Insect natural products and processes: New treatments for human disease

Insect Biochemistry and Molecular Biology 41 (2011) 747e769

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Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb

Review

Insect natural products and processes: New treatments for human disease Norman A. Ratcliffe a, b, *, Cicero B. Mello c, Eloi S. Garcia a, Tariq M. Butt b, Patricia Azambuja a a

Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro, 21045-900, RJ, Brazil Department of Biosciences, College of Science, Swansea University, Singleton Park, Swansea, SA2 8PP Wales, UK c Laboratório de Biologia de Insetos, Departamento de Biologia Geral, Universidade Federal Fluminense, Niterói, RJ, Brazil b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2011 Received in revised form 16 May 2011 Accepted 20 May 2011

In this overview, some of the more significant recent developments in bioengineering natural products from insects with use or potential use in modern medicine are described, as well as in utilisation of insects as models for studying essential mammalian processes such as immune responses to pathogens. To date, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey and silk, for at least 4e7000 years, and have featured in folklore medicine for thousands of years. Particular examples of Insect Folk Medicines will briefly be described which have subsequently led through the application of molecular and bioengineering techniques to the development of bioactive compounds with great potential as pharmaceuticals in modern medicine. Insect products reviewed have been derived from honey, venom, silk, cantharidin, whole insect extracts, maggots, and blood-sucking arthropods. Drug activities detected include powerful antimicrobials against antibiotic-resistant bacteria and HIV, as well as anti-cancer, anti-angiogenesis and anti-coagulant factors and wound healing agents. Finally, the many problems in developing these insect products as human therapeutic drugs are considered and the possible solutions emerging to these problems are described. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Insect natural products Medicinal drugs Folk medicine Honey Venom Silk Cantharidin Maggots Antimicrobial factors Anticoagulants Anti-cancer agents

“Insects represent an inexhaustible source for pharmaceutical substances of the future” (Pravda online, 21.04.2005)

1. Introduction Except for honey as food, silk for clothing and pollination of plants, most people regard insects as pests with little thought ever given to the benefits of insects in their lives. Clearly, if entomological research is to flourish, the public perception of insects needs improving and the profiles of beneficial species must be raised. This is especially important as more and more natural products are being extracted from insects and knowledge of their physiological processes is used to benefit people’s lives. The fact that in some countries with strong traditions in entomology, such as the UK and France, the study of insects is now sadly neglected will not help to facilitate these goals (Leather, 2009).

* Corresponding author. Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro, 21045-900, RJ, Brazil. Tel.: þ5521 38658134; fax: þ5521 38658200. E-mail address: [email protected] (N.A. Ratcliffe). 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.05.007

In this overview, we consider some of the more significant recent developments in identifying and bioengineering natural products from insects with use or potential use in medicine, as well as in utilising insects as models for studying essential mammalian processes such as immune responses to pathogens. The reader is also referred to recent reviews by Cherniak (2010) and Dossey (2010) which include details of the use of medicinal insects throughout the World and aspects of their potential for drug discovery. Insects make up about 75% of all animal species and must have produced many natural factors that assisted their survival during the numerous environmental insults which would have occurred over the last 4e500 million years of their evolution. Recently, this fact has been recognised by a number of companies set up to exploit natural products from insects utilising modern molecular and biochemical techniques. Some of this research has been termed “Drugs from Bugs” (goliath.ecnext.com/.../Drugs-from-bugs-thepromise.html), and companies involved include a French consortium formed to develop, amongst other things, insect natural products by means of a “Strategic Alliance of Three French Bioclusters” to form a “Life Science Corridor” to produce the “Therapeutic Innovations of Tomorrow” (www.alsace-biovalley.com/). In

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addition, in 2009, the Chinese Government began investing more than 12 billion US dollars over 5 years in new drug development, including drugs against infectious diseases (www.nature.com/nrd/ journal/v9/n8/full/nrd3238.html), and insects as potential sources of such drugs have not been overlooked by Chinese companies such as Changchun Protelight Pharmaceutical & Biotechnology Co., Ltd (www.protelight.com). Although insects are highly diverse and successful, the fact is that the most successful drugs from natural products have been isolated from plants, microbes and marine organisms. Such drugs include aspirin, codeine, quinine, artemisinin, simvastatin, penicillin, cyclosporine and cytarabine (Cytostar-U) (eg. Borel and Kis, 1991; Gurib-Fakim, 2006; Thomas et al., 2010). 2. Early natural products from insects Thus, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey (beeswax, propolis, pollen and Royal Jelly) and silk, for at least 4e7000 years (Crane, 1983), and have featured in folklore medicine for thousands of years too (Feng et al., 2009). 2.1. Insect natural products in folklore medicine Folklore Medicine seems a strange topic to include in a 21st Century scientific paper, however, the importance of retaining an open mind in science is illustrated below. The use of insects in folk medicine has been particularly common in China and in the State of Bahia in Brazil (Costa-Neto, 2002; Feng et al., 2009) but is also present in many other counties including Mexico, India, Africa and South Korea (Pemberton, 1999; Costa-Neto, 2005a,b; Dossey, 2010). Insects have been used in Chinese medicine for over 3000 years and for at least 1000 years in South America. An estimated 300 insect species are used to produce 1700 traditional Chinese medicines while 42 species have been used in Bahian Folk Medicine (Costa-Neto, 2002; Feng et al., 2009). Often the medicines are extracted from the stings of bees and wasps, or by grinding up the toasted bodies of insects or from their secretions, and then teas made for drinking or ointments for external use (Dossey, 2010). Sometimes the extracts have been used to increase sexual vigour or as love charms (Costa-Neto, 2002)! Particular examples of Insect Folk Medicines which have subsequently proven to have great potential as pharmaceuticals in modern medicine include:i. Honey products for treating wounds and infections. ii. Bee, wasp and ant venom for cancer and all sorts of infections eg. TB, flu, and colds.

iii. Silk prescribed in Chinese medicine for flatulence, dissolving phlegm, and relieving spasms. iv. Cantharidin from blister beetles, and other insect defensive secretions, used in Chinese medicine for treating cancer. v. Whole body extracts of many bees, wasps, flies, butterflies, moths, cockroaches, beetles etc as anti-viral, anti-bacterial and anti-cancer agents. vi. Maggots for treating wounds to enhance healing and reduce infections. vii. Horseflies and other blood-sucking insects to treat blood problems. The logic in the use of some of these arthropods in folk medicine is that the characteristic of the insect relates to its medicinal use. Thus, blister beetles are used to treat skin problems and centipedes with numerous legs are used to treat leg, foot and joint problems (Pemberton, 1999). 3. Honey Honey production worldwide is now worth at least 1.7 billion dollars per year. The harvesting of honey has been featured in rock paintings of more than 6000 yr ago. Honey was also recognised in ancient Egypt for its healing medicinal properties (www.beehexagon.net/files/honey/history.pdf). To quote from the Edwin Smith Ancient Egyptian Surgical Papyrus (www.nim.nih.gov/news/ turn_page_egyptians.html):“Thou shouldst bind [the wound] with fresh meat the first day [and] treat afterwards with grease, honey [and] lint every day until he recovers”. A summary of the medicinal functions of honey products and their activities, which have been determined by modern scientific methods and can mainly be assigned to specific factors involved, is given in Table 1. There has recently been a renewed interest in the healing properties of honey, particularly of Manuka honey derived from the Manuka bush, Leptospermum scoparium, which grows wild in New Zealand. Several clinical trials have been undertaken and the results, although limited, indicated some increase in healing times (Tonks et al., 2007; Cherniak, 2010), although only for mild to moderate superficial burns (Jull et al., 2008). In addition, there are reports of increased granulation tissue in wounds treated with honey (Moran, 1999), and, after incubation of epidermal keratinocytes with honey, of the stimulation of TNF-a, IL-1b and TGF-b and matrix metalloproteinase-9 (MMP-9) cytokines which are involved in the re-epithelialization of skin wounds (Majtan et al., 2010). Real progress in understanding the healing properties of honey and

Table 1 Summary of the medicinal functions of various honey products and of specific factors involved. Honey product

Activity

Specific factors

References

1. Honey and royal jelly

Not determined

Majtan et al., 2010

Apalbumin 1(¼ major royal jelly protein-1)

Majtan et al., 2006

5.8 kDa honey fraction

Tonks et al., 2007

4. Honey

Wound healing by increasing TNF-a, IL-1b, TGF-b and MMP-9 cytokines from keratinocytes and re-epithelialization of skin wounds Wound healing by release of cytokine TNF-a from murine macrophages Wound healing by release of cytokine TNF-a from murine and human monocytes/macrophages via TLR4 Killing of antibiotic-resistant bacteria including MRSA

Kwakman et al., 2010

5. 6. 7. 8. 9.

Blocking the PA-IIL lectin of P. aeruginosa Anti-angiogenesis Neurite growth stimulation Anti-allergic responses Killing human pancreatic cancer PANC-1 cells

Synergistic action of sugar, hydrogen peroxide, methylglyoxal, and defensin-1 Apalbumin 1 The fatty acid, 10-hydroxy-2-decenoic acid Adenosine monophosphate N1 oxide Apalbumin 3 (¼ major royal jelly protein 3) Cycloartane-type triterpenes C30H44O3

2. Honey and royal jelly 3. Honey

Royal jelly Royal jelly Royal jelly Royal jelly Propolis

Lerrer et al., 2007 Izuta et al., 2009 Hattori et al., 2010 Okamoto et al., 2003 Li et al., 2009

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monocytes. Tonks et al. (2007), using MALDIeTOF spectrometry, also identified a 5.8-kDa honey component which in human monocyte cultures stimulates production of the cytokine TNF-alpha via TLR4. This latter fraction has a mass of 5.8 kDa and is heat labile and quite distinct from possible LPS contaminants of the honey. Since monomeric apalbumin in royal jelly and honey has a mass of 55 kDa (Majtan et al., 2006), it is probably quite different from the 5.8 kDa factor in honey (Tonks et al., 2007). This work could potentially lead to the development of novel therapeutics to improve wound healing. Honey has been reported to have many other uses including antibacterial activity and for the treatment of skin and gut disorders as well as allergic rhinitis (reviewed by Cherniak, 2010). Unequivocal proof is available for the anti-bacterial activity of honey against a range of bacteria including clinical strains of antibiotic-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VREF), extended-spectrum b-lactamase-producing Escherichia coli (E. coli ESBL) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) (Kwakman et al., 2010). The killing properties of the Revamil medical grade honey used were shown to be due to the presence of sugar, hydrogen peroxide, methylglyoxal and bee defensin-1 which act synergistically to effect killing (Kwakman et al., 2010). The value of antimicrobial peptides of insects, including defensins, as natural products is discussed below (see, Section 7).

Fig. 1. Morphological changes of PANC-1 human pancreatic cancer cells (white arrow: nucleus fragmentation and condensation; black arrow: membrane bleb) in NDM (nutrient-deprived medium) after 24 h exposure with 6.25 mM of compound 1 from propolis. Figure reprinted with permission from Li et al., “Chemical constituents of propolis from Mynanmar and their preferential cytotoxicity against a human pancreatic cancer cell line”. Li et al., J. Nat. Prod., 72, 1283e87, 2009. Copyright, 2010, American Chemical Society.

royal jelly was made when scientists identified, using murine macrophage cultures, that a major protein from these honey bee products, apalbumin, stimulates the release of the cytokine TNFalpha (Majtan et al., 2006). TNF-alpha is an inflammatory cytokine involved in tissue repair and regeneration and is produced by

Table 2 Summary of major componentsa of bee, wasp and ant venom with potential for development as drugs. Insectb

Component

Activityc

% Whole venom

Amino acidd sequence and ca. mass

References

Honey bee and waspi

Melittin peptide

Kills bacteria and cancer cells, anti-inflammatory

40e60 in bee

GIGAVLKVLTTGLPALISWIKRKRQQ 2.84 kDa

Gevod and Birdi, 1984

Bombolitins peptide

Antimicrobial

Major peptide

LNLKKILGKIGVMLSHLN 1.99 kDa

Choo et al., 2010

Apamin peptide

Treat muscular dystrophy and kill tumours?

2e3

CNCKAPETALCARRCQQH-NH2 2.04 kDa

Lange et al., 1994; Son et al., 2007

Honey and bumble beeiv

Mast cell degranulation peptide (MCD)

Analogues inhibit IgE binding to mast cells and allergies

2e3

MCICKNGKPLPGFIGKICRKICMMGGTHNH2 2.58 kDa

Argiolas et al., 1985

Honey bee

Adolapin and other polypeptides

Analgesic and anti-inflammatory

1

Adolapin contains 103 amino acids 11.5 kDa

Chen and Lariviere, 2010

Honey and bumble beeiv, waspv and antvi

Hyaluronidase enzyme

Enhance cancer chemotherapy?

