurotransmitters, antigens, modulators of cell surface receptors etc. Simple peptide structures have Among post-translational modifications C-terminal α-amidation certain limitations to be developed and used as drugs. is known to be the most important and essential for biological rapidly degrade peptides by cleaving peptide bonds which activity. About 50% of mammalian peptide hormones and more ultimately leads to a loss of biological activity (3-5). Oral delivery than 80% of insect hormones are α- amidated at the C-termini of peptides is difficult due to the instability of bonds under (7). It is generally accepted that N-terminal plays a physiological conditions. The gastrointestinal tract (GI) doesn’t role in regulating stability and membrane targeting. promote the delivery of peptide drugs to target areas. Cellular Experimental data indeed indicated that with peptidases from mucosal cells and the brush-border membranes acetylated N-termini were more stable in vivo than non- of epithelial cells form a major enzymatic blockade to peptides acetylated proteins (8). Chemically synthesized peptides carry (6). , aminopeptidases and free amino and carboxy termini unless special methods are from GI tract breakdown the sequences from N- and adopted during the synthesis. The in vivo half-life of the C-terminals, whereas endopeptidases recognize cleavage sites N-terminal acetylated somatostatin analogue was improved to within the linkages. The combination of these types more than 400 minutes compared to the natural peptide with a of enzymes facilitates rapid and efficient degradation and is half-life of only 3 minutes (9). Amidation of peptides enhances significant barrier to the administration of peptide therapeutics activity of peptide hormones, and also prolong their shelf live. through the GI route. In addition, the instability of peptides The terminal acetylation/amidation will generate a closer mimic toward peptidases in the systemic blood circulation causes of the native protein from which a peptide drug has been rapid elimination (i.e., short half-life). Researchers have tailored thus increasing the permeability of the peptides to cells. developed several methods to alter the physicochemical N-terminal acetylation of peptide is performed at the end of properties of peptides to improve drug delivery and enhance the synthesis once all of the amino acids have been assembled. biological activity of therapeutic peptides (e.g. stability, C-terminal peptide are made either by an amidation transportation and distribution, affinity and targeting, controlled reaction or by using special solid phase resins such as Rink release, and immunogenicity). In this review methods such as amide aminomethyl resin.

Monographic supplement series: Oligos & Peptides - Chimica Oggi - Chemistry Today, Vol. 32(2) - March/April 2014 35 CYCLIZATION

Cyclization is one of the most widely used method to increase the conformational stability of peptides. Naturally occurring cyclic structures exist in many native peptides such as somatostatin, oxytocin, and insulin. Cyclization confers a structural constraint, which reduces conformational flexibility and also may enhance potency, selectivity, stability and bioavailability as well as membrane barrier permeability (10- 12). Peptide loops formed by cyclization also mimic protein secondary structures thereby increasing binding potency and selectivity. Peptides can be cyclized in a number of ways utilizing different locations of Table 1. Methods of increasing metabolic stability and bioavailability of peptide molecules the peptide main chain and side chains. These include head to tail, to side chain, the advantage of a thio-ether bridge has been reported by head to side chain, side chain to tail, backbone to backbone, Kluskens and co-workers by modifying angiotensin [Ang-(1-7)]. side chain to back bone, backbone to side chain, head to The resulting cyclized Ang-(1-7) is fully resistant against purified backbone and backbone to tail. Head- to- tail cyclization angiotensin-converting , has significantly increased forming an extra amide bond and side chain- to- side chain stability in homogenates of different organs and in plasma cyclization forming an amide bond or bond are the derived from pig, and displays a strongly (34-fold) enhanced most commonly used methods. In backbone cyclization, a survival in Sprague-Dawley (SD) rats in vivo (19). Another class of is constructed by interconnecting atoms in the peptides known as bicyclic peptides, with two circular units in backbone (N and/or C) of a target linear peptide to form a ring the structure are emerging as potential therapeutic entities as (13). A significant advantage of backbone cyclization is the they offer better specificity, binding properties, rigidity, fact that unlike the usual cyclization techniques (e.g., side enzymatic stability and good tissue penetration compared to chain- to- side chain and side chain- to- amino or carboxy their linear counterparts (20). ends) the bridge/interconnection is formed between backbone atoms and not side chains, which are generally essential for biological activity. This method has been used on various ALKYLATION OF AMIDE bioactive peptides and has been shown to improve the pharmacological selectivity of a given peptide as The N-alkylation of an amide bond (CO-NH, hydrogen replaced demonstrated for substance P24 and somatostatin analogs. It by an alkyl group) induces a restriction in the conformational has also been shown to dramatically enhance the metabolic flexibility of a peptide there by improving the stability under the stability of peptides to intestinal and serum as well as influence of proteolytic enzymes. For example, a number of to improve their paracellular permeability(14). N-methylated analogues of enkephalin have been prepared Cyclic peptides are called homodetic if the linkage that several years ago, most of which retain activity while exhibiting contributes to the ring is a peptide bond and heterodetic if the clear improvement in stability (21). It is worth noting that about 7 cyclization includes other bridges such as disulfide, out of 11 peptide bonds in cylcosporin A, a natural peptide (lactone), ether or thio-ether bridges (15-17). One or multiple used an immunosuppressant, are N-methylated and the disulfide bridges are formed by compound has an excellent cyclization through groups on pharmacokinetic profile (Figure 1). residues or in special cases via To demonstrate the applicability of the thiol group of penicillamine (e.g. N- to highly active but poorly enkephalin peptide analogues). This bioavailable peptides, Kessler’s research strategy requires in many cases group performed a full N-methyl scan of incorporation of cysteine residues the cyclopeptidic somatostatin analog resulting in the modification of peptide cyclo(-PFwKTF-), known as the Veber- sequence. One has to be careful in not Hirschmann peptide. It was shown that sacrificing the activity of a peptide by the simple approach of multiple incorporating cysteine residues. The N-methylation can drastically improve conformation-restricted cyclic the metabolic stability and intestinal encephalin analogue is extremely permeability of peptides, for example, resistant to enzymatic degradation, is resulting in 10% oral bioavailability for a rapidly distributed throughout the body Figure 1. Cyclosporin A, a natural peptide has tri-N-methylated Veber-Hirschmann seven N-methyl groups (shown in green arrows) and crosses the blood brain barrier (BBB) which adds to the stability of this cyclic peptide peptide analog (22). N-methylated efficiently (18). An interesting example of analogues of substance P, a hypotensive

