Novel Approaches to the Treatment of Systemic Anthrax

REVIEW ARTICLE
Novel Approaches to the Treatment of
Systemic Anthrax
Andrew W. Artenstein and Steven M. Opal
Center for Biodefense and Emerging Pathogens, Department of Medicine, Memorial Hospital of Rhode Island, Pawtucket, and The Warren Alpert
Medical School of Brown University, Providence, Rhode Island
Systemic anthrax, defined as invasive Bacillus anthracis
infection associated with bacterial dissemination or
toxin-mediated multi-organ dysfunction, may be secondary to any of the well-described forms of clinical
disease. Once a zoonotic disease of major economic
importance, it was controlled in the developed world
through widespread livestock vaccination [1]. Although
systemic anthrax is uncommon in humans, it is highly
lethal; of the 250 patients who developed the disease
during the twentieth century, more than 90 percent
died [2]. It remains a persistent threat, both as a cause
of deliberate and natural outbreaks of disease. Eleven
cases, five of them fatal, resulted from a series of
bioterrorist attacks in the U.S. in 2001 [3]. More
recently, a novel form of systemic anthrax associated
with injected heroin caused at least 54 infections in the
Received 30 October 2011; accepted 9 December 2011.
Correspondence: Andrew W. Artenstein, MD, Physician-in-Chief, Department of
Medicine, Director, Center for Biodefense and Emerging Pathogens, Memorial
Hospital of Rhode Island, Professor of Medicine and Health Services, Policy and
Practice, The Warren Alpert Medical School of Brown University, 111 Brewster St.
Pawtucket, RI 02860 (artenstein@brown.edu).
Clinical Infectious Diseases 2012;54(8):1148–61
Ó The Author 2012. Published by Oxford University Press on behalf of the
Infectious Diseases Society of America. All rights reserved. For Permissions,
please e-mail: journals.permissions@oup.com.
DOI: 10.1093/cid/cis017
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Artenstein & Opal
U.K. and Europe with an attendant mortality rate of
33 percent [4–7].
Improvements in mortality may be partially attributed to the empiric deployment of antimicrobials with
activity against B. anthracis. Current treatment recommendations involve the use of either a quinolone or
doxycycline in combination with at least one or two
other agents against which the organism is typically
susceptible, such as vancomycin, rifampin, carbapenems, penicillin, ampicillin, or chloramphenicol [3].
Clindamycin has been used in combination therapy
due to its theoretical inhibition of anthrax toxin synthesis [8] and its demonstrated clinical activity in toxic
shock syndromes caused by other Gram-positive organisms [9]. However, no efficacy data for clindamycin
have been reported in anthrax, and in combination
with ciprofloxacin, it significantly decreased the survival of gamma-irradiated, spore-challenged mice when
compared with either drug alone [10].
Despite the use of appropriate antimicrobials and
advances in supportive care, individuals with systemic
anthrax remain at high risk of death due to the deleterious effects of two secreted exotoxins and other
virulence factors [3, 11]. Thus, new therapeutic strategies that target pathogenic events in the disease
process are being investigated. This review describes
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Anthrax continues to generate concern as an agent of bioterrorism and as a natural cause of sporadic disease
outbreaks. Despite the use of appropriate antimicrobial agents and advanced supportive care, the mortality
associated with the systemic disease remains high. This is primarily due to the pathogenic exotoxins produced
by Bacillus anthracis as well as other virulence factors of the organism. For this reason, new therapeutic
strategies that target events in the pathogenesis of anthrax and may potentially augment antimicrobials are
being investigated. These include anti-toxin approaches, such as passive immune-based therapies; nonantimicrobial drugs with activity against anthrax toxin components; and agents that inhibit binding, processing,
or assembly of toxins. Adjunct therapies that target spore germination or downstream events in anthrax
intoxication are also under investigation. In combination, these modalities may enhance the management of
systemic anthrax.
approaches to the treatment of systemic anthrax that may
augment antimicrobials.
ANTHRAX PATHOGENESIS
Anthrax Toxin Activities
SEARCH STRATEGY AND SELECTION
CRITERIA
Virulence Factors and Toxin Formation
In order to determine what novel therapeutic strategies should
be used against anthrax, it is important to understand the details
of its pathogenesis. B. anthracis owes its lethality to its capacity
to generate environmentally resistant spores that persist. It has
an exocapsule, which allows the organism to escape immune
clearance and disseminate, and it secretes two potent exotoxins –
lethal toxin (LT) and edema toxin (ET) [1, 12]. After the initial
spore uptake by local tissue macrophages, the organism germinates into its vegetative, replicative form [13–15], and rapid
generation times contribute to its capacity to overwhelm innate
host defenses [12].
The pathogen uses bicarbonate detection via an ATP-binding
cassette transporter to sense a permissive host environment [16].
Such environmental cues induce the synthesis of the transacting, virulence gene regulator atxA, which activates the
genes for capsular assembly and toxin synthesis [17]. Similar
to other bacterial pathogens, the anthrax bacillus senses its own
population density by a quorum-sensing apparatus, allowing
the coordination of its virulence factors to its rapidly expanding
density [18].
