To
the extent that the cholic acid derivatives mimic the behavior of
PMB, the compounds may indicate the functionality necessary for the
activity of PMB. This functionality can be distilled down to an
array of amines (or other basic groups, such as guanidines) oriented
on one face of a hydrophobic scaffolding, with an attached acyl or
alkyl chain facilitating self-promoted transport through the outer
membrane. Permeabilizers, such as compounds 4 and 5,
may be useful in synergistic combination with antibiotics, such as erythromycin
or rifampin, in inhibiting the growth of gram-negative bacteria,
whereas alone the antibiotics are ineffective. Derivatives with a
hydrophobic side chain, such as compounds 6, 7, and 8,
alone display low MICs with gram-negative and gram-positive strains
of bacteria. However, their systemic use may be limited by their
hemolytic activity. Nevertheless, due to their potent activity and
simplicity, they may be well suited for topical applications5.
Against
Gram-positive bacteria, the compounds all displayed antimicrobial activity. In fact,
the compounds were generally more active against the multidrug-resistant
strains as compared with standard strains. Compound 1, with the hydrophobic
octylamine group, was especially active. The E. faecalis strains were
moderately resistant to compounds 2 and 3, which lack a hydrophobic chain6.
Against the Gram-negative organisms, the compounds exhibited a range of
activities. It was
noted that a hydrophobic chain, such as the one found in 1, is
required for the compound to traverse the outer membranes of Gram negative
bacteria, gaining access to the cytoplasmic membrane and causing cell death 7. Consequently, expected 1 to be more active than 2 or 3 against
Gram-negative organisms. Compound 1 was very active against each of the
multidrug-resistant strains including the P. aeruginosa strains, while 2 and 3
were much less active alone. Nevertheless, 2 and 3 were potent sensitizers of
Gram-negative bacteria to a hydrophobic antibiotic (erythromycin). With only a
few exceptions, these compounds are able to permeabilize the outer membranes of
drug-resistant bacteria. And, as noted among the MIC data, in many cases the
compounds were more active against the drug-resistant strains.Results with the
resistant strains of K. pneumoniae were particularly interesting. Strain
40-6564A was more sensitive to 2 and 3 than strain 21-2751C. In
contrast, the former strain was poorly sensitized to erythromycin as compared
with the latter strain. To determine whether strain 40-6564 displayed a
specific resistance mechanism for erythromycin, FICs were determined using 2
and 3 in combination with novobiocin and rifampicin. With novobiocin,
FICs with 2 and 3 were 0.24 and 0.58, respectively, and with
rifampicin the FIC values were 0.19 and 0.13, respectively. These FICs are much
higher than those measured with other K. pneumonia strains. The inherent
resistance of strain 40-6564A may be due to a change in its membrane structure
or the action of efflux pumps.
The
MHC values for 1 and 2 (6 and 100 mg/L, respectively) have
previously reported . In an effort to increase the
selectivity of cholic acid derivatives for negatively charged prokaryotic
membranes over their electrically neutral eukaryotic counterparts 8,
an additional positive charge was included in 3 (as compared with 2).
As expected, the MHC of 3 (170 mg/L) was much greater than those of 1
and 2. The fact that the MHCs of the cholic acid derivatives increased
upon addition of another positive charge may indicate a general means of
increasing the prokaryote/eukaryote selectivity of this type of compound. As
might be expected for membrane active antimicrobial agents, compounds 1–3
are active against multi drug resistant organisms. This behaviour, coupled with
the idea that haemolytic activity is markedly decreased in compound 3,
may make these compounds useful in treating resistant bacterial infections. In
addition, because compounds 2 and 3 permeabilize bacterial
membranes at low concentrations, they may prove valuable in enlarging the
arsenal of antibiotics that can be used against Gram negative organisms and
allow use of hydrophobic antibiotics that alone ineffectively traverse the
outer membrane 9.
