Drug Discovery Today: Technologies
Vol. 11, 2014
Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Arlington, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Drug resistance
A need for new generation antibiotics against MRSA resistant bacteria Cornelia Pasberg-Gauhl Xilicate GmbH, Richard Neutra Gasse 5, 1210 Vienna, Austria
New antibiotics are highly needed due to continuously emerging resistances, in particular for methicillin-resistant Staphylococcus aureus (MRSA). Only a few new generation antibiotics with new mechanisms of action are available or in development in the recent years. Promising emerging drug candidates with a new mechanism of action are the synthetic guanidine-based polymers based on Akacid. They are highly potent against a wide range of microorganisms and have a beneficial safety profile as reflected in their excellent tolerability when applied to skin, mucosa or eyes. There is a high potential for topical and systemic use of Akacid as an antibiotic in humans and animals, in particular in cases of resistant microorganisms.
Introduction Antibiotics are drugs which prevent the growth of or destroy pathogenic bacteria, usually by intervening with crucial steps of metabolic pathways, and are given to patients for the treatment of an infectious disease caused by bacteria [1]. Most antibiotics have highly target specific mechanisms of action and interfere with one particular cellular function such as cell wall synthesis, protein or RNA synthesis, DNA replication or energy metabolism [2–4]. Antibiotics are widely used in humans and animals, resulting in the increasing emergence of resistant bacterial strains. Bacteria can be intrinsically resistant to certain substances or overcome susceptibility by genetic adaptation. Multi-drug-resistant (MDR) E-mail address: ([email protected]) 1740-6749/$ ß 2014 Elsevier Ltd. All rights reserved.
Section editors: Ju¨rgen Moll – Boehringer-Ingelheim, Vienna, Austria. Gemma Texido´ – Nerviano Medical Sciences S.r.l, Nerviano, Italy. pathogens lose their susceptibility to more than one antibiotic [3]. Selection of antibiotic resistances and the spread of antibiotic-resistant pathogens are a dynamic process [5–7] and cause a serious health problem. For example, in the US, at least 2 million people become infected with antibiotic-resistant bacteria and at least 23,000 people die each year as a direct result of these infections [8]. Hence the development of new generation antibiotics is a high unmet medical need. Worldwide about 80 different antibiotics, grouped into different classes, have been developed so far and are essential tools in modern medicine since the introduction of penicillin in the 1940s. Until the 1970s, the pharmaceutical industry provided a steady flow of new antibiotics, including several with new mechanisms of action that circumvented the problems caused by bacterial resistance to earlier agents (Table 1). Thereafter, the number of new classes of antibiotics reaching the markets has been significantly dropping, leaving fewer options to treat resistant bacteria (Table 2) [8–10]. Antibiotics are among the most commonly prescribed drugs used to control bacterial diseases in humans and animals [8]. Most of them are synthesized from a small set of basic chemical molecules and their derivatives. Just four of such chemical classes, including cephalosporins, macrolides, penicillins and quinolones, account for 73% of the antibiotics introduced between 1981 and 2005 [11]. Over the past 10–20 years multi-drug resistance has emerged in many frequently encountered pathogenic bacteria [5,6,8,9]. Actually, the most common single multi-drug-resistant bacterium in
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Table 1. Year of official introduction of major antibiotics still in use (EU, USA) and resistances identified [8,9] Year
Antibiotic class introduced
1928
Discovery of penicillin
1936
Sulfonamides
Important antibiotics (examples)
Sulfamethoxazole
1940 1943
Penicillin-R Staphylococcus Beta-lactamase inhibitors Subgroup: penicillins Subgroup: cephalosporins (1953) Subgroup: carbapanems (1985) Subgroup: monobactams Subgroup: beta-lactamase inhibitors
Penicillin (G), Flucloxacillin, Ampicillin, Methicillin Cefazolin, Cefalexin, Cefotiam, Cefuroxin, Ceftazidim Impenem, Meropenem, Ertapenem Aztreonam Clavulanic acid, Sulbactam, Tazobactam
1944
Aminoglycosides
Streptomycin, Kanamycin, Gentamycin
1947
Polymyxins
Polymyxin, Colistin
1948
Tetracylines
Tetracycline, Oxytetracycline, Doxycycline, Tigecycline
1949
Amphenicol
Chloramphenicol
1952
Macrolides Subgroup: ketolides (2001)
Erythromycin, Azithromycin, Clarithromycin Telithromycin
1958
Glycopeptides
Vancomycin, Teicoplanin
1962
Streptogramines
Quinupristin and Dalfopristin in combination
1962
Gyrase inhibitors Subgroup: quinolones Subgroup: fluoroquinolones (1983)
Nalidixic acid Ciprofloxacin, Ofloxacin, Levofloxacin, Moxifloxacin
Lincosamides
Clindamycin
1959
1964
Antibiotic resistance indentified
Tetracycline-R Shigella Methicillin-R Staphylococcus
1965
Penicillin-R Pneumococcus
1968
Erythromycin-R Streptococcus
1979
Gentamicin-R Enterococcus
1987
Ceftazidim-R Enterobacteriaceae
1988
Vancomycin-R Enterococcus
1996
Levofloxacin-R Pneumococcus
1998
Imipenem-R Enterobacteriaceae
2000
Oxazolidinones
Linezolid
XDR-tuberculosis
2001
Linezolid-R Staphylococcus
2002
Vancomycin-R Staphylococcus
2003
Lipopeptides
Daptomycin, Caspofungin
2004/2005
PDR-Acinetobacter and Pseudomonas
2009
Ceftazidime-R Neisseria gonorrhoeae
2009
PDR-Enterobacteriaceae
2011
Ceftaroline-R Staphylococcus
Europe is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to the action of methicillin, and related beta-lactam antibiotics [12,13]. Public health authorities have been working on a strategy to foster the development and coordination of sufficient drug development capacities to tackle all areas concerning antimicrobial resistance [5,6,8,14,15]. 110
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The search for new agents that are active on MRSA strains and that do not select easily for resistant strains becomes increasingly important [16]. Alternatives to environmentally critical disinfectants and existing antibiotics are antimicrobial polymers [17]. Promising new candidates under development as alternatives for antibiotics are the synthetic guanidine-based polymers (Akacids), with a new physico-chemical mechanism
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Drug Discovery Today: Technologies | Drug resistance
Table 2. Antibiotics actually in development (or recently introduced) that at least have reached phase III (Status: 09.07.13; without tuberculosis medicines [9]) Active ingredient
Status in the EU
Application
Ceftarolin (cephalosporins)
Since October 2012 commercially
Bacterial infections with MRSA and other Gram-positive bacteria, including complicated skin infections and pneumonia
Fidaxomicin (macrocyclic antibiotic, new class)
Since January 2013 commercially
Infections caused by Clostridium difficile and vancomycin-resistant Enterococcus, in antibiotic-induced colitis
Ceftobiprol (cephalosporins)
In the approval process
Pneumonia caused by Gram-positive and Gram-negative bacteria, including MRSA
Dalbavancin (lipoglycopeptide)
Phase III
Complicated skin infections caused by Gram-positive bacteria, including MRSA
Oritavancin (lipoglycopeptide)
Phase III
Complicated skin and soft tissue infections, including MRSA
Tedizolid = torezolid (oxazolidinones)
Phase III
Complicated skin and soft tissue infections with Gram-positive bacteria, including MRSA
Nemonoxacin (non-fluorinated quinolones)
Phase III
Pneumonia; complicated skin and soft tissue infections, including MRSA
Finafloxacin (fluoroquinolones)
Phase III
Otitis media, urinary tract infections, Helicobacter pylori infections
Surotomycin (lipopeptide)
Phase III
Clostridium difficile
HT-61 (quinolone derivate, new class for topical use)
Phase III
Infections with S. aureus, including MRSA, also effective against other Grampositive bacteria
Ceftazidim + avibactam (cephalosporins + new b-lactamase inhibitor)
Phase III
Infections with Gram-negative bacteria including Pseudomonas
of action and favourable toxicological properties. In this review the properties of Akacid and its derivates and their future potential as antibiotics, in treating patients suffering from infections with resistant bacteria, are discussed.
