Medical countermeasures for unwanted CBRN exposures: Part I chemical and biological threats with review of recent countermeasure patents

ABSTRACT

Introduction: The threat of chemical, biological, radiological, and nuclear (CBRN) warfare has been addressed as the uppermost risk to national security since the terrorist attacks on 11 September 2001. Despite significant scientific advances over the past several decades toward the development of safe, non-toxic and effective countermeasures to combat CBRN threats, relatively few countermeasures have been approved by the US Food and Drug Administration (US FDA). Therefore, countermeasures capable of protecting the population from the effects of CBRN attack remain a significant unmet medical need. Chemical and biological (CB) threat agents can be particularly hazardous due to their effectiveness in small quantities and ease of distribution.

Area covered: This article reviews the development of countermeasures for CB threats and highlights specific threats for which at least one countermeasure has been approved following the FDA Animal Rule. Patents of CB countermeasures since 2010 have been included.

Expert opinion: Nine CB countermeasures have received FDA approval for use in humans following the Animal Rule, and a number of promising CB countermeasures are currently under development. In the next few years, we should expect to have multiple countermeasures approved by the FDA for each indication allowing for more flexible and effective treatment options.

KEYWORDS:

• Animal Rule
• chemical and biological threats
• countermeasures
• US Food and Drug Administration

1. Introduction

A weapon of mass destruction is defined as a chemical, biological, radiological, and nuclear (CBRN) or other weapon capable of generating significant harm to a large number of humans [1,2]. Despite considerable investments prompted by the US 9/11 terrorist attacks in 2001, much work remains to prepare nations against CBRN threats. Based on the frequency of credible reports by journalists, threats of CBRN weapons being deployed by terrorist groups or by rogue nations clearly seem to be on the rise globally [3]. Unfortunately, nations are currently limited by both the number of the currently approved medical countermeasures, as well as by the appropriateness of defense systems needed to guard against potential CBRN exposures. These medical countermeasures include not only drugs and vaccines, but also medical devices (including diagnostic tests, equipment, and supplies) crucial in responding to public health emergencies [4]. Challenges of developing these medical countermeasures include the requirement of extensive government resources, the collaboration amongst public and private sector institutions, as well as the navigation and the compliance with the Animal Rule.

The development and regulatory approval of medical countermeasures for CBRN events remain a high priority of the US Department of Defense (DoD) and the Department of Health and Human Services (DHHS) which includes the FDA, the principal regulatory agency in this undertaking. The FDA is currently using a three-pillar approach to address key challenges of the regulatory review process of medical countermeasures; these involve but are not limited to gaps in regulatory science for medial countermeasure development and evaluation, as well as the legal regulatory and policy framework for an effective public health response [5]. The three-pillar approach launched in 2010 with the Medical Countermeasure Initiative consists of (1) improving the medical countermeasure regulatory review process; (2) moving regulatory science for medical countermeasure development and evaluation forward; and (3) updating the legal, regulatory, and policy framework for a successful public health response. The aim behind such an approach was to improve capability to respond faster and more effectively to CBRN and emerging infectious disease threats. Filling in the gaps of the medical countermeasure enterprise is a major national undertaking and as such requires the collaborative cooperation of all stakeholders, including federal, corporate, and research organizations [6,7].

The FDA’s Animal Rule only permits the approval of drugs that have proven efficacy to counter CBRN health effects in animal models, proper safety profiles, and sufficient evidence that these drugs will yield a reasonable health benefit in humans [8–10]. Furthermore, there needs to be an understanding of the mechanisms of injury, drug efficacy, and the utilization of efficacy biomarkers [11]. The Animal Rule can be applied to cases of infections or exposure to select types of agents with low natural incidences or exposure frequencies (anthrax or mustard gases, respectively) but would be unethical to expose human subjects to for the sake of efficacy testing. It is highly likely that additional new countermeasures will be approved by a combination of the Animal Rule and traditional approval processes. The Project BioShield Act specifically encourages and appropriates funding for the development of countermeasures against agents that may be used in terrorist attacks [12]; it also created the Strategic National Stockpile (SNS) to store considerable quantities of drugs and medical supplies to protect the US population in case of a public health crisis [13,14]. The 2006 US Pandemic and All-Hazards Preparedness Act built upon the 2004 Project BioShield Act and offered the DHHS additional authority to fund the development and procurement of medical countermeasures against CBRN threats [15].