The anthrax genome comprises a single, covalently closed
chromosome accompanied by two essential virulence plasmids: pXO1 and pXO2 [19]. The smaller, 95.3 kilobase pair
(kbp) pXO2 carries the capBCAE gene operon, responsible for
the synthesis of the poly-c-D-glutamic acid exocapsule [19].
The capsule contributes to pathogenesis by several distinct
mechanisms (Table 1); loss of capsular function by plasmid
deletion is the basis of attenuation for the live, Sterne vaccine
strain used in veterinary settings [1].
The 184.5 kbp pXO1 plasmid carries the atxA regulatory
gene and three others responsible for toxin synthesis [19].
LF is a zinc metalloproteinase enzyme that inactivates MAPKK
[59–61]; LT affects numerous cellular targets, impairing both
innate and adaptive immune functions (Table 1) [24–28,
33–35]. EF alters the transcriptional programs of target cells
by inducing excess intracellular levels of cAMP through calciumand calmodulin-dependent adenylyl cyclase activity [36]. ET
causes tissue edema, resulting in local changes associated with
cutaneous anthrax lesions and contributing to the pleural effusions and massive fluid shifts seen in patients with systemic
disease [37, 62]. ET, like LT, adversely impacts numerous host
functions (Table 1).
Hypotension, vasodilation, tachycardia, and reduced myocardial performance are characteristic of experimental intoxication
with LT; at least additive hemodynamic derangements are noted
with the combination of LT and ET in animal models [30, 31,
37, 42]. Shock also accompanied fatal cases of systemic anthrax
in the 2001 U.S. attacks and recent U.K. outbreak [5, 6, 62].
The deleterious hemodynamic effects of anthrax intoxication
in experimental models occur largely in the absence of inflammatory mediators such as pro-inflammatory cytokines or
chemokines [30–33, 35]. However, a more pronounced cytokine
response and rapidly progressive, generalized inflammatory response, similar to that seen in other forms of bacteremia and
septic shock, is observed following spore challenge or systemic
infection with vegetative forms of B. anthracis [14, 15, 34]. This
is likely related to the effects of the capsule and exotoxins in
concert with TLR 2/6 ligand stimulation by outer membrane
components of the organism [43] (Table 1).
NOVEL APPROACHES TO THERAPY
New approaches to the therapy of systemic anthrax derive from
in vitro and animal studies, the latter serving as surrogates for
Novel Treatments for Anthrax
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References were identified through iterative searches of the
PubMed database using the terms ‘‘systemic anthrax,’’ ‘‘anthrax
lethal toxin,’’ ‘‘anthrax edema toxin,’’ and ‘‘inhibitors’’ of the
aforementioned terms. As anthrax toxins were initially identified
in the 1950s, most references were retrievable from PubMed.
More than 97 percent of the 2989 articles identified were published in English. Articles and references cited therein were selected by their relevance to pathogenesis or potential treatment
strategies other than antimicrobials; vaccines or other preventive
measures were not considered relevant.
Anthrax exotoxins are binary, A/B-type bacterial toxins
comprising a nontoxic enzyme (A) moiety—lethal factor (LF)
or edema factor (EF), each associated with a common binding
(B) component—protective antigen (PA), named for its role
in engendering protective immunity in experimental models
[44]. In combination, the two components form LT—PA plus
LF, and ET—PA plus EF.
PA is a pore-forming protein that requires post-translational
processing and assembly to become functional (Figure 1). After
PA monomers bind to surface receptors and undergo proteolytic processing by host-derived, cell-associated, serine
endoproteases of the proprotein convertase (PC) family
[49, 50], they form a membrane-associated ‘prepore,’ which is
an efficient delivery vehicle for either LF or EF [44, 52]. After
receptor-mediated endocytosis and a series of subsequent
endosomal events, anthrax toxins are released into the cytosol.
