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Therapeutic utility of antibacterial peptides in wound healing Expert Rev. Anti Infect. Ther. Early online, 1–11 (2015)

Laszlo Otvos Jr*1 and Eszter Ostorhazi2 1 Temple University, Philadelphia, PA, USA 2 Semmelweis University, Budapest, Hungary *Author for correspondence: [email protected]

Cationic antimicrobial peptides were first thought to fight infection in animal models by disintegrating bacterial peptides and later by inhibiting bacteria-specific intracellular processes. However, ever increasing evidences indicate that cationic peptides accumulate around and modulate the immune system both systemically and in cutaneous and mucosal surfaces where injuries and infections occur. Native and designer antibacterial peptides as well as cationic peptides, never considered as antibiotics, promote wound healing at every step of cutaneous tissue regeneration. This article provides an introductory list of examples of how cationic peptides are involved in immunostimulation and epithelial tissue repair, eliminating wound infections and promoting wound healing in potential therapeutic utility in sight. Although a few antimicrobial peptides reached the Phase II clinical trial stage, toxicity concerns limit the potential administration routes. Resistance induction to both microbiology actions and the integrity of the innate immune system has to be carefully monitored. KEYWORDS: acne vulgaris . anti-inflammatory . burn wounds . immunostimulation . macrophage . re-epithelialization .

skin infections

.

toxicity

Expanding the effects of antimicrobial peptides on bacteria

The time has arrived when the terms ‘antibacterial peptide’ and ‘antimicrobial peptide’ (AMP) have to be replaced with alternative designations that more closely reflect the true activity of these peptidic molecules during infection models in vivo. The classic, and still most frequently cited, mode of action is the accumulation of the mostly cationic peptides on the negatively charged bacterial membrane up to a threshold concentration, which leads to membrane permeation/disintegration [1]. However, the observation that several so-called AMPs fail to modulate membrane permeability at their microbiologically active concentration forced investigations on alternative bacterial targets [2]. Recently, many additional activities were described on bacteria, including inhibition of RNA synthesis [3], lipid complexation [4], inactivation of protein synthesis [5] and protein folding [6], bacterial toxin deactivation [7] or activation of the own autolytic systems of the microbes [8]. In fact, all the bacterial targets of peptides exhibiting

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protective abilities against infections are too many to list here [9]. The move from bacterial targets to host defense actions

Simultaneously, in the past few decades, research was extended to host responses to cationic peptides with immunostimulation [10] via activation of macrophage performance [11] or upregulation of anti-inflammatory cytokine production [12] being the primary focus. In general, tissue damaging inflammatory responses, together with stimulating antimicrobial immune responses point to complex immunomodulatory properties of AMPs [13]. Many of the activities of AMPs target mammalian membranes and their receptors to influence diverse host cellular responses [14]. Finally, the conclusion was made that these peptides were misclassified as antimicrobials when their primary role was to upregulate chemotaxis, or growth factor activation [15]. One of the most frequently studied AMP, LL-37, induces angiogenesis, a process essential to host defense and tissue repair that takes us to wound healing [16]. Indeed, the profound


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anti-infective and selective anti-inflammatory properties of AMPs culminate in remarkable adjuvant and wound healing efficacies in animal models [17]. Cathelicidins and defensins led the way to the attempted therapeutic use of peptides once called AMPs in skin infections and tissue repair [18]. Cutaneous wound healing starts with migration of keratinocytes across the damaged area to re-establish the epithelial barrier followed by fibroblast-mediated replacement of the provisional matrix and revascularization of the damaged area by endothelial cells [19]. The stages of wound healing consist of inflammation (neutrophil and macrophage invasion), tissue formation (angiogenesis and epithelialization) and remodeling (collagen synthesis) [20]. This response is a complex process involving structural proteins, growth factors and proteinases [21], all of which can be influenced by AMPs. The original observation that the activity of even blood-borne AMPs and proteins extends to immune modulation and wound repair [22] was later used to explain diminished skin integrity in newborns who lack significant AMP production [23] and correlate protease activity of wound fluids with infection control by AMP [24]. Cutaneous cellular activities of cationic peptides