1.5e2

Contains 349 amino acids in honey bee 39 kDa

Son et al., 2007; Monteiro et al., 2009; Hoffman, 2010

Honey and bumble beeiv, waspvii

PhospholipasesA2 (PLA2) enzyme

Kills cancer cells and inhibits malaria

10e12

Contains 134 amino acids in honey bee 19 kDa

Monteiro et al., 2009

Waspv and antvi

Phospholipases A1 (PLA1) enzyme

Sting diagnosis and immunotherapy

6e14

Contains 304 amino acids in some species 34 kDa

Monteiro et al., 2009

Waspv and antvi

Antigen 5 polypeptide

Sting diagnosis and immunotherapy. Anti-cancer?

ca. 8

Contains 202e212 amino acids in wasps 23 kDa

Hoffman, 1993; Hoffman, 2010

Waspviii

Mastoparans peptide

Antimicrobial and anti-cancer

Major peptides

INWLKLGKKVSAIL-NH2 1.6 kDa

Xu et al., 2006; Mendes et al., 2005; Murata et al., 2009

Waspviii, beeix and antx

Kinins eg. bradykinin and other neurotoxins

Pain control and neurological diseases

Major peptides in some species

DKNKKPIRVGGRRPPGFTPFR-OH 2.4 kDa

Mendes and Palma, 2006; Mortari et al., 2007

Antvi

Solenopsins alkaloids

Neurological disease and anti-angiogenesis

Major alkaloids

253D

Leclercq et al., 1994; Arbiser et al., 2007; Chen et al., 2009a

Bumble beeii Honey bee, wasp

iii

a Many other components, such as the amines, histamine, dopamine, and small peptides and enzymes, are not included here as they are either not unique to venom or knowledge of their activities and properties is limited. See: Palma (2006), Monteiro et al. (2009), Matysiak et al. (2011) and references in Table 2 for more details. b These species are just examples as many others contain these components. i Polistes hebraeus, Vespa velutina nigrithorax, Vespa magnifica and Vespula maculifrons (Shi et al., 2003); ii Bombus ignites; iii Pimpla turionellae; iv Bombus pennsylvanicus; v Polistes annularis; vi Solenopsis invicta; vii Agelaia pallipes pallipes; viii Protopolybia exígua; ix Apis melífera; x Dinoponera australis. c Additional references for drug activities are given in text below. d The amino acid sequence is given for just one example of each venom component since, as with wasp mastoparans, the venom may contain several such factors varying slightly in their sequences.

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The major proteins in royal jelly, and to a lesser extent in honey, the apalbumins, have also been shown to act as decoys to block the PA-IIL lectin of P. aeruginosa which is responsible for biofilm formation and the adhesion of bacteria to animal cells (Lerrer et al., 2007). In addition, royal jelly has other properties including antiangiogenesis (ie. anti-cancer metastasis) by means of the component fatty acid 10-hydroxy-2-decenoic acid (Izuta et al., 2009), neurite growth stimulation due to the presence of adenosine monophosphate N1 oxide (Hattori et al., 2010), and anti-allergic responses due to apalbumin (Okamoto et al., 2003). Finally, the flavonoids in Myanmar (Burma) propolis have been reported to kill human pancreatic cancer PANC-1 cells in vitro, inducing apoptosis-like morphological changes for PANC-1 cells within 24 h of treatment (Fig. 1; Li et al., 2009). The active factors from propolis were extracted, identified and include cycloartanetype triterpenes. The most potent factor, designated compound 1, was a white, waxy substance, and its molecular formula was determined by high-resolution fast atom bombardment mass spectrometry to be C30H44O3. All these properties of honey products vary considerably depending upon the source of honey used for the experiments (Li et al., 2009). Significantly, work on these properties of honey products is ongoing using modern scientific methodology which should help to satisfy medical regulatory bodies governing approval of their use in conventional medicine. 4. Bee, wasp and ant venoms “Pharmaceutical companies are currently funding extensive research into the potential of venom as the next generation of cancer fighting drugs” (Son et al., 2007). Stinging insects such as bees, wasps and ants produce venom containing a complex cocktail of different chemicals including peptides, enzymes, neurotoxins, biogenic amines, amino acids and lipids with a range of pharmacological functions (eg. Palma, 2006; Son et al., 2007; Monteiro et al., 2009; Chen and Lariviere, 2010). It has been estimated that venom of some ants may contain as many as 75 different components (Hoffman, 2010) while the venom of a parasitoid wasp was recently shown to have 79 proteins (Danneels et al., 2010). Bee venom therapy has been used in Chinese medicine, as well as in ancient Greece and Egypt, for thousands of years for the treatment of arthritis, rheumatism, other auto-immune diseases, cancer, skin diseases, pain and infections (Son et al., 2007; Cherniak, 2010; Chen and Lariviere, 2010). Some of the more important components of Hymenopteran venoms for potential drug development and their activities are summarised in Table 2. This list only includes components of venom that have been purified, characterised and often synthesised, and tested using modern scientific methodology. Only by such methods will it be possible to identify and characterise which venom components have remedial value in medicine and allow these to gain approval for development as drugs. Despite the widespread use of bee venom therapy, it has yet to be approved by drug and safety authorities or widely accepted by conventional medicine. There are, however, some crude pharmaceutical formulations available, such as Apiven in France, made from crude honey bee venom (Matysiak et al., 2011). Honey bee venom alone contains over 20 chemicals including melittin, apamin, adolapin, MCD, phospholipase A(2), histamine, hyaluronidase, catecholamine and serotonin. The components of the venom of social bees, wasps and ants have evolved as deterrents, protecting the colony from predators by inflicting a painful sting resulting in oedema swelling and even death. In solitary wasps, however, the venom paralyses the victim for egg-laying and

for feeding, and may also be antimicrobial to prevent bacterial contamination of the food. The components of the venom therefore reflect the lifestyle of the insect with social forms containing chemicals, such as melittin, PLA2 and MCD, causing maximal pain and cell lysis, while the solitary forms have neurotoxins and antimicrobial factors (Libersat, 2003; Palma, 2006). In Table 2 (above), most research on insect venom components as potential drugs has focused on bee melittin, although progress is also described for bee bombolitin, apamin, adolapin, MCD, hyaluronidase, and PLA2, as well as for wasp mastoparans and kinins, and for ant solenopsins. 4.1. Melittin There is extensive interest in developing melittin as a medicinal drug and this is reflected in the vast literature on this major venom component of the honey bee which is also present in some wasps (Uçkan et al., 2004). Melittin is an important allergen, forming about 40e60% of bee venom and is the principal toxin causing pain, inflammation and hypersensitivity (Son et al., 2007). It is a watersoluble, cationic, amphipathic, a-helical polypeptide molecule, composed of 26 amino acids with a tetrameric structure in the venom sac (Terwilliger and Eisenberg, 1982). The amphipathic and cationic nature of melittin mediate its binding by electrostatic interactions with a range of anionic cell membranes from bacteria to cancer cells. Subsequent to binding, melittin has powerful cytolytic activity for most membranes including, unfortunately, those of normal cells, and this has delayed its development for therapeutic use against cancer due to toxicity in vivo (Hoskin and Ramamoorthy, 2008). Many in vitro assays have shown that melittin binding results in inhibition or killing of different types of cancers, including, leukaemic, prostrate, ovarian, mammary, hepatic and ovarian cells (reviewed in Son et al., 2007). Scientists, endeavouring to harness the anti-cancer properties of melittin, have found that cancer cells, with their higher anionic surface charge, are much more sensitive to melittin than normal cells, as are transformed cells with high levels of expression of the rasoncogene (Sharma, 1992). Thus, it is possible to dilute melittin to levels that can kill lung cancer cells in vitro without affecting normal cells (Zhu et al., 1991). More recent work on the potential anti-cancer use of melittin has concentrated on novel delivery systems to the tumour cells in order to avoid damage to normal cells in the body. These systems include using a synthetic lytic peptide based on melittin conjugated to the beta chain of human chorionic gonadotropin (called hecateCGb). This targets cells with up-regulated expression of luteinizing hormone receptors (LHR), such as ovarian, testicular and adrenocortical tumours in mice in vivo (eg. Vuorenoja et al., 2008). These results could lead to new therapies for hormone dependent cancers (Son et al., 2007). Other strategies have involved gene therapy by the transfection of the melittin gene into tumours as well as the use of melittin immunoconjugated with radiolabels and antibodies, all of which have been shown to cause inhibition and apoptosis of several cancers (Son et al., 2007). The most exciting study, however, has recently shown, using nanotechnology, that melittin can be delivered specifically in vivo in nanoparticles to kill melanomas (Fig. 2) and other cancers without any cytotoxicity for normal tissues (Soman et al., 2009). The concentration of melittin injected was 4 times the LD of that for free melittin and targeting was mediated with nanoparticles incorporating a avb3 integrin-binding ligand (Soman et al., 2009). Other scientists have also targeted melittin to cancer cells by utilising their over-expression of matrix metalloproteinase-2 (MMP), which specifically cleaves a melittineavidin conjugate at the site of MMP expression to reactivate melittin and lyse the cancer cells (Holle et al., 2003).

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Fig. 2. Showing therapeutic efficacy of melittin-loaded nanoparticles in syngeneic B16F10 mouse melanoma tumours. Graph shows the increase in tumour volume of B16F10 melanoma tumours during the course of treatment with melittin-loaded nanoparticles (8.5 mg/kg) or controls (saline or nanoparticles alone; n ¼ 5 each group). Photos show the dramatic differences in tumour volume at day 14 after 4 doses of melittin-loaded nanoparticles in comparison with the saline control. Data are represented as mean  SD. **P < 0.01. Figure used with permission of American Society for Clinical Investigation and from, “Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumour cells in mice, reducing growth”, Soman et al., Journal of Clinical Invest, 119, 2830e2842, 2009, permission conveyed through Copyright Clearance Center, Inc.

Regarding the mode of action of melittin, it perturbs and lyses cell membranes and apparently kills cells by a number of mechanisms including direct activation of a Ca2þþ influx to induce TRAIL-mediated apoptosis (Chu et al., 2007; Wang et al., 2009) (TRAIL ¼ tumour necrosis factor related apoptosis-inducing ligand), and also indirectly by activation of phospholipase A2 (PLA2) which then leads to a Ca2þ influx and apoptosis (reviewed in Chu et al., 2007). Melittin, however, not only kills cancer cells but also prevents cancer metastasis by inhibiting the gene expression of the endopeptidase, matrix metalloproteinase-9 (MMP-9), involved in destruction of the extracellular matrix, upstream from the activator protein-1 and NF-kB (Park et al., 2010).