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B. anthracis Components
Macrophages, DCs,
Lymphocytes
Neutrophils
Cardiac Tissue
Vascular Effects
Pro-inflammatory Cytokines
Poly-c-D-glutamic
acid capsule [12, 20, 21]
Promotes PA binding to cell
membranes; potentiates
LT activity; aggregates
bacterial cells into
microcolonies on hepatic
endothelium
Impairs phagocytosis; hinders
immune surveillance of
cell surface antigens
Increases LT activity
by inducing
myocardial
dysfunction
Acts synergistically with
LT to induce hypotension
May potentiate cytokine
generation by LT
Lethal Toxin (LT) [22–35]
Macrophage cell line
cytotoxicity via activation
of the cytosolic
inflammasome pathway
leading to caspase-1mediated pyroptosis;
impairs DC maturation,
lymphocyte activation,
B cell proliferation, and
antigen recognition,
processing, and
presentation to T cells
Impairs tissue phagocytosis
via effects on actin-based
mobility
High dose directly
impairs myocardial
function
YCardiac output, Ystroke
volume, diastolic
dysfunction, vasodilation
Promotes low level release
of cytokines late in anthrax,
secondary to the necrosis
of tissue macrophages
Edema Toxin (ET)
[25, 30, 31, 36–42]
Promotes migration of
infected macrophages
to the lymph nodes;
facilitates systemic
dissemination of
intracellular bacteria;
immune evasion via
inhibition of T-cell
activation
[cAMP impairs phagocytosis
and oxidative burst
[Inotropic and
chronotropic effects
via [cAMP (indirect)
Hypotension; vasodilation;
Ystroke volume via
Ypreload; diuresis
Immune suppression,
related to cytokine
dysregulation; antiinflammatory effects
that limit cytokine
release by cAMPinduced COX-2
synthesis
Cell wall components
[12, 43]
Activation via TLR 2/6
Activation via TLR 2/6
Decreased myocardial
performance as seen
in sepsis
YVascular resistance;
induces hypotension
[Cytokines and
chemokines as seen
in sepsis
Abbreviations: cAMP, cyclic adenosine monophosphate; COS-2, cyclo-oxygenase 2; DCs, dendritic cells; PA, protective antigen; TLR, Toll-like receptor.
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CID 2012:54 (15 April)
Table 1. The Immunologic and Hemodynamic Effects of Bacillus anthracis and Its Toxin Components [12, 20–43]
human trials in a disease that rarely occurs in the U.S. The FDA
promulgated the ‘‘animal efficacy rule’’ in 2002 to permit the
regulatory approval of novel drugs and biological products
for life-threatening conditions related to biothreat agents based
on data derived from animal studies when well-controlled
clinical studies are not feasible [63].
In order for evidence to qualify under the rule, certain provisions must be met: the pathophysiology of the illness
must be understood and must justify the use of the product;
a favorable effect must either be demonstrated in more than
one species with a response predictive of that expected in
humans or in a single well-characterized animal model
predictive of human responses; animal study endpoints must
generally involve either survival or significant morbidity
benefits; and pharmacodynamic data must be sufficient
to allow for appropriate dose correlation and selection in
humans. Separate clinical safety and pharmacokinetic evaluations are required.
Hemodynamic Support Maneuvers
Illumination of the hemodynamic derangements induced by
anthrax exotoxins and structural components suggests a role
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Figure 1. Schematic representation of events in the pathogenesis of anthrax and the potential therapeutic opportunities (identified as numbers) that
relate to each event. Entities within parentheses represent examples of agents that may inhibit or block a particular pathogenic event (see text): (1) spore
germination and bacterial growth (antimicrobials; anthrax phage-derived lysin therapy; quorum-sensing inhibitors, anti-spore antibody, 6-thioguanosine,
retrocyclins, CXC chemokines); (2) poly-c-D-glutamic acid exocapsule as a virulence factor (anti-capsule mAbs); (3) bacterial protein synthesis
(clindamycin); (4) PA83 binding to either a low- (ANTXR1, formerly tumor endothelial marker 8) or high-affinity (ANTXR2, formerly capillary morphogenesis
protein 2) type 1 transmembrane receptor expressed on human cells [44–48] (anti-PA mAbs, soluble ANTXR2 receptor); (5) proteolytic processing of PA83
to PA63 and PA20 by furin or other PCs [49, 50] (IaIp, synthetic furin inhibitors); (6) assembly of the PA prepore on the surface of infected cells via
heptamerization or octamerization of proteolytically processed PA monomers [44, 51, 52] (dominant negative PA monomers, cisplatin); (7) LF or EF binding
to PA prepore (anti-LF or –EF mAbs); (8) prepore clusters congregate on lipid rafts in a process that may [53] or may not [54] require a co-receptor—LRP6
(dominant negative co-receptor variants) and are internalized in clathrin-coated pits via receptor-mediated endocytosis to the early endosomal
compartment [44, 55, 56]; (9) acidification of the endosome results in conformational changes that transform the prepore into a transmembrane, cytosolicdelivery pore that translocates partially denatured LF and EF across its aperture into vesicles that fuse with late endosomal membranes, releasing active
toxins into the cytosol, where they exert their deleterious effects on the host [44, 57, 58] (chloroquine, amiodarone, niclosamide); (10) assembly and
release of EF/ET and its downstream effects (nifedipine, indomethacin, cromolyn, adefovir); (11) assembly and release of LF/LT and its downstream effects
via the inflammasome system (N-acetyl-L-cysteine, auranofin); (12) LF effects via MAPKK pathways (verapamil, dantrolene, protamine, neomycin,
quinidine, cisplatin, chemically modified tetracyclines, green tea polyphenols). EF, edema factor; LF, lethal factor; PA83, the initial 83 kD monomeric form of
the protective antigen synthesized by B. anthracis; ANTXR1/2, anthrax receptor type 1 and type 2; PA63 and PA20, the 63 kD and 20 kD post-proteolytic
forms of protective antigen, respectively. Abbreviations: cAMP, cyclic adenosine monophosphate; CRE, cAMP-response elements; CREB, cAMP response
element binding protein; ET, edema toxin; IaIp, inter-alpha-inhibitor proteins; LRP6, low-density lipoprotein receptor-related protein 6; LT, lethal toxin;
mAbs, monoclonal antibodies; MAPKK, mitogen-activated protein kinase kinases; PCs, proprotein convertases.