Such resistant strains cause not only human tragedies by killing patients, but also considerable costs for the society. In Europe, overall societal costs of infection due to antibioticresistant bacteria are estimated at about 1.5 billion Euros each year [5].
Multi-drug-resistant bacteria MRSA are often sub-categorized as hospital-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA), depending upon the circumstances of the environment in which the resistance appeared originally. On the basis of current data, these resistant bacteria are distinct bacterial sub-strains of the original species [13]. The rising number of MRSA or MRSE (methicillin-resistant Staphylococcus epidermidis) and the appearance of vancomycinresistant Staphylococcus aureus (VRSA), Enterococcus spp. (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) are alarming. MDR Mycobacterium tuberculosis and the manifestation of extremely drug-resistant (XDR) strains are growing threats all over the world. MDR ESBL (extended spectrum beta-lactamases)-positive strains of Gram-negative bacteria are responsible for dangerous, almost untreatable infections. Particularly Enterobacteriaceae such as Enterobacter spp., Escherichia coli and Klebsiella spp., Acinetobacter spp. and Pseudomonas aeruginosa can become MDR or pan-drug resistant (PDR) to commonly used antibiotics such as carbapenems, cephalosporins, monobactams, penicillins, polymyxins, quinolones and tetracyclines [6,8,11,14].
Cationic antimicrobial polymers New antibiotics with a new mode of action are urgently needed to combat resistant bacterial strains, but progress in developing them has been slow [5,8,11] since most pharmaceutical drug developing companies have another indication focus. One of the emerging classes of antibiotics belongs to the cationic antimicrobial polymers which have been in use mainly as antiseptics, surface disinfectants and topical antimicrobials for over 80 years within clinical, industrial and domestic hygiene delivery [18]. The number of antimicrobial polymers has significantly increased in the past decade and the state of the art has been summarized in different reviews [17–21]. These include different groups of active substances such as quaternary ammonium-based molecules (QUATs, e.g. benzalkonium chloride, cetrimide) or polymeric biguanides (e.g. chlorhexidine, polyhexanide), which are the active ingredients, for example, of many disinfectants, cosmetics or pharmaceutical products such as topical or oral washes, wound dressings, and so on [21,22]. Cationic antimicrobial polymers are polycondensation products of a guanidinium salt with diamines [21]. Marked www.drugdiscoverytoday.com
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NH
•
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HCl
R
R’
O N H
N H
O
n
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Figure 1. Akacid (oligo-2-(2-ethoxy)-ethoxyethyl-guanidiniumhydrochloride) is the starting material for all commercially relevant Akacid substances and a condensation product of guanidine and oxyalkylendiamine.
antibacterial activity was noted for mixtures of poly-hexamethylene-biguanide (PHMB) salts produced by a reaction of hexamethylene-bis-dicyanodiamide and hexamethylenediamine [23]. Polymeric molecules bear positive charges or are synthesized in the presence of cationic substances, incorporated in their backbone and/or as side chains [21,24,25]. Depending on the choice of components different polymeric guanidines are formed. Water soluble PHMB is a well-known antimicrobial cationic polymer. Antimicrobial activity is based on a biguanide group attached to a hexamethylene group. PHMB is commercially available all over the world and has been marketed for more than forty years as the active ingredient of many disinfectants and preservatives, for example polyhexanide [22,26]. In spite of its long-term use, only little information is available regarding the physical and chemical properties of PHMB. In 2011, De Paula et al. [26] studied and characterized this molecule. By the consensus group of wound management PHMB is considered as one of the most important topical antiseptic/antimicrobial agents to reduce clinical bacterial burden since there are no new antibiotic therapies emerging in the near future [27]. Akacid and Akacid Plus are new candidates as antibiotics under development with a new physico-chemical mechanism of action. Akacid is an oligo-2-(2-ethoxy)-ethoxyethylguanidinium-hydrochloride (OEEG, Fig. 1) and is derived from a condensation product of guanidine and oxyalkylendiamine [28]. A second derivative is Akacid Plus, which was developed with the aim to enhance antimicrobial activity against a broader spectrum of pathogens combined with a significantly better tolerability. Akacid Plus is a poly-hexamethylene-biguanide (PHMB), product of a polycondensation process of a guanidinium salt with two different diamines [28,29].
Mechanism of action Cationic antimicrobial polymers possess a broad physicochemical multi-site mechanism of action which is very difficult to overcome for microorganisms, in contrast to most of the common antibiotics actually in use, which have a very target specific mechanism of action, which facilitates the 112
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emergence of resistance by pathogens [12,21,30]. Despite the long and widespread use of cationic antimicrobial polymers, there has been a lack of experimental evidence with respect to their antimicrobial mechanism of action [21]. However, over the past 10 years research focused on cationic polymers has been intensified [18,25,31–34] and some of the basic mechanisms have been discovered. The outermost surface of bacterial cells carries a net negative charge. Therefore cationic antimicrobials with their net positive charges have a high binding affinity for bacterial cells. They attach to the surface of pathogens and bind to negatively charged sites of the membrane, which are then neutralized [21,35]. A reversal of the negative charge of the microbial membrane occurs and subsequent damage follows, documented as leakage of potassium ions followed by other cytoplasmic materials, resulting in the death of the pathogen [31,32,36]. Razzaghi-Abyaneh et al. [37,38] demonstrated the dose-dependent efficacy of Akacid Plus in growth inhibition against Aspergillus parasiticus by destroying the plasma membrane and major cytoplasmic organelles of the fungus.
Akacid and Akacid Plus properties Initially, Akacid Plus was introduced as a biocidal and disinfection agent, which was developed especially for quick and easy decontamination of rooms, surfaces and areas with high risk of infections [39]. With respect to the favourable physicochemical properties of Akacid and Akacid Plus and their strong inhibitory effects against many different microorganisms, they are promising candidates for pharmaceutical use, for example, for application against human and animal bacterial or fungal pathogens [40–43]. Liquid OEEGs and PHMGs, including Akacid and Akacid Plus, are highly water soluble and their antimicrobial effects are active throughout a wide pH range. Their high stability against UV-light and extreme temperatures ( 708C up to 2008C tested) makes these compounds easy to transport and to store with a long shelf life, which is an advantage for usage, for example, in developing countries. Toxicological studies carried out with Akacid and Akacid Plus, following OECD guidelines for chemical testing [44], showed a high tolerability. Acute oral toxicity (OECD 423) and acute dermal toxicity (OECD 402) studies in rodents demonstrated LD50 doses of Akacid or Akacid Plus higher than 2000 mg/kg body weight [41]. An acute dermal irritation/corrosion study (OECD 404), a skin sensitization study using a local lymph node assay (OECD 429) and an acute eye irritation/corrosion study (OECD 405) with rodents revealed a high tolerability of Akacid Plus in vivo (our unpublished results).