This is the first part of a two article series; here, we communicate countermeasures for CB threats, while in the second article, we discuss countermeasures for RN threats. Nine medical countermeasures have been approved by the FDA following the Animal Rule for various CB indications (Figures 1 and 2). In this report, we have highlighted only those CB threats/agents and recent patents for which at least one countermeasure has been approved by the FDA under the agency’s Animal Rule. These CB threats/agents include organophosphorus nerve agents, cyanide, mustard gas, bacteria-induced botulism, anthrax, and plague [8,9]. We have reviewed patents for CB countermeasures from 2010 to 2015. All of these patents are summarized in Tables 1–5.

Table 1. Patents of countermeasures for several indications.

Patent number*	Year of publication	Inventor(s)	Molecule/Compound details	Properties	Indications
US9149536 B2	2015	Baasov et al.	Aminoglycosides and non-ribosomal active antibodies	Conjugated antimicrobial	1, 2, 3
EP2343304 B1	2015	Baker et al.	Boronophtalide compounds	Antimicrobial	1, 2, 3
US9006174 B2	2015	Bikker et al.	CMAP27 peptide	Antimicrobial; low hemolytic activity	1, 2, 3
EP2340307 B1	2015	Carter et al.	Rugged dsRNA	High specificity TLR3-binding	1, 2, 3
EP2245056 B1	2015	Cerami et al.	Peptides containing pyroglutamate	Protects against tissue damage	1, 2, 3, 4
US8962891 B2	2015	Dagan et al.	Ceramide analogs	Treats cell proliferative diseases	1, 2, 3
US9186400 B2	2015	Dickey et al.	C CpG Oligodeoxynucleotide (ODN2395, ODN M362 or ODN10101) and PAM2CSK4	TLR9 and TLR2/6 agonists stimulate innate resistance	1, 3
US9005599 B2	2015	Ennis et al.	Genetically modified human umbilical cord perivascular cells	Expresses polypeptide of choice	1, 3
US9139626 B2	2015	Farris	Mitrecin A polypeptide	Antibacterial	1, 2, 3
US9028841 B2	2015	Henn et al.	Ecobiotic-purified bacterial population	Inhibits pathogenic bacteria	1, 2, 3
US8932846 B2	2015	Horwitz et al.	Recombinant BCG immunogenic compositions	Induces immune response; no antibiotic resistance markers	1, 3
US9216186 B2	2015	Hu et al.	4-Methyl-8-phenoxy-1-(2-phenylethyl)-2,3-dihydro-1H-pyrrolo[3,2-c]quinoloine	Combined with aminoglycoside antimicrobial agent	1, 2, 3
US9066978 B2	2015	Ilyinskii et al.	Nanocarrier combination vaccines	Produces high antibody response	1, 2, 3
US8975289 B2	2015	Keppler et al.	Benzyl aralkyl ether compounds	Antimicrobial	1, 2
US9029318 B2	2015	Kim et al.	Aquatic organism/moss peptides	Inhibits biofilm formation	1, 2, 3
US9101666 B2	2015	Langer et al.	Poly(β-amino esters)	Delivers nanoparticles	1, 2, 3
US9017681 B2	2015	Levin et al.	Adenylyl cyclase modulator	Causes reverting to nonpathogenic state	1, 2, 3
US9138468 B2	2015	Lien et al.	Modified conspecific bacteria	Produces high-potency lipopolysaccharide	1, 2, 3
US9181290 B2	2015	Liu et al.	1,2,3,4,6-Penta-O-galloyl-d-glucopyranose	Inhibits biofilm formation	1, 2, 3
CA2797589C	2015	Locklin	Sulfated quaternary polyethylenimine	Topical antimicrobial	1, 3
EP2346516 B1	2015	Nygaard et al.	Copepod extract	Antibacterial	1, 2, 3
US9018216 B2	2015	O’Dowd et al.	Heterocyclic phosphorus compounds	Antibacterial	1, 3
US9163065 B2	2015	Peoples et al.	Depsipeptides	Antibacterial with broad activity	1, 2, 3
US8962842 B2	2015	Roussel et al.	2-pyridone compounds	Inhibits Type II topoisomerase	1, 3
US9187529 B2	2015	Schlievert	Cationic peptides	Inhibits exotoxin production	1, 2, 3
US9023352 B2	2015	Shoemaker et al.	Heteromultimeric neutralizing binding protein	Recombinant, antibody-bound	1, 2
