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European Biopharmaceutical Review

First Class

The rise of antimicrobial resistance and the emergence of new pathogens means there is an urgent need to develop novel classes of antimicrobials. Antimicrobial peptides (AMPs) represent one of these, although, to date, this is an underevaluated area of R&D which lags behind the resurgence in protein/peptide drug development. Despite the reduction in antibiotic R&D at pharmaceutical companies, the top two therapeutic areas for peptides in Phase 3 clinical trials by mid-2013 were oncology and infectious disease, with around 10 per cent of peptide drug candidates in development being antimicrobials (see Figure 1) (1,2).

Marketed Peptide Therapeutics
R&D of therapeutic peptides has seen significant interest since the late 1990s and has led to the marketing approval of six peptide therapeutics as new molecular entities in 2012. Additionally, at least 12 candidate peptides are in late-stage clinical trials (1). The current market for protein/peptide drugs is approximately $40 billion per annum and the market share is growing faster than other pharmaceutical class (3). In 2010, around 75 per cent of marketed peptide drugs were less than 20 amino acids long (4). Examples of successfully marketed peptide drugs are given in Table 1.

Problems and Solutions
Large-scale chemical synthesis of peptides (~85 per cent of peptide drugs are produced this way) has significantly advanced in the last decade. Developments in chemical peptide synthesis have resulted in a variety of strategies including solid-phase synthesis, convergent synthesis and chemical ligation, allowing the synthesis of hundreds of kilograms (5).
A problem with peptide therapeutics is the lack of oral bioavailability; therefore, peptides often require administration through injection or inhalation. Advances in peptide medicinal chemistry – for example, the use of non-natural amino acids, cyclisation, constrained peptides, stapled peptides and peptidomimetics – and delivery systems should lead to an increased number of orally bioavailable peptide drugs (4). Indeed, cyclosporin A is available orally and calcitonin is available as an intranasal spray.

Antimicrobial Peptides
AMPs have been discovered in all forms of life and they form an important part of the animal and plant immune system. AMPs are highly evolutionarily conserved, and there has been relatively little resistance to this class of antimicrobials. To date, more than 2,000 AMPs have been identified (6).
AMPs are often broad-spectrum, exhibiting antibacterial, antifungal, anti-parasitic and antiviral properties. Natural AMPs often have additional roles in the innate immune system, including inflammation, regulation of the adaptive immune system, wound repair, angiogenesis and maintaining homeostasis. Many hypothesise that the primary function of natural AMPs is immunomodulation, rather than direct antimicrobial effects. These functions may be undesirable when developing new antimicrobials, and most approaches concentrate on their antimicrobial properties.
A disadvantage of peptide drugs is the difficulty in intracellular delivery. Most AMPs are membranolytic, negating this problem – although difficulties may arise for AMPs targeting intracellular pathogens. A number of reports have described synergistic effects between AMPs and traditional antibiotics. Additional mechanisms of action (MoAs) have been described for AMPs taken up by target cells, including inhibition of DNA/RNA synthesis, protein synthesis, cell wall synthesis and cell division. The specificity of AMPs for particular membrane regions – for example lipid rafts – has not been investigated in detail, although the peptidomimetic LTX109 was membranolytic versus Saccharomyces cerevisiae in a sphingolipid-dependent manner (7).
Due to these multiple MoAs, AMPs demonstrate a low potential for inducing de novo resistance. Nevertheless, all AMPs depend on an initial interaction with the microbial cell membrane (8,9). Recent US federal government incentives endowing additional market exclusivity (GAIN Act), and Food and Drug Administation (FDA) assistance regarding priority review and fast-tracking (QIDP status), have provided further motivation for the development of  MPs as antimicrobials. Some of the advantages and disadvantages of AMPs as drug candidates are outlined in Table 2.
Most AMPs that are in development are targeted at treating bacterial infections. Table 3 provides a summary.