Melittin has also been shown to have other properties which enhance its potential for drug development. It is a powerful antimicrobial peptide (AMP) (see Section 7.1, below), killing bacteria such as E. coli, and S. aureus, as well as the spirochaete causing Lyme disease, Borrelia burgdorferi, the fungus, Candida albicans and even has anti-viral, HIV1, activity (eg. Lubke and Garon, 1997; Aliwaga et al., 2001; Meenakshisundaram et al., 2009; Park and Lee, 2010). Again the lysis of normal mammalian cells by melittin, and many other AMPs, has impeded its therapeutic development. However, melittin has now been bioengineered and short peptide analogues have been designed and sythesised, often combined with cecropin A, with significantly (2-fold) increased anti-bacterial

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activity and markedly (8-fold) reduced lysis of mammalian erythrocytes (eg. Zhu et al., 2007; Ferre et al., 2009). Finally, melittin has also been reported to have both nociceptive and anti-nociceptive effects (pain induction and pain reduction effects) (Son et al., 2007). It is also anti-inflammatory and anti-arthritic by inactivation of the JNK pathway (Park et al., 2008) and inhibiting O2, i-NOS as well as COX-2 genes and pro-inflammatory cytokines which may cause tissue damage in inflammatory diseases such as arthritis and neurodegeneration (Somerfield et al., 1986; Moon et al., 2007). These latter properties of melittin would partially explain the anti-arthritic properties of bee venom. 4.2. Other, mainly bee, venom components (Table 2) 4.2.1. Bombolitins As the name implies, have been isolated from bumble bees in which they make up ca. 50% of the venom dry weight and are structurally and biologically similar to melittin. Thus, bombolitins, like melittin, are amphipathic a-helical polypeptides able to interact with cell membranes, lytic for mammalian erythrocytes and activators of PLA2 (Argiolas and Pisano, 1985). Recently, the bombolitin of Bombus ignitus has been cloned and shown to have antimicrobial activity against both Gram-positive and negative bacteria as well as plant pathogenic fungi (Choo et al., 2010). 4.2.2. Apamin Has also been reported in wasps (Uçkan et al., 2004), is a neurotoxic inhibitor of the highly sensitive calcium activated potassium channels (KCa2) and can cross the bloodebrain barrier to induce hyperexcitability (reviewed in Son et al., 2007). Results of work by Lamy et al. (2010) on the nature of the apamin-blocking of the KCa2 channels may help to design drugs for the treatment of dementia, depression and muscular dystrophy. In addition, apamin can be grafted to the oncoproteins, MDM2 and MDMX, which negatively regulate the tumour suppressor protein p53, to form a novel class of potent p53 activators with potential antitumour activity (Li et al., 2009). 4.2.3. Mast cell degranulation peptide (MCD) Is a bee venom component and, like apamin, is a neurotoxin but with two sites for interaction in the mammalian body, the brain and the mast cells. In the brain, MCD binds to voltage-dependent potassium channels (Kondo et al., 1992,), increasing the firing rate of brain hippocampal neurons which may induce epilepsy (Stutzmann et al., 1997). Interest, however, has focused on the binding of MCD to mast cells and the subsequent interaction with histamine release. At low concentrations, MCD induces mast cell degranulation and histamine release while at higher concentrations, and in the presence of IgE, it inhibits histamine discharge (Buku et al., 2005). Thus, MCD has the potential of binding to the FceRI mast cell receptor to inhibit IgE binding to these cells and thus prevent crosslinking of the bound IgE molecules by allergens which would activate histamine, proteases and cytokines release resulting in Type 1 hypersensitivity reactions (Buku et al., 2005, 2008). MCD analogues, such as MCD [Val6, Ala12] 7, have now been synthesised which compete with IgE for binding to the FceRI mast cell receptor at mm concentrations (Buku et al., 2008). These analogues have considerable therapeutic potential for treating certain types of allergies. 4.2.4. Adolapin This is a ca. 11.5 kDa polypeptide present in honey bee venom which has been reported to have analgesic, anti-inflammatory and anti-pyretic activity. These activities of adolapin probably result from the inhibition of PLA2, cyclooxygenase and, subsequently,

prostaglandins by this compound (Shkenderov and Koburova, 1982; Koburova et al., 1985). Bee venom has been widely used for treating arthritis (Son et al., 2007), and the analgesic and antiinflammatory properties of adolapin probably play an important role in this treatment. 4.2.5. Phospholipases and hyaluronidase These enzymes are major allergens present in many bees, wasps and ants (eg. King and Spangfort, 2000) and most work on their potential as medicinal drugs has been undertaken on bee PLA2. PLA2, as mentioned above, is activated by melittin to kill tumour cells (reviewed in Son et al., 2007). In addition, Putz et al. (2007) have shown that bee venom PLA2, together with 3-phosphorylated phosphatidylinositol (a membrane phospholipid)-homologues, are capable of acting synergistically to kill a range of tumours including renal, breast, prostate and lung cells in vitro. The killing process involves membrane disruption and interference with signal transduction which is not surprising considering the normal role of membrane-associated PLA2 in the cell and the fact that the phosphatidylinositol-homologues are converted to lytic molecules by the PLA2. The subsequent tumour lysates are also a potential source of tumour antigens for vaccination against cancer (Putz et al., 2007). Another role for bee venom PLA2 includes use by transgenesis as an effector gene to stop the development of malaria in Anopheles stephensi mosquitoes. Expression of the PLA2 gene in the gut of mosquitoes reduced Plasmodium berghei oocyst formation by ca. 87% and greatly impaired parasite transmission to naive mice (Moreira et al., 2002). There is great industrial and pharmaceutical interest in phospholipases due to their association with many human disorders so that the recent production of recombinant bee PLA2 in an insect cell line is significant (Shen et al., 2010). Similarly, hyaluronidases have many uses in medicine including helping the spread of drugs in tissues and as anti-cancer agents, so that the production of a recombinant honey bee hyaluronidase may provide a new drug with great commercial potential (Reitinger et al., 2008). 4.3. Wasp mastoparans, kinins and other neutotoxins 4.3.1. Wasp mastoparans These, like bee MCD and melittin, induce the degranulation and release of histamine from mast cells, although there is only limited sequence homology between these molecules (King et al., 2003). Mastoparans are the most abundant peptides in wasp venom and are amphipathic, a-helical polypeptides and therefore potentially antimicrobial. Several different mastoparans may be present in some species of wasps, such as Protopolybia exigua (Mendes et al., 2005), in which they have been synthesised and divided into two groups based on their biological activities. Protopolybia-MP I apparently perturbs the mast cell membrane to effect degranulation while Protopolybia-MP II and III bind to G protein coupled receptors to activate a cascade culminating in mast cell degranulation (Mendes et al., 2005) or the release of many other products from different target cells (Monteiro et al., 2009). Cerovsky et al. (2008) have recently isolated 4 mastoparans from neotropical social wasps, synthesised 40 analogues of these and discovered very potent antimicrobial activity against E. coli, Bacillus subtilis and S. aureus. Likewise, a mastoparan, polybia-MPI, has been identified from the social wasp, Polybia polista, synthesised and shown not only to have antibacterial activity but also to inhibit prostrate and bladder cancer cells in vitro (Wang et al., 2008a). Most importantly, polybia-MPI has low cytotoxicity for normal erythrocytes and fibroblasts. 4.3.2. Kinins Are present in wasps, bee and ant venom (Mendes and Palma, 2006) and are bradykinin (BK)-related molecules and often

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referred to as pain-producing or nociceptive peptides. Most research on these kinins has involved those isolated from solitary and social wasps. They are neurotoxins, paralysing their insect victims by presynaptically blocking nicotinic synaptic transmission in the insect brain (Piek et al., 1990). In the solitary wasp, Cyphononyx fulvognathus, four such BK-related peptides have been reported including the commonly occurring threonine6-bradykinin (Thr6-BK; Picolo et al., 2010). The fact that Thr6-BK purified from the social wasp, Polybia occidentalis, induces a potent antinociceptive effect approximately twice that of morphine will no doubt stimulate further studies of BK-related peptides (Mortari et al., 2007). 4.3.3. Other neurotoxins Wasp venom also contains other non-kinin, neurotoxins such as philanthotoxin (PhTX), a polyamine amide from the solitary wasp Philanthus triangulum, which is a non-competitive inhibitor of nicotinic acetylcholine and glutamate receptors in the mammalian brain (Brier et al., 2003). This toxin has been used to label and study Ca2þ-permeable glutamate receptors which are implicated in ischaemic conditions, epilepsy and growth of glioblastomas (Osswald et al., 2007). Numerous analogues have also been synthesised with therapeutic potential for treating neurodegenerative disease or drug addiction (eg. Jayaraman et al., 1999).

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have been used for detoxification and treating bacterial infections causing sore eyes, swollen throat and loss of speech (www. aumec2009.info/detoxification-part-vii-how-to-detoxify-yourbody-with-chinese-herbs), as well as for impotence (Ahn et al., 2008). Neither silk nor silkworms are probably prescribed in conventional medicine today but, even so, and as a testament to human inventiveness, silkworm extracts are being developed for use in modern medicine. For example, Bombyx mori pupae have been used to extract a vasorelaxation substance, a dimethyladenosine compound, which inhibits phosphodiesterase and greatly enhances NO production in endothelial cells. This compound is a pharmaceutical candidate for development to treat vasculogenic impotence in men (Ahn et al., 2008). In addition, B. mori larvae have been used as bioreactors to express high levels of the antioxidant, Mn-superoxide dismutase, then vacuum dried at 56  C and homogenised to produce a powder. This silkworm powder has subsequently been used to treat mice and shown to enhance various immune parameters such as activation of NK cells to effectively inhibit hepatoma 22 tumours in vivo. Treatment of mice with the silkworm powder is totally non-toxic, in contrast to chemotherapy drugs normally used, so that this antioxidant powder provides a potential new medicine for development in the future (Yue et al., 2009). 5.2. Use of silk in modern medicine

4.4. Ant solenopsin The venom of most ants, in contrast to the proteinaceous venoms of other Hymenopterans, is composed mainly of alkaloids (Chen et al., 2009a). In the red imported fire ant, Solenopsis invicta, six of these piperideine alkaloids have been characterised. One of these alkaloids, Solenopsin A, has been shown to have antiangiogenic activity when tested in the SVR angiogenesis assay which uses ras-transformed murine, endothelial cells. In particular, Solenopsis A suppressed the downstream effector, Akt, of the PI3K signalling pathway which plays a crucial role in angiogenesis (Arbiser et al., 2007). P13K and Akt are amplified or overexpressed in various malignancies such as melanoma and ovarian cancers. Akt is an important target for cancer therapy and Solenopsis A could well be developed as a specific drug for treating advanced cancers (Arbiser et al., 2007). 5. Silk Silk production is about 5000 years old and one legend about the discovery of the silkworm’s silk concerns an ancient empress called, Si Ling-Chi. She was drinking tea under a mulberry tree when a silk cocoon fell into her tea and began to unravel. She noticed the shiny threads, began to wrap them around her finger and realised how strong they were. The silk ran out to reveal the silkworm larva in the centre of the cocoon. She worked out that the larva was responsible for weaving the threads and taught this to her people so that silk farming was born (www.chinatravel.com/.../ sericulture-and-silk-craftsmanship-of-china.htm). The economic value of silk production in 2010 is estimated to be more than 42 billion US dollars with 74% produced in China. Each pupa is made up of 300e900 m of silk thread and 2e3000 pupae are needed to make a pound of silk. Chinese output of silk cocoons was 785,000 tons in 2007 (www.articleblast.com/pushing_high-grade_ silk) which is equivalent to the output from ca. 4.5  1013 larvae!!!! 5.1. Medicines from silkworms Silk has been prescribed in Chinese medicine for treating flatulence, dissolving phlegm and relieving spasms, while silkworms

Concerning the use of silk in modern medicine, prior to the introduction of synthetic polymers, silk proteins were used for years as medical sutures because of their mechanical properties and biocompatibility, although sericin, the glue holding the silk fibroin fibres together, has been shown to be immunogenic (MacIntosh et al., 2008). In the last ten years, rapid progress has been made in understanding silk genetics, structure and biophysics, especially with the publication of the genome of the silkworm, B. mori (The International Silkworm Consortium, 2008). Cloning and expression of native and synthetic silks have been achieved in a variety of host systems and now silk proteins modified by genetic engineering can be produced as biomaterials. These biomaterials can be used in medicine to deliver drugs or genes to specific sites in the body (Numata et al., 2009; Numata and Kaplan, 2010) and in tissue engineering (MacIntosh et al., 2008). Silk biomaterials are tough, flexible, biodegradable, biocompatable and can be prepared under aqueous conditions to avoid loss of activity of the drugs or genes being delivered. They can be produced in many forms including tubes, scaffolds, films, coatings, hydrogels and nanoparticles, and even their rate of degradation can be controlled to regulate the release of their carrier drugs in the body (Wang et al., 2008b; Mandal et al., 2009; Numata and Kaplan, 2010). For the preparation of silk solutions for forming silk-based biomaterials, B. mori cocoons are boiled in 0.02 M Na2CO3 to remove the sericin glue and to release the silk fibroin structural proteins in solution which can then be processed by a range of techniques into different forms (Numata and Kaplan, 2010). The potential use of these silk biomaterials is the subject of intense research and will no doubt lead to many applications in medicine (reviewed by Numata and Kaplan, 2010). For example, multi-layered silk coatings incorporating heparin and other drugs have been applied to vascular stents to prevent adverse reactions. These silk coatings successfully inhibit platelet adhesion initiated by vascular injury and could eliminate the need for restenosis (stent replacement) (Wang et al., 2008b). Silk implants enclosing adenosine-containing microspheres have also been inserted in the brains of rats and resulted in a partial inhibition of epilepsy (Szybala et al., 2009). In addition, silk-based nanoparticles containing curcumin have been shown to have potential for treating

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Fig. 3. Schematic presentation of primary structure of a silk-based block copolymer and functions of each module of the copolymer. Functional peptides can be added at the both ends of molecules for targeted delivery or therapeutic effects. Molecular weight and the secondary structures of silk multimer sequences can control sizes, enzyme resistance, degradation rates, and drug/gene release rates. Polylysine sequences affect sizes as well as surface charge (zeta potential) of the silk-based complexes. Published in Advanced Drug Delivery Reviews, Volume 62, Numata, K., and Kaplan, D.L., “Silk-based delivery systems of bioactive molecules”, pages 1497e1508, 2010. Copyright Elsevier (2010), with permission.