The native human cathelicidin AMP LL-37 is expressed in a number of tissues and is present in squamous epithelia where its expression is regulated in several inflammatory conditions [25]. In addition to direct antimicrobial activity, LL-37 acts as a chemoattractant and stimulates receptor-mediated endothelial cell proliferation [26]. Its porcine analog, the proline-rich peptide PR-39 induces proteoglycan synthesis important for wound repair [27]. While human a-defensins increase the expression of the pro-inflammatory cytokines TNF-a and IL-1 in human monocytes [28], b-defensins appear to be chemotactic for immature monocytes and memory T cells [29]. Human b-defensins are overexpressed in cutaneous surgical site infections and their presence in the postoperatively inflamed epithelium suggests a target for pharmaceutical interventions [30]. In contrast, treatment of immortalized macrophages with a non-cationic AMP, the fishoriginated epinecidin-1, does not induce TNF-a production but epinecidin-1 inoculation into mice elevates the plasma level of the anti-inflammatory cytokine IL-10 [31]. In our hands, using human peripheral blood mononuclear cells the designer proline–arginine rich peptide dimer A3-APO upregulates the expression of anti-inflammatory cytokines IL-4 and IL-10 by 4–6-fold. As A3-APO is unable to kill some of those infectious strains in vitro to which it shows potent in vivo efficacy in lung and cutaneous animal infection models, we suggested that one of the mechanisms mediating the in vivo protecting effects of A3-APO might be the prevention of inflammation around bacterial infiltration [32]. Anti-inflammatory peptides play certainly a role during bacterial infections. Continuous parenteral administration of the anti-lipopolysaccharide peptide Pep2.5 is able to reduce sepsisinduced cytokine release and tissue inflammation [33]. The N-terminal fragment of the iron-binding protein human lactoferrin, hLF1-11, is an arginine-rich AMP with very diverse doi: 10.1586/14787210.2015.1033402

activities in bacterial and mammalian cells and a widely studied pharmaceutical agent in animal models of infection [34]. LF1-11 binds and penetrates human monocytes and subsequently inhibits the enzymatic activities of myeloperoxidase [35]. Peptide hLF1-11 is also responsible for directing GM-CSFdriven monocyte differentiation toward macrophages that produce both pro- and anti-inflammatory cytokines [36]. The molecular components of postoperative wound fluid were identified as histone fragments, thymosin and members of two AMP families: a-defensins and human cathelicidins [37]. Expression of the cell surface proteoglycans, the syndecans, is induced in mesenchymal cells during wound repair, a process that can be mimicked by PR-39, itself isolated from wound fluid [38]. After injury, myeloid cells infiltrate the wound site and induce smooth muscle actin production with subsequent formation of a myofibroblastic scaffold [39]. These myofibroblastic cells, macrophages and adjacent epithelial cells all proliferate early in the wound healing process and appear to be essential in wound repair and formation of a fibrovascular scar. BMAP28, a bovine cathelicidin AMP, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes [40]. In vivo peritoneal protection exerted by BMAP28 appears to be mediated through neutrophil recruitment and activation of macrophages for bacterial clearance [41]. Some basic animal studies of non-classical AMPs have already pointed to potential later therapeutic approaches of more traditional AMP analogs in skin infections and wound healing. A cationic immunomodulatory peptide derived from a bactenecin-based library (deliberately not called AMP) enhances chemokine induction in vitro that correlates with stronger protective activity in vivo in a Staphylococcus aureus-invasive infection model [42]. The protective activity of the peptide is associated with augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. A mixture of temporin B and jellein-originated peptides exhibits an ability to curb the synthesis of the proinflammatory cytokines TNF-a and IFN-g to the same extent as acetylsalicylic acid in a cellular model [43]. In Staphylococcus epidermidis-infected mice, the mixture sterilizes the animals in 11 days and inhibits the expression of genes encoding COX-2 and CD64, two important parameters of inflammation. Synthetic mimics of AMPs reduce cytokine (both proand anti-inflammatory) production in response to S. aureus in vitro and the induction of pro-inflammatory TNF and IL-6 in vivo while simultaneously reducing bacterial loads by 4 log10 units and other signs of infection [44]. Efficacy of peptide antibiotics in skin & burn infections

The potential for AMPs in the treatment of wound infections was initiated by the observations of these peptides in skin diseases. Patients with diabetic foot ulcers have lower levels of AMP at the lesion area, contributing to the impairment of wound healing [45]. Primary cultures from skin biopsies treated with dihydroxyvitamin D3 increase the production of human Expert Rev. Anti Infect. Ther.