36 Monographic supplement series: Oligos & Peptides - Chimica Oggi - Chemistry Today, Vol. 32(2) - March/April 2014 and smooth-muscle contracting compound, have been shown alpha-helical conformation through site-specific introduction of to be resistant to the action of various proteolytic enzymes while a chemical brace, an all-hydrocarbon staple (30). The idea of maintaining potency (23). peptide stapling was introduced to overcome the limitations of Soto et al introduced several combinations of single, double two broad classes of therapeutic agents (small molecules and and triple N- at the major sites of proteolytic proteins-biologics) in targeting intracellular protein-protein degradation of a beta-sheet breaker peptide, developed for interactions. Creation of a staple into a peptide thus entails the the treatment of Alzheimer’s disease (24). All of the derivatives incorporation of two appropriately spaced, α-methyl, present a better in vitro and in vivo stability in blood and brain α-alkenylglycine residues, having defined stereo chemical than the parental peptide. But only one derivative exhibited a configuration and alkene chain length, followed by ruthenium- similar in vitro activity. The authors attributed the lower of activity mediated olefin metathesis on the synthesis resin and then of other analogs to the decreased hydrogen bonding ability release from the resin and deprotection to yield the stapled and cis-amide formation. The N-alkylation of an amide bond peptide. Three different types of all hydrocarbon staples are induces a restriction in the conformational flexibility of a shown in the figure demonstrating the creation of stabilized peptide. Hence these factors should be carefully considered in α-helix in a peptide (Figure 2). Bird et al have recently shown evaluating the consequences of this modification. that that peptide double-stapling confers striking resistance that translates into markedly improved pharmacokinetic properties, including oral absorption (31). INCORPORATION OF D-AMINO ACIDS AND UNNATURAL AMINO ACIDS MODIFICATION OF PEPTIDE BONDS The incorporation of unnaturally occurring α-amino acids into a peptide sequence can enhance or reduce conformational Complete replacement or substitution of some of the atoms constraints within the molecule, affect hydrogen-bonding participating in the peptide backbone formation can lead to a capacities and change electronic interactions. Most class of peptides known as pseudo-peptides. Examples of such importantly they will increase the metabolic stability of an peptides include , azapeptides and amide bond analog by introducing groups that cannot be recognized by surrogates. Peptoids, or poly-N-substituted , are a class of enzymes. This approach has been utilized in many peptide pseudopeptides whose side chains are appended to the nitrogen drugs which are in the market. A substance P heptapeptide atom of the peptide backbone, rather than to the α-. analogue, methylated on phenylalanine and residues, Peptoids generally exhibit greater proteolytic stability and has proven to be quite active and very resistant to breakdown bioavailability than their respective peptides (32). Azapeptides by brain extract (25). Backbone rigidity introduced by unnatural are peptide analogs in which one or more of the amino residues is amino acids has great potential of improving the metabolic replaced by a semicarbazide. This substitution of a nitrogen for the stability of a peptide. For example, α-aminoisobutyric acid (Aib) α- center results in conformational restrictions, which bend has the ability to stabilize local secondary structure of peptide the peptide about the aza-amino acid residue away from a linear (26). Svenson’s research group has synthesized short geometry. In biologically active peptide analogs, the aza- antimicrobial peptides containing unnatural analogs of substitution has led to enhanced activity and selectivity as well as and displaying a high selectivity against bacterial improved properties, such as prolonged duration of action and cells with great stability toward chymotryptic degradation (27). metabolic stability (33). Amide bond surrogates consist of Substituting some or all of the L-amino acids of a peptide molecules in which some amide bonds have been replaced by sequence with their corresponding D-amino acids can increase other chemical groups. The introduction of such modifications to the resistance to proteolytic degradation and decrease the sequence completely prevents protease degradation of the immunogenicity. Recently it has been shown that substitution of amide bond. Improvements of peptide chemistry technology glycine with a D-amino acid in a bicyclic peptide inhibitor of the over the past several years have allowed the introduction of cancer-related protease urokinase-type plasminogen activator various and innovative types of amide bond surrogates, (uPA), resulted in increased binding affinity and proteolytic substantially increasing the number of chemical groups that may resistance (28). An interesting strategy to further improve the be considered as replacements. The reduction of the carbonyl metabolic stability of a D-amino acid containing peptide is known as retro-inversion. Retro-inversion reverses both the primary structure and replaces L-amino acids with D-amino acids in a peptide sequence. This method is useful especially when D-amino acid analog of a peptide is inactive due to conformational/structural change. Taylor et al have shown that retro-inversion of a 14-amino-acid neurotrophic peptide not only retain the biological activity but also remained intact in brain or serum for 60 min after i.v. administration and was transported across the blood-brain barrier (29).