Peptide Antibiotics
Peptide antibiotics have had a long and successful market history. Vancomycin (tricyclic glycopeptide) and colistin (cyclic lipopeptide) have been in use since the 1950s. Vancomycin is used intravenously for the treatment of complicated skin and skin structure infections (cSSSI), hospital-acquired pneumonias and sepsis where meticillinresistant Staphylococcus aureus (MRSA) is suspected. It is also used orally (for example Vancocin®; ViroPharma) for the treatment of Clostridium difficile associated conditions and staphylococcal enterocolitis. Generic IV vancomycin is widely available and generic oral vancomycin recently gained FDA approval. Although resistance to peptide antibiotics is slow to develop, widespread use of vancomycin has probably contributed to the emergence of vancomycin-resistant strains of S. aureus and enterococci.
The polymyxin colistin has been used therapeutically since the late 1950s for the treatment of Gram negative infections, including Pseudomonas aeruginosa and Acinetobacter baumanni (10). In recent years one new antibiotic peptide – daptomycin (Cubicin®; Cubist) – has come to the market as an alternative to vancomycin for the treatment of meticillin-sensitive S. aureus/MRSA cSSSIs. A lipopeptide, daptomycin – with a different MoA to vancomycin – acts on the cell membrane, causing the formation of holes and leakage of ions from the cell without causing cell lysis. It has an improved biological half-life compared to vancomycin, allowing treatment of some outpatients (11).
Other peptide therapeutics in development include Surotomycin (Cubist), which is in Phase 3 trials and has a similar MoA to daptomycin. Other lipoglycopeptides include Dalbavancin (Durata Therapeutic) and Oritivancin (The Medicines Company).

Antibacterial AMPs
AMP MoAs vary, but the main membranolytic mechanisms are described in Figure 2 (see page 58). Many AMPs have broad spectrum antimicrobial activity, which can be beneficial when treating infections based on clinical diagnosis prior to microbiological confirmation. However, they may be problematic if they have detrimental effects on the commensal microbiota. C16G2 (C3 Jian) is a specifically targeted AMP with a highly specific target, Streptococcus mutans. It is in development Phase 2 trials for dental plaque reduction and caries prevention (12).
The synthetic analogue of magainin (Locilex™; Pexiganan) has been through Phase 3 clinical trials for the treatment of diabetic foot ulcers. Developed in 1990s by Magainin Pharmaceuticals, it was then acquired by MacroChem (Access Pharmaceuticals) in 2009. Current owners, Dipexium Pharmaceuticals, have filed a patent application based on formulation improvements.
Lytyxar® (Lytix Biopharma), being developed as a prophylaxis to remove MRSA from the nose, is in Phase 2 trials because carriage presents a risk during scheduled surgery and for transmission to susceptible individuals. Iseganan (Ardea Biosciences) – a broad spectrum protegrin-1 analogue – demonstrates activity against bacteria, fungi and viruses. It was developed as a treatment for mucositis, but this ended after the failure of a Phase 3 trial and a Phase 2/3 trial for ventilatoracquired pneumonia in 2004. Despite this, Polyphor have since adapted the molecule to create a highly-specifc cyclic peptide, POL7080 which targets Pseudomonas aeruginosa – an opportunistic pathogen responsible for many hospital-acquired pneumonias and a significant cause of morbidity and mortality in cystic fibrosis patients.

Antifungal Peptides

Increasing antifungal resistance development means there is a pressing need for new therapies for which AMPs are ideal candidates. The three main classes of antifungal drugs – echinocandins (such as caspofungin), azoles (for example fluconazole) and polyenes (such as amphotericin B) – have associated problems, including resistance and/or toxicity.
The potential of the AMPs as antifungal drugs has been uncovered by looking to nature, and many AMPs have promising preclinical results. Pacgen Life Science Corporation has a 12 aa peptide – PAC-113 – derived from a histatin protein which is naturally found in saliva and is effective against oral candidiasis. PAC-113 acts on the fungal cell membrane, resulting in permeability, the leakage of cellular contents and, ultimately, death. Phase 2b data from the clinical trial of PAC-113 reported that PAC-113 was effective, well-tolerated and generally safe, resulting in a 34 per cent increase in clinical cure at day 19.
Other antifungal peptides going through clinical trials include NovaBiotics’ Novexatin® – a brush-on treatment for onychomycosis. This cyclic cationic peptide has been proven to be safe and well-tolerated, and is to begin Phase 2b clinical trials. Another preclinical antifungal peptide within the NovaBiotics portfolio is Novamycin®, which is effective against Candida and Cryptococcus species.
Other antifungal peptides have huge potential, including hybrid peptide-polyketides, which are active against C. albicans, produced by the fungus Culvularia geniculate. There are also derivatives of the HM-1 killer toxins produced by Williopsis saturnus that are active against Candida and Cryptococcus species (13,14). Amphibian skin has long been known to be a rich source of antifungal peptides. Bombina species produce Bombinin and Bombinin-like peptides, which are active against Candida species in vitro (15).
Although not generally considered AMPs due to their size, the echinocandins are peptide antifungals. The echinocandins are synthetically modified lipopeptides that were discovered in the fermentation broths of fungi (16). These semi-synthetic lipopeptides are fungicidal against many fungi, including many Candida and Aspergillus species. Recently, fungal resistance to the echinocandins has been seen and, therefore, newer antifungals such as AMPs are good candidates.