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Therapeutic utility of antibacterial peptides in wound healing

b-defensin and LL-37 in the culture supernatant. Lucifensins are defensins isolated from the medicinal larvae of blowflies that contribute to wound healing during a procedure known as maggot debridement therapy, which is routinely used at hospitals worldwide [46]. Free-range larvae of Lucilia sericata eliminate most Gram-positive, and some Gram-negative bacterial strains in patients with infected diabetic foot ulcers. Methicillin-resistant S. aureus (MRSA) is a frequent pathogen associated with skin injuries (TABLE 1). In a skin trauma-mediated MRSA infection, while untreated mice and animals treated with methicillin die within 4 days, mice treated with epinecidin-1 survive the infection [47]. Microscopically, the peptide decreases MRSA bacterial counts in the wounded region, enhances wound closure and increases angiogenesis at the injury site. In agreement with the cellular studies, treatment with epinecidin-1 decreases the serum levels of pro-inflammatory cytokines, including TNF-a and IL-6. The bactenecin-derived peptide IDR-1018 demonstrates significantly accelerated wound healing in methicillin-sensitive S. aureus infected porcine and non-diabetic but not in diabetic murine wounds [48]. However, no significant differences in bacterial colonization are observed demonstrating that in addition to the immunomodulatory activities IDR-1018 promotes wound healing independent of direct antibacterial activity. Treatment of diabetic and acute skin flap wounds of rats with peptide SR-0379 significantly accelerates wound healing when compared to fibroblast growth factor 2 [49]. The beneficial effects of SR-0379 on wound healing can be explained by enhanced angiogenesis, granulation tissue formation and proliferation of endothelial cells and fibroblasts. Nisincontaining nanofiber wound dressings reduce S. aureus bioluminescence in vivo and viable cell numbers in a murine excisional skin infection model [50]. The bacterial burden of wounds treated with nisin-containing nanofiber wound dressings was 4 log10 CFU/wound less compared to untreated lesions at the end of the 7-day trial. In addition, the nisin-containing wound dressing stimulates non-infected wound closure. Mice were infected with S. aureus and the 13.6 kDa snake toxin CaTx-II was injected into the wounds. CaTx-II-treated mice show significant wound closure and complete healing in 16 days when compared to untreated controls [51]. Histological examination documents enhanced collagen synthesis and neovascularization after treatment with CaTx-II compared to fusidic acid ointment-treated controls. Measurement of tissue cytokines reveals that IL-1b expression in CaTx-II treated mice is significantly suppressed versus untreated controls. We extended the investigation of AMP actions to burn wound infections [52]. The most common pathogens in blast wounds are Acinetobacter baumannii strains followed by Escherichia coli and Pseudomonas spp as well as Klebsiella spp, Enterobacter spp and Proteus spp in lesser proportion [53]. CD-1 mice were inflicted with burn wounds and different inoculums of A. baumannii, isolated from an injured soldier, were placed into the wound sites [52]. The designer proline–arginine rich peptide dimer A3-APO and control colistin and imipenem antibiotics were added intramuscularly 1–5-times. A dose of informahealthcare.com

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5 mg/kg A3-APO improves survival and reduces the bacterial counts in the blood and the wounds significantly better than any other antibiotic treatment. Later, we studied the efficacy of A3-APO in two additional mouse models: in a MRSA and a mixed Klebsiella pneumoniae–A. baumannii–Proteus mirabilis wound infection model mimicking blast infections and foot ulcers, respectively [32]. While the peptide practically does not kill the strains in vitro, when administered intramuscularly, it remarkably extends the survival time and reduces the bacterial counts at the infection site. The blast wounds of treated animals, unlike untreated counterparts, lack pus and inflammation signs. In the foot ulcer model, the peptide improves survival compared to untreated animals, likely by inhibition of bacterial toxin production. A3-APO similarly extends survival time by inactivating lethal toxins in a systemic Bacillus anthracis infection model [7] suggesting the utility of the peptide alone or in synergy with other types of antimicrobial modes in combating resistant or life-threatening topical or systemic bacterial or fungal infections. Acne vulgaris

