Peptide-based Drug Discovery & Development

Sponsored by Senn

Common Misconceptions

Although biotechnology investors and preclinical pharmacology experts may not always concur, peptide therapeutics offer demonstrable commercial success [1]. More than 50 peptide drugs, with annual sales in excess of $1 billion each, were marketed in 2010. Primary therapeutic indications include cancer and metabolic disease, but a wide range of novel uses such as vaccines, antimicrobials, and contraceptives are actively under development.

Skepticism is routed primarily in physical attributes. Peptides fall between small molecule and protein drugs in size, but when assessed by the typical criteria of these two drug classes, peptides fall short. However, they represent very tight, single receptor site binding ligands and therefore offer the potential of low dose administration without major side effects. These characteristics are directly responsible for their impressive success rate. Once Investigational New Drug application hurdles are overcome, peptide-based therapeutics are more likely to clear early clinical safety trials than most other drug candidates.

The four most significant concerns voiced about peptide-based drugs are: potential immunogenicity, poor pharmacokinetics (PK), lack of oral activity, and high manufacturing costs. The last issue is greatly dependent upon a variety of factors, which will be discussed in an upcoming article, Peptide-based Drug Research and Development, Relative Costs, Comparative Value. This article features a detailed discussion of the first three factors.

Misconception 1: Peptides Elicit Undesired Immune Response

The Foreign Antigen Requirement

The normal immune system mounts response to foreign antigens. This principle eliminates immunogenic concerns when using unmodified human peptide hormones/protein fragments as receptor agonists/ antagonists in medicine. We are then left with peptides of non-human (insect, amphibian, crustacean) origin, designer peptides, and modified human sequences as potential immune response threats.

The MHC-Binding Requirement

To induce cellular immune responses, antigens must bind major histocompatibility complex (MHC) proteins. Class I MHC recognizes 9-mer native peptides, and class II MHC binds slightly longer amino acid stretches in very specific side-chain orientation. Considering there are approximately 15,000 annotated human proteins (and a similar number in animals e.g. Drosophila melanogaster), each containing an average 360 amino acid residues, we can calculate more than 5,000,000 tenamino acid stretches in any eukaryotic proteome (partially disregarding conserved repetitive domains). With less than 10,000 potential MHCbinding peptides in existence [2], it is quite unlikely that any non-human natural ligand of a human receptor, or other therapeutically important target, includes an MHC-binding motif.

To increase stability and/or receptor binding, contemporary peptide drug development strategies commonly utilize non-natural amino acid moieties or modified backbones in peptide drug leads. These synthetic modifications decrease rather than increase MHC-binding potential by eliminating either the proper side-chain topochemistry or the required hydrogen bonding pattern [3].

The T-Cell Support Requirement

Although complete non-human proteins are able to induce antibody response, antibody production against small- or medium-sized synthetic peptides (such a peptide-based drug) is more an art form than a common event [4]. Short peptides are poor immunogens, unable to elicit antibody production without the support of T helper cells. This fact poses a challenge to vaccine manufacturers desiring a strong immune response. To begin with, peptide antigens must be conjugated to carrier proteins, or one or more class II MHC epitopes, to enhance immunogenicity. Once such structural alterations are implemented, the peptide constructs must be coadministered with adjuvants to produce mammalian antibody response. Alternatively, vaccine developers add one or more, non polyamide-based, modules (e.g. lipids, carbohydrates). These non-adjuvant approaches experience variable success [5]. Notably, although peptidomimetic backbones, side-chains, and stereochemical replacements do render peptides increasingly immunogenic, the effect is still insufficient to induce humoral immune response [6].

The Antibacterial Peptide Experience

In the author’s laboratory, the antimicrobial peptide lead A3-APO failed to mount antibody response despite extended size (41 residues) and 3 modified amino acid residues [7]. Moreover, inducing antibodies to any insect-derived antibacterial peptide failed when attempting to identify the precursor proteins using monoclonal antibodies. Even after adorning a 4-channel influenza M2 peptide vaccine candidate with multiple promiscuous T helper cell epitopes and non-covalent adjuvants, the neutralizing inbred mouse antibody response could not be reproduced in outbred mice [8].

Misconception 2: Poor Pharmacokinetics Equates to Poor Pharmacodynamics

Serum Instability

Poor stability and PK parameters may be even more pervasive argument than the immunogenicity misconception. Since the early nineties, stability studies in various serum preparations have become the most important secondary screening assays in peptide-based drug development [9]. The author of this article initially promoted this notion [10]. Ultimately, however, the shortcomings of serum stability were realized.

Peptides Circulate Rapidly

Most peptides degrade in undiluted serum within an hour. Human whole body blood circulation occurs in less than one minute. Circulation time is even shorter for smaller species. In the same time, high concentrations of peptide drugs reach cell surface receptors long before decomposing to smaller fragments. As a side note, it should be mentioned that even peptidic metabolites may retain the intended biological activities [7].