Broad antimicrobial spectrum Akacid and Akacid Plus are active against a wide range of Grampositive and Gram-negative bacteria, yeasts, filamentous
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Table 3. Minimum inhibitory concentration (MIC) of Akacid, Akacid Plus and commercially used medicines, tested on bacteria and fungi [41,43,48,49] Pathogens
Minimum inhibitory concentration (mg/L)
Species (no. of strains)
Akacid
Akacid Plus
VRE (5)
Range MIC50 MIC90 Range MIC50 MIC90 Range Range MIC50 MIC90 Range
2–8 4 8 2–8 4 8 0.5–2 32–64 64 64 32–64
0.06–0.5 0.125 0.25 0.06–0.5 0.125 0.25 0.06–0.25 2–16 8 16 4–16
Spores Spores of B. subtilis (1) Spores of B. anthracis (1)
Range Range
0.5 0.5
Proteus spp. (7)c Salmonella spp. (6)d Shigella spp. (2)e Yersinia enterocolitica (1) Acinetobacter spp. (4)f Stenotrophomonas maltophilia (4)
Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 Range Range Range Range Range Range
8–32 16 32 8–16 16 16 16–32 16 32 32–128 64 64 8–128 8–16 16–32 32 8–32 128–256
1–8 2 4 1–8 2 8 1–8 2 8 4–32 8 32 4–32 1–2 2–4 2 1–8 8–32
Fungi Candida spp. (10)g Aspergillus spp. (7)h
Range Range
0.25–64 16 to >256
0.125–4 1–16
Gram-positive bacteria MSSA (36)
MRSA (62)
MRSE (9) Enterococcus faecalis (27)
Gram-negative bacteria Escherichia coli (65)
Klebsiella spp. (45)a
Enterobacter spp. (20)b
Pseudomonas aeruginosa (59)
Species (no. of strains)
Dermatophytic fungi Candida albicans (46)
Candida non-albicans (44)
Trichophyton rubrum (17)
Trichophyton non-rubrum (11)
0.125 0.125
Chlorhexidine digluconate 0.06–1 0.25 0.5 0.5–2 2 2 0.5–2 2–16 8 8 4–16 1 1
Mupirocin
0.06–1 0.125 0.25 0.06 to >256 0.125 8 0.25–0.5 32–128 64 64 32–64 1 1
2–8 2 8 4–32 8 16 8–32 8 32 8–32 16 32 8–64 2–4 1–2 32 2–32 16–32
128–256 128 256 32 to >256 256 >256 128 to >256 256 >256 32 to >256 >256 >256 256 to >256 128–256 128–256 256 32 to >256 256 to >256
1–16 8–64
32–256 64 to >256
Fluconazole
Minimum inhibitory concentration (mg/L)
Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90
Akacid Plus
Chlorhexidine digluconate
Clotrimazole
0.5–8 1 4 0.03–4 1 4 8–32 16 32 16–32 16 32
8–32 16 32 4–64 16 64 16–32 32 32 16–32 16 32
0.03–8 0.125 0.5 0.03–1 0.125 1 0.125 0.5 0.5 0.06–0.125 0.06 0.125
0.25 1 64 0.125 4 32 4–64 4 32 16–64 16 64
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Table 3 (Continued ) Species (no. of strains)
Minimum inhibitory concentration (mg/L) Akacid Plus
Chlorhexidine digluconate
Other fungi Trichoderma longibrachiatum (15) Trichoderma harzianum (1)
Range Range
Species (no. of strains)
Minimum inhibitory concentration (mg/L)
0.125–0.5 0.25
Akacid Plus Aspergillus spp. (7)
Range MIC50 MIC90
Other moulds (7)
Range MIC50 MIC90
2–16 8 16 0.25 1 8
1–8 8
Amphotericin B 1–8 4 8 1–16 4 8
Clotrimazole
Fluconazole
not determined not determined
64–256 1024
Voriconazole
Caspofungin
0.5–8 8 8
16 to >256 128 256
0.25–32 0.5 1
16 to >256 128 256
a
Includes Klebsiella pneumoniae and Klebsiella oxytoca. Includes Enterobacter aerogenes and Enterobacter cloacae. c Includes Proteus mirabilis and Proteus vulgaris. d Includes Salmonella enteritidis and Salmonella typhimurium. e Includes Shigella sonnei and Shigella flexneri. f Includes Acinetobacter baumannii and Acinetobacter iwoffii. g Includes Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis. h Includes Asperillus niger, Aspergillus flavus and Aspergillus fumigatus. b
fungi, viruses and spores, most notably MRSA, MRSE and ESBLproducing Gram-negative bacteria. About 600 clinical and non-clinical ATCC strains have been tested up to date [39– 43]. Table 3 demonstrates the in vitro antimicrobial profile of Akacid and Akacid Plus in comparison with the widely known chlorhexidine digluconate and mupirocin, which are used topically against bacterial infections including MRSA and MRSE. A total of 369 resistant (including MDR) clinical fungal and bacterial isolates from patients with documented infections in hospitals were tested using the microdilution method with the determination of the minimal inhibitory concentrations (MICs) according to CLSI criteria (Clinical and Laboratory Standards Institute, USA; before known as NCCLS) [45– 47]. Killing curves of S. aureus ATCC 29213 and E. coli ATCC 35218 demonstrated an impressive in vitro efficacy of Akacid polymers [40]. In vitro activity of Akacid Plus against dermatophytes and moulds was compared to those of chlorhexidine and conventional antifungal drugs including fluconazole, clotrimazole, amphotericin B, voriconazole and caspofungin (Table 3). MICs of Akacid Plus against the dermatophytes Trichophyton rubrum and non-rubrum were in the range of 8–32 mg/ L. Chlorhexidine and fluconazole reached similar MIC values between 4 and 64 mg/L. The MIC range of Akacid Plus, amphotericin B and voriconazole against tested moulds was 0.25–32 mg/L. Higher MIC values (16 to >256 mg/L) against Aspergillus spp. and non-Aspergillus species were found for caspofungin [43]. The fungal pathogen Trichoderma spp. shows increasing medical importance, particularly in immunocompromised patients. Akacid Plus demonstrated higher activity against 114
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all clinical Trichoderma strains tested in comparison to chlorhexidine or fluconazole (Table 3) [48]. It is worth mentioning that Akacid Plus was active in vitro across all tested fungal species.
Lack of resistance development against Akacid In vitro selection of resistance to Akacid Plus was carried out on one fungal and 24 different bacterial strains. It was hypothesized that the physico-chemical mode of action of Akacid Plus prevents the development of resistance, which was demonstrated in resistance selection tests with sub-inhibitory concentrations. Even after 30 passages, no reduced susceptibility against clinically relevant Staphylococcus spp. (including MRSA and MRSE), Enterococcus faecalis (including VRE strains), Klebsiella spp., E. coli (including ESBL-positive strains), P. aeruginosa (including ESBL-positive strains) and Acinetobacter spp. was observed [40]. With Candida krusei ATCC 6258, 15 passages were carried out with Akacid Plus and no resistance development was observed. In contrast to Akacid, C. tropicalis ATCC 750 developed resistance to fluconazole; a fourfold increase in the MIC could be detected after 15 passages (S. Tobudic, PhD thesis, University of Vienna, 2006). Using optimal concentrations of Akacid Plus, no induction of resistance in the multi-passage models against the tested bacteria or yeasts was observed.