In addition to S. aureus, the second major Gram-positive pathogen in skin and soft-tissue infections is Propionibacterium acnes that also develops resistance to current antibiotics at an alarming rate [54]. Actually, P. acnes cutaneous infections are recognized targets for cationic AMPs and peptidomimetics even in the clinical setting [55]. We compared the efficacies of A3-APO and its monomeric metabolite in mouse models of S. aureus and P. acnes intradermal infections following their administration intramuscularly or as topical treatments [56]. In spite of being inactive against the strains in vitro, in vivo the skin conditions of the mice were dramatically improved upon treatment with both proline-rich peptides regardless of the applied dose or administration mode. In the P. acnes model, A3-APO statistically significantly reduces ear thickness and ear bacterial counts. The amounts of ear connective tissue and epithelial macrophages correlate with therapy success. In the S. aureus study, both peptides eliminate wound bacteria to almost background levels, although the monomer appears to be somewhat more successful. These experiments verify two earlier suggestions, made based on in vitro observations, regarding the use of topical AMP therapy in acne vulgaris. First, synthetic granulysin-derived peptides kill P. acnes in isolated human microcomedone preparations and have potential anti-inflammatory effects, as demonstrated by suppression of P. acnes-stimulated cytokine release, indicating that peptides may be useful as topical therapeutic agents, providing alternatives to current acne therapies [57]. Second, it has been suggested that future therapeutic applications may prefer topical rather than systemic administration because peptides applied to infected skin lesions in the form of sprays or creams can penetrate into the stratum corneum to kill microorganisms [58]. The honey bee apidaecin-based proline–arginine rich peptide Api88 inhibits LPS-induced TNF-a production in a doi: 10.1586/14787210.2015.1033402

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Table 1. Examples of peptides efficacious in animal models of skin and burn infections. Peptide

Origin/family

Infectious agent/animal model

Readout

Less active comparator

Epinecidin-1

Fish – 21 aa

Staphylococcus aureus/mouse full-thickness skin abrasion

Survival time Wound closure

Methicillin

[47]

IDR-1018

Bactenecin – cationic – 12 aa

S. aureus/mouse and pig fullthickness skin abrasion

Wound closure

LL-37, defensin

[48]

SR039

Metabolite of AG30 – 20 aa

S. aureus/rat full-thickness skin abrasion

Wound closure

[49]

Nisin

Lantibiotics – 34 aa

S. aureus/mouse fullthickness skin abrasion

Wound closure Bacterial load

[50]

A3-APO

Proline–arginine rich dimer 41 aa

Acinetobacter baumannii/ mouse skin burn

Wound closure Bacterial load

A3-APO

Proline–arginine rich dimer – 41 aa

S. aureus/mouse skin burn Klebsiella pneumoniae – A. baumannii – Proteus Mirabilis mouse skin burn

Wound closure and bacterial load Survival time

[32]

A3-APO

Proline-arginine rich dimer – 41 aa

S. aureus/mouse skin burn Propionibacterium acnes/ mouse intradermal ear

Wound closure and bacterial load Ear thickness and bacterial load

[56]

Chex1-Arg20

Metabolite of A3-APO – 20 aa

S. aureus/mouse skin burn P. acnes/mouse intradermal ear

Wound closure and bacterial load Ear thickness and bacterial load

[56]

CaTx-II

Snake toxin – 13.6 kDa

S. aureus/mouse fullthickness skin abrasion

Collagen synthesis, neovascularization

[51]

Imipenem Colistin

Ref.

[52]

aa: Amino acids.

concentration-dependent manner but resting monocytes do not respond to Api88 [59]. Apparently, Api88 is a multifunctional peptide that can modulate biological responses of human monocytes and mast cells as well as kill bacteria in vitro. An application to acne vulgaris, frog-derived AMPs were shown to efficiently mitigate the growth of P. acnes as well as inhibit the release of pro-inflammatory cytokines IFN-g and TNF-a and stimulate the release of anti-inflammatory cytokines TGF-b, IL-4 and IL-10 in peripheral blood mononuclear cells [58]. Mucosal fluids

As discussed above, mounting evidence suggests that AMPs have more positive effects on host responses to injuries than just eliminating bacteria microbiologically. AMP production and protease activity are elevated in burn margin of mice but skin extracts from burn margin do not exhibit changes in the ability to inhibit bacterial growth [60]. Peptide levels are also altered in the lung and bladder that are the common sites of secondary infection in burn-injured patients. A few additional in vitro studies on mucosal fluids, distantly related to wound injuries further support this idea. Human tears contain a long list of peptide components and peptide precursors, including a- and b-defensins and lactoferrin [61]. The level of these antibiotics increases in infections and wound healing and decreases in dry eye. In wounded human