The Allo-aca Experience

A compelling example of the poor relationship between peptide serum stability/PK data and potential in vivo efficacy can be found with our leptin receptor antagonist peptide (peptidomimetic). Allo-aca is a linear nonapeptide containing 3 non-natural amino acid residues. It reduces leptin-dependent growth and signaling in hormone-positive and negative breast cancer cell lines with IC50 values of 50-200 pM [11]. Allo-aca is orexigenic in mice when added intraperitoneally or subcutaneously (sc).

It also suppresses growth of orthotopic human breast cancer xenografts in immunocompromised mice. In mice inflicted with triple negative breast cancer, Allo-aca is more efficacious than any other current therapy regimen as indicated by survival figures [11]. Moreover, Allo-aca reduces the extent of rheumatoid arthritis development markers in mice, owing to the similarity of the molecular mechanisms in arthritis and cancer. All this positive in vivo activity data are due to rapid and effective peptide biodistribution including penetration into the brain [12]. These results would have never been realized if research was halted due to poor stability and PK data. Allo-aca, similarly to other peptide drugs and pro-drugs, exhibits in vitro and in vivo half-lives measurable in minutes [13, 14]. Notably, the estimated affinity of Allo-aca to the ligand binding domain of the leptin receptor is in the low-mid pM range with a peptide-receptor complex half-life measurable in hours. Allo-aca, and other peptide drugs excel in terms of high activity and target selectivity despite poor serum stability and pharmacokinetics. These drugs may modify receptor responses significantly longer than standard stability analyses indicate.

Mouse PK is Irrelevant

Because peptides are rapidly excreted through the kidney, in vitro serum stability studies are not representative of true turnover. The 5-10 minutes Tmax of peptide drug leads in mouse PK measurements should be higher in humans where the renal clearance rate is 10-fold longer [15]. Therefore, mouse PK parameters are of little value to predict the duration of human peptide circulation.

Pharmacodynamics is Instructive

It is generally accepted that pharmacodynamics (PD) parameters, including duration and extent of action, may be more indicative of peptide drug utility than plasma drug concentration. For example, the peptide Allo-aca produces weight gain in normal mice two days after a 0.1 mg/kg bolus (sc administration) despite very short-lived blood levels. Nonetheless, without a clearly measurable PK:PD relationship, greater efforts to educate investors, traditional pharmacologists, and regulatory agencies on (low dose) efficacy and low toxicity risk, are necessary.

Misconception 3: Lack of Oral Bioavailability is a Non-starter

Lifestyle Drugs

With the new millennium, so-called lifestyle drugs became the dream of pharmaceutical investors and manufacturers. The idea of an oral once-a-day pill to improve non-threatening, but otherwise undesirable, conditions is very appealing. The lifestyle drug market is currently estimated at $30B, including treatments for weight-loss, smoking, erectile dysfunction, wrinkles, and baldness [16]. The demand is significant. We received as many calls from body builders as from pharmaceutical companies, inquiring about our leptin receptor agonist. But most peptidebased therapeutics do not fit the single pill-a-day narrative.

Lifestyle-Compatible Drug Delivery

It is a fact that most peptide drugs are not orally bioavailable. But this is not a deal-breaker.

Synthetic insulin has been prescribed for at-home subcutaneous administration for more than thirty years. Continuous technology improvements have successfully reduced biohazard risk and dosing discomfort associated with peptide drug delivery. Other peptide hormones and their analogs, including amylin, somatostatin, and human growth hormone are now also available in patient-friendly packaging ready for self-administration.

Chemical and physical modifications that can improve oral bioavailability of peptide drugs include conjugation to passive and active transport enhancers. Use of micro- and nanoparticles further expand the dose delivery options. In effect, as soon as a peptide drug lead is identified, research is initiated to improve its oral availability and/or optimize dose form and delivery. For example, mucoadhesive devices, made of carbopol, pectin and sodium methylcarboxy cellulose, in enteric coated capsules, significantly improve the oral bioavailability and PD parameters of yet another existing peptide drug, salmon calcitonin [17].

Without an exhaustive review of current approaches, a few significant peptide drug delivery developments are worth mention: subcutaneous and intranasal. Leuprolide, a synthetic nonapeptide agonist of the luteinizing hormone-releasing hormone receptor, is currently available in a oncea- year implantable device for commercial use. At a research scale, the transdermal delivery of leuprolide can be improved with microneedles and/or iontophoresis [18]. Also at the research scale, somatostatin now can be formulated for release over a 5-week period in guinea pigs, rats and rabbits [19]. Optimal intranasal efficacies for peptides, comparable to those measured after injection, are also achieved by using alkylsaccharide transmucosal delivery agents [20]. Polymeric controlled release formulations such as hydrogels are also gaining increased importance in peptide-based drug delivery as mucosal absorption-enhancing additives. The market is currently saturated with personalized, reusable, and virtually painless transdermal or intranasal medical devices for drug administration. Yet the technology continues to improve. For example, electronic autoinjectors are available for first line therapies against multiple sclerosis. RebiSmart™ specifically offers several innovative features targeting improving adherence during subcutaneous administration of interferon ß-1a [21].

