**10.Clinical use and commercialization**

### **10.1. Companies**

diture of metabolic energy by the microbe. In most cases, the resistance that arises is very

In Gram negative organisms such as *Salmonella* and *Pseudomonas*, the PhoP-PhoQ two com‐ ponent sensor system has been shown to control resistance to aminoglycosides, polymyx‐ in B, and cationic antimicrobial peptides (Macfarlane et al., 1999; Macfarlane et al., 2000; Groisman, 2001). In *Pseudomonas*, CAPs and the polymyxins are capable of inducing the pmrA-pmr genes and the putative LPS modification operon, thus increasing resistance to these agents (McPhee et al., 2003). The homologous basR-basS system of *E. coli* is also induced by exposure to sublethal concentrations of the proline-rich CAP, Bac7(1-35), sug‐ gesting that it may also mediate resistance (Tomasinsig et al., 2004). Resistance to Gram positive organisms such as *Staphylococcus* is mediated by the Aps three component sen‐ sor system, and interestingly, some CAPs are inducers of this system (Li et al., 2007). Recently, additional resistance genes in other bacteria such as *Clostridium difficile* (McBride and Sonenshein, 2011) and *Vibrio* (Shen et al., 2010) have been discovered, emphasizing the

Solid phase peptide synthesis is expensive and companies seeking regulatory approval for peptides in their pipelines require suppliers that adhere to good manufacturing practice (GMP) regulations, which also adds to the cost. However, there have been many advances in synthetic chemistry that have significantly reduced the cost of not only peptides but also non-peptide mimics and peptoids, and the lower operating costs of suppliers in Asia has re‐

Although CAPs represent a promising class of therapeutics, they have several *in vivo* draw‐ backs such as salt inactivation, protease degradation, and poor bioavailability. As described above, a number of approaches can be used to mitigate these limitations. Serum stability can be overcome by cyclisation or incorporation of amino acid analogs and some CAPs, particu‐ larly those such as pleurocidin that originated from marine sources, are active at physiologi‐

Some CAPs exhibit *in vivo* toxicity and for others there are unknown toxicity profiles (see section 5.7). Nephrotoxicity has been associated with polymixin cyclic peptides (Mogi and Kita, 2009). Since some CAPs can stimulate growth factor receptors, induction of tumorigen‐ esis must be considered. Similarly, excessive release of histamine from mast cells must be

low compared to conventional antibiotics.

138 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

importance of this phenomenon.

sulted in more affordable peptides (Eckert, 2011).

cal salt concentrations (Mai et al., 2011; Patrzykat et al., 2003).

**9.3. Cost**

**9.4. Stability**

**9.5. Side effects**

avoided to minimize adverse reactions.

A number of CAPs and synthetic CAP analogs such as pexiganan™ (magainin derivative), omiganan™ (indolicidin derivative), novispirin™ (cathelicidin derivative) and iseganan™ (protegrin-1 derivative) are now in commercial development. Several recent reviews de‐ scribe the companies, their products and their progression through clinical trials (Eckert, 2011; Kindrachuk and Napper, 2010; Yeung et al., 2011; Zhang and Falla, 2010). Some candi‐ dates show great promise in the treatment of such disorders as HIV-associated oral candi‐ diasis, acne, wound infections, diabetic foot ulcers, and oral biofilms. However, the inability to demonstrate advantage over existing therapeutics has resulted in failure of a number of CAPs at Phase III clinical trials. As appropriate formulations, bioassays and endpoints are established, the probability that these promising products will receive regulatory approval will improve.