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Table 2. Approved Drugs Currently or Previously Used for Indications Other Than Anthrax That Possess Anti-anthrax Toxin Activity
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In vitro Antitoxin
Activity
Amiodarone [87] LT, murine Mu and
CHO cells
In vivo Antitoxin
Activity
Potential Mechanism
Increases survival in
Blocks PA pore
LT-challenged rats
formation; raises
at high concentrations endosomal pH
Current Clinical
Uses
Cardiac arrhythmias
Serious Toxicities
Therapeutic Antitoxin
Levels
Pulmonary fibrosis
LT IC50 5 3.5 lM
Pneumonitis
Thyroid dysfunction
Usual serum 5 3.75 lM
Toxicity . 5 lM
Drug Interactions
Drugs that
prolong QTc
d
CYP3A4 inducers
or inhibitors may
Yor [levels,
respectively
LT, murine peritoneal Enhances survival in
Mu
BALB/c mice with
pre- or post-LT
challenge Rx
Raises endosomal
pH
Anti-malarial
Blurred vision
15–30 ng/ml
Retinopathy
(Anti-toxin effect at
achievable serum
and tissue levels)
Same as
Amiodarone
Dysrhythmias
N-acetyl-Lcysteine [89]
(Mucomyst)
LT, murine Mu
Enhances survival
in pretreated, LT
challenged BALB/c
mice
Antioxidant:
abrogates
cytolysis by
reactive oxygen
intermediates
Acetaminophen
toxicity
Anaphylactoid
reaction
ND
Nitrates (additive
hypotension)
Emesis
Niclosamide [90] LT, murine Mu
NA
Blocks PA pore via
alterations to
endosomal pH
Antihelminthica
Abdominal pain, anorexia,
dysgeusia, diarrhea
ND
Quinidine [91]
LT, rat Mu
NA
K1 channel
inhibition
Cardiac arrhythmias
Dysrhythmias
ND
Verapamil [92]
LT, murine Mu
NA
Ca21 channel
blockade (LT
requires Ca21)
Anti-hypertensive
Dysrhythmias
Nifedipine [93]
ET, CHO cells
NA
Ca21 channel
blockade (ET
requires Ca21)
Anti-hypertensive
Dysrhythmias
ND
Same as
Amiodarone
Cinchonism
ND but .10x usual
serum levels needed
for antitoxin effect
CYP3A4
metabolism as
per amiodarone;
additive effects
with other
antihypertensive
agents
Cerebral ischemia
Cerebral ischemia
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Arrhythmia
Chloroquine [88]
ND
As per verapamil
Table 2 continued.
In vitro Antitoxin
Activity
Neomycin [94]
LF, murine Mu
In vivo Antitoxin
Activity
NA
Potential Mechanism
Binds to the
polyamine class
of glutamate
receptors on LF;
competitively
inhibits substrate
cleavage
Current Clinical
Uses
Antimicrobial
(topical form)
Serious Toxicities
Nephrotoxic
Therapeutic Antitoxin
Levels
ND
Drug Interactions
Additive effects
with other
nephrotoxic
agents
Ototoxic
Bowel decontamination Prohibitively toxic
(oral form) as
when used
adjunctive Rx for
systemically
hepatic
encephalopathy
Protamine [95]
Statins [96]
(simvistatin,
fluvastatin)
LF, in cell-free
activity assay
LT, murine Mu
NA
NA
Zinc chelation vs
competitive LF
binding
Counteracts
heparin effects
Novel Treatments for Anthrax
Disrupts
Lipid-lowering
posttranslational
agents
processing of
precursors via
modulating the
Rho-family of
GTPase activity;
affects trafficking
and/or localization
of LT once
internalized
Bleeding
Serum levels 5 4–100 lM Neutralizes
heparin
Anaphylactoid
reaction
Antitoxin effect at 10 lM
Myopathy
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ND
CYP3A4
metabolism
as per
amiodarone
ND
Additive bleeding
risk when
used with
anticoagulants
or antiplatelet
agents
Hepatic transaminase
elevations
Acute kidney injury
Indomethacin [97] NA
Improved ET-associated Reduces ETmorbidity (ie, edema)
induced vascular
with pretreatment of
leakage by
rabbits, but no effect
possible
on survival noted
interference with
ET action on
inflammatory
mediators
Anti-inflammatory,
analgesic
Acute kidney injury
GI hemorrhage
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Table 2 continued.