Antiparasitic and Antiviral Peptides

Antiviral and antiparasitic AMPs are generally at much earlier stages of development than antibacterial and antifungal peptides and are beyond the scope of this review. Good references featuring antiparasitic and antiviral are available. A notable exception is the HIV-inhibitor Fuzeon® (enfuvirtide), a complex 36-amino acid peptide that blocks the fusion of HIV with the host cell, which generated almost $900 million sales in 2010.

Successful Development
Recently, much of the focus on AMPs has been to improve antimicrobial activity, but it is becoming increasingly clear that for a broader application, modifications to the peptides and their mechanisms of delivery will need to be thoroughly understood. The ability of medicinal chemists and other researchers to design and synthesise AMPs in which structure-activity relationships can be better understood, and the ability to modify the constituent amino acids, intramolecular bonds and/or additional groups, will lead to the successful development of AMPs as marketed drugs. A better understanding of the detailed cellular targets of AMPs – rather than simply the membrane – will allow greater focus and specificity to be designed into the next generation of peptide antimicrobials.

References
1. Kaspar AA and Reichert JM, Future directions for peptide therapeutics development, Drug Discovery Today 18(17/18): pp807-817, 2013
2. Spellberg B et al, Infectious Diseases Society of America, Combating antimicrobial resistance: policy recommendations to save lives, Clinical Infectious Diseases 52(S5): pp397-428, 2011
3. Craik DJ et al, The future of peptide-based drugs, Chemical Biology and Drug Design 8: pp136-147, 2013
4. Vlieghe P et al, Synthetic therapeutic peptides: science and market, Drug Discovery Today 15: pp40-56, 2010
5. Glaser V, Scaling up peptide drugs, Genetic Engineering and Biotechnology News 33(7), 2013
6. Wang G, Li X and Wang Z, APD2: the updated antimicrobial peptide database and its application in peptide design, Nucleic Acids Research 37: ppD933-D937, 2009
7. Bojsen R et al, The synthetic amphipathic peptidomimetic LTX109 is a potent fungicide that disrupts plasma membrane integrity in a sphingolipid dependent manner, PLOS One 8(7): e69483, 2013
8. Gellatly SL et al, Multifunctional cationic host defence peptides and their clinical applications, Cellular and Molecular Life Sciences 68: pp2,161-2,176, 2011
9. Fox JL, Antimicrobial peptides stage a comeback, Nature Biotechnology 31(5): pp379-382, 2013
10. Spapen H et al, Renal and neurological side effects of colistin in critically ill patients, Ann Intensive Care 1: p14, 2011
11. Steenbergen JN et al, Daptomycin: A lipopetide antibiotic for the treatment of serious Gram positive infections, J Antimicrob Chemother, 55: pp283-288, 2005
12. Eckert R, Sullivan R and Shi W, Targeted antimicrobial treatment to re-esablish a healthy microbial flora for long-term protection, Adv Dent Res, 24: pp94-97, 2012
13. Chomcheon P et al, Curvularides A-E: Antifungal hybrid peptide-polyketides from the endophytic fungus Curvularia geniculat, Chem Eur J 16: pp11,178-11,185, 2010
14. Kabir EM et al, Peptide derived from anti-idiotypic singlechain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide, Appl Micriobiol Biotechnol 91: pp1,151-1,160, 2011
15. Simmaco M, Kriel G and Barra D, Bombinins, antimicrobial peptides from Bombina species, Biochem Biophys Acta 1788: pp1,551-1,555, 2009
16. Denning D, Echinocandins: a new class of antifungal, J Antimicrob Chemother 49: pp889-891, 2002

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About the authors

Dr Vanessa Duncan joined NovaBiotics Ltd in 2006 as a Senior Research Associate. She studied Microbiology and Biochemistry at the University of Aberdeen, before carrying out her PhD and post-doctoral research in the laboratories of the Aberdeen Fungal Group, University of Aberdeen.

Dr Douglas Fraser-Pitt is a Senior Research Associate at NovaBiotics Ltd. He studied Molecular Biology at the University of Liverpool. Douglas went on to gain an MSc in Medical and Molecular Microbiology at the University of Manchester and his PhD at the University of Aberdeen. He has since worked at the Moredun Research Institute for Animal Health in Edinburgh.

Dr Derry K Mercer is the Principal Scientist at NovaBiotics Ltd, with more than 20 years’ experience within the areas of microbiology, molecular biology, protein biochemistry and cell biology. He began his career at the University of Liverpool. Derry's range of skills and experience drive forward the R&D strategies of NovaBiotics Ltd. He is an Honorary Professor at Robert Gordon University.
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Dr Vanessa Duncan
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Dr Douglas Fraser-Pitt
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Dr Derry K Mercer
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