Mimics
of squalamine and polymyxin B (PMB) have been prepared from cholic acid in hope
of finding new antimicrobial agents. The squalamine mimics include the
polyamine and sulphate functionalities found in the parent antibiotic, however,
the positions relative to the steroid nucleus have been exchanged. The PMB
mimics include the conservation of functionality among the polymyxin family of
antibiotics, the primary amine groups and a hydrophobic chain. Although the
squalamine and PMB mimics are morphologically dissimilar, they display similar
activities. Both are simple to prepare and demonstrate broad spectrum
antimicrobial activity against Gram-negative and Gram-positive organisms.
Specific examples may be inactive alone, yet effectively permeabilise the outer
membranes of Gram-negative bacteria rendering them sensitive to hydrophobic
antibiotics. Problems associated with some of the squalamine and PMB mimics
stem from their haemolytic activity and interactions with serum proteins,
however, examples exist without these side effects which can sensitise
Gram-negative bacteria to hydrophobic antibiotics 10.
Few
studies have been conducted on antimicrobial resistance in
lactobacilli, presumably because of their nonpathogenic nature as
anaerobic commensals. Resistance was assessed in 43 type strains and
isolates representing 14 species by using agar disk diffusion and
MIC analysis in MRS medium. Most noteworthy were two general phenotypes
displayed by nearly every strain tested: (i) they were more
susceptible (up to 256-fold in some cases) to the deconjugated bile
acid cholic acid than to the conjugate taurocholic or taurodeoxycholic
acid, and (ii) they became susceptible to aminoglycosides when
assayed on agar medium containing 0.5% fractionated bovine bile (ox
gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus
plantarum WCFS1, at increasing concentrations of ox gall (0 to
30.3 mg/ml) displayed corresponding decreases in resistance to all
of the aminoglycosides tested and ethidium bromide. This effect was
clinically relevant, with the gentamicin MIC decreasing from
>1,000 to 4 µg/ml in just 3.8 mg of ox gall per ml. In uptake
studies at pH 6.5, [G-3H]gentamicin accumulation
increased over control levels when cells of this strain were exposed
to bile acids or reserpine but not when they were exposed to
carbonyl cyanide m-chlorophenylhydrazone. The effect was
dramatic, particularly with cholic acid, increasing up to 18-fold,
whereas only modest increases, 3- and 5-fold, could be achieved with
taurocholic acid and ox gall, respectively. Since L. plantarum,
particularly strain WCFS1, is known to encode bile
salt hydrolase (deconjugation) activity, data indicate that mainly
cholic acid, but not taurocholic acid, effectively permeabilizes the
membrane to aminoglycosides. However, at pHs approaching
neutral conditions in the intestinal lumen, aminoglycoside resistance
due to membrane impermeability may be complemented by a potential
efflux mechanism 11.
The
prevalence of drug-resistant bacteria drives the quest for new antimicrobials,
including those that are not expected to readily engender resistance. One option
is to mimic Nature’s most ubiquitous means of controlling bacterial growth,
antimicrobial peptides, which have evolved over eons. In general, bacteria
remain susceptible to these peptides. Human antimicrobial peptides play a
central role in innate immunity, and deficiencies in these peptides have been
tied to increased rates of infection. However, clinical use of antimicrobial
peptides is hampered by issues of cost and stability. The development of
nonpeptide mimics of antimicrobial peptides may provide the best of both
worlds: a means of using the same mechanism chosen by Nature to control
bacterial growth without the problems associated with peptide therapeutics. The
ceragenins were developed to mimic the cationic, facially amphiphilic
structures of most antimicrobial peptides. These compounds reproduce the
required morphology using a bile-acid scaffolding and
appended amine groups. The resulting compounds are actively bactericidal
against both Gram-positive and Gram-negative organisms, including drug-resistant
bacteria. This antimicrobial activity originates from selective association of
the ceragenins with negatively charged bacterial membrane components.