d
In vivo Antitoxin
Activity
Potential Mechanism
NA
Improved ET-associated Reduces ETmorbidity (ie, edema)
induced vascular
with pretreatment of
leakage by
rabbits, but no effect
possible
on survival noted
interference with
ET action on
inflammatory
mediators
Cromolyn [97]
NA
Ketotifen [98]
NA
d
Celecoxib [97]
Artenstein & Opal
Cisplatin [99]
LT, mouse Mu
Current Clinical
Uses
Anti-inflammatory,
analgesic
Serious Toxicities
Therapeutic Antitoxin
Levels
Drug Interactions
Possible increased
risk of myocardial
infarction and stroke
ND
Additive bleeding
risk when
used with
anticoagulants
or antiplatelet
agents
Improved ET-associated Inhibits mast cell
Anti-allergic (used in
morbidity (ie, edema)
degranulation,
systemic
with pretreatment of
thereby affecting
mastocytosis)
rabbits, but no effect
histamine activity,
on survival noted
and thus
supporting a role
for histamine in
ET morbidity
None reported
ND
None reported
LT, no direct LT
Inhibits mast cell
Allergic conjunctivitis
inhibition; reduces
degranulation,
(only approved in
vascular leakage
thereby affecting
ophthalmic form)
(LT-induced) in mice;
histamine activity
extends time to rat
death
Protects BALB/c mice Inhibits LT cleavage Anti-neoplastic
or Fischer rats when
of MEKs without
given with LT; pre-Rx
affecting LF
does not protect
proteolysis;
renders PA unable
to form
heptamers,
thereby impacting
LF and EF
translocation
CNS or respiratory
depression
ND
None reported
Nephrotoxicity
LT effects are at
Cumulative
biologically relevant
nephrotoxicity
doses; however, not
with
feasible in current
aminoglycosides;
form because only
additive ototoxicity
effective when given
with loop diuretics
concurrently with LT
(cisplatin inefficient in
presence of competing
thiol groups)
Ototoxicity
Peripheral neuropathy
Bone marrow
suppression
Adefovir [100]
EF, mouse Mu
NA
Adefovir metabolite Antiviral (hepatitis B)
binds in the
catalytic site of EF
Bone marrow suppression EF inhibition at
None reported
relevant antiviral doses
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In vitro Antitoxin
Activity
FDA-approved but no longer marketed in U.S.
a
Crystalluria
Abbreviations: CHO, Chinese hamster ovary; CNS, central nervous system; CYP, cytochrome; EF, edema factor; ET, edema toxin; GI, gastrointestinal; IC, inhibitory concentration; LF, lethal factor; LT, lethal toxin; MEKs,
mitogen-activated protein kinase kinases; Mu, macrophages; NA, not assessed; ND, not determined; PA, protective antigen; QTC, corrected QT interval; Rx, treatment.
Additive CNS
depression with
other agents;
cardiovascular
toxicity with Ca21
channel blockers
Usual serum levels
up to 2.5 lM; antitoxin effect at 10 lM
Malignant hyperthermia, Muscle weakness
Blockade of
neuroleptic malignant
intracellular Ca21
store mobilization
syndrome, chronic
muscle spasticity
NA
Dantrolene [102] LT, mouse Mu
Drug Interactions
Additive
hepatotoxicity
with other toxic
agents
ND
Dysrhythmias
Antiprotozoal
Inhibition of Ca dependent
phospholipase
A2 activity
NA
Quinacrine [101] LT, mouse Mu
21
Therapeutic Antitoxin
Levels
Serious Toxicities
Current Clinical
Uses
Potential Mechanism
In vivo Antitoxin
Activity
In vitro Antitoxin
Activity
Passive Immunotherapy
Toxin-neutralizing antibodies, either in conjunction with antimicrobial agents or as salvage therapy in those not responsive to
antimicrobials, target the intoxication responsible for the excessive mortality of systemic disease. Anthrax immune globulin
(AIG), an unlicensed polyclonal preparation of antibodies derived from the pooled plasma of healthy, anthrax-vaccinated
donors, has been anecdotally used in the management of a small
number of cases [67, 68]. Although a clinical trial examining
its safety and pharmacodynamics in human volunteers has
recently been completed, efficacy studies are lacking. Nonetheless, AIG is a component of the U.S. Strategic National
Stockpile and has been recommended for possible early use
in conjunction with appropriate antimicrobials and/or surgical
debridement in the management of recent cases in Scotland [69].
PA has been a primary focus of passive immunotherapy based
on several lines of evidence: it is a shared component of both
LT and ET, and thus its neutralization would theoretically
derail the pathogenesis of systemic anthrax; it is the major
immunogen of the currently licensed U.S. and U.K. anthrax
vaccines [1, 70]; and anti-PA antibody has been validated as
a serologic correlate of protection in a rabbit inhalational
anthrax model [71]. Polyclonal antibodies directed against
PA protect guinea pigs from the lethal effects of B. anthracis
spores [72].