breast cancers in vivo (Numata and Kaplan, 2010), and gel films have been made from sericin and successfully tested as wound dressings (Teramoto et al., 2008). DNA recombinant techniques have also been used to produce hydrogel co-polymers, for example, of silk and elastin (¼SELP, silkelastin-like proteins) or silk with poly (L-lysine) domains which have been used for drug and gene delivery systems (Greish et al., 2009; Numata et al., 2009). The advantage of these recombinant silks is that they can be engineered for specific target delivery and can contain cell-binding motifs (eg. RGD), cell-penetration peptides, viral signal peptides or tumour-homing peptides (Fig. 3) (Numata and Kaplan, 2010). For gene therapy, silk hydrogels with poly (L-lysine) domains interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency (Numata et al., 2009). The development of such non-viral gene therapy would be optimal to avoid any side effects resulting from the introduction of living viruses. Finally, silk is flexible and can resist tensile and compressive forces and can be exploited in tissue engineering (MacIntosh et al., 2008). Silk scaffolds seeded with mesenchymal stem cells differentiate along osteogenic or chondrogenic lineages resulting in the synthesis of bone-like or cartilage-like tissues (Meinel et al., 2006; MacIntosh et al., 2008). The implantation of such osteogenic scaffolds across bone fractures enhanced bone healing in vivo (Meinel et al., 2006). Such techniques coupled with the use of immobilised bone/cartilage growth factors are very promising for development of new bioengineering techniques in the future (MacIntosh et al., 2008). There seems to be no end to the versatility of insect silk as it can even be modified, as with caddis fly silk, to act as a glue under water and may therefore be useful for closing sutures during surgery (Stewart and Wang, 2010). Silkworm silk has approval from the FDA for use in the human body. 6. Cantharidin and other insect defensive secretions Insects produce a huge number of defensive secretions against predators and these molecules represent an enormous pool of mainly small molecules with potential as medicinal drugs (eg. Eisner et al., 2005; Dossey, 2010). One such defensive secretion is cantharidin which is synthesised by some coleopteran species belonging to the Meloidae and Oedemeridae (Gullan and Cranston, 2010). Those beetles producing cantharidin are often termed “blister beetles” as cantharidin is a toxic terpenoid (exo, exo-2, 3dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride; Southcott, 1989) which in contact with the skin causes blistering. Examples are Mylabris phalerata and Mylabris cichorii, the dried bodies of which have been used in Chinese folk and

Chinese traditional medicine for the treatment of cancer for over 2000 years (Wang, 1989). In addition, cantharidin has been prescribed for numerous other conditions including rabies, dropsy, warts and impotence (Dorn et al., 2009). Cantharidin in the form of crushed beetles, Lytta vesicatoria, has also been used for centuries as an aphrodisiac powder called “Spanish Fly”. Not only is the efficacy of this powder questionable but it is also highly toxic although less toxic analogues such as a demethylated form, norcantharidin, have now been synthesised (Wang, 1989). It is ironic that in nature cantharidin is offered during courtship as a pre-copulatory gift by male beetles and only those with high concentrations of cantharidin are acceptable by the female for mating (Eisner et al., 1996). The female impregnates her eggs with cantharidin to protect them from predators (Gullan and Cranston, 2010). Cantharidin or its derivatives have been shown to kill a variety of tumour cells in vitro as well as in animal models in vivo including hepatomas (Mack et al., 1996; Chen et al., 2003), leukaemia (Dorn et al., 2009), breast cancer (Huang et al., 2009), melanoma (An et al., 2004), bladder and gall bladder carcinomas (Fan et al., 2006; Huan et al., 2006), colorectal carcinoma (Peng et al., 2002) and pancreatic cancer (Li et al., 2010). Research has shown that cantharidin is a selective inhibitor of protein phosphatase 2A (PPA2), arresting the growth of cancer cells at the G2/M phase so that they enter mitosis but then undergo apoptosis. Inhibition of PPA2 has been shown to induce the JNK pathway which increases the expression of p21, TNF-a, Bad and Bax which further trigger G2/ M arrest and apoptosis mediated via the mitochondrial caspase cascade (summarised in Dorn et al., 2009; Li et al., 2010). Particularly important is the ratio between the proteins produced by the pro-apoptotic Bax gene and the anti-apoptotic Bcl-2 gene as these have been shown to be significant regulators of cell proliferation and apoptosis (Huang et al., 2009). Fig. 4 shows that treatment of highly-metastatic human breast cancer cells in vitro with norcantharidin down-regulates the expression of the anti-apoptotic protein Bcl-2 and, although not obvious in Fig. 4, the proapoptotic protein Bax is also up-regulated. The result is a reduction in the ratios of the Bcl-2/Bax proteins which would trigger cell death (Huang et al., 2009). There still remain concerns about the toxicity of cantharidin and its analogues, particularly for the urinary system of mammals, and as a result these factors have had limited development in the clinical situation (Liu and Chen, 2009), with trials confined to treatments for skin problems such as common warts. Scientists have, however, persisted in their efforts to develop cantharidin as an anti-cancer drug for several reasons. For example, cantharidin and its analogues have now been shown not only to kill multidrugresistant cancer cells (Rauh et al., 2007) but, in contrast to many anti-cancer drugs in use today, may be less toxic to bone marrow cells (see, however, Kok et al., 2006). In addition, recently, norcantharidin has been shown to effectively inhibit aggressive pancreatic tumour cells and induce apoptosis while being mildly toxic to normal pancreatic cells (Li et al., 2010). May be, as with melittin, the answer lies in confining cantharidin to nanoparticles for specific targeted delivery in the body (see, Section 4, above) or using it in low doses in combination therapy (Zhang et al., 2010a). There are many other insect defensive secretions with potential development as medicinal drugs. For example, in the stick insects or phasmids which when disturbed emit an irritating spray of chemicals from a pair of prothoracic glands. One such chemical identified is parectadial from Parectatosoma mocquerysi which structurally has similarities to perillyl alcohol extracted from plants such as lavender. Perillyl alcohol has been shown to have anti-cancer activity against breast and lung cancer so that further study of the cytotoxic properties of parectadial is merited (Dossey, 2010).

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Fig. 4. Effects of norcantharidin (NCTD) on protein expressions of Bcl-2 and Bax in human breast cancer cells. The expressions of Bcl-2 and Bax proteins were analyzed by Western blotting. The cells were treated for 48 h with NCTD at the indicated concentrations; b-Actin was used in Western blot analyses as a sample loading control. The ratio of Bcl-2 and Bax (the ratio of relative density of each band normalized to b-actin), shown as mean  SD (Bar) is relative to that of zero (vehicle) as the control (designated as 1.0). Figure from “Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action” Huang et al., 2009, Cytotechnology, 59, 201e208. Permission from Springer.

Many such insect defensive secretions and other natural products may well be derived from the plants on which they feed of from symbiotic bacteria in the gut and other tissues. Thus, in the volatile glandular secretions released by the brassy willow leaf beetle, Phratora vitellinae, used to combat pathogens in the microenvironment, the main component is salicylaldehyde which is synthesised by sequestration and catabolization of the plantborne precursors salicin and saligenin (Kuhn et al., 2004; Gross et al., 2008). An example of an insect defensive secretion produced by symbiotic bacteria and with anti-tumour and antiviral activity is pederin produced in the rove beetle Paederus spp. Pederin is a non-protein, cytotoxic polyketide which disrupts DNA, has great potential as a cancer therapeutic agent and for which chemical synthesis has been successfully achieved (Liu et al., 2009). Strong molecular evidence shows that the polyketide synthase gene cluster belongs to a bacterial symbiont of the genus Pseudomonas (Kellner, 2002; Zimmermann et al., 2009; Liu et al., 2009). Unfortunately, to date it has been impossible to culture such symbionts to produce pederin in large quantities in vitro. 7. Whole body extracts of many bees, wasps, flies, butterflies, moths, cockroaches, beetles etc. as anticancer and anti-bacterial agents “We are in danger of returning to a pre-antibiotic era” The Royal Society, 2008 (royalsociety.org/Innovative-mechanisms-fortackling-antibacterial-resistance) Insect body extracts have been used widely in folk medicine, and in Chinese traditional medicine, for treating throat and ear infections, tuberculosis, influenza, cancer and many other diseases and ailments (Pemberton, 1999; Costa-Neto, 2002, 2005a,b; Feng et al., 2009). In China, insects from 77 species, 14 families and 8 orders have been traditionally used to treat tumours and cancer (Jiang, 1990). Considering some of the challenging lifestyles and

microbe-infested niches occupied by many insects and their success, in terms of huge numbers and diversity, it is not surprising that they should possess very effective immune systems producing powerful antimicrobial/cytotoxic factors. Examples are maggots, beetles and fruit flies feeding on decomposing corpses, dung or rotting fruit, respectively, or large numbers of social insects living in confined spaces with elevated temperatures. All these are situations ideal for massive microbial growth and invasion which must be countered by effective immune defences in order to survive. Insects possess innate immune defences mediated by interacting cellular and humoural reactions (Feldhaar and Gross, 2008). The cellular arm involves haemolymph coagulation, phagocytosis and encapsulation processes resulting from co-operating haemocyte types (Anggraeni and Ratcliffe, 1991), following recognition of pathogen-associated molecular patterns (PAMPs) by a range of haemocyte surface receptors (eg. Lavine and Strand, 2002; SchmidHempel, 2005; Jiravanichpaisal et al., 2006; Mamararis and Lampropoulou, 2009). The humoural arm includes constitutive and inducible antimicrobial peptides as well as haemolymph coagulation, melanisation, reactive oxygen and nitrogen species, opsonins and eicosanoid generation (eg. Lavine and Strand, 2002; Bulet and Stocklin, 2005; Jiravanichpaisal et al., 2006; Mamararis and Lampropoulou, 2009; Stanley et al., 2009; Wang, 2010). The fact that lymphocytes and specific antibodies are absent in insects indicates a lack of adaptive or acquired immunity of the sort present in higher vertebrates. Evidence, however, is gathering that some insects not only have a form of long-term acquired memory (eg. Schmid-Hempel, 2005) but also specificity in their immune systems (Bowden et al., 2007; Pham et al., 2007). Obviously, these innate immune processes are effective since insects make up about 75% of all animal species! Of the components of the insect innate immunity, it is the antimicrobial peptides that are potentially the most important sources of natural products. This has resulted from the rise of antibiotic-resistant bacteria, such as methicillin-resistant S. aureus

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(MRSA) and Clostridium difficile, and the urgent need for new weapons with which to kill these organisms. In the USA, MRSA alone is estimated to kill ca. 20,000 people per year and to have increased more than 7 fold in the community in the last few years (Klein et al., 2009). The World Health Organisation identified infections from bacteria resistant to antimicrobials as the largest worldwide pharmaceutical gap (www.who.int/). 7.1. Insect antimicrobial peptides Insect Antimicrobial Peptides (AMPs) were first detected by modern scientists in the 1950s (Briggs, 1958) but not described in detail until Hans Boman’s work on Drosophila and cecropia moths in the 1970s (Boman et al., 1976). Subsequently, over 800 AMPs have been isolated from natural sources (Antimicrobial Peptide Database, http://aps.unmc.edu/AP/main.php) from a range of bacteria, a diversity of invertebrates, fungi, plants, vertebrates and humans. Approximately, half of all these AMPs have been described from insects. Most AMPs contain 5e100 amino acid residues, are amphipathic, and cationic (although anionic AMPs also exist) which assist in their interaction with negatively charged microbial membranes (Otvos, 2002). The AMPs detailed in this section are those freely circulating in the haemolymph or associated with epithelia such as in the gut. The polypeptides, melittin, bombolitin and mastoparans, present in bee and wasp venoms, are also amphipathic and cationic AMPs but have been described in Section 4, above. A few AMPs, such as lysozyme, occur constitutively in the haemolymph without the need for induction but the majority of AMPs are induced following exposure to the PAMPS (pathogen-associated molecular patterns) of invading parasites or pathogens (Bulet and Stocklin, 2005). AMPs are synthesised and released into the haemolymph not only by the fat body but also by the haemocytes, epithelia, and particularly by the gut in vector species (Ratcliffe and Whitten, 2004; Bulet and Stocklin, 2005; Boulanger et al., 2006). Any insect, such as Drosophila, is capable of producing multiple AMPs and can discriminate between different invaders and produce the appropriate peptide (Hancock and Chapple, 1999). For example, in Drosophila (Fig. 5), Gram-negative bacteria elicit diptericin, drosocin, cecropin and attacin, Gram-positive bacteria elicit defensin,