Association has been studied with synthetic models of bacterial membrane
components, with bacterial lipopolysaccharide, with vesicles derived from
bacterial phospholipids, and with whole cells. Comparisons of the antimicrobial
activities of ceragenins and representative antimicrobial peptides suggest that
these classes of compounds share a mechanism of action. Rapid membrane
depolarization is caused by both classes as well as blebbing of bacterial
membranes. Bacteria express the same genes in response to both classes of
compounds. On the basis of the antibacterial activities of ceragenins and
preliminary in vivo studies, we expect these compounds to find use in
augmenting or replacing antimicrobial peptides in treating human disease 12.
Ceragenins are a group of cholic acid derivatives that
have been chemically modified to make them cationic amphiphiles. Several of
these derivatives exhibit antimicrobial activity against a broad range of
bacteria. These compounds have advantages over cationic amphipathic peptides in
that they are resistant to proteolysis and they incorporate stably into
membranes. Although some forms of ceragenins are effective against both
Gram-negative and Gram-positive bacteria, they are generally more potent
against Gram-positive bacteria. Surprisingly, it is not the cell wall, but the
high content of phosphatidylethanolamine in most Gram-negative bacteria that
endow them with resistance. Ceragenins have the unusual property of forming
complexes with phospholipids. Factors contributing to the mechanism of action
of these agents are discussed. The ceragenins are a class of agents with many
properties to make them favorable for application as anti-infective agents 13.
Ceragenins
are unique, small molecular weight compounds that have potent bactericidal
activity against both Gram-negative and -positive bacteria. Previous data have
suggested potent activity of CSA-13 against Gram-positive pathogens such as
methicillin-resistant S. aureus
and glycopeptide-intermediate and -resistant S. aureus. However, ceragenin data
on Gram-negative organisms are limited. Although a collection of Gram-negative
pathogens including Acinetobacter spp., Escherichia coli and Klebsiella
pneumoniae were evaluated elsewhere, the number of organisms tested
for susceptibility for each species was 10. Here, we report an MIC50
of 16 mg/L for 50 P. aeruginosa
strains, and an MIC50 of 8 mg/L for a subset of carbapenem-resistant
isolates. This is similar to previous data as reported by Savage who observed
an MIC50 of 8 mg/L in two separate small studies 14,15.
Indeed, the CSA-13 antimicrobial activity is lower in
this population of Gram-negative pathogens compared with results previously
reported on Gram-positive organisms. This could be attributed to a high content
of phosphatidylethanolamine and may inhibit CSA-13 from inducing leakage of
aqueous contents from phosphatidylethanolamine-rich liposomes 16.
Our study also showed that CSA-13 has an MIC50/MBC50
ratio of 1, suggesting that the bactericidal activity is close to the
inhibitory concentration. Indeed, varying CSA-13 concentrations at, below and
above the MIC demonstrated concentration-dependent antimicrobial activity,
similar to previous published data on glycopeptide-resistant S. aureus. These
findings suggest that CSA-13 exhibits concentration-dependent activity against
both Gram-positive and -negative organisms. In addition, CSA-13 can be used to
enhance other antimicrobial agents against P. aeruginosa. Combination
time–kill studies against four clinical strains, three of which were meropenem
intermediate to resistant, demonstrated synergy or additive effect with the
addition of cefepime or ciprofloxacin, achieving early synergy or additive
effect at 4–8 and 1–4 h, respectively. However, we were only able to
demonstrate synergy with the combination of tobramycin and CSA-13 in one strain
(711). Evaluating synergy in a time–kill assay would be technically difficult
in this case given that the tobramycin MICs for three of the strains were
≤1 mg/L, with the exception of strain 711 which has an MIC of 4. The
addition of CSA-13 may have enhanced the bacterial activity of tobramycin
against this relatively resistant strain and thus perhaps explains the observed
synergy. In addition, tobramycin has been used as a synergistic agent against Pseudomonas
in vitro and enhanced activity is usually best observed in β-lactam
combinations 17, 18.
Previous
preliminary data have studied the potential synergy of a similar ceragenin,
CSA-8 (with less activity than CSA-13) in combination with rifampicin against
tobramycin-resistant P. aeruginosa.