Polyclonal preparations comprise antibodies against a broad
array of microbial epitopes and therefore may be useful in the
setting of a laboratory-manipulated binding site mutation, as
might occur in a bioterrorism scenario. However, monoclonal
antibodies (mAbs) can be engineered to enhance specificity,
affinity, and durability; their relative purity may be associated
with improved safety profiles [73]. Among mAbs, humanderived products are less likely than their animal-derived
counterparts to be associated with hypersensitivity reactions.
Multiple, distinct, recombinant, humanized or fully human
mAbs directed against PA have demonstrated efficacy in preexposure protection of LT-challenged rats [74–76] and in
pre- and post-exposure protection of spore-challenged rabbits
[77]. Several products, including raxibacumab, a recombinant
human IgG1k mAb that binds PA with high affinity and
Novel Treatments for Anthrax
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Table 2 continued.
for specific supportive measures. Volume support with crystalloid infusion appears to worsen mortality in a continuous,
LT-infusion rat challenge model, as compared with its beneficial effects in gram-negative bacterial sepsis, perhaps related
to endothelial dysfunction [64]. The impact of vasopressors
on shock associated with systemic anthrax has received scant
attention but has not been shown to affect survival in LTchallenged rats [65]. Additionally, corticosteroids, which are
used in cases of sepsis, may have deleterious consequences in
anthrax intoxication [66].
Approved Agents for Indications Other Than Anthrax With Antitoxin Activities
A number of drugs approved for use in diseases other than
anthrax possess activity against anthrax toxins (Table 2). These
therapeutic agents generally have well-described pharmacokinetics in humans, and their safety profiles have been established
in human studies and/or in post-marketing surveillance. Many
are available in oral form, and they could be ready for immediate
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use in the event of a biological attack or natural outbreak.
However, most have been studied in pathogenesis, not therapeutic models.
Several compounds have in vitro activity against either
LT/LF or ET/EF. Verapamil [92] and nifedipine [93] act through
calcium channel blockade of LT and ET, respectively. Dantrolene
[102], used in malignant hyperthermia and chronic spasticity,
and quinacrine [101], an antiprotozoal agent, also leverage the
calcium dependence of LT but through blockades of calcium
mobilization and phospholipase A2 activity, respectively. Others
disrupt distinct steps in toxin pathogenesis, such as the competitive binding of LF [94, 95] or EF [100], the processing events
in intracellular trafficking [96], or pore formation, which is
impaired by raising the endosomal pH [87, 88, 90].
In vivo data derived from small animal experimentation are
available for very few agents. N-acetyl-L-cysteine [89], used in
acetaminophen toxicity, and the antineoplastic agent cisplatin
[99] affect distinct, downstream events in LT pathogenesis.
Selected, non-steroidal, anti-inflammatory agents and mast
cell stabilizers have been shown to improve edema but not
mortality in a rabbit model through their effects on inflammatory mediators incited by ET [97].
Chloroquine, an antimalarial with an established safety
profile in humans, has demonstrated both in vitro and in vivo
data in support of a potential therapeutic, anti-toxin role in
anthrax. It improves the survival of LT-challenged mice at
clinically relevant doses and serum/tissue levels when given
either before or after systemic intoxication (Table 3) [88].
Additionally, chloroquine modulates the deleterious effect of
LT on T cells [103] and demonstrates an enhanced anti-toxin
effect when combined with amiodarone [87] or furin inhibitors [104]. However, its potential use may be complicated
by the in vitro observation that it may reduce the sporicidal
efficiency of macrophages [105]; this will only be resolved
through additional animal experimentation.
Several experimental agents have anti-toxin activity. Selected,
non-antimicrobial, chemically modified tetracyclines competitively inhibit LF-mediated cleavage of MAPKK in vitro at doses
achieved in phase I/II human cancer trials [106]. These agents
may be active after LF has entered cells. Polyphenols found in
green tea non-competitively inhibit the proteolytic activity of
LF in vitro and protect rats from LT-induced death, but optimal therapeutic concentrations have not been determined
and the mechanism used by the polyphenols is unclear [107].
Conversely, aspirin and other antiplatelet agents are associated
with increased mortality in LT-challenged mice and thus may
be contraindicated in systemic anthrax [108]. Further discovery of novel, small molecule LF inhibitors is complicated
by several issues: as metalloproteinase inhibitors, these compounds may adversely affect the function of essential host
metalloproteinases; many candidate drugs appear to lose
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inhibits binding to its cellular receptor, have been developed for
human use [78, 79].
A single bolus of raxibacumab significantly improved survival
in prophylactic and therapeutic studies in rabbits and cynomolgus macaques challenged with aerosolized spores [80].
Intravenous raxibacumab, given up to 48 hours after spore
challenge and concurrent with bacteremia detection, conferred a significant survival advantage in monkeys; nearly
two-thirds of the animals in the mAb group survived [80]. No
significant safety concerns were observed among human volunteers achieving drug levels that correlated with survival
benefits in two animal models [80]. Since the serum half-life
of raxibacumab in humans is significantly longer than that
in monkeys, humans may maintain therapeutically effective
levels for at least 28 days after infusion. Based on these data,
raxibacumab is in the Strategic National Stockpile.