Fig. 5. Adult Drosophila showing the logistical production of AMPs at the main openings of the body. Figure modified from “Antimicrobial peptides in Drosophila: structures, activities and gene regulation” Imler, J.-L., Bulet, P. 2005, in: Kabelitz, D., Schröder JM (Eds), Mechanisms of Epithelial Defence, Chem Immunol Allergy, Karger, Basel, 86, pp. 1e21. With authors permission.

and fungi result in drosomycin and metchnikowin (Imler and Bulet, 2005; Lemaitre and Hoffmann, 2007). AMPs not only kill bacteria and fungi but also protozoans, viruses and cancer cells (Slocinska et al., 2008). Numerous reviews have been published giving details of the mode of action of AMPs in killing their targets and the processes involved may vary according to the AMP structure and composition (eg. Sang and Blecha, 2008; Diamond et al., 2009). In Table 3, the cationic AMPs are loosely classified on the basis of biochemistry and structure into 3 groups, together with a few examples of important members in terms of potential development as drugs. 7.1.1. Linear a-helical AMPs Cecropins are the most abundant group of insect linear, a-helical AMPs and are commonly found in dipterans and lepidopterans and have, for example, been reported in Drosophila melanogaster, Aedes aegypti, Anopheles gambiae, Musca domestica, B. mori and Helicoverpa armigera as well as in vertebrates and parasitic nematodes (Lee et al., 1989; Bulet and Stocklin, 2005; Pillai et al., 2005; Jin et al., 2010; Wang et al., 2010). Often, as with D. melanogaster, B. mori and H. armigera, cecropin multifamily genes are present which are expressed at different times during development as well as in response to different pathogens (eg. Wang et al., 2010). In H. armigera, HaCec-1 and HaCec-3 are greatly up-regulated after fungal infection while HaCec-2 is up-regulated by bacteria (Slocinska et al., 2008; Wang et al., 2010). The cecropins are amongst the best studied AMPs and can kill Gram-positive and Gram-negative bacteria, fungi, protozoans, viruses, nematode worms as well as tumour cells (eg. Chalk et al., 1995; Slocinska et al., 2008; Suttmann et al., 2008; Kokoza et al., 2010). A cecropin B1 analogue showed potent cytolytic activity against human leukaemia cell lines at concentrations which fail to lyse normal fibroblasts or erythrocytes (Hoskin and Ramamoorthy, 2008). Both bacterial and tumour cell membranes have negative surface charges due to LPS and anionic lipids in bacteria, and by anionic lipids and increased glycosylation in tumour cells. These anionic molecules facilitate binding of cecropin to bacteria and tumour cells by electrostatic interactions while the normal cells with their neutral surface charge remain unharmed (Hoskin and Ramamoorthy, 2008). The killing mechanisms probably involve a combination of membrane disruption and pore formation resulting in mitochondrial damage and apoptosis (Schweizer, 2009). Cecropins have great promise for use as anti-cancer drugs especially in combination with conventional chemotherapeutic drugs to lower the dosage of these and reduce their harmful side effects (Hoskin and Ramamoorthy, 2008), or with melittin (see Section 4.1). In addition, in another approach to use AMPs and to control mosquito-borne diseases, cecropins A and defensin A genes have been overexpressed in transgenic A. aegypti mosquitoes and shown to completely block the transmission of Plasmodium gallinaceum (Kokoza et al., 2010). Likewise, paratransgenesis, in which symbiotic Rhodococcus rhodnii were transformed to express Cecropin A in the kissing bug, Rhodnius prolixus, successfully reduced hindgut infection rates of Trypanosoma cruzi by 99% (Durvasula et al., 1997; Beard et al., 2002; Coutinho-Abreu et al., 2010). Sarcotoxin 1A is a linear, cecropin-like, a-helical AMP from the flesh fly Sarcophaga peregrina and has bactericidal activity with Gram-negative bacteria more susceptible than Gram-positive forms. Sarcotoxin 1A has mainly been utilised to engineer transgenic plants such as tobacco (Natori, 2010). 7.1.2. Linear proline/glycine-rich AMPs Of these, the short, proline-rich AMPs are of particular clinical interest as they have a bacteria-specific intracellular target and are generally non-toxic to mammalian cells (Otvos, 2002). Examples of

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Table 3 Three main groups of insect cationic antimicrobial peptides together with representative examples.a Peptide group

Examples

Insects

Activity against

Structure

Linear a-helical

Cecropin A Cecropin B Sarcotoxin 1A

Hyalophora cecropia Sarcophaga peregrina

Bacteria, fungi, protozoa, víruses, nematodes cancer Gram-negative bacteria

KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR

Linear proline/ glycine-rich

Pyrrhocoricin Drosocin

Pyrrhocoris apterus Drosophila melanogaster

Gram-negative bacteria Gram-negative bacteria

VDKGSYLPRPTPPRPIYNRN GKPRPYSPRPTSHPRPIRV

Cysteine-stabilised

Sapecin B Defensin A

Sarcophaga peregrina Phormia terraenovae

Gram-positive bactéria, fungi Gram-positive bacteria

LTCDLLSGEIDRSLCLLHCRLKGYLRAYCSQQKVCRCVQ ATCDLLSGTGINHSACAAHCLLRGNRGGYCNGKGVCVCRN

a

For additional references to other insect AMPs see, Bulet and Stocklin, 2005; Wiesner and Vilsinskas (2010).

proline-rich AMPs are pyrrhocoricin, drosocin, apidaecin and formaecin, with the names reflecting the insect of origin. They effectively kill mainly Gram-negative bacteria by binding to the 70 kDa heat shock protein, DnaK, and inhibiting protein folding (Cudic et al., 2002). The short chain proline-rich AMPs take 6e12 h to kill bacteria, in contrast to the a-helical (see above) and most of the cysteine-stabilised forms (see below), which can kill within 1 h (Cudic et al., 2002). Pyrrhocoricin has been the subject of much research and this AMP and a synthetic dimeric analogue have been shown in vitro to kill antibiotic-resistant Salmonella typhimurium, E. coli, Haemophilus influenzae, Klebsiella pneumoniae and Moraxella catarrhalis with only very low minimal inhibitory concentrations required for killing (Cudic et al., 2002). In addition, it was also possible to successfully treat systemic E. coli infections and H. influenzae lung infections in mice with pyrrhocoricin analogues or with the native peptide, at doses of 10 or 20 mg/kg (Otvos et al., 2000; Cudic et al., 2002). Subsequently, as a result of their earlier work, Otvos and his colleagues have produced a proline-rich designer anti-bacterial peptide dimer, A3-APO, with improved stability in vivo for clinical development (Noto et al., 2008; Rozgonyi et al., 2009). Pyrrhocoricin and the other proline-rich AMPs have considerable potential for drug development as there is a significant difference in the AMP-binding regions of the DnaK heat shock proteins of bacteria and mammals (Otvos, 2002). Finally, pyrrhocoricin has also been shown to have potential as a drug delivery vehicle for treating intracellular, resistant parasites such as Cryptosporidium parvum (Boxell et al., 2008). Drosocin is similar in sequence to pyrrhocoricin with both AMPs having a threonine residue carrying a disaccharide motif which may be important for the anti-bacterial activity (Bulet et al., 1999) although this has been challenged (Bikker et al., 2006). Like most proline-rich AMPs, drosocin is mainly active against Gram-negative bacteria of the Enterobacteriacae family (Otvos, 2002). Drosocin is, however, unstable in mammalian blood, and in the 25e100 mg/kg dose range fails to protect mice from systemic E. coli infection so that research has favoured pyrrhocoricin for drug development. 7.1.3. Cysteine-stabilised AMPs These are small cationic peptides, 33e46 amino acids long, with an even number of cysteines residues forming 3e4 disulphide bridges to stabilise the molecules (Bulet and Stocklin, 2005). Their secondary structures consist of a mixture of a and b strands linked by disulphide bonds. They are present in most insects and this group includes the sapecins, phormicins, royalisin, spodoptericin, gallerimycin, heliomycin and drosomycin which are often grouped together as defensins (Bulet and Stocklin, 2005). They are mainly active against Gram-positive bacteria and/or filamentous fungi (Bulet and Stocklin, 2005; Dassanayake et al., 2007). The defensins have also been reported to be antiparasitic in some vector insects (reviewed in Ratcliffe and Whitten, 2004; Boulanger et al., 2006). They have been shown to be inactivated by physiological saline

solutions which can compromise both their in vitro activity (Bulet and Stocklin, 2005) and their therapeutic potential. Despite this, synthetic versions of defensins have considerable potential both as antibiotics and anti-cancer drugs. For example, Cho et al. (1999) and Saido-Sakanaka et al. (2005) have shown, respectively, that the short, synthetic, anti-bacterial peptides, KLKLLLLLKLK-NH2, based on sapecin of S. peregrine, and ALYLAIRRR-NH2, based on the defensin of the rhinoceros beetle, Oryctes rhinoceros, strongly inhibited MRSA both in vivo and in vitro. In addition, one of the O. rhinoceros synthetic peptides, D-peptide B, also disrupted myeloma cells in vitro with no effects on normal leucocytes (Iwasaki et al., 2009). Finally, the antifungal role of many defensins, such as drosomycin and heliomicin, is well-known and their potential for development as anti-mycotics is now recognised (Thevissen et al., 2007). 7.2. Other miscellaneous factors Apart from the AMPs described above, insects also produce other factors which do not belong to the 3 AMP groups in Table 3 but with killing activities (reviewed in Slocinska et al., 2008). Some of these are large proteins, including pierisin-1 from Pieris spp. at 98 kDa (Koyama et al., 1996) and a S. peregrina lectin at 190 kDa (Itoh et al., 1986). Others are much smaller, such as seraticin from Lucilia sericata at only 365 Da (Bexfield et al., 2008), alloferon from Calliphora vicina at 1265 Da (Chernysh et al., 2002) and an N-myristoylated peptide at 916 Da from Heliothis virescens (Ourth, 2004). Greater details of seraticin and alloferons from dipterans are given below in Section 8 in which the functions of maggot secretions for treating human disease are discussed. Pierisin-1 has been shown to induce apoptosis and cytotoxicity in a range of mammalian cell lines including gastric cancer, cervical carcinoma and HeLa cells (Kono et al., 1999). In vivo, pierisin-1 has anti-cancer activity but is acutely cytotoxic in rats and mice (Shiga et al., 2006) which, as with melittin, can be overcome by bioengineering (Orth et al., 2010) and developing specific delivery systems (see “melittin” in Section 4.1, above). S. peregrina lectin not only functions in insect larval development (Natori, 2010) but also has anti-tumour activity in vitro and in vivo and has cytokine-like activity to stimulate mouse macrophage-like cells to produce a TNF-like factor (reviewed in Slocinska et al., 2008). 7.3. Therapeutic use of AMPs Despite the first description of an insect AMP nearly 35 years ago (Pye and Boman, 1977), clinical trials have been limited and no AMPs have yet been approved for systemic use in humans (Bommarius et al., 2010). This is surprising considering the large range of bioactive factors, described in previous sections, capable of killing a range of microorganisms and cancer cells. The situation with AMPs derived from non-insectan vertebrate sources is more

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promising with a few AMPs having advanced to the last stages of clinical trials or having already been approved for external use (eg. Zaiou, 2007; Nijnik and Hancock, 2009; Wang, 2010; Wiesner and Vilsinskas, 2010). These include AMPs in skincare products for acne, as food preservatives and mouthwashes for gingivitis, as well as for treating catheter-related and hospital infections (Marr et al., 2006). As the incidence of antibiotic resistance in hospital and in the community continue to increase so will the pressure to find new antibiotics and to overcome the barriers to the development of the AMPs. Some problems and possible solutions in developing AMPs as drugs are presented in Table 4 and discussed more fully below.

cost is that screening of natural products is time consuming and wasteful, only yielding one marketable drug out of 20 candidate drugs identified. Alternative methods of identifying candidate drugs involving target-based genomic approaches have been attempted although these have not yet been successful (www.euro. who.incentives). AMPs present a particular problem since they occur at very low concentrations naturally and the cost of peptide production by solid phase peptide synthesis is very high. This cost can be reduced by using truncated derivatives and recombinant technology (Nijnik and Hancock, 2009), although production in bacteria has been hindered by their anti-bacterial activity and proteolytic degradation (Bommarius et al., 2010). Good progress, however, has been reported using recombinant technology to scale up the production of cecropin for clinical trials (Shen et al., 2007; Chen et al., 2009b). Chen et al. (2009b) obtained highly efficient expression of recombinant cecropin AD in B. subtilis yielding a pure, highly stable, peptide with potent antimicrobial activity at low MICs (minimal inhibitory concentrations) and applicable to industrial production. There are many other reasons for increased costs for development of AMPs and other antibiotics (see, www. euro.who.incentives, for detailed discussion), including changes in the regulatory rules requiring the demonstration of relative efficiency within tighter statistical parameters, and these extra costs have eliminated incentives to invest in R&D of antibiotics (Power, 2006). No doubt other countries such as China and Russia are taking full advantage of these difficulties to advance AMPs for clinical use!