19 The authors concluded that CSA-8 and CSA-13 can permeabilize the
outer membrane of Gram-negative organisms thus resulting in sensitization to
antimicrobials. This mechanism may explain the synergy that we observed with
cefepime and ciprofloxacin in combination with CSA-13 20.
Charged
trident: A new facial amphiphile (see
structure) based on cholic acid and with a permanent ionic character was
prepared. The aggregation of this three-headed surfactant into small micelles
and its inhibitory effect on bacterial growth are presented 21.
Novel
cholic acid-derived antimicrobial agents that decompose under mildly basic
conditions have been prepared. These compounds range in biological properties
from potent antibacterial activity to effective permeabilization of the outer
membranes of Gram-negative bacteria 22.
Synthesis
of novel 1,2,3-triazole-linked β-lactam–bile acid conjugates 17–24
using 1,3-dipolar cycloaddition reaction of azido β-lactam and terminal
alkyne of bile acids in the presence of Cu(I) catalyst (click chemistry) have
been realized. These molecules were evaluated in vitro for their antifungal and
antibacterial activities. Most of the compounds exhibited significant
antifungal and moderate antibacterial activity against all the tested strains 23.
Tetrapeptides
derived from glycine and β-alanine were hooked at
the C-3β position of the modified cholic acid to realize novel linear
tetrapeptide-linked cholic acid derivatives. All the synthesized compounds were
tested against a wide variety of microorganisms (Gram-negative bacteria,
Gram-positive bacteria and fungi) and their cytotoxicity was evaluated against
human embryonic kidney (HEK293) and human mammary adenocarcinoma (MCF-7) cell
lines. While relatively inactive by themselves, these compounds interact
synergistically with antibiotics such as fluconazole and erythromycin to
inhibit growth of fungi and bacteria, respectively, at 1–24 μg/mL.
The synergistic effect shown by our novel compounds is due to their inherent
amphiphilicity. The fractional inhibitory concentrations reported are
comparable to those reported for Polymyxin B derivatives.
Generally,
spermine was found to be the most effective polyamine side chain among
the compounds with the same steroidal backbone, and among the compounds with
spermine, SM-7, SM-19, SM-25 and SM-34, based on deoxycholic, ursocholanic, lithocholic, and
chenodeoxycholic acid, respectively, were highly active, while SM-13 and SM-28,
based on cholic acid and hyodeoxycholic acid had lesser activity. SM-7, SM-13
and SM-25 that substituted their C-3 position to the non-sulfate residue were
found to have more activity than SM-8, SM-14, and SM-22 in which the C-3
position was sulfated 24.
REFERENCES:
1) Li, C., L. P. Budge, C. D. Driscoll, B. M. Willardson, G. W. Allman, and
P. B. Savage.; J. Am. Chem. Soc. , 1999,121:931-940.
2) Hancock, R. E. W.; Annu. Rev. Microbiol. , 1984, 38 :237-264
3) Matsuzaki, K., K. Sugishita, N. Fujii, and K. Miyajima.; Biochemistry, 1995, 34:3423-3429.
4) Vaara, M.; Microbiol. Rev. , 1992, 56 : 395-411.
5) Chunhong Li, Matthew R.Lewis,
Amy B. Gilbert, Mark D.Noel, David H. Scoville, Glenn W. Allman, Paul B.Savage,
Antimicrobial Agents and Chemotherapy, June 1999, Vol.43, No.6, 1347-1349.
6) Li,
C., Lewis, M. R., Gilbert, A. B., Noel, M. D., Scoville, D. H., Allman, G. W.
et al. , Antimicrobial Agents and Chemotherapy, 1999, 43, 1347–9.
7) Li,
C., Budge, L. P., Driscoll, C. D., Willardson, B. M., Allman, G. W. &
Savage, P. B., Journal of the American Chemical Society , 1999, 121, 931–40.
8) Matsuzaki,
K., Sugishita, K., Fukii, N. & Miyajima, K. ;
Biochemistry, 1995, 34, 3423–9.
9)
Erica J. Schmidta,
J. Scott
Boswella,Joshua P.