Despite successful treatment of established infection in
animals, clinical evidence of systemic disease might be an
insensitive trigger for the initiation of raxibacumab, as the
median length of time from exposure to symptom onset in U.S.
anthrax cases was 4 days [62]. Raxibacumab has demonstrated
some efficacy when deployed late, after the onset of hemodynamic decompensation in LT-challenged rats [81]. Nonetheless, based on the clinical suspicion of exposure, passive
immunotherapy would likely need to be initiated empirically,
early, and at the time of combination antimicrobial therapy.
Importantly, raxibacumab does not appear to alter the pharmacokinetics of concurrently used antimicrobials or the natural
immune response to anthrax infection [80].
Because of the perceived importance of diversity in protective
immune responses to anthrax, mAbs directed against other
pathogenic elements of the organism have received attention.
Both fully human mAbs and humanized chimpanzee mAbs
targeting LF demonstrated efficacy in protecting sporechallenged mice and LT-challenged rats, respectively; both
may provide additive effects with concurrent anti-PA mAbs
[82, 83]. A humanized mAb targeting EF was shown to protect
mice against the local and systemic effects of ET [84]. Immunologic approaches targeting the ANTXR2 receptor [85]
and capsule [86] represent potential strategies that could be
combined with anti-toxin mAbs for passive protection against
systemic anthrax.
Table 3. Chloroquine Enhances Survival in BALB/c Mice
Challenged With Anthrax Lethal Toxin
Day 3
Treatment Group
Survivors/
Total
Day 7
Pa
Survivors/
Total
Pb
CQ controls
5/5
.
5/5
.
LT controls
1/17
.
0/17
.
(CQx2)c plus LT
8/16
.007
5/16
.018
(CQx1)c plus LT
3/16
.335
2/16
.227
4/16
15/48
.175
.049
4/16
11/48
.044
.054
LT plus (CQx1)c
Total CQ-treated
plus LT-challenged
Abbreviations: CQ, chloroquine; LT, lethal toxin.
a
Compared with LT controls at day 3 (Fisher’s exact test).
b
Compared with LT controls at day 7 (Fisher’s exact test).
c
Position of parentheses, either preceding or after LT, refers to the timing of
CQ relative to challenge; and (1 or 2) refers to the number of doses of CQ.
Adapted from reference [88], by permission of Oxford University Press.
Proprotein Convertase Inhibitors
Furin, a member of the proprotein convertase (PC) family of
serine proteases, is largely responsible for proteolytic processing of PA at the cell surface [110, 111]. Inter-alpha inhibitor proteins (IaIp) represent a family of endogenous
serine protease inhibitors that exist in several isoforms in
human plasma and urine [112]. Their broad-spectrum activity
during severe inflammatory states against many potentially
detrimental proteases found in the plasma, such as complement
components, coagulation and fibrinolytic enzymes, and granzymes, and the inverse correlation between plasma IaIp levels
and mortality in such states, has fueled interest in IaIp as
a possible therapy for bacterial sepsis [113–115].
IaIp have also been shown to inhibit furin and to protect
experimental animals when administered up to 24 hours after
anthrax spore challenge, enhancing the effect of antimicrobials
(Figure 2) [112, 116]. In systemic anthrax, IaIp may inhibit
furin-mediated toxin processing and address sepsis-induced
functional derangements by blocking excess plasma protease
activity. Since IaIp are physiologic molecules and are found
in high concentrations in normal human plasma, they are
likely to be safe. IaIp are currently undergoing animal
challenge studies to confirm their efficacy in established
systemic anthrax infection. Other synthetic furin inhibitors
have demonstrated potential for use in anthrax intoxication
adjunctive therapy [110, 117].
Mutant variants of PA monomers have dominant negative
(DN) activity and are able to prevent the assembly of wild-type
PA monomers into functional prepores, even when present
in low concentrations [22, 118]. DN mutants co-assemble
with wild-type PA monomers, disrupting pore formation and
thereby blocking the delivery of toxins to the intracellular
compartment. Such mutants are protective in a rat intoxication
model [118]. The low likelihood that sufficient quantities of
DN mutants could be administered in time to improve the
outcome in ill patients with systemic anthrax makes it improbable that they will advance to clinical application in their
current forms.
Other Adjunct Therapy
Novel antimicrobial strategies are in development to supplement or replace antibiotics against anthrax bacilli or other
gram-positive bacteria with lysins, which are lytic enzymes
derived from bacteriophages [119, 120]. Lysin therapy could be
used for infections caused by genetically engineered, antibioticresistant B. anthracis strains.
Quorum-sensing inhibition represents a potential novel
approach to disrupt communication between anthrax bacilli
[121]. A number of naturally occurring or synthetic, halogenated furanones have been shown to impair virulence gene
expression and growth of B. anthracis [17, 18].
The soluble form of the high affinity anthrax toxin receptor,
ANTXR2, has been used as a molecular sponge to adsorb
prepores and thus prevent PA binding and assembly on cell
membranes [26]. A synthetic, polyvalent inhibitor of PA,
based upon the activity of a 12-amino acid peptide discovered
in a phage display process, prevents PA pores from translocating EF or LF into the cytosol [122].