HYDROCARBON STAPLING

High conformational instability of peptides leading to proteolytic cleavage and low bioavailability can be tackled via Figure 2. Three different types of stapled peptides formed by ring conformational stabilization of α-helical structure. Hydrocarbon closing metathesis (RCM) reaction. stapled peptides are mini-proteins locked into their bioactive

Monographic supplement series: Oligos & Peptides - Chimica Oggi - Chemistry Today, Vol. 32(2) - March/April 2014 37 portiony b replacement of CONH with a CH2SH or CH2NH surrogate is a way to detect a possible functional role of the . A CH2SH surrogate has been introduced in encephalin analogues, being more efficient at the ends than in interior positions of the peptide sequence (34). Octapeptide renin inhibitors, containing the reduced bond CH2NH, have shown greatly increased affinity for renin and resistance to enzyme (35). The ability of the surrogate to mimic the steric, electronic Figure 3. A 1,2,3-triazole function formed by click reaction between an azide and alkyne moieties resemble an amide bond and solvation properties of the amide bond is certainly the most important characteristic determining the potency of pseudopeptide analogues. In principle, a CH=CH enhanced enzymatic degradation in the gut wall by a variety surrogate would represent the best replacement to maintain the of peptidases expressed at the enterocytes brush border and planarity and rigidity of the amide bond. Due to its relative poor intestinal permeation. In addition, the instability of peptides planarity, strong dipole moment (~5 D) and hydrogen bonding toward peptidases in the systemic blood circulation causes ability, the 1, 2, 3-triazole function formed by click reaction rapid elimination (i.e., short half-life). A number of chemical between an azide and alkyne (Figure 3) bears a physicochemical modification techniques have been developed over the years resemblance to the amide bond. Consequently, the triazole to increase the metabolic stability of peptides. Methods such as linkage has been used as an amide bond surrogate (36). The conjugation, N-methylation, stapling, peptide bond triazole unit is resistant to enzymatic degradation, hydrolysis, and modification etc. have the potential to address the above oxidation, making it an attractive moiety to replace more labile mentioned drawbacks in drug development and delivery. By linkers in biologically active compounds. combining different methods it will be possible to make peptide drugs orally available provided the biological activity and potency are preserved. CONJUGATION