Walsha,
Matthew M. Schellenberga,Timothy
W. Wintera,
Chunhong Lib,
Glenn W. Allmana
and Paul B. Savageb,; Journal of Antimicrobial
Chemotherapy , Volume 47, Issue
5, pp. 671-674.
10) Savage PB, Li C., Expert Opin
Investig Drugs. , 2000 Feb; 9(2):263-72.
11) Christopher A. Elkins and Lisa
B .Mullis; Applied and Environmental Microbiology ,
December 2004,Vol.70, No.12, 7200-7209.
12) Xin-Zhong Lai, Yanshu Feng,
Jacob Pollard, Judy N.Chin , Michael J. Rybak , Robert Bucki, Raquel F. Epand,
Richard M. Epand and Paul B. Savage; Acc. Chem. Res., 2008, 41 (10), 1233- 1240
13)
Richard
M. Epand, Raquel F. Epand and Paul B. Savage; Drug News Perspect , 2008, 21(6): 307.
14) Savage PB,
Chambers HF, Genberg C, et al. Abstracts
of the Forty-fifth Interscience Conference on Antimicrobial Agents and
Chemotherapy; 2005; Washington, DC.
Washington,DC, USA: American Society for Microbiology; .
15) Savage PB,
Chambers HF, Genberg C, et al. Abstracts
of the Forty-fifth Interscience Conference on Antimicrobial Agents and
Chemotherapy; 2005
; Washington, DC, USA. Washington, DC, USA:
American Society for Microbiology;
.
16) Epand RF, Savage PB, Epand RM,
Biochim Biophys Acta, 2007;1768:2500-9.
17) Owens RC Jr, Banevicius MA,Nicolau DP, et al. , Antimicrob Agents Chemother , 1997;41: 2586-8.
18) Tam VH, Schilling AN, Lewis
RE, et al., Antimicrob Agents
Chemother 2004;48:4315- 21.
19) Savage PB, Orsak T, Burns JL.
Abstracts of the 2005 Annual
Conference on Antimicrobial Resistance; 2005; Bethesda, MD. Bethesda, MD, USA:
National Foundation for Infectious Diseases ; Abstract
P12.
20)
Judy N. Chin1,Ronald N. Jones, Helio S. Sader,Paul B. Savage and Michael
J. Rybak1, Oxford Journals Medicine Journal of Antimicrobial Chemotherapy,Volume61, Issue2,
365- 370.
21)
Hendra M. Willemen, Louis
C.P.M. De Smet, Arie Koudijs, Marc C.A. Stuart Dr, Ineke G.A.M. Heikamp-De
Jong, Antonius T.M. Marcelis Dr, Ernst J.R. Sudholter Prof. Dr ; Angewandte
Chemie International edition, November 15, 2002, Volume 41,Issue 22,4275-4277.
22) Qunying Guan, Chunhong Li,
Erica J. Schmidt, J. Scott Boswell, Joshua P. Walsh, Glenn W. Allman and Paul
B. Savage , Org. Lett., 2000,2(18), 2837-2840.
23) Namdev S. Vatmurge, Braja G. Hazra, , Vandana S. Pore, Fazal Shirazi, Pradnya S. Chavan and
Mukund V. Deshpande, Bioorganic
& Medicinal Chemistry Letters, 15 March 2008, Volume 18, Issue 6,
2043-2047.
24) Sudhir N. Bavikar, Deepak B. Salunke, Braja G. Hazra,
, Vandana S. Pore , Robert H. Dodd ,Josiane Thierry, Fazal
Shirazi, Mukund V. Deshpande, Sreenath Kadreppa and Samit Chattopadhyay , Bioorganic
& Medicinal Chemistry Letters,15 October 2008, Volume 18, Issue 20,
5512-5517
25) International Publication No.
WO 08/452,846; Methods for the
manufacture and use of antimicrobial sterol conjugates; based upon
International Patent Application No 08/877618; Inventors Regen, Steven L.;
Applicant: Lehigh University (Bethlehem, PA); 11/10/1998.