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and macaque corneas, b-defensins are expressed during re-epithelialization suggesting their involvement in ophthalmic wound healing [62]. Topical administration of a human neutrophil-derived peptide, in addition to accelerating corneal wound healing, effectively clears Pseudomonas aeruginosainduced corneal infection in mice [63]. Peptide LL-37 can protect the integrity of the gingival tissue by inhibiting biofilm formation by Aggregatibacter actinomycetemcomitans (a major pathogen in periodontal disease) in a concentration when it cannot kill the microorganism [64]. LL-37 acts as an opsonin or agglutinin that greatly enhances A. actinomycetemcomitans clearance by neutrophils and macrophages. In fact, saliva contains many of the same antimicrobials as tears including lysozyme, lactoferrin, secretory leukocyte peptidase inhibitor, mucins and AMPs [65]. Wound healing by peptides without bacterial infection

In mouse burn wounds without experimental infection (environmental bacterial contamination cannot be excluded), a homogeneous eosinophilic and necrotic material is observable under the epithelium that contains polymorphonuclear pus cells and is surrounded with loose granulation tissue [52]. In the tissue of A3-APO-treated mice, there are clear signs of a regenerated epithelial layer under the necrotic surface without pus cells and decreased levels of inflamed granulation (TABLE 2). The frog

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Table 2. Wound healing in animal models by selected peptides without microbial infection. Peptide

Origin/family

Wound type/ animal model

Administration mode/readout

Mechanistic explanation

Ref.

A3-APO

Proline–arginine rich dimer – 41 aa

Skin burn wound in mouse

Intramuscular/wound closure

Prophylactic antibiotic

[52]

Tiger17

Frog-based designer – 11 aa cyclic

Mouse full-thickness skin abrasion

Topical/wound closure

Macrophage recruitment, pro-inflammatory cytokine release

[66]

HB-107

Cecropin B – 19 aa

Dermal biopsy in mouse

Topical/wound size

Leukocyte infiltration, chemotaxis

[70]

SHAP1

Designer amphipathic – 19 aa

Mouse skin excisional wound

Topical/wound closure

Transactivation of EGFR

[73]

QL6

Artificial QL repeats – 16 aa

Spinal cord injury in rats

Topical/tissue preservation

Reduced inflammation, increased apoptosis

[75]

LL-37

Cathelicidin – 37 aa

Rabbit muscle

Local/vessel growth and histology

Angiogenesis, arteriogenesis

[82]

LL-37

Cathelicidin – 37 aa

Mouse skin excisional wound

Topical/wound regeneration

Vascularization and re-epithelialization

[83]

aa: Amino acids.

antibiotic-based peptide Tiger17 exerts significant effects on three stages of wound healing progresses in a murine fullthickness skin wound model, including recruitment of macrophages to the wound site during the inflammatory reaction stage, promotion of the migration and proliferation of both keratinocytes and fibroblasts leading to re-epithelialization and granulation tissue formation and tissue remodeling through the release of transforming TGF-b1 and IL-6 in macrophages and activating mitogen-activated protein kinase signaling pathways [66]. Indeed, temporins, another AMP group from frog skin, promote wound healing in a monolayer of HaCaT keratinocytes, with front speed migrations between 12 and 19 mm/h [67]. Migration is inhibited by mitomycin C and involves the EGFR signaling pathway. A proper balance in stimulating immune functions but suppressing excessive inflammation poses a challenge in dosing of cationic peptides. These are frequently toxic drug leads, with potentially unwanted systemic responses in serum concentrations that appear to be non-toxic in cellular assays [68]. In our experience, topical or intramuscular administration of A3-APO poses significantly lower risk of systemic toxicity than intravenous or intraperitoneal A3-APO addition represents [52,68]. Interestingly, when a host defense peptide cocktail was administered intramuscularly to E. coliinfected birds, a trend towards protection against E. coli-related mortality was observed, and a significant number of birds had a reduced bacterial load on cellulitic lesions but there was no reduction in straight bacteremia death or lesion size [69]. In contrast, after subcutaneous peptide administration, the surviving birds had not only better bacteremia rates but also large cellulitic lesions. The insect AMP cecropin-derived peptide HB-107 lacks antimicrobial activity, but when applied to mouse wounds, it informahealthcare.com

induces keratinocyte hyperplasia and increased leukocyte infiltration [70]. In addition, HB-107 stimulates IL-8 secretion from cultured endothelial cells, an effect that may explain the increase in leukocyte migration. It was also observed that chitin coated with AMPs may serve as a scaffold for hemocyanin binding, and the resulting phenoloxidase activity appears to function as a trigger of exoskeleton wound healing [71]. SHAP1 is a fully artificial peptide having a sequence similar to amphipathic lysine/arginine–leucine-containing AMPs and is endcapped for helix stability [72]. SHAP1 treatment accelerates closure and healing of full-thickness excisional wounds in mice without infection, and maintains this activity when S. aureus is inoculated into the skin injury [73]. Peptides not even resembling AMP in sequence features have wound healing potential. Nanofiber scaffolds made up from self-assembling peptides are used in a wide range of innovative medical technologies, including regenerative medicine [74]. One of these peptides named QL6 when injected into the spinal cord tissue of rats 24 h after clip compression spinal cord injury leads to a significant reduction in post-traumatic apoptosis, inflammation and astrogliosis [75]. Peptide QL6 also promotes spinal cord tissue preservation and axonal preservation and regeneration. Integrity of the skin barrier