Pharmaceutical applications of Akacid and Akacid Plus Promising scientific results relating to Akacid’s anti-infective efficacy form the basis for its preclinical development as a pharmaceutical. Through several proprietary formulation
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technologies, several pharmaceutical relevant Akacid products are currently in development. Several in vitro and in vivo efficacy studies as well as a set of toxicological studies show great promise for the indications as systemic antiinfectives or dermal anti-infectives against internal and external pathogenic bacteria, yeasts, fungi, spores and viruses (our unpublished results). In addition, Akacid is also active against typical oral pathogens in tooth cavities (M. Mathe, PhD thesis, University of Vienna, 2008; G. Zips, MSc thesis, University of Vienna, 2008). Akacid Plus is in preclinical development for use as an aqueous solution for topical or oral administration. The in vivo efficacy of Akacid Plus was evaluated in a guinea pig model of experimental skin infection using a mupirocinsensitive clinical MRSA. Different concentrations of Akacid Plus in Ultraphil (Hecht Pharma, Stilstedt, Germany) cream vehicle and a commercially available mupirocin 2% cream formulation (Bactobran; Smith-Kline Beecham Pharmaceuticals, Crawley, United Kingdom) were compared for their activity against bacterial infections of humans. Topical applications of Akacid Plus cream demonstrated a dose-dependant activity. At concentrations of 0.5% and above, Akacid Plus was as effective as mupirocin 2% cream in the treatment of superficial skin infection with MRSA [49]. Skin infections caused by Candida albicans are among the most common fungal infections of humans. Antifungal creams, lotions or sprays are used to treat superficial mycoses. Quantitative suspension tests demonstrated efficacy of Akacid at a concentration of 0.1% during 60 min against C. albicans [41]. Antifungal activity was also studied in guinea pigs with artificial C. albicans infections. Skin treatment with Akacid in Ultrasicc cream (Hecht Pharma, Stilstedt, Germany) at concentrations of 0.5%, 1% and 2% produced better results than clotrimazole 1% cream (Canesten, Bayer, Leverkusen, Germany), a standard antimycotic product. Akacid 0.5% cream eradicated 62.5% of C. albicans compared to 56% with clotrimazole 1% cream [50]. These results prove the efficacy of Akacid in an experimental skin infection model with C. albicans. Oule´ et al. demonstrated in detail the sporicidal efficacy of PHMG hydrochloride against spores of Bacillus subtilis [51].
Akacid Medical Formulation (AMF) for systemic use Akacid given i.v. causes necrosis, which is often seen with cationic polymeric compounds. A new fully purified Akacid with a molecular weight of 1000 Da was separated by nanofiltration [28] and encapsulated in liposomes for systemic use [36]. This high molecular fraction was called AMF and was successfully developed for intravenous administration with a proprietary binding technology to avoid vein irritation and occlusion at higher doses. A tolerance study conducted in mice showed that in contrast to free Akacid the liposomal encapsulated AMF formulation is well tolerated at a daily
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intravenous dosing regimen at a dose of 2.5 mg/kg body weight (our unpublished results).
Conclusions Over the past 10 years a rising number of MDR pathogens have been identified as a major threat to human health [5,8,12]. In several EU Member States, multi-drug resistance has reached 25% or more among Gram-positive and Gramnegative bacteria that cause serious infections in humans. Every year more than 25,000 patients die due to an infection with a MDR bacterium [5]. In healthcare settings, such as hospitals or nursing homes, MRSA is characterized as causing more severe problems compared to MSSA (methicillin-sensitive Staphylococcus aureus), such as bloodstream infections, pneumonia and surgical site infections [5,8], resulting in extra days of hospitalization and more deaths. New antibiotics are highly needed due to continuously emerging resistances in particular for MRSA. Only a few new generation antibiotics with new mechanisms of action are available or in development in the recent years. Akacid and Akacid Plus are potential promising antimicrobials, anti-infectives and anti-proliferatives under development for topical and systemical use in humans and animals with a low toxicity profile and are well tolerated when applied on skin, mucosa or eyes. Compared to existing antibiotics, Akacid and Akacid Plus act through a new mechanism of action and, interestingly, no resistance development was observed so far. In the field of dermatology, Akacid and Akacid Plus have the potential for the treatment of larger skin defects such as diabetic foot and traumatic defects. In the future these molecules could become a powerful treatment of nosocomial and other infections even with MDR pathogens, which are nowadays difficult to treat.
Conflict of interest statement The author is a full time employee of Xilicate GmbH, Vienna, Austria.
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7. ECDC. Surveillance report. Antimicrobial resistance surveillance in Europe; 2011, http://www.ecdc.europa.eu. 8. CDC – Centers for Disease Control and Prevention, U.S. Department of Health and Human Services. Antibiotic resistance threats; 2013, http:// www.cdc.gov.drugresistance/threat-report-2013. 9. Verband der Forschenden Pharma-Unternehmen (vfa). Neue Antibiotika: ¨ ber den Bakterien wahren; 2013, http:// Den Vorsprung gegenu www.vfa.de/. 10. Coates AR, et al. Novel classes of antibiotics or more of the same? Br J Pharmacol 2011;163:184–94. 11. Fischbach MA, Walsh CT. Antibiotics for emerging pathogens. Science 2009;325:1089–93. 12. Levy SB, Marshall B. Review: antibacterial resistance worldwide: causes, challenges and responses. Nat Med 2004;10:122–9. ¨ ck R, et al. Methicillin-resistant Staphylococcus aureus (MRSA): burden of 13. Ko disease and control challenges in Europe; 2010, http:// www.eurosurveillance.org/. 14. ECDC. Updated public health microbiology strategy and work plan 2012– 2016; 2013, http://www.ecdc.europa.eu. 15. World Health Organization. Antimicrobial resistance. Fact sheet no. 194; 2013, http://www.who.int/mediacentre/factsheets/fs/en/. 16. Tan CM, et al. Restoring methicillin-resistant Staphylococcus aureus susceptibility to b-lactam antibiotics. Sci Transl Med 2012;4:1–11. 17. Siedenbichel F, Tiller JC. Antimicrobial polymers in solution and on surfaces: overview and functional principles. Polymers 2012;4:46–71. 18. Moore LE, et al. In vitro study of the effect of cationic biocides on bacterial population dynamics and susceptibility. Appl Environ Microbiol 2008;74:4825–34. 19. Tew GN, et al. De novo design of antimicrobial polymers, foldamers and small molecules: from discovery to practical applications. Acc Chem Res 2010;43:30–9. 20. Kenaway ER, et al. The chemistry and applications of antimicrobial polymers: a state-of-the-art review. Biomacromolecules 2007;8:1359–84. 21. Gilbert P, Moore LE. Cationic antiseptics: diversity of action under a common epithet. J Appl Microbiol 2005;99:703–15. 22. SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks). Antibiotic resistance effects of biocides. European Commission; 2009, http://ec.europa.eu/health/ph_risk/risk_en.htm. 23. Rose FL, Swain G. Bisguanides having antibacterial activity. J Chem Soc Part IV 1956;4422–5. 24. Ivanov I, et al. Characterization of non-biological antimicrobial polymers in aqueous solution and at water–lipid interfaces from all-atom molecular dynamics. J Am Chem Soc 2006;128:1778–9. 25. Samal SK, et al. Critical review: cationic polymers and their therapeutic potential. Chem Soc Rev 2012. http://dx.doi.org/10.1039/c2cs35094g, http://pubs.rsc.org. 26. De Paula GF, et al. Physical and chemical characterization of poly (hexamethylene biguanide) hydrochloride. Polymers 2011;3:928–41. 27. Barret S, et al. PHMB and its potential contribution to wound management. Wounds UK; 2010. 28. Feiertag P, et al. Structural characterization of biocidal oligoguanidines. Macromol Rapid Commun 2003;24:567–70. 29. Albert M, et al. Structure–activity relationships of oligoguanidines – influence of counterion, diamine and average molecular weight on biocidal activities. Biomacromolecules 2003;4:1811–7. 30. Furi L, et al. Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory generated mutants of Staphylococcus aureus. J Antimicrob Chemother 2013;5:3488–97.