Because the transformation of B. anthracis from its infective
spore form to its invasive, vegetative form initiates pathogenesis, agents that interfere with the germination process
may be useful if deployed early after exposure. The anti-cancer
agent 6-thioguanosine, a nucleoside analog of the germinant
inosine, blocks spore germination and cytotoxicity in murine
macrophages [123]. Selected retrocyclins, synthetic peptides
derived from defensin effectors of the macaque innate immune
response, have shown both sporicidal and antitoxin activity
[124]. Recently, the murine CXC chemokines CXCL9, CXCL10,
and CXCL11 were shown to disrupt B. anthracis spore germination and vegetative outgrowth in vitro [125, 126], suggesting
a potential role for these effectors in novel therapeutic approaches to anthrax.
Auranofin, an organo-gold compound used in the treatment
of rheumatoid arthritis, blocks LT-induced caspase-1 synthesis
and significantly delays mortality in the Fischer rat model of
anthrax intoxication [23]. These findings raise the possibility
Novel Treatments for Anthrax
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potency under physiologic conditions; and drugs must act
intracellularly, and therefore, be of high affinity to be therapeutically viable [109]. The availability of crystal structures of
experimentally potent LF inhibitors may facilitate the design
of future candidates.
Dominant Negative Mutants
that inflammasome inhibitors or inhibitors of caspase-1 immune products, such as the IL-1 receptor antagonist, may have
a role in the management of systemic anthrax [127].
CONCLUSIONS
Although antibacterial agents with activity against B. anthracis
are effective at clearing bacteremia, optimal therapy for systemic
disease will require intervention with toxin inhibitors and perhaps other adjuncts. Some of the more promising existing drugs
with anti-toxin activity, such as chloroquine, and endogenous
furin inhibitors, such as IaIp, should be rigorously studied in
combination with antibacterial agents, anti-PA immune-based
therapy, or both in animal models of systemic anthrax. However, it must be acknowledged that host-signaling and immunebased therapies may appear efficacious in animal models of
infection and intoxication, yet fail to improve outcomes in
clinical trials of severely ill patients [128]. To prove efficacy,
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these experimental agents will need to be studied in comparative
clinical trials that will treat anthrax victims during an outbreak.
Although credible research during epidemics of naturally
occurring or intentionally released pathogens is challenging, it
must be done in real-time in a systematic and ethical manner
and will likely require the development of international, collaborative, clinical research networks [129].
Notes
Authors’ contributions. A. W. A. and S. M. O. both contributed to the
literature search, the writing of the manuscript, and the construction of
Tables and Figures in this work.
Acknowledgments. The authors acknowledge Kristina Ward, PharmD,
Thomas Hill, and Brian Kurt for information regarding drug pharmacology, and Margo Katz for administrative assistance with the manuscript.
Potential conflicts of interest. A. W. A. has no conflicts of interest;
S. M. O. has a patent application pending for the use of IaIp as therapy for
anthrax.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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Figure 2. Inter-alpha inhibitor proteins (IaIp) are endogenous inhibitors that prevent the proprotein convertase furin from proteolytically activating PA at
the cell surface; IaIp protect against experimental anthrax. A, Concentration-dependent inhibition of furin activity in a fluorometric assay (reproduced with
permission from the American Society for Microbiology) [112]; B, IaIp administration immediately before a lethal toxin challenge of BALB/c mice results
in significant protection against toxin-induced lung tissue injury, with reduced pulmonary edema as measured by wet/dry weight, and significant protection
against toxin-induced injury in the spleen, with reduced splenic pathology as blindly scored by a pathologist (reproduced with permission from the American
Society for Microbiology) [112]; C, Kaplan-Meier survival plot of A/J mice injected with a lethal intraperitoneal dose of B. anthracis Sterne strain (ie, lacking
the pXO2 plasmid encoding the bacterial capsule) spores and subsequently administered 30 mg/kg of IaIp either 1 h or 24 h later with or without concomitant
moxifloxacin or PBS. Mice receiving IaIp in combination with moxifloxacin at either time point after infection demonstrated a significant improvement in
survival (86% in the 1-h group; 65% in the 24-h group, P , .001 for both vs PBS controls) as compared with animals receiving either agent alone or PBS
control animals (by permission of the Shock Society) [116]; D, Gram-stained lung tissue (200X) from a control group mouse showing alveolar capillary spaces
with clumps of Gram-positive bacilli and red blood cells (by permission of the Shock Society) [116]; E, Representative hematoxylin and eosin-stained lung
tissue (200X) from an IaIp- and moxifloxacin-treated mouse showing intact airspaces, minimal cellular infiltrates, and an absence of bacterial invasion (by
permission of the Shock Society) [116]. Abbreviations: C, control group; PBS, phosphate-buffered saline; W/D, wet-to-dry weight ratio.
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