As all of these reports show, cationic peptides play an important role in maintaining the integrity of the skin barrier and are involved in cutaneous tissue restoration during injury [76]. The presence of these peptides in the skin forms a barrier against microbial pathogenesis and promotes attractive functions during wound repair, contact dermatitis and psoriasis [77]. The conclusion was made that AMP or similar peptides are promising candidates for new therapeutic approaches in wound doi: 10.1586/14787210.2015.1033402

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healing. Lactoferrin and peptidic fragments of lactoferrin were first studied as potential wound healing agents, without bacterial infections, due to their ability to modulate inflammatory responses, improve keratinocyte functions, wound re-epithelialization as well as fibroblast proliferation and migration [78]. However, the most studied peptide in both therapeutic and diagnostic aspects of wound healing is LL-37 due to its anti-biofilm, antibacterial and immunological effects within the wound environment [79]. Growth factors important in wound healing, such as IGF-1 and TGF-a, induce the expression of a series of peptides, such as hCAP-18/LL-37, human b-defensin 3, neutrophil gelatinase-associated lipocalin and secretory leukocyte protease inhibitor in human keratinocytes [80]. Although LL-37 has little or no antimicrobial activity in tissue-culture media, the peptide is not only upregulated at sites of infection but also under the same conditions, demonstrates immunomodulatory effects on epithelial cells, monocytes and dendritic cells [81]. These effects include induction of chemokine production in epithelial cell lines and monocytes and profound alterations of dendritic cell differentiation, resulting in the capacity to enhance a T helper cell Type 1 response. In vivo, LL-37 induces neovascularization in a rabbit model of hind limb ischemia [82]. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18 shows that cathelicidinmediated angiogenesis is important for cutaneous wound neovascularization in vivo [82]. Peptide LL-37 induces endothelial cell proliferation, migration and tubule-like structure formation in vitro and vascularization and re-epithelialization in cutaneous wounds of dexamethasone-treated mice [83]. The expression of two b-defensin variants is lower in both lesional and adjacent nonlesional skin from dogs with atopic dermatitis in comparison to normal skin [84]. A recombinant version of a third canine b-defensin (CBD103) and its human analog shows potent and comparable antimicrobial killing properties against both methicillin-susceptible and methicillinresistant Staphylococcus pseudintermedius [85]. However, skin biopsy specimens from dogs with atopic dermatitis revealed CBD103 expression levels similar to those in healthy controls. The dominant black coat color of dogs, seen in Labrador retrievers and 37 different breeds, can be explained by a single amino acid sequence alteration in mature b-defensin CBD103 [86]. The modified defensin peptide works as a neutral antagonist in the melanocortin system implying yet additional, non-canonical functions of defensins [87]. The influence of AMPs on keloid scar formation

Keloids are benign, proliferative fibromas that form following skin injuries. They consist of fibroblasts exhibiting abnormally increased activity. In the process of wound healing, a balance is maintained for the control of fibroblast proliferation and extracellular matrix synthesis. An increase in cell proliferation with a concomitant reduction in apoptosis may lead to keloid formation [88]. Keloid fibroblasts exhibit a significantly increased proliferative and anti-apoptotic ability [89] as well as a distinct doi: 10.1586/14787210.2015.1033402

increase in collagen fiber synthesis and secretion of matrix metalloproteinase inhibitors [90] compared to normal skin fibroblasts. Keloid fibroblasts secrete cytokines and chemokines, such as granulocyte–macrophage colony-stimulating factor, stem cell factor, TNF-a, nerve growth factor, IL-8 and eosinophil-activating factors [91]. Cathelicidin LL-37 has inhibitory effects on collagen production in fibroblasts, but its expression is downregulated in keloids [92]. Psoriasin (S100A7) and koebnerisin (S100A15) are released by keratinocytes during physiological wound healing. The production of these AMPs is markedly decreased in keloid scar tissue. These peptides inhibit the expression of collagen I and III, fibronectin-1, a-smooth muscle actin and TGF-b by fibroblasts, and they suppress fibroblast proliferation. The anti-fibrotic effects of AMPs may lead to novel preventive and therapeutic strategies for fibroproliferative diseases [93]. Into the marketplace