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31. Oule´ MK, et al. Polyhexamethylene guanidine hydrochloride-based disinfectant: a novel tool to fight methicillin-resistant Staphylococcus aureus and nosocomial infections. J Med Micobiol 2008;57:1523–8. 32. Zhou ZX, et al. Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences. J Appl Microbiol 2010;108:898–907. 33. Timofeeva L, Kleshcheva N. Antimicrobial polymers: mechanism of action, factors of activity and applications. Appl Microbiol Biotechnol 2011;89:475–92. 34. Zhou ZX, et al. Extensive in vitro activity of guanidine hydrochloride polymer analogs against antibiotic-resistant clinically isolated strains. Mater Sci Eng C 2011;31:1836–43. 35. Maillard JY. Bacterial target sites for biocide action. J Appl Microbiol 2002;92:16–7. 36. Neuwirt H, et al. Akacid-medical-formulation, a novel biocidal oligoguanidine with antitumor activity reduces S-phase in prostate cancer cell lines through the Erk½ mitogen-activated protein kinase pathway. Int J Oncol 2006;29:503–12. 37. Razzaghi-Abyaneh M, et al. Inhibitory effects of Akacid (plus) on growth and aflatoxin production by Aspergillus parasiticus. Mycopathologia 2006;161:245–9. 38. Razzaghi-Abyaneh M, et al. Ultrastructural evidences of growth inhibitory effects of a novel biocide Akacid (plus) on an aflatoxigenic Aspergillus parasiticus. Toxicon 2006;48:1075–82. 39. Kratzer C, et al. Validation of Akacid plus as a room disinfectant in the hospital setting. Appl Environ Microbiol 2006;72:3826–31. 40. Buxbaum A, et al. Antimicrobial and toxicological profile of the new biocide Akacid plus. J Antimicrob Chemother 2006;58:193–719. 41. Kratzer C, et al. Antimikrobielles Wirkprofil der beiden neuen polymerischen Guanidine Akacid und Akacid plus. Antibiotika Monitor 2006;22:2–11. 42. Kratzer C, et al. In vitro antimicrobial activity of the novel polymeric guanidine Akacid plus. J Hosp Infect 2006;63:316–22. ¨ ber Spross43. Tobudic S, et al. In vitro-Wirksamkeit von Akacid Plus gegenu ¨ sen Pilzen im Vergleich zu konventionellen Antimykotika und filamento und Chlorhexidin. Antibiotika Monitor 2006;22:13–20. 44. OECD (Organisation of Economic Co-operation and Development). OECD guidelines for the testing of chemicals: Section 4: Health effects; 2013, http://www.oecd.org/. 45. National Committee for Clinical Laboratory Standards. Reference method for broth dilution. Antifungal susceptibility testing of yeasts. 2nd ed. Standard M27-A2; 2002. 46. National Committee for Clinical Laboratory Standards. Reference method for broth dilution. Antifungal susceptibility testing of conidium-forming filamentous fungi. Standard M38-A; 2002. 47. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 6th ed. Standard M7-A6; 2003. 48. Kratzer C, et al. In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp. J Antimicrob Chemother 2006;58:1058–61. 49. Kratzer C, et al. In vivo activity of a novel polymeric guanidine in experimental skin infection with methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2007;51:3437–9. 50. Kratzer C, et al. In vivo Wirksamkeit von Akacid: Candida albicans-Infektion. Jatros Infektiologie 2008;1:10–2. 51. Oule´ MK, et al. Akwaton, polyhexamethylene-guanidine hydrochloridebased sporicidal disinfectant: a novel tool to fight bacterial spores and nosocomial infections. J Med Microbiol 2012;61:1421–7.
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Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Arlington, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Drug resistance
A need for new generation antibiotics against MRSA resistant bacteria Cornelia Pasberg-Gauhl Xilicate GmbH, Richard Neutra Gasse 5, 1210 Vienna, Austria
New antibiotics are highly needed due to continuously emerging resistances, in particular for methicillin-resistant Staphylococcus aureus (MRSA). Only a few new generation antibiotics with new mechanisms of action are available or in development in the recent years. Promising emerging drug candidates with a new mechanism of action are the synthetic guanidine-based polymers based on Akacid. They are highly potent against a wide range of microorganisms and have a beneficial safety profile as reflected in their excellent tolerability when applied to skin, mucosa or eyes. There is a high potential for topical and systemic use of Akacid as an antibiotic in humans and animals, in particular in cases of resistant microorganisms.
Introduction Antibiotics are drugs which prevent the growth of or destroy pathogenic bacteria, usually by intervening with crucial steps of metabolic pathways, and are given to patients for the treatment of an infectious disease caused by bacteria [1]. Most antibiotics have highly target specific mechanisms of action and interfere with one particular cellular function such as cell wall synthesis, protein or RNA synthesis, DNA replication or energy metabolism [2–4]. Antibiotics are widely used in humans and animals, resulting in the increasing emergence of resistant bacterial strains. Bacteria can be intrinsically resistant to certain substances or overcome susceptibility by genetic adaptation. Multi-drug-resistant (MDR) E-mail address: ([email protected]) 1740-6749/$ ß 2014 Elsevier Ltd. All rights reserved.
Section editors: Ju¨rgen Moll – Boehringer-Ingelheim, Vienna, Austria. Gemma Texido´ – Nerviano Medical Sciences S.r.l, Nerviano, Italy. pathogens lose their susceptibility to more than one antibiotic [3]. Selection of antibiotic resistances and the spread of antibiotic-resistant pathogens are a dynamic process [5–7] and cause a serious health problem. For example, in the US, at least 2 million people become infected with antibiotic-resistant bacteria and at least 23,000 people die each year as a direct result of these infections [8]. Hence the development of new generation antibiotics is a high unmet medical need. Worldwide about 80 different antibiotics, grouped into different classes, have been developed so far and are essential tools in modern medicine since the introduction of penicillin in the 1940s. Until the 1970s, the pharmaceutical industry provided a steady flow of new antibiotics, including several with new mechanisms of action that circumvented the problems caused by bacterial resistance to earlier agents (Table 1). Thereafter, the number of new classes of antibiotics reaching the markets has been significantly dropping, leaving fewer options to treat resistant bacteria (Table 2) [8–10]. Antibiotics are among the most commonly prescribed drugs used to control bacterial diseases in humans and animals [8]. Most of them are synthesized from a small set of basic chemical molecules and their derivatives. Just four of such chemical classes, including cephalosporins, macrolides, penicillins and quinolones, account for 73% of the antibiotics introduced between 1981 and 2005 [11]. Over the past 10–20 years multi-drug resistance has emerged in many frequently encountered pathogenic bacteria [5,6,8,9]. Actually, the most common single multi-drug-resistant bacterium in
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Table 1. Year of official introduction of major antibiotics still in use (EU, USA) and resistances identified [8,9] Year
Antibiotic class introduced
1928
Discovery of penicillin
1936
Sulfonamides
Important antibiotics (examples)
Sulfamethoxazole
1940 1943
Penicillin-R Staphylococcus Beta-lactamase inhibitors Subgroup: penicillins Subgroup: cephalosporins (1953) Subgroup: carbapanems (1985) Subgroup: monobactams Subgroup: beta-lactamase inhibitors
Penicillin (G), Flucloxacillin, Ampicillin, Methicillin Cefazolin, Cefalexin, Cefotiam, Cefuroxin, Ceftazidim Impenem, Meropenem, Ertapenem Aztreonam Clavulanic acid, Sulbactam, Tazobactam
1944
Aminoglycosides
Streptomycin, Kanamycin, Gentamycin
1947
Polymyxins
Polymyxin, Colistin
1948
Tetracylines
Tetracycline, Oxytetracycline, Doxycycline, Tigecycline
1949
Amphenicol
Chloramphenicol
1952
Macrolides Subgroup: ketolides (2001)
Erythromycin, Azithromycin, Clarithromycin Telithromycin
1958
Glycopeptides
Vancomycin, Teicoplanin
1962
Streptogramines
Quinupristin and Dalfopristin in combination
1962
Gyrase inhibitors Subgroup: quinolones Subgroup: fluoroquinolones (1983)
Nalidixic acid Ciprofloxacin, Ofloxacin, Levofloxacin, Moxifloxacin
Lincosamides
Clindamycin
1959