7.3.1. The withdrawal of large pharmaceutical companies from research and development of AMPs This has been an important factor in delaying the appearance of AMPs as new antibiotics. The 1960s and 1970s were considered the golden age of antibiotics with many new compounds discovered and developed. However, by the late 1980s, despite the appearance of multiple antibiotic-resistant S. aureus and Streptococcus pneumoniae, many large pharmaceutical companies had withdrawn from research on new antibiotics. By 1989 approximately half of the US and Japanese pharmaceutical companies had ceased or decreased anti-bacterial research efforts (Shlaes et al., 2004). Between 1998 and 2004, the major pharmaceutical companies only included 4 new anti-bacterial drugs out of 290 listed as under development in the new drug pipeline. One important reason for this withdrawal was because the potential financial return on an anti-infective, usually requiring only a short course of treatment, is likely to be less than on other types of drugs treating chronic diseases, such as cancer and depression, which need long-term medication (Shlaes et al., 2004). This reduction in research has been the subject of much concern and prompted the publication of a joint paper from WHO and various European Organisations (www.euro.who.incentives) in which the provision of incentives was proposed to encourage the development of new antibiotics with novel mechanisms of action (MoA). AMPs do indeed have novel MoAs as many disrupt bacterial membranes in contrast to most conventional antibiotics which usually target an intracellular metabolic enzyme (Sang and Blecha, 2008).

7.3.3. Toxicity towards mammalian host cells and instability These result from high salt, high ionic composition or protease breakdown and are also serious problems with many insect AMPs, undermining their therapeutic efficacy in vivo. Some of these problems can be reduced by optimising conditions for AMP antimicrobial activity in vitro in terms of salt and ionic composition of the media so that the poor correlation between in vitro and in vivo activities can also be minimised. The real solution to these problems involves bioengineering AMPs to produce analogues with the properties required such as stability in serum (Nguyen et al., 2010). For example, dimeric analogues of pyrrochorocin were designed that were less toxic to mice than the native molecule, maintained bacterial killing in vitro and in vivo, were not vulnerable to protease breakdown in mammalian serum, and successfully treated mice infected with H. influenzae (Cudic et al., 2002). Likewise, an 11-mer analogue of sapecin B composed of leucine and lysine residues had enhanced antimicrobial activity against MRSA and other bacteria

7.3.2. The cost of developing any new antibiotic drug To reach the point of regulatory approval of a new drug, it has been estimated to cost approximately US$ 800 million although this is debateable (www.euro.who.incentives). One reason for this

Table 4 Summary of problems and possible solutions in developing AMPs for therapeutic use. Problems

Possible solutions

Many large pharmaceutical companies ceased R & D for new antibiotics

Implement incentive schemes for new drug development

Development and production costs too high

Introduce new recombinant methodologies for large scale production

High cytotoxicity of many AMPs in mammals

Bioengineer less toxic analogues using SARS.a Also use new specific delivery systems such as nanoparticles and/or combination drug therapy to reduce MIC and side effects

Instability in mammalian blood due to high salt or ionic composition and protease breakdown

Bioengineer analogues using SARSa able to withstand mammalian salt/ionic concentrations in blood and other body fluids and resistant to protease degradation

Development of bacterial resistance

Many AMPs act at the microbial membrane and may have multiple sites of activity so resistance is less likely

Multifunctional so side effects likely

Increase specificity by designing analogues using SARSa and by use of new delivery systems.

a

SARS ¼ structureeactivity relationship studies.

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(Alvarez-Bravo et al., 1994). In addition, a mutational analysis with the insect antifungal defensin, ARD1, showed that single amino acid substitutions not only increased cationicity and hydrophobicity of ARD1 but also antifungal activity by 2e4 fold (Thevissen et al., 2007). Many other studies, often referred to as “SARS” (structuree activity relationship studies) exist describing the production of synthetic or truncated analogues of a-helical AMPs designed to enhance the killing properties and stability in salt or serum without increasing toxicity in comparison with the native molecule (eg. Bulet and Stocklin, 2005; Schweizer, 2009). High-throughput peptide synthesis combined with mathematical and modelling techniques in quantitative structureeactivity relationships (QSAR) analysis is now being introduced to optimise correlation of chemical structure to biological activity and to facilitate the extensive screening required (Hilpert et al., 2008). An alternative approach to reduce toxicity has been described in Section 4.1, above, in which the cytotoxic bee venom peptide melittin was shown to be delivered specifically in vivo in nanoparticles to kill melanomas (Fig. 2) and other cancers without any cytotoxicity for normal tissues (Soman et al., 2009). Cecropin A has also now been bioengineered with short peptide analogues of melittin with significantly (2-fold) increased anti-bacterial activity and markedly (8-fold) reduction of lysis of mammalian erythrocytes (eg. Zhu et al., 2007; Ferre et al., 2009). Another promising development with an insect AMP has been to combine cecropin A with cancer chemotherapeutic drugs to produce a synergistically enhanced killing with the lower level of drug required resulting in reduced side effects (Hoskin and Ramamoorthy, 2008). 7.3.4. Development of bacterial resistance This is generally less likely to occur with AMPs than with conventional antibiotics. Zhang et al. (2005) showed that P. aeruginosa serially transferred 30 times in sub-MICs of a range of synthetic cationic peptides showed only minimal and transient increases in MIC values. In contrast, resistance against the aminoglycoside antibiotic gentamicin increased 190 fold under the same conditions (Steinberg et al., 1997). In addition, in insects such as Drosophila, any infection elicits the production of a number of AMPs which will work together synergistically to kill the bacteria. These AMPs may interact with different sites in the bacterial cell so that resistance is unlikely to occur. In addition, the primary site of interaction of AMPs is with the bacterial outer membrane with killing often occurring in seconds so that re-configuration of this membrane would present some difficulty for the bacteria. Even so bacterial resistance to AMPs has been reported and includes destruction of AMPs by secreted bacterial proteases, changing the AMP target, and removal of AMPs from their site of action (Andreu and Rivas, 1998; Otto, 2009). Strains of P. aeruginosa and Bacillus larvae are just two bacterial species able to produce cecropindegrading enzymes (Andreu and Rivas, 1998). In strains of S. aureus, the teichoic acid cell wall component is substituted by D-alanine to reduce the negative surface charge and make them less attractive to cationic AMPs, while in Neisseria gonorrhoeae, efflux pumps such as the MtrCE system may remove toxic AMPs from the cell (reviewed by Otto, 2009). 7.3.5. The multifunctional role of AMPs This property is well-known for both vertebrate and invertebrate AMPs. Thus, the S. peregrina AMP, sapecin, also plays a role in embryonic development (Natori, 2010), and many vertebrate AMPs, such as the b-defensins, are immunomodulatory (Easton et al., 2009). From one aspect, this multifunctional role of AMPs is advantageous as they can potentially be developed as immunotherapeutic agents for treating inflammation. This multifunctional role, however, may delay the development of the AMPs due to

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concerns over the consequences of injecting factors with multiple roles and receptor sites in the body. Extensive use of computational methods for designing immunomodulatory peptides will be required together with extensive animal testing since small perturbations of the immune system can be magnified and potentially lethal (Easton et al., 2009). In addition, the development of vertebrate AMPs for systemic therapy, with notable exceptions, is also being favoured now. One reason for this probably relates to concerns over possible side effects of introducing foreign molecules, such as insect AMPs into the mammalian system. Much is already known about the roles of mammalian AMPs and their interactions with the body so toxic side effects should be minimised. Even so, major therapeutic contributions of insect AMPs to medicine can potentially be made as illustrated by the use of a synthetic insect defensin to treat silk sutures and effectively kill MRSA (Saido-Sakanaka et al., 2005). 7.4. Use of Drosophila as a model for human disease processes Until recently, due to the slow development of AMPs as drugs (see above), it has not been the therapeutic potential of AMPs which has provided the most exciting benefit for human medicine but the knowledge of the signalling pathways involved in the expression of the insect AMPs. Research led by Jules Hoffmann, Bruno Lemaitre, Dan Hultmark and many colleagues, using Drosophila as a model, has worked out how the fruit fly AMP genes in the fat body are activated by pathogens (eg. Engstrom et al., 1993; Lemaitre et al., 1995; Hoffmann, 2003). They showed that Gram-negative bacteria activate the IMD pathway while Gram-positive bacteria and fungi activate the Toll pathway. Activation of either of these pathways leads to a cascade of events resulting in an increase in NFkB analogues (DIF/ Dorsal and Relish, respectively, for the Toll and IMD pathways) which are translocated to the nucleus to target and activate the specific AMPs genes. Many excellent reviews with details of these pathways have been published (eg. Naitza and Ligoxygakis, 2004; Lemaitre and Hoffmann, 2007; Ferrandon et al., 2007). What is so special about this work is the fact that it directly led to the discovery of the role of Toll-like signalling cascades in mammalian cells and raised the profile of innate immunity. In 1997, Medzhitov et al. reported the presence of Toll-like receptors (TLRs) in a mammalian cell line which could lead to NFkB activation. Soon after, the TLRs were shown to be sensors of microbial cell wall components and inducers of innate immunity (Poltorak et al., 1998; Rock et al., 1998). Subsequently, at least 13 TLRs have been described in mammalian cells, sensing viruses, bacteria, protozoans and fungi (Uematsu and Akira, 2008). The TLRs not only mediate innate immunity and AMP production in mammals but also the production of cytokines which lead to adaptive immunity and apoptosis (Ishii et al., 2006; Kawai and Akira, 2007; Iwasaki and Medzhitov, 2010). Subsequently, various human TLRs have been discovered to be involved in inflammation, cancer, allergy, sepsis, atherosclerosis and autoimmunity and, as a result, the potential for a new therapy targeting the TLRs for the treatment of disease is an exciting development for the future (Hennessy et al., 2010). The importance of TLRs to medicine was indicated by a meeting of The Royal Society of Medicine, London, on Monday 9 February 2009, entitled “Targeting toll-like receptors: A new approach in the treatment of cancer, infection and autoimmunity” (www.rsm.ac.uk/ academ/cmg102.php). The full significance of the discovery of the Toll/IMD signalling pathways in Drososphila and their equivalences in mammals will not be realised until this latter therapy has been fully developed and tested. In addition, as a result of the discovery of the role of mammalian TLRs and signalling pathways raising the profile of innate immunity, as well as the publication of the Drosophila (Adams et al., 2000)

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Table 5 Examples of the use of Drosophila as a model for human diseases. Disease or organism tested

Results

References

Pseudomonas aeruginosa Mycobacterium marinum Cryptococcus neoformans Vibrio cholorae Candida spp. Enterococcus faecalis Listeria monocytogenes Zygomycetes fungi Alzheimer’s disease

Pil chp genes identified as important virulence factors for both flies and probably mammals Provides an ideal model for studying the interaction of mycobacteria with phagocytes Toll pathway shown to be necessary for clearance of yeast An accurate model for elucidation of hostepathogen interaction A promising model for studying virulence mechanisms and antifungal drug activity Virulence factors important in mammals verified in the Drosophila model New host genes identified involved in immunity and pathogenesis Genes identified of importance in hostepathogen interaction The Toll > NFkB pathway promotes the neuropathological effect of the Alzheimer’s peptide Ab42