Antibacterial peptides entered clinical trials early in the biotechnology revolution in the form of pexiganan, a synthetic 22-amino acid version of the skin-derived AMP magainin-2 [94]. Topical pexiganan and oral ofloxacin treatments against foot ulcer are similarly efficacious in clinical improvement rates (85–90%), overall microbiological eradication rates (42–47%) and wound healing rates [95] in a Phase II randomized trial. No significant resistance to pexiganan emerged during the treatment period. Although ensuing Phase III clinical trials showed activity in healing of diabetic foot ulcer wounds with few side effects [96], the drug did not obtain regulatory approval because of lack of advantage over standard care of that time. Later it was shown that S. aureus does evolve resistance to pexiganan that is costly in terms of impaired growth rate [97]. More worrisome, in vitro, resistance to pexiganan equips S. aureus with cross-resistance to human neutrophil defensin-1, a key component of the innate immune response to infection. While the search for resistance mechanisms to AMPs has traditionally focused on membrane or intracellular components [98], the drastic undermining of the innate immune system projects serious potential risks of clinical AMP therapies [97]. Interestingly, a review article published as late as 2013 still lists pexiganan acetate in Phase III clinical trials against diabetic foot ulcers [99]. The parenteral use of AMP in the clinical setting is historically considered hampered by inadequate safety margins and frequently by rapid clearance leaving them suitable only for topical applications [100]. In 2006, omiganan, a bovine indolicidin-based peptide, was in Phase II clinical trials against mild-to-moderate acne [94]. Omiganan gel formulations have been demonstrated to be rapidly bactericidal and fungicidal, with significant dose-dependent activity against a broad spectrum of infectious organisms [101]. Omiganan is currently in Phase II trials against rosacea [99] due to its ability to prevent the inflammatory cascade. After 9 weeks of treatment, oncedaily omiganan 2.5% gel showed superior lesion count reductions and treatment success, compared to 1% omiganan and vehicle [102]. Expert Rev. Anti Infect. Ther.

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Currently, AMPs enjoy a second life in the clinics due to their ability to favorably modulate immunity and enhance wound healing [99]. Along these lines, novexatin, a cationic cyclic heptapeptide, has shown significant promise as a treatment for fungal infections of the toenail (onychomycosis) in preclinical and Phase IIa clinical testing given as a topical treatment [103]. While not classical AMPs, a combination of polymyxin B and gramicidin S (neomycin) is frequently used as a topical wound cream, eye drop and ear drop [94]. Ongoing clinical trials use AMPs not only at therapeutic agents but also as diagnostics or measures of treatment success of different types of drugs in skin diseases and wound healing [104]. One study examined whether administration of oral vitamin D3 given over 21 days changes cathelicidin and human b-defensin expression in the skin or saliva of subjects with atopic dermatitis. Another study aims to find the appropriate dose of LL-37 that can stimulate the immune system to help control melanoma. A study withdrawn before start was designed to establish the tolerability of treatment with the immunomodulatory peptide hLF1-11 administered intravenously as a single dose given for 10 consecutive days to patients with bacteremia due to Staphylococcus epidermidis. LF1-11 was also tried to prevent infections during neutropenia and mucosal barrier injury. Expert commentary

Translational antibacterial peptide research suffered a setback when predominantly bacterial membrane-active AMPs were found unsatisfactorily efficacious compared to traditional antibiotics in human applications or needed doses too high to be considered safe and economical for human use. In general, peptides are not absorbed through the intestinal mucosa, restricting their bioavailability during oral administration [105]. These limitations pushed AMPs as daily therapeutics against communal infections to the sideline and prompted the study of AMPs as acute life-saving modalities in multi-drug resistant nosocomial infections. With the discovery of novel and widespread modes of action, in recent years reports mushroom for the successful systemic use of AMPs, mostly against Enterobacteriaceae, in animal models. However, with the trend of pharmaceutical companies moving out of antimicrobial research, and public policy makers favoring prevention and frugal use of existing antibiotics instead of research and development of new alternatives, AMPs have little chance to be systemic game changers. Biotechnology was historically good for AMP drug development, but currently investors tend to target low investment projects with the expectation of a 30 return from their few successful ventures [106]. Given that simple formula, a 30–100-step peptide synthesis is certainly less appealing than the 8–10-step process typical of most small molecule drugs in terms of cost-of-goods calculations. It is the diverse activity profile, most importantly the upregulation of host defenses and anti-inflammatory activity, that paved way for cutaneous use of AMPs. Be it skin infections, wound healing or just protecting the integrity and physical attributes of the skin, the ability of AMPs to restore cutaneous informahealthcare.com