1964
Antibiotic resistance indentified
Tetracycline-R Shigella Methicillin-R Staphylococcus
1965
Penicillin-R Pneumococcus
1968
Erythromycin-R Streptococcus
1979
Gentamicin-R Enterococcus
1987
Ceftazidim-R Enterobacteriaceae
1988
Vancomycin-R Enterococcus
1996
Levofloxacin-R Pneumococcus
1998
Imipenem-R Enterobacteriaceae
2000
Oxazolidinones
Linezolid
XDR-tuberculosis
2001
Linezolid-R Staphylococcus
2002
Vancomycin-R Staphylococcus
2003
Lipopeptides
Daptomycin, Caspofungin
2004/2005
PDR-Acinetobacter and Pseudomonas
2009
Ceftazidime-R Neisseria gonorrhoeae
2009
PDR-Enterobacteriaceae
2011
Ceftaroline-R Staphylococcus
Europe is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to the action of methicillin, and related beta-lactam antibiotics [12,13]. Public health authorities have been working on a strategy to foster the development and coordination of sufficient drug development capacities to tackle all areas concerning antimicrobial resistance [5,6,8,14,15]. 110
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The search for new agents that are active on MRSA strains and that do not select easily for resistant strains becomes increasingly important [16]. Alternatives to environmentally critical disinfectants and existing antibiotics are antimicrobial polymers [17]. Promising new candidates under development as alternatives for antibiotics are the synthetic guanidine-based polymers (Akacids), with a new physico-chemical mechanism
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Table 2. Antibiotics actually in development (or recently introduced) that at least have reached phase III (Status: 09.07.13; without tuberculosis medicines [9]) Active ingredient
Status in the EU
Application
Ceftarolin (cephalosporins)
Since October 2012 commercially
Bacterial infections with MRSA and other Gram-positive bacteria, including complicated skin infections and pneumonia
Fidaxomicin (macrocyclic antibiotic, new class)
Since January 2013 commercially
Infections caused by Clostridium difficile and vancomycin-resistant Enterococcus, in antibiotic-induced colitis
Ceftobiprol (cephalosporins)
In the approval process
Pneumonia caused by Gram-positive and Gram-negative bacteria, including MRSA
Dalbavancin (lipoglycopeptide)
Phase III
Complicated skin infections caused by Gram-positive bacteria, including MRSA
Oritavancin (lipoglycopeptide)
Phase III
Complicated skin and soft tissue infections, including MRSA
Tedizolid = torezolid (oxazolidinones)
Phase III
Complicated skin and soft tissue infections with Gram-positive bacteria, including MRSA
Nemonoxacin (non-fluorinated quinolones)
Phase III
Pneumonia; complicated skin and soft tissue infections, including MRSA
Finafloxacin (fluoroquinolones)
Phase III
Otitis media, urinary tract infections, Helicobacter pylori infections
Surotomycin (lipopeptide)
Phase III
Clostridium difficile
HT-61 (quinolone derivate, new class for topical use)
Phase III
Infections with S. aureus, including MRSA, also effective against other Grampositive bacteria
Ceftazidim + avibactam (cephalosporins + new b-lactamase inhibitor)
Phase III
Infections with Gram-negative bacteria including Pseudomonas
of action and favourable toxicological properties. In this review the properties of Akacid and its derivates and their future potential as antibiotics, in treating patients suffering from infections with resistant bacteria, are discussed.
Such resistant strains cause not only human tragedies by killing patients, but also considerable costs for the society. In Europe, overall societal costs of infection due to antibioticresistant bacteria are estimated at about 1.5 billion Euros each year [5].
Multi-drug-resistant bacteria MRSA are often sub-categorized as hospital-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA), depending upon the circumstances of the environment in which the resistance appeared originally. On the basis of current data, these resistant bacteria are distinct bacterial sub-strains of the original species [13]. The rising number of MRSA or MRSE (methicillin-resistant Staphylococcus epidermidis) and the appearance of vancomycinresistant Staphylococcus aureus (VRSA), Enterococcus spp. (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP) are alarming. MDR Mycobacterium tuberculosis and the manifestation of extremely drug-resistant (XDR) strains are growing threats all over the world. MDR ESBL (extended spectrum beta-lactamases)-positive strains of Gram-negative bacteria are responsible for dangerous, almost untreatable infections. Particularly Enterobacteriaceae such as Enterobacter spp., Escherichia coli and Klebsiella spp., Acinetobacter spp. and Pseudomonas aeruginosa can become MDR or pan-drug resistant (PDR) to commonly used antibiotics such as carbapenems, cephalosporins, monobactams, penicillins, polymyxins, quinolones and tetracyclines [6,8,11,14].
Cationic antimicrobial polymers New antibiotics with a new mode of action are urgently needed to combat resistant bacterial strains, but progress in developing them has been slow [5,8,11] since most pharmaceutical drug developing companies have another indication focus. One of the emerging classes of antibiotics belongs to the cationic antimicrobial polymers which have been in use mainly as antiseptics, surface disinfectants and topical antimicrobials for over 80 years within clinical, industrial and domestic hygiene delivery [18]. The number of antimicrobial polymers has significantly increased in the past decade and the state of the art has been summarized in different reviews [17–21]. These include different groups of active substances such as quaternary ammonium-based molecules (QUATs, e.g. benzalkonium chloride, cetrimide) or polymeric biguanides (e.g. chlorhexidine, polyhexanide), which are the active ingredients, for example, of many disinfectants, cosmetics or pharmaceutical products such as topical or oral washes, wound dressings, and so on [21,22]. Cationic antimicrobial polymers are polycondensation products of a guanidinium salt with diamines [21]. Marked www.drugdiscoverytoday.com
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NH
•
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HCl
R
R’
O N H
N H
O
n
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Figure 1. Akacid (oligo-2-(2-ethoxy)-ethoxyethyl-guanidiniumhydrochloride) is the starting material for all commercially relevant Akacid substances and a condensation product of guanidine and oxyalkylendiamine.
antibacterial activity was noted for mixtures of poly-hexamethylene-biguanide (PHMB) salts produced by a reaction of hexamethylene-bis-dicyanodiamide and hexamethylenediamine [23]. Polymeric molecules bear positive charges or are synthesized in the presence of cationic substances, incorporated in their backbone and/or as side chains [21,24,25]. Depending on the choice of components different polymeric guanidines are formed. Water soluble PHMB is a well-known antimicrobial cationic polymer. Antimicrobial activity is based on a biguanide group attached to a hexamethylene group. PHMB is commercially available all over the world and has been marketed for more than forty years as the active ingredient of many disinfectants and preservatives, for example polyhexanide [22,26]. In spite of its long-term use, only little information is available regarding the physical and chemical properties of PHMB. In 2011, De Paula et al. [26] studied and characterized this molecule. By the consensus group of wound management PHMB is considered as one of the most important topical antiseptic/antimicrobial agents to reduce clinical bacterial burden since there are no new antibiotic therapies emerging in the near future [27]. Akacid and Akacid Plus are new candidates as antibiotics under development with a new physico-chemical mechanism of action. Akacid is an oligo-2-(2-ethoxy)-ethoxyethylguanidinium-hydrochloride (OEEG, Fig. 1) and is derived from a condensation product of guanidine and oxyalkylendiamine [28]. A second derivative is Akacid Plus, which was developed with the aim to enhance antimicrobial activity against a broader spectrum of pathogens combined with a significantly better tolerability. Akacid Plus is a poly-hexamethylene-biguanide (PHMB), product of a polycondensation process of a guanidinium salt with two different diamines [28,29].