D’Argenio et al., 2001 Dionne et al., 2003 Apidianakis et al., 2004 Blow et al., 2005 Chamilos et al., 2006 Cox and Gilmore, 2007 Ayres et al., 2008 Chamilos et al., 2008 Tan et al., 2008

and human (Lander et al., 2001) genome sequences, and the discovery that as many as 70% of human disease genes match Drosophila genes (Reiter et al., 2001), scientists realised just how similar and conserved are many of the Drosophila and human physiological processes and pathways. In consequence, Drosophila is now being used widely as a model for human diseases, particularly for invading pathogens (Scully and Bidochka, 2006). Such models are not confined to Drosophila but have been extended to other insect species, including the wax moth, Galleria mellonella (eg. Mylonakis et al., 2005; Mukherjee et al., 2010; Vilcinskas, 2010a; 2011) and the silkworm, B. mori (Kurokawa et al., 2009). G. mellonella has numerous advantages over Drosophila models since their larger size makes injection into the haemolymph easier, they have numerous haemocytes for studying cellular immunity and proteome assays, and they can be maintained at 37  C to produce reliable estimates of microbial virulence. In addition, over 70% of the Galleria transcriptome has been determined so that powerful RNAi knockout and microarray techniques can be applied (Kavanagh and Reeves, 2007; Mukherjee et al., 2010). Vilcinskas (2011) describes a Galleria metalloproteinase inhibitor (IMPI) which has specific and potent activity against thermolysin-like microbial metalloproteinases including those of human pathogens and such inhibitors provide promising templates for the design of new drugs. Table 5 gives some examples of Drosophila models for human diseases probably involving the innate immune system and Toll/IMD signalling pathways. Many other human diseases from muscular atrophy to cardiac disease are also being modelled in Drosophila (eg. Botas, 2007). 8. Maggots for treating wounds to enhance healing and reduce infections “If a house fly falls in the drink of anyone of you, he should dip it (in the drink), for one of its wings has a disease and the other has the cure for the disease.” A saying of the Prophet Muhammad about 1400 years ago (Sahih Al-Bukhari: Volume 4, Book 54, No. 537). Several reviews have been published on maggot therapy (MT) (Mumcuoglu et al., 1999; Sherman et al., 2000; Sherman, 2003, 2009; Nigam et al., 2006a,b; Vilcinskas, 2010b) and so this section emphasises factors produced by maggots with potential as medicinal drugs. The use of maggots to treat wounds is thousands of years old and is recorded in ancient folk medicine, for example, by Mayan Indians and Australian aborigines (Church, 1996). Maggot efficacy at wound cleaning was also noted in the Napoleonic Wars and the American Civil War in the 19th Century (Sherman et al., 2000). However, it was not until after the First World War in the 1920’s that William Baer, a clinical professor of orthopaedic surgery at John Hopkins, introduced MT for the treatment of infected wounds into hospitals in the USA (Baer, 1931). Baer had served on the battlefield during the First World War and had noted the efficacy of maggot infestations at cleaning and healing wounds. By the 1930’s

and 40’s there were over 300 hospitals in the USA and Canada as well as in Europe using this technique. Only with the introduction of antibiotics in the 1940’s did MT lose favour until its resurgence in the 1980’s with the rise of antibiotic-resistant bacteria such as methicillin-resistant S. aureus (MRSA) (Enright, 2003). MT is now used widely in the USA, Israel and Europe for the treatment of bed sores, leg ulcers, diabetic foot wounds, primary burns, osteomyelitis, and postoperative infections. The sterile maggots are applied directly to the wound or else encased in bags to prevent their escape (Evans, 2002). In many cases, MT has been used for chronic wounds not responding to conventional nursing and antibiotics (Sherman et al., 2000) and success rates for wound healing may be as high as ca. 68% (Steenvoorde et al., 2007). The larvae of the green-bottle, L. sericata, are most commonly used in MT as they are facultative parasites normally feeding on carrion so that in humans they prefer necrotic rather than living tissues (Church, 1996). Many other species of flies, including Lucilia cuprina, C. vicina and Phormia regina, have been used for MT (Sherman et al., 2000) and so the potential production of drug candidates by a range of Dipteran species is also mentioned below. In 2004, the value of L. sericata larvae in the treatment of wounds was recognised by both the US Food and Drug Administration and by the UK Prescription Pricing Authority so that sterile maggots can now be officially prescribed (www.medicaledu.com/maggots.htm). The previous section dealing with insect antimicrobial peptides has emphasised the urgent need for new classes of antibiotics to treat antibiotic-resistant pathogens like MRSA. The emergence of vancomycin-resistant MRSA in the USA and around the World (Tiwari and Sen, 2006) underlines this requirement and since maggot secretions can kill MRSA (Thomas et al., 1999; Bexfield et al., 2004, 2008) they may well provide new drugs to meet this need. MT includes three main processes, namely, 1. debridement, 2. accelerated wound healing and 3. wound disinfection. All these stages are mediated by factors produced by the maggots and have potential for development as drugs. 8.1. Debridement Debridement or wound cleaning begins immediately maggots are applied to a wound and results in the removal of necrotic tissue so that granulation tissue forms and healing can occur. Maggots clean all areas of the wound which could only be matched conventionally by micro-surgery (Mumcuoglu et al., 1999). Debridement by L. sericata larvae involves the extracorporeal secretion of proteolytic enzymes to digest any bacterial slough and necrotic tissue and form a soup upon which the maggots feed (Fig. 6) (Chambers et al., 2003). The enzymes secreted include trypsin-like and chymotrypsin-like serine proteases, a metalloproteinase and an aspartyl proteinase. The maggots also secrete ammonia into the wound and this raises the pH of the environment which is optimal for the activity of the serine proteases. The first instar larvae produce the most active enzymes. Testing the

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Other factors: Apart from maggot NES, other factors appear to stimulate the growth of human fibroblasts in vitro including human epidermal growth factor, the insect hormone 20hydroxyecdysone, as well as L. sericata hemolymph and alimentary secretions (Prete,1997). Maggot NES, however, would probably contain both hemolymph and gut secretions and their component proteases. In addition, specific amino acid derivatives and fatty acid extracts of L. sericata have been shown, respectively, to be mitogenic for human endothelial cells but not fibroblasts (Bexfield et al., 2010), and to enhance angiogenesis and wound healing (Zhang et al., 2010b). This information might prove useful for developing products from maggots for accelerating wound healing.

8.3. Maggot modulation of the mammalian leucocytes Fig. 6. Showing final instar Lucilia sericata larvae feeding on liver and producing copious amounts of extracorporeal secretions which can be collected for testing. Photograph by kind permission of Mr. I.F. Tew, Swansea University.

substrate specificity of the maggot secretions revealed that the chymotrypsin-like serine protease was the most active in degrading ECM components, like collagens, laminin and fibronectin, while solubilisation fibrin clots involved the non-trypsin-like enzyme and the metalloproteinase (Chambers et al., 2003). The removal of the fibrin clots and ECM components allows the initiation of the healing process. Subsequently, Pritchard and his colleagues have been working on the incorporation of these enzymes into hydrogel bandages to accelerate debridement and healing of wounds (Smith et al., 2006; Pritchard et al., 2007). Their results indicate that the controlled delivery of maggot secretory factors may be therapeutic for wounds by stimulating tissue regeneration (Smith et al., 2006). The enzymes present in the native excretions/secretions (NES) of L. sericata maggots that are involved in debridement are also probably responsible for the disruption of biofilms formed by S. aureus, P. aeruginosa and Staphylococcus epidermidis (Van der Plas et al., 2008; Harris et al., 2009). These biofilms form around medical devices and necrotic tissue to protect bacteria from antibiotics and the host’s immune response and are critical to bacterial survival. S. epidermidis can produce two different types of biofilms, one mainly composed of polysaccaharides and the other proteinaceous. The former is disrupted by a glycosidase present in the NES while the latter is disrupted by the proteases, such as those described above, and others, the genes of which have been detected in L. sericata (Altincicek and Vilcinskas, 2009; Harris et al., 2009). 8.2. Accelerated wound healing This has been reported during maggot therapy (eg. Sherman et al., 2000; Armstrong et al., 2002; Sherman, 2003, 2009; Parnes and Lagan, 2007), although not in every study (Dumville et al., 2009). Several factors have been implicated in actively promoting wound healing by maggots including:Physical movement of the maggots in the wound: This is believed to stimulate healing (Buchman and Blair, 1932). Ammonium bicarbonate and high pH, allantoin and urea: These are given off by maggots (Sherman et al., 2000). The high pH of the wound would activate the serine proteases produced by the maggots and have been shown to be involved in remodelling the ECM and ECM-fibroblast interactions to potentially initiate new tissue formation and wound healing (Chambers et al., 2003; Horobin et al., 2003). The proteases or other NES components have also been shown to enhance the migration and co-ordination of action between fibroblasts and other cells (Horobin et al., 2006).

This has also been shown to occur and this would result in an enhancement of wound healing. Thus, Lucilia spp. NES inhibits the migration and pro-inflammatory responses of monocytes/macrophages and neutrophils (Van der Plas et al., 2007, 2009a) and prevents lymphocyte activation (Elkington et al., 2009) in vitro. The production of type 2, pro-angiogenic macrophages, rather than type 1, pro-inflammatory macrophages, would result in the secretion of basic fibroblast growth factor (bFGF) and the vascular endothelial growth factor (VEGF) from the type 2 cells, both of which are involved in endothelial cell migration and division leading to angiogenesis and wound healing (Van der Plas et al., 2009b). The inhibition of neutrophil migration would suppress the tissue damage elicited by these cells in chronic wounds (Van der Plas et al., 2007). Finally, the inhibition of lymphocyte activation by a maggot 56 kDa serpin protein (Elkington et al., 2009), probably as part of the maggot evasion of the host response, would also protect the wound site from tissue damage resulting from an adaptive immune response against the maggot secretions. Some of the factors involved might provide new drugs for facilitating wound healing and immune suppression. 8.4. Wound disinfection Disinfection by maggot secretions occurs throughout maggot therapy. There are many reports showing that the secretions of maggots have anti-bacterial properties (eg. Simmons, 1935; Pavillard and Wright, 1957; Thomas et al., 1999; Kawabata et al., 2010). Kawabata et al. (2010) showed that some of these factors are induced in L. sericata by an infected environment. A few of the anti-bacterial factors involved have now been identified and characterised (Table 6). In Table 6, only a few representative Dipteran AMPs are given and, apart from lucifensin, have briefly been described in Section 7.1 (above). Lucifensin was identified in L. sericata, the most commonly used species in MT, and is active against Gram-positive bacteria including Streptococcus spp., MRSA and GISA (glycopeptides intermediate S. aureus) (Andersen et al., 2010; Cerovsky et al., 2010). Andersen et al. (2010) believe that since lucifensin is active against clinically relevant Streptococcus species then it has potential as a pharmaceutical drug candidate. Other interesting factors produced by maggots have molecular masses <1300 Da with the majority <600 Da (Table 6). They include:Two variants of alloferon: These were originally isolated from bacteria-challenged C. vicinae haemolymph, are 12e13 amino acids long and have little homology with known peptides (Chernysh et al., 2002). Synthetic alloferons have been shown to have anti-tumour and anti-virus properties in mice in vivo

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Table 6 Summary of anti-bacterial and anti-cancer factors produced by Dipteran larvae. Maggot factors

Dipteran species

Activity against

Structure

References

Antimicrobial peptides Sarcotoxin 1A

Sarcophaga peregrina

Gram-negative bacteria

ca. 4000 Da GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR

Natori, 2010

Sapecin B

Sarcophaga peregrina

Gram-positive bactéria, MRSA, fungi, cancer cells

ca. 4080 Da LTCDLLSGEIDRSLCLLHCRLKGYLRAYCSQQKVCRCVQ

Natori, 2010

Defensin A

Phormia terranovae

Gram-positive bacteria

ca. 4000 Da ATCDLLSGTGINHSACAAHCLLRGNRGGYCNGKGVCVCRN

Dimarcq et al., 1990

Lucifensin

Lucilia sericata

Gram-positive bacteria, MRSA

ca. 4113 Da ATCDLLSGTGVKHSACAAHCLLRGNRGGYCNGRAICVCRN

Andersen et al., 2010; Cerovsky et al., 2010

Smaller antimicrobial factors Alloferon 1

Calliphora vicina

Viruses and anti-cancer with cytokine activity

ca. 1265 Da HGVSGHGQHGVHG GVSGHGQHGVHG

Chernysh et al., 2002

p-hydroxybenzoic acid p-hydroxyphenylacetic acid Octahydro-dipyrrolo [1,2-a;10 ,20 -d]

Lucilia sericata

Bacteria

138 Da 152 Da 194 Da

Huberman et al., 2007

Seraticin

Lucilia sericata

Bacteria, MRSA

365 Da

Bexfield et al., 2008

5-S-GADa

Sarcophaga peregrina

Bacteria and anti-cancer

573 Da

Leem et al., 1996

Alloferon 2

a

5-S-GAD ¼ N-b-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine.

and to stimulate human NK lymphocytes in vitro. They also have strong immunomodulatory roles in vivo and induce interferon production in mouse and human models. The crucial role of the NK cell/network in controlling viruses and cancer is well-known (Chernysh et al., 2002). Proof of alloferon’s immunomodulatory and anti-herpes simplex and antihuman papilloma virus activities has been provided clinically (Ryu et al., 2008). Alloferon potentiates immune cells via activation of the NF-kappaB signalling pathway through downregulation of antioxidant proteins (Ryu et al., 2008). The Allopharm Company was formed in Russia to produce pharmaceuticals based on insect immune molecules and an alloferon-derived product, Allomedin, was marketed in 2005 for the treatment of cold sores, genital herpes and gingivitis (www.allomedin.ru/english.shtml). Three low molecular mass factors from non-sterile L. sericata larvae: These lyse both Micrococcus luteus and P. aeruginosa when used in combination (Huberman et al., 2007). The factors include two phenol derivatives and one cyclic dimer of proline. Since the maggots were non-sterile and L. sericata carries Proteus mirabilis, which is known to produce phenylacetic acid (Erdman and Khalil, 1986), then it seems possible that these antibiotics are produced by the contaminating bacteria and not the larvae. However, at the alkaline pH of the maggot secretions, the anti-bacterial activity of phenylacetic acid would probably be low (Erdman, 1987). 5-S-GAD (N-b-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine): This was isolated from immunized S. peregrina and shown to have activity against both Gram-positive and Gramnegative bacteria via generation of H2O2 (Leem et al., 1996). Subsequently, 5-S-GAD has been shown to have multiple activities including inhibition of some cancer cells in vitro and of angiogenesis in mice sarcoma cells in vivo, as well as protecting rat retinal ganglion cells against apoptosis induced by oxygen radicals and other conditions associated with glaucoma (Koriyama et al., 2009). Even more fascinating is that 5-S-GAD prevents cataract formation in the eye (Akiyama et al., 2009), so that it has potential as a pharmaceutical drug for use in eye drops (Kawada et al., 2009).