Conjugation to a carrier molecule could have several favorable REFERENCES effects on peptides: it can enhance the solubility, improve cellular uptake, provide prolonged blood circulation, alter 1. P. Vlieghe, V. Lisowski, J. Martinez, M. Khrestchatisky, Drug Discovery biodistribution, and also in some cases it can diminish toxicity. Today, 15, pp. 40-56 (2010). Different strategies can be utilized for conjugation: the 2. A.. K Sato, et al., Curr. Opin. Biotechnol., 17, pp. 638–642 (2006). 3. L..Shihong, C. Schöneich, R. T. Borchardt, Pharmaceutical News, 2, pp. biologically active compound can be attached directly to the 12 (1995).J. Brownlees, C. H. Williams, J. Neurochem., 60, pp. 793 (1993). carrier molecule, or it is possible to use a spacer structure in 4. J.L. Fauchère, C. Thuriau, C. Adv. Drug Res., 23, pp. 127 (1992). between. The most widely used of these methods, so far, is 5. J.. F Woodley, Crit. Rev. Ther. Drug Carrier Syst., 11, pp. 61-95 (1994). PEGylation, the conjugation of polyethylene glycol to proteins. 6. K.. H Kim, B. L. Seong, Biotechnology and Bioprocess Engineering, 6, pp This process has been shown to increase the protease stability, 244-251 (2001). solubility, and half-life of circulating (37). 7. H., A Heller et al., Proc Natl Acad Sci U S A, 81, pp. 7021–7025 (1984). The attachment of a lipid moiety has shown to protect unstable 8. M. Benuck, N. Marks, Life Sci., 19, pp. 1271 (1976). LHRH peptides from enzymatic degradation (38). The lipid was 9. F.. W Okumu et al., Pharm. Res., 14, pp. 169 (1997) attached via either a stable linkage, or a labile one, creating a 10. R.. T Borchardt, J. Controlled Release, 62, pp. 231 (1999). pro-drug. This use of a pro-drug strategy enhanced the 11. S.. J Weber, Pharmacol. Exp. Ther., 263, pp. 1308 (1992). 12. C. t Gilon e al., , 31, pp. 745 (1991). pharmacological characteristics of the peptide without 13. G. t Byk, e al., J. Med. Chem., 39, pp. 3174 (1996). decreasing its activity or selectivity. N-terminal conjugation with 14. S.. A Kates, et al., In Peptides: Design, synthesis, and biological activity lipidic amino acids increased the half-life of LHRH by (Basava, C. and Anantharamaiah, G. M., Ed.), 4, pp. 39-58 (1994). approximately 30-fold in Caco-2 cell homogenates. 15. R.. A Wiley, D. H. Rich, Med. Res. Reviews, 13, pp. 327 (1993). Liposaccharide-based delivery systems were designed to 16. M. Goodman, S. Ro, In Burger’s Medicinal Chemistry and Drug improve bioactive peptides and were shown to enhance their Discovery, (Wolff, M. E., Ed.; John Wiley and Sons, Inc: New York), 20, bioavailability (39). Schellenberger et al have shown that pp. 803-861 (1995). genetic fusion of an unstructured recombinant polypeptide of 17. S.. J Weber, et al., J. Pharmacol. Exp. Ther., 263, pp. 1308 (1992). 864 amino acids, called XTEN, to a peptide or protein provides 18. L.. D Kluskens, et al., J Pharmacol Exp Ther. 328, pp. 849-54 (2009). an apparently generic approach to extend plasma half-life (40) 19. V. Baeriswyl, C. Heinis, Chem. Med. Chem., 8, pp. 377-84 (2013). 20. C.. R Beddell, Proc. R. Soc. Lond. B. Biol. Sci., 198, pp. 249 (1977). . Recently a method known as “NMEGylation”-the covalent 21. J. Chatterjee, et al., Acc. Chem. Res., 41, pp. 1331-42 (2008). attachment of an oligo-N-methoxyethylglycine (NMEG) chain” 22. R. Laufer, R. et al., FEBS Lett., 123, pp. 291 (1981). has been shown to enhance the bioavailability of short 23. C. Adessi, et al., J. Biol. Chem., 278, pp. 13905 (2003). peptides (41). Hruby’s research group has utilized 24. B.. E Sandberg, et al., Eur. J. Biochem., 114, pp. 329 (1981). using O-beta-glycosylated (Ser(Glc)) to obtain a 25. R. Nagaraj, P. Balaram, FEBS Lett., 96, pp. 273 (1978). metabolically more stable analgesic peptide derivative (42). 26. R. Karstad, et al., J. Med. Chem., 53, pp. 5558–5566 (2010). 27. S. t Chen, e al., Chembiochem., 14, pp. 1316-22 (2013). 28. W.. M Taylor, et al., JPET , 295, pp.190–194 (2000). CONCLUSIONS 29. Z. t Guo, e al., Chem Biol Drug Des., 75: pp. 348–359 (2010). 30. G.. H Birda, et al., PNAS, 107, pp. 14093-14098 (2010).

Oral administration of peptides often results in low bioavailability Readers interested in a full list of references due to their inactivation in the gastro–intestinal tract followed by are invited to visit our website at www.teknoscienze.com

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