Mechanism of action Cationic antimicrobial polymers possess a broad physicochemical multi-site mechanism of action which is very difficult to overcome for microorganisms, in contrast to most of the common antibiotics actually in use, which have a very target specific mechanism of action, which facilitates the 112
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emergence of resistance by pathogens [12,21,30]. Despite the long and widespread use of cationic antimicrobial polymers, there has been a lack of experimental evidence with respect to their antimicrobial mechanism of action [21]. However, over the past 10 years research focused on cationic polymers has been intensified [18,25,31–34] and some of the basic mechanisms have been discovered. The outermost surface of bacterial cells carries a net negative charge. Therefore cationic antimicrobials with their net positive charges have a high binding affinity for bacterial cells. They attach to the surface of pathogens and bind to negatively charged sites of the membrane, which are then neutralized [21,35]. A reversal of the negative charge of the microbial membrane occurs and subsequent damage follows, documented as leakage of potassium ions followed by other cytoplasmic materials, resulting in the death of the pathogen [31,32,36]. Razzaghi-Abyaneh et al. [37,38] demonstrated the dose-dependent efficacy of Akacid Plus in growth inhibition against Aspergillus parasiticus by destroying the plasma membrane and major cytoplasmic organelles of the fungus.
Akacid and Akacid Plus properties Initially, Akacid Plus was introduced as a biocidal and disinfection agent, which was developed especially for quick and easy decontamination of rooms, surfaces and areas with high risk of infections [39]. With respect to the favourable physicochemical properties of Akacid and Akacid Plus and their strong inhibitory effects against many different microorganisms, they are promising candidates for pharmaceutical use, for example, for application against human and animal bacterial or fungal pathogens [40–43]. Liquid OEEGs and PHMGs, including Akacid and Akacid Plus, are highly water soluble and their antimicrobial effects are active throughout a wide pH range. Their high stability against UV-light and extreme temperatures ( 708C up to 2008C tested) makes these compounds easy to transport and to store with a long shelf life, which is an advantage for usage, for example, in developing countries. Toxicological studies carried out with Akacid and Akacid Plus, following OECD guidelines for chemical testing [44], showed a high tolerability. Acute oral toxicity (OECD 423) and acute dermal toxicity (OECD 402) studies in rodents demonstrated LD50 doses of Akacid or Akacid Plus higher than 2000 mg/kg body weight [41]. An acute dermal irritation/corrosion study (OECD 404), a skin sensitization study using a local lymph node assay (OECD 429) and an acute eye irritation/corrosion study (OECD 405) with rodents revealed a high tolerability of Akacid Plus in vivo (our unpublished results).
Broad antimicrobial spectrum Akacid and Akacid Plus are active against a wide range of Grampositive and Gram-negative bacteria, yeasts, filamentous
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Table 3. Minimum inhibitory concentration (MIC) of Akacid, Akacid Plus and commercially used medicines, tested on bacteria and fungi [41,43,48,49] Pathogens
Minimum inhibitory concentration (mg/L)
Species (no. of strains)
Akacid
Akacid Plus
VRE (5)
Range MIC50 MIC90 Range MIC50 MIC90 Range Range MIC50 MIC90 Range
2–8 4 8 2–8 4 8 0.5–2 32–64 64 64 32–64
0.06–0.5 0.125 0.25 0.06–0.5 0.125 0.25 0.06–0.25 2–16 8 16 4–16
Spores Spores of B. subtilis (1) Spores of B. anthracis (1)
Range Range
0.5 0.5
Proteus spp. (7)c Salmonella spp. (6)d Shigella spp. (2)e Yersinia enterocolitica (1) Acinetobacter spp. (4)f Stenotrophomonas maltophilia (4)
Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 Range Range Range Range Range Range
8–32 16 32 8–16 16 16 16–32 16 32 32–128 64 64 8–128 8–16 16–32 32 8–32 128–256
1–8 2 4 1–8 2 8 1–8 2 8 4–32 8 32 4–32 1–2 2–4 2 1–8 8–32
Fungi Candida spp. (10)g Aspergillus spp. (7)h
Range Range
0.25–64 16 to >256
0.125–4 1–16
Gram-positive bacteria MSSA (36)
MRSA (62)
MRSE (9) Enterococcus faecalis (27)
Gram-negative bacteria Escherichia coli (65)
Klebsiella spp. (45)a
Enterobacter spp. (20)b
Pseudomonas aeruginosa (59)
Species (no. of strains)
Dermatophytic fungi Candida albicans (46)
Candida non-albicans (44)
Trichophyton rubrum (17)
Trichophyton non-rubrum (11)
0.125 0.125
Chlorhexidine digluconate 0.06–1 0.25 0.5 0.5–2 2 2 0.5–2 2–16 8 8 4–16 1 1
Mupirocin
0.06–1 0.125 0.25 0.06 to >256 0.125 8 0.25–0.5 32–128 64 64 32–64 1 1
2–8 2 8 4–32 8 16 8–32 8 32 8–32 16 32 8–64 2–4 1–2 32 2–32 16–32
128–256 128 256 32 to >256 256 >256 128 to >256 256 >256 32 to >256 >256 >256 256 to >256 128–256 128–256 256 32 to >256 256 to >256
1–16 8–64
32–256 64 to >256
Fluconazole
Minimum inhibitory concentration (mg/L)
Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90 Range MIC50 MIC90
Akacid Plus
Chlorhexidine digluconate
Clotrimazole
0.5–8 1 4 0.03–4 1 4 8–32 16 32 16–32 16 32
8–32 16 32 4–64 16 64 16–32 32 32 16–32 16 32
0.03–8 0.125 0.5 0.03–1 0.125 1 0.125 0.5 0.5 0.06–0.125 0.06 0.125
0.25 1 64 0.125 4 32 4–64 4 32 16–64 16 64
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Table 3 (Continued ) Species (no. of strains)
Minimum inhibitory concentration (mg/L) Akacid Plus
Chlorhexidine digluconate
Other fungi Trichoderma longibrachiatum (15) Trichoderma harzianum (1)
Range Range
Species (no. of strains)
Minimum inhibitory concentration (mg/L)
0.125–0.5 0.25
Akacid Plus Aspergillus spp. (7)
Range MIC50 MIC90
Other moulds (7)
Range MIC50 MIC90
2–16 8 16 0.25 1 8
1–8 8
Amphotericin B 1–8 4 8 1–16 4 8
Clotrimazole
Fluconazole
not determined not determined
64–256 1024
Voriconazole
Caspofungin
0.5–8 8 8
16 to >256 128 256
0.25–32 0.5 1
16 to >256 128 256
a
Includes Klebsiella pneumoniae and Klebsiella oxytoca. Includes Enterobacter aerogenes and Enterobacter cloacae. c Includes Proteus mirabilis and Proteus vulgaris. d Includes Salmonella enteritidis and Salmonella typhimurium. e Includes Shigella sonnei and Shigella flexneri. f Includes Acinetobacter baumannii and Acinetobacter iwoffii. g Includes Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis. h Includes Asperillus niger, Aspergillus flavus and Aspergillus fumigatus. b
fungi, viruses and spores, most notably MRSA, MRSE and ESBLproducing Gram-negative bacteria. About 600 clinical and non-clinical ATCC strains have been tested up to date [39– 43]. Table 3 demonstrates the in vitro antimicrobial profile of Akacid and Akacid Plus in comparison with the widely known chlorhexidine digluconate and mupirocin, which are used topically against bacterial infections including MRSA and MRSE. A total of 369 resistant (including MDR) clinical fungal and bacterial isolates from patients with documented infections in hospitals were tested using the microdilution method with the determination of the minimal inhibitory concentrations (MICs) according to CLSI criteria (Clinical and Laboratory Standards Institute, USA; before known as NCCLS) [45– 47]. Killing curves of S. aureus ATCC 29213 and E. coli ATCC 35218 demonstrated an impressive in vitro efficacy of Akacid polymers [40]. In vitro activity of Akacid Plus against dermatophytes and moulds was compared to those of chlorhexidine and conventional antifungal drugs including fluconazole, clotrimazole, amphotericin B, voriconazole and caspofungin (Table 3). MICs of Akacid Plus against the dermatophytes Trichophyton rubrum and non-rubrum were in the range of 8–32 mg/ L. Chlorhexidine and fluconazole reached similar MIC values between 4 and 64 mg/L. The MIC range of Akacid Plus, amphotericin B and voriconazole against tested moulds was 0.25–32 mg/L. Higher MIC values (16 to >256 mg/L) against Aspergillus spp. and non-Aspergillus species were found for caspofungin [43]. The fungal pathogen Trichoderma spp. shows increasing medical importance, particularly in immunocompromised patients. Akacid Plus demonstrated higher activity against 114
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all clinical Trichoderma strains tested in comparison to chlorhexidine or fluconazole (Table 3) [48]. It is worth mentioning that Akacid Plus was active in vitro across all tested fungal species.