Seraticin: This is also produced in the NES by sterile larvae of L. sericata (Bexfield et al., 2004, 2008). Initially, the NES was shown to have significant anti-bacterial activity against both Gram-positive and Gram-negative potential pathogens including S. aureus, Bacillus thuringiensis, MRSA, P. aeruginosa, E. coli and Enterobacter cloacae. The fact that the NES samples with the highest pH had the highest anti-bacterial activity probably precludes phenylacetic acid from commensal P. mirabilis as the factor responsible (Erdman, 1987). Subsequent fractionation of the NES revealed two anti-bacterial factors:The first is in the 0.5e10 kDa fraction, with activity against S. aureus but not MRSA (in our hands!). This may be lucifensin, a typical 4 kDa defensin-like AMP recently identified in L. sericata larvae but with activity against MRSA (Andersen et al., 2010; Cerovsky et al., 2010). The second, in the <500 Da range, has activity against S. aureus and MRSA as well as a range of Gram-positive and Gramnegative bacteria, including 10 strains of MRSA (Bexfield et al., 2008). The <500 Da fraction has been investigated further because of its activity against MRSA and C. difficile. The factor has been isolated, characterised, an empirical formula derived and the <500 Da molecule designated as “seraticin”. Intensive mass spectrometry and NMR studies have been undertaken as well as successful synthesis experiments to produce active fractions with similar antimicrobial activities to native seraticin (patent pending). A similar <1000 Da active molecule with activity against MRSA has also been reported from sterile larvae of L. sericata and exhibited great stability essential for commercialization (Kerridge et al., 2005). In conclusion, many such small anti-bacterial molecules have been reported previously from insects (eg. Meylaers et al., 2003). Frequently, they are phenols, quinones or benzoic acids associated with the cuticular glands of the body wall to produce repulsive secretions or pheromones for defence against predators but even so could represent a reservoir of important antibiotics awaiting discovery. Finally, Altincicek and Vilcinskas (2009), screened for L. sericata SPP genes that are differentially expressed as a result of septic wounding and discovered 65 novel, immune inducible genes. Some

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of these genes have close homology to Drosophila genes, such as lysozyme and transferrin, while others are more specific to L. sericata, such as 3 proline-rich AMPs. This study therefore reveals that the NES of L. sericata potentially contains additional factors, yet to be studied, and which facilitate the beneficial effects of MT and which could form the basis of new improved drug therapy. 9. Horseflies and other blood-sucking insects to treat blood problems “The clot thickens: clues provided by thrombin structure,” (Stubbs and Bode, 1995) The main groups of blood-sucking arthropods are soft (Agasidae) and hard (Ixodidae) ticks, Diptera, Hemiptera, Anoplura, and Siphonaptera (Koh and Kini, 2009). The anticoagulants and other components in the salivary glands of blood-sucking insects, for example in horseflies, have been used for hundreds of years as anti-thrombosis treatments in Eastern Medicine (Yang et al., 2000). Only recently, however, has the full potential for drug development from the enormous diversity of pharmaceutical active factors present in the saliva of blood feeding insects been realised. This final section is included just as an indicator of the enormous potential of these factors as new anti-coagulant drugs and immunomodulators. During blood feeding, the host tissue damage resulting from probing by the insect mouthparts, and the presence of the foreign antigens in the injected saliva, induce haemostatic and inflammatory reactions in the host. The saliva of blood feeding insects contains a huge variety of bioactive factors to counteract these responses. The host responses can be almost immediate and include increased platelet stickiness and adhesion to form a plug, blood coagulation, as well as the production of histamine and serotonin affecting vascular permeability and resulting in oedema and damage to the blood feeding insect. Neutrophils are also activated within a short time to degranulate and release PAF (platelet activating factor) which enhances platelet aggregation (Ribeiro and Arca, 2009). Longer-term host responses involving antibody production against the salivary gland proteins may not only reduce insect fitness but can increase mortality too (Tabachnick, 2000). In addition, in longer-feeding arthropods, such as hard ticks (Ixodidae), the level of the host immune response can be extensive as the ectoparasite may remain attached to the host for one week or more (Francischetti et al., 2009). The number and diversity of bioactive factors in insect saliva aimed at speeding up the blood feeding process and providing protection against the host response is a virtual Aladdin’s Cave of potential pharmaceutical drugs. For example, in blood-sucking Nematocera (mosquitoes, blackflies and sandflies), it has been calculated from published sialotranscriptomes that there could be as many as 1280 protein families associated with the bloodsucking lifestyle (Ribeiro et al., 2010). The functions of many of these saliva components are unknown as they have only been detected by recent progress in transcriptome research. Some of these proteins together with other non-protein molecules such as NO, adenosine and prostaglandins present in saliva (Ribeiro and Arca, 2009) have, however, been shown to act as:i. inhibitors of platelet aggregation, ADP, arachidonic acid, thrombin, and PAF. ii. anticoagulants iii. vasodilators iv. vasoconstrictors v. antihistamines vi. sodium channel blockers

vii. viii. ix. x. xi. xii. xiii.

763

immunomodulators complement inhibitors pore formers inhibitors of angiogenesis anaesthetics AMPs and microbial pattern recognition molecules. Parasite enhancers/activators

More details of these factors are given by Francischetti et al. (2005), Santos et al. (2007), Ribeiro and Arca (2009), Andersen (2010), Francischetti (2010) and Ribeiro et al. (2010). Three of the most important roles in the above list are:i. anti-haemastasis ii. immunomodulation iii. parasite enhancement/activation Anti-haemostasis, involving anticoagulants such as apyrases, lipocalins, serine proteases and serpins, occurs immediately upon injection of the saliva (eg. Ribeiro and Arca, 2009). Blood feeding arthropods have also been shown to modulate the immune system of their hosts in many other ways to facilitate feeding and reduce detection. Thus, in R. prolixus, histamine of the host mast cells is bound by nitrophorins to reduce the inflammatory response (eg. Ribeiro and Walker, 1994). Ticks too modulate the cytokine network, for example, in Ixodes scapularis saliva, Salp15 binds to CD4 cells to prevent their activation (eg. Tabachnick, 2000; Francischetti et al., 2009; Hajnická et al., 2011). Parasites, however, can take advantage of this loss of immune homeostasis to sneak past the normally vigilant host defences to enhance their chances of infection. Examples of this include experiments showing that injection of sandfly saliva into mice leads, by chemokine manipulations, to potent neutrophil recruitment, and that these cells act as hosts for early stage infection by Leishmania major (De Moura et al., 2010). Also, with A. aegypti transmitting West Nile virus, the insect saliva disregulates cytokine signalling of the the vertebrate antigen-presenting cells in the skin, IL-10 levels are enhanced and virus replication and invasion increase (Schneider et al., 2010).

9.1. Therapeutic development of salivary gland components From the above brief summary, the therapeutic potential of many of the components of the saliva of blood feeding insects is apparent. To date, as far as the authors are aware, there are few, if any, drugs on the market developed from these salivary gland secretions. Some progress has been made with ticks, and a tick anticoagulant peptide (TAP) from Ornithodoros moubata and another anti-coagulant, Ixolaris from I. scapularis, have been tested in vivo with animal models (Maritz-Olivier et al., 2007). In addition, new NO-releasing drugs are required that only unload their NO in tissues undergoing acidosis following, for example, cardiovascular events since drugs presently available release their NO in all tissues and can cause hypotension. The nitrophorins of R. prolixus are of interest as model systems in this regard due to their unique pHdependent binding and release of NO (Montfort et al., 2000). There are many reasons for this lack of therapeutic development of insect salivary gland components including:Development costs: These would be even higher than for the AMPs (Section 7.3, above) due to the added complexity of clinical trials testing new anticoagulants against cardiovascular events.

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New synthetic non-insectan anticoagulants: Are now being produced by SARS-type (structureeactivity relationship studies) and are small, highly specific molecules able to modulate coagulation at almost every stage (Hirsh et al., 2007). Functions have yet to be assigned to many of these molecules: The latest molecular and data analysis techniques have shown the true diversity of these salivary gland molecules (Ribeiro et al., 2010). This contrasts with other blood-sucking animals such as leeches in which the thrombin inhibitor, hirudin, was first isolated from Hirudo medicinalis over 40 years ago (Markwardt and Walsmann, 1967) and, subsequently, derivatives have been marketed (Tanaka-Azevedo et al., 2010). Interestingly, some arthropod salivary gland factors with additional properties, not directly associated with blood feeding, might justify their accelerated development as drugs. It has now been reported in human cancer biopsies that there is a direct correlation between aggressive tumour behaviour and the expression of tissue factor (TF), a co-factor for initiation of blood coagulation (reviewed in Carneiro-Lobo et al., 2009). Using a U87-MG human glioma cell line, nude mice were inoculated subcutaneously with tumour cells and then 3 days later injected with the anti-coagulant, Ixolaris, and tumour growth then monitored for 20 days. The results showed a significant reduction in tumour weight following Ixolaris injections as well as a downregulation of VEGF (vascular endothelial growth factor) and a reduced tumour vascularisation (CarneiroLobo et al., 2009). Malignant gliomas are aggressive cancers and refractory to therapy so that Ixolaris provides a potential treatment inhibiting tumour growth, angiogenesis and and metastasis (Carneiro-Lobo et al., 2009). Likewise, Amblyomin X, from the hard tick, Amblyomma cajennense, targets melanoma cells, inducing apoptosis and tumour regression (Chudzinski-Tavassi et al., 2010). Finally, even if all this information accumulating about salivary gland components in blood feeding arthropods does not yield new drugs immediately, it can be utilised in other ways. Thus, vaccines can be produced against the salivary components of vector arthropods which create hostile conditions for the transmitted parasites at the site of feeding (eg. Willadsen, 2004; Labuda et al., 2006). In addition, the host immune response, such as IgG levels, to salivary antigens may be a useful epidemiological tool (Remoue et al., 2006). In high risk areas with large number of vectors and bites, the immune response will be elevated even before the disease has appeared so that therapy could be instigated. The only problem is that many of the salivary components of different vectors may cross-react and give inaccurately elevated recordings (Ribeiro and Arca, 2009). 10. Concluding remarks We hope that this review will not only be informative to those interested in mining insects for medicinal drugs but also to those with a lifelong admiration for this amazing group. The plan of this essay acknowledges the wisdom often revealed in the use of ancient remedies and attempts to link the basics from folk medicine to the development by modern technology of new drugs for the future. It is of concern that although many insect natural products have great promise as sources of new drugs, there are very few such products on the market. The prime example is the slow advance in developing antimicrobial peptides as new antibiotics/anti-cancer drugs. It is our belief that both political and public pressure is mounting for such drugs and that failures to obtain approval in the past should not deter the scientific pioneers working in this area today. In addition, in China, Brazil and other countries with a long tradition in the use of plant and animal extracts as medicines, the

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