Currently available devices, and future iterations, eradicate the stigma of peptide drug administration and significantly improve patient compliance—thereby eliminating one more misconception.


  1. Goodwin, D., Simerska, P., Toth, I. (2012) Peptides as therapeutics with enhanced bioactivity. Curr. Med. Chem. 19, 4451-4461.
  2. Meydan, C., Out, H.H., Sezerman, O.U. (2013) Prediction of peptides binding to MHC class I and class II alleles by temporal motif mining. BMC Bioinform. 14, S13.
  3. Herve, M., Maillere, B., Mourier, G., et al. (1997) On the immunogenic properties of retro-inverso peptides. Total retro-inversion of T-cell epitopes causes a loss of binding to MHC II molecules. Mol. Immunol. 34, 157-163.
  4. Hancock, D.C., O’Reilly, N.J. (2005) Synthetic peptides as antigens for antibody production. Meth. Mol. Biol. 295 (Immunochemical Protocols), 13-25.
  5. Zheng, W., Azzopardi, K., Hocking, D., et al. (2012) A totally synthetic lipopeptide-based self-adjuvanting vaccine induces neutralizing antibodies against heat-stable enterotoxin from enterotoxigenic Esherichia coli. Vaccine 30, 4800-4806.
  6. Lozano, J.M., Lesmes, L.P., Careno, L.F., et al. (2010) Development of designed site-directed pseudopeptide-peptidomimetic immunogens as novel minimal subunit vaccine candidates for malaria. Molecules 15, 8856-8889.
  7. Noto, P.B., Abbadessa, G., Cassone, M., et al. (2008) Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo. Protein Sci. 17, 1249-1255.
  8. Wolf, A.I., Mozdzanowska, K., Williams, K.L., et al. (2011) Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice. PloS One 6, e28445.
  9. Powell, M.F., Grey, H., Gaeta, F., et al. (1992) Peptide stability in drug development: a comparison of peptide reactivity in different biological media. J. Pharm. Sci. 81, 731-735.
  10. Powell, M.F., Stewart, T., Otvos, L. Jr., et al. (1993) Peptide stability in drug development. II. Effect of single amino acid substitution and glycosylation on peptide reactivity in human serum. Pharmacol. Res. 10, 1268-1273.
  11. Otvos, L. Jr., Surmacz, E. (2011) Targeting the leptin receptor: a potential new mode of treatment for breast cancer. Expert Rev. Anticancer Ther. 11, 1147-1150.
  12. Beccari, S., Kovalszky, I., Wade, J.D., et al. (2013) Designer peptide antagonist of the leptin receptor with peripheral antineoplastic activity. Peptides 44, 127-134.
  13. Otvos, L. Jr., Kovalszky, I., Scolaro, L., et al. (2011) Peptide-based leptin receptor antagonists for cancer treatment and appetite regulation. Biopolymers 96, 117-125.
  14. Janssen, S., Rosen, D.M., Ricklis, R.M., et al. (2006) Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandural kallikrenin 2 (hK2)-activated thapsigargin prodrug. The Prostate 66, 358-368.
  15. Sakamoto, H., Hatano, K., Higashi,Y., et al. (1993) Animal pharmacokinetics of FK037, a novel parenteral broad-spectrum cephalosporin. J. Antibiot. 46, 120-130.
  16. Atkinson, T. (2002) Lifestyle drug market booming. Nat. Med. 8, 909.
  17. Gupta, V., Hwang, B.H., Lee, J., et al. (2013) Mucoadhesive intestinal devices for oral delivery of salmon calcitonin. J. Controlled Rel. 172, 753-762.
  18. Sachdeva, V., Zhou, Y., Banga, A.K. (2013) In vivo transdermal delivery of leuprolide using microneedles and iontophoresis. Curr. Pharm. Biotechnol. 14, 180-193.
  19. Wang, S., Wu, M., Li, D., et al. (2012) preparation, characterization and related in vivo release, safety and toxicity studies of long acting lanreotide microspheres. Biol. Pharm. Bull. 35, 1898-1906.
  20. Maggio, E.T. (2006) Intravail: highly effective intranasal delivery of peptide and protein drugs. Expert Opin. Drug Deliv. 3, 529-539.
  21. Lugaresi, A. (2013) RebiSmart TM (version 1.5) device for multiple sclerosis treatment delivery and adherence. Expert Opin. Drug Deliv. 10, 273-283.

Professor Laszlo Otvos’ current research focuses on the development of antimicrobial peptides to resistant infections as well as agonists and antagonists to adipokine receptors. His first-of-kind and optimized peptide analogs show promising preclinical advantages over conventional therapy against not only bacterial infections, but also metabolic and cardiovascular diseases, certain cancer types and arthritis forms. Currently Laszlo serves as Councilor for the American Peptide Society, Regional Editor for Protein and Peptide Letters and Senior Editor for Biochemical Compounds. His research papers have been cited more than 10000 times.

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