Lack of resistance development against Akacid In vitro selection of resistance to Akacid Plus was carried out on one fungal and 24 different bacterial strains. It was hypothesized that the physico-chemical mode of action of Akacid Plus prevents the development of resistance, which was demonstrated in resistance selection tests with sub-inhibitory concentrations. Even after 30 passages, no reduced susceptibility against clinically relevant Staphylococcus spp. (including MRSA and MRSE), Enterococcus faecalis (including VRE strains), Klebsiella spp., E. coli (including ESBL-positive strains), P. aeruginosa (including ESBL-positive strains) and Acinetobacter spp. was observed [40]. With Candida krusei ATCC 6258, 15 passages were carried out with Akacid Plus and no resistance development was observed. In contrast to Akacid, C. tropicalis ATCC 750 developed resistance to fluconazole; a fourfold increase in the MIC could be detected after 15 passages (S. Tobudic, PhD thesis, University of Vienna, 2006). Using optimal concentrations of Akacid Plus, no induction of resistance in the multi-passage models against the tested bacteria or yeasts was observed.
Pharmaceutical applications of Akacid and Akacid Plus Promising scientific results relating to Akacid’s anti-infective efficacy form the basis for its preclinical development as a pharmaceutical. Through several proprietary formulation
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technologies, several pharmaceutical relevant Akacid products are currently in development. Several in vitro and in vivo efficacy studies as well as a set of toxicological studies show great promise for the indications as systemic antiinfectives or dermal anti-infectives against internal and external pathogenic bacteria, yeasts, fungi, spores and viruses (our unpublished results). In addition, Akacid is also active against typical oral pathogens in tooth cavities (M. Mathe, PhD thesis, University of Vienna, 2008; G. Zips, MSc thesis, University of Vienna, 2008). Akacid Plus is in preclinical development for use as an aqueous solution for topical or oral administration. The in vivo efficacy of Akacid Plus was evaluated in a guinea pig model of experimental skin infection using a mupirocinsensitive clinical MRSA. Different concentrations of Akacid Plus in Ultraphil (Hecht Pharma, Stilstedt, Germany) cream vehicle and a commercially available mupirocin 2% cream formulation (Bactobran; Smith-Kline Beecham Pharmaceuticals, Crawley, United Kingdom) were compared for their activity against bacterial infections of humans. Topical applications of Akacid Plus cream demonstrated a dose-dependant activity. At concentrations of 0.5% and above, Akacid Plus was as effective as mupirocin 2% cream in the treatment of superficial skin infection with MRSA [49]. Skin infections caused by Candida albicans are among the most common fungal infections of humans. Antifungal creams, lotions or sprays are used to treat superficial mycoses. Quantitative suspension tests demonstrated efficacy of Akacid at a concentration of 0.1% during 60 min against C. albicans [41]. Antifungal activity was also studied in guinea pigs with artificial C. albicans infections. Skin treatment with Akacid in Ultrasicc cream (Hecht Pharma, Stilstedt, Germany) at concentrations of 0.5%, 1% and 2% produced better results than clotrimazole 1% cream (Canesten, Bayer, Leverkusen, Germany), a standard antimycotic product. Akacid 0.5% cream eradicated 62.5% of C. albicans compared to 56% with clotrimazole 1% cream [50]. These results prove the efficacy of Akacid in an experimental skin infection model with C. albicans. Oule´ et al. demonstrated in detail the sporicidal efficacy of PHMG hydrochloride against spores of Bacillus subtilis [51].
Akacid Medical Formulation (AMF) for systemic use Akacid given i.v. causes necrosis, which is often seen with cationic polymeric compounds. A new fully purified Akacid with a molecular weight of 1000 Da was separated by nanofiltration [28] and encapsulated in liposomes for systemic use [36]. This high molecular fraction was called AMF and was successfully developed for intravenous administration with a proprietary binding technology to avoid vein irritation and occlusion at higher doses. A tolerance study conducted in mice showed that in contrast to free Akacid the liposomal encapsulated AMF formulation is well tolerated at a daily
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intravenous dosing regimen at a dose of 2.5 mg/kg body weight (our unpublished results).
Conclusions Over the past 10 years a rising number of MDR pathogens have been identified as a major threat to human health [5,8,12]. In several EU Member States, multi-drug resistance has reached 25% or more among Gram-positive and Gramnegative bacteria that cause serious infections in humans. Every year more than 25,000 patients die due to an infection with a MDR bacterium [5]. In healthcare settings, such as hospitals or nursing homes, MRSA is characterized as causing more severe problems compared to MSSA (methicillin-sensitive Staphylococcus aureus), such as bloodstream infections, pneumonia and surgical site infections [5,8], resulting in extra days of hospitalization and more deaths. New antibiotics are highly needed due to continuously emerging resistances in particular for MRSA. Only a few new generation antibiotics with new mechanisms of action are available or in development in the recent years. Akacid and Akacid Plus are potential promising antimicrobials, anti-infectives and anti-proliferatives under development for topical and systemical use in humans and animals with a low toxicity profile and are well tolerated when applied on skin, mucosa or eyes. Compared to existing antibiotics, Akacid and Akacid Plus act through a new mechanism of action and, interestingly, no resistance development was observed so far. In the field of dermatology, Akacid and Akacid Plus have the potential for the treatment of larger skin defects such as diabetic foot and traumatic defects. In the future these molecules could become a powerful treatment of nosocomial and other infections even with MDR pathogens, which are nowadays difficult to treat.
Conflict of interest statement The author is a full time employee of Xilicate GmbH, Vienna, Austria.
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