Review

tissues finally allowed these biopolymers to find their niche in the drug and cosmetics agent repertoire. In addition to be lifesavers in blast or burn injuries, the idea of a cream or bandage to improve non-threatening, but otherwise undesirable, conditions is very appealing [107]. The lifestyle drug market, currently estimated at US$ 30 billion and which includes treatments for weight loss, smoking, erectile dysfunction, wrinkles and baldness [108] can easily accommodate host defense peptides for a long range of cutaneous tissue restoration uses. Five-year view

The successful middle-stage clinical trials with omiganan against rosacea should provide encouragement for both researchers and investors to continue the development of host defense peptides in skin conditions. While 2 years ago peptide A3-APO was considered too expensive to be included in a traditional wound dressing, increased marketing potential together with novel polymeric carriers utilizing micro- and nanofibrous drug delivery systems [109] may change the pharmacoeconomics view of AMPs alone, or in combination with other peptides or small molecule drugs as therapy regimens or consumer product additives. While many cationic peptides contain non-natural amino acid residues or polymeric attachment bonds, in principle, they are still natural products and thus considered ‘green drugs’. This feature will make them especially attractive in the food and cosmetics industries where public pressure requests biological rather than chemical treatment modalities. For entering clinical trials and gaining drug approval, however, regulatory agencies have to be educated about the benefits of peptides in general and host defense peptides in particular. With above 4 mg/l MIC in purely microbiology terms in the eyes of regulatory agencies, the target strains are considered resistant. Then an in vitro immunological or biochemical readout point, convincing for the ensuing clinical efficacy of the peptides, has to be selected and presented. Likewise, efforts have to be made to educate the research and investment communities about the lack of pharmacokinetic and pharmacodynamic relationships of peptide drugs [105]. It is now generally accepted that pharmacodynamic parameters, including duration and extent of action, are more informative of peptide drug utility than plasma drug concentration [110]. Peptide drugs excel in terms of high activity and target selectivity, regardless of poor serum stability and pharmacokinetics. These drugs may modify biological responses significantly longer than standard stability analyses indicate. In spite of the varying past success at the clinics, cationic peptides have a key modulatory role in the innate immune response and being effective adjuvants are synergistic with other immune effectors [111]. Effects on the production of both proinflammatory and anti-inflammatory cytokines by peritoneal macrophages and peripheral blood mononuclear cells have been observed [112] so that clinical applications as anti-inflammatory, immunosuppressive and immunostimulatory agents are possible. As a result, they are very attractive drug leads in injuries involving wounds and skin abrasions. Moreover, their ability to neutralize doi: 10.1586/14787210.2015.1033402

Review

Otvos & Ostorhazi

sepsis and endotoxemia will raise them above conventional antibiotics in a number of potential clinical applications [94]. Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial

conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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Key issues .

Many cationic antibacterial peptides exhibit significantly better protection in animal models of infection than their microbiology activities in vitro would indicate. Frequently, they are successful in eliminating certain bacteria in animals that are resistant to the same peptides in culture media sensitivity assays.

.

Cationic peptides recruit macrophages and neutrophils and, in general, stimulate the immune system around the site of infection. Native antimicrobial peptides accumulate at infection sites and are highly expressed at cutaneous and mucosal surfaces that are prone to injury and ensuing microbial infections.

.

Native antimicrobial peptides are beneficial effectors of each step of the wound healing process: inflammation (neutrophil and macrophage invasion), tissue formation (angiogenesis and epithelialization) and remodeling.

.

In rodent models, cationic peptides show very impressive efficacy in treating wound and burn infections as read by the re-epithelialization of the injury site and the extent and time required for wound closure. Even uninfected wounds heal better if cationic peptides are administered systemically or topically.

.

As both pro- and anti-inflammatory properties are observed, in vivo toxicity is a valid concern. Topical and intramuscular administration

.

Another concern is the induction of resistance not only to the various modes of microbiology actions (membrane disintegration,

appears to be the safest treatment modes. inhibition of protein synthesis and folding, etc.) but also to the integrity of the innate immune system.

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