#### **10.2. Formulations**

Because of systemic toxicity and bioavailability issues as well as the diverse and complex mechanisms of action, the majority of CAPs have been developed as topical formulations e.g. for diabetic foot ulcers (Lipsky et al., 2008) and skin infections (Falla and Zhang, 2010 ). Depending on the application, a number of different ways of formulating CAPs can be envi‐ sioned (Eckert, 2011). CAPs have been incorporated into lens preservation and artificial tear solutions (Huang et al., 2005) and aerosols (Lange et al., 2001). Inclusion of CAPs in coatings (Kazemzadeh-Narbat et al., 2010), polymers (Gao et al., 2011), hydrogels (Roy and Das, 2008), liposomes (Lange et al., 2001), micro- and nanoparticles (Bi et al., 2011; Garlapati et al., 2011) and even chewing gum (Faraj et al., 2007) also represent promising formulations. Long-term activity has been achieved by covalent immobilization of gramicidin A on func‐ tionalized gold surfaces and resulted in inhibition of Gram-positive and Gram-negative bac‐ teria and the yeast *Candida albicans* for up to 6 months as well as delayed development of bacterial biofilm for 24 hours (Yala et al., 2011). Surface modifications by CAPs will be im‐ portant in preventing colonization of medical devices such as catheters and bioimplants.

CAPs can also be used in combination therapies with each other or with approved antibiot‐ ics. For example, cecropin B enhanced the activities of beta lactams in rat septic shock mod‐ els (Ghiselli et al., 2004). Cecropin A and magainin 2 administered together showed *in vitro* synergy against *S. aureus* in a mouse sepsis model (Cirioni et al., 2006). Magainin 2 adminis‐ tered with vancomycin showed the highest efficacy in this model.

Pleurocidin showed synergistic activity with inducible histone peptides and lysozyme in a salmonid *in vivo* infection model (Patrzykat et al., 2001). Studies with *S. mutans* showed that the efficacy of a targeted pleurocidin was increased in the presence of EDTA and sodium fluoride, two commonly used components of oral rinses (Mai et al., 2011).

#### **10.3. Targeted delivery**

CAPs with cell-penetrating properties are under development as vectors for translocation of bioactive cargos with inherently poor membrane-crossing abilities into eukaryotic cells (Splith and Neundorf, 2011). Delivery of CAP-modified liposomes containing therapeutics to bacteria has also been reported, e. g. bacteria-targeted delivery of photodynamic antimi‐ crobial chemotherapy to improve efficiency against MRSA and *P. aeruguinosa* in local infec‐ tions (Yang et al., 2011).

### **11.Conclusions**

CAPs show exciting promise as novel therapeutic agents, particularly in the fight against an‐ tibiotic-resistant bacteria and cancer and as immunostimulants. However, translation to clin‐ ical use has been hampered by concerns over stability, cost, systemic administration, known toxicity, and unknown long-term toxicity. The applications that show the most promise are those involving topical applications, particularly in combination with established antibiot‐ ics. Deeper understanding of the varied mechanisms of action of these diverse peptides and the production of cost-effective, stable, and highly selective CAPs will aid in bringing these molecules closer to the clinic.

### **Author details**

Susan Douglas\*

Address all correspondence to: susan.douglas@nrc.ca

Institute for Marine Biosciences, Canada

## **References**


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CAPs with cell-penetrating properties are under development as vectors for translocation of bioactive cargos with inherently poor membrane-crossing abilities into eukaryotic cells (Splith and Neundorf, 2011). Delivery of CAP-modified liposomes containing therapeutics to bacteria has also been reported, e. g. bacteria-targeted delivery of photodynamic antimi‐ crobial chemotherapy to improve efficiency against MRSA and *P. aeruguinosa* in local infec‐

CAPs show exciting promise as novel therapeutic agents, particularly in the fight against an‐ tibiotic-resistant bacteria and cancer and as immunostimulants. However, translation to clin‐ ical use has been hampered by concerns over stability, cost, systemic administration, known toxicity, and unknown long-term toxicity. The applications that show the most promise are those involving topical applications, particularly in combination with established antibiot‐ ics. Deeper understanding of the varied mechanisms of action of these diverse peptides and the production of cost-effective, stable, and highly selective CAPs will aid in bringing these

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