US9198928 B2	2015	Warner Gisborne et al.	Poly-N-acetylglucosamine nanofibers	Stimulates Akt, cytokines, and defensin expression	1, 2, 3
US8877208 B2	2014	Baker et al.	Nanoemulsion vaccines	Stimulates immune response	1, 2, 3
US8841100 B2	2014	Benjamin et al.	Methylsulfonyl methane	Treats drug-resistant microbes	1, 2, 3
US8633311 B2	2014	Bommer et al.	Porphyrin	Antimicrobial	2, 3
CA2672261C	2014	Fiala	Fosfomycin	Antibiotic to treat respiratory bacterial infection and cystic fibrosis	1, 2, 3
US8715641 B2	2014	Filutowicz et al.	One or more species of amoebae phylum Mycetozoa	Spores kill or slows growth of infectious microorganisms	1, 2, 3
US8815838 B2	2014	Griffith et al.	Aerosolized fluoroquinolones	Antimicrobial with increased bioavailability	2, 3
US8772197 B2	2014	Hatton et al.	Substrate, metallic cluster, and reactive group composition	Decomposes organophosphate agents through nucleophilic hydrolysis	1, 2, 3
CA2526150C	2014	Holladay et al.	Colloidal silver composition	Antimicrobial	1, 3
EP2094711 B1	2014	Kuhne et al.	Pro-rich polypeptides	Inhibits proline-rich motif binding domains	1, 2, 3
US8691242 B2	2014	Lara et al.	Staphylococcus aureus Div1B polypeptide	Antigenic vaccine	1, 2, 3
US8648035 B2	2014	Llano-Sotelo et al.	Peptide SEQ ID NO:28	Inhibits protein synthesis by binding to ribosomal subunit	1, 3
US8709447 B2	2014	Lowell et al.	Proteasome and liposaccharide compounds	Stimulates immune system	1, 3
US8785499 B2	2014	Mackerell et al.	Nicotinate mononucleotide adenylyltransferase inhibitor	Inhibits NAD biosynthesis	1, 2, 3
US8653133 B2	2014	Ogbourne	Crystalline form of ingenol mebutate	Disrupts mitochondria; primary necrosis	1, 2, 3
US8901071 B2	2014	O’Neil et al.	Arg-Phe peptides	Antimicrobial	1, 2, 3
US8722064 B2	2014	Reed et al.	Synthetic glucopyranosyl lipid adjuvant	Immunomodulator	1, 2, 3
US8673557 B2	2014	Scharenberg et al.	Endonuclease with end-processing enzyme	Targeted mutagenesis	1, 2, 3
US8389021 B2	2013	Baker	Homogeneous microparticulate suspensions of bismuth–thiol compound	Treats microbial infections in epithelial tissues (chronic or acute wounds) and biofilms	1, 3
US8546373 B2	2013	Benjamin et al.	Dimethyl sulfoxide combined with methylsulfonyl methane	Treats antimicrobial-resistant bacteria	1, 2, 3
CA2519429C	2013	Berman et al.	Composition of two or more select antibiotic agents, synergistic	Targets Fabl (bacterial fatty acid synthesis)	1, 2, 3
US8481055 B2	2013	Klinman et al.	CpG-containing polypeptide	Enhances vaccine immunogenicity	1, 2, 3
US8586035 B2	2013	Kopf et al.	Combination treatments	IL-23 antagonist with at least one other therapeutic modality	1, 2, 3
US8354451 B2	2013	Moran et al.	NTBC	HPPD-inhibitor, enhances macrophage/neutr-ophils phagoly-sosomal fusion	1, 2, 3
US8604184 B2	2013	Mullis et al.	Thyolated aptamer molecule that links immune response component to target molecule/compound	Provides immediate immunity to any molecule or compound	1, 2
US8470769 B2	2013	O’Neil	Lys/Arg polypeptide	Disrupts pathogen cell membranes	1, 2, 3
US8613934 B2	2013	Raviv et al.	Membrane proteins, UV ir5, and detergents	Inactivates viral proteins	1, 2, 3
US8546423 B2	2013	Surber et al.	Aerosolized fluoroquinolones	Inhaled antimicrobial	2, 3
US8241562 B1	2012	Betty et al.	DF-200	Decontaminates surfaces	1, 3
US8263078 B2	2012	Rachamim et al.	MAB – targets gram-negative bacteria flagellin	High affinity, neutralizes flagellin reactivity	1, 2, 3
US8143372 B2	2012	Theil	Synthetic ferritin pore regulators	Reduces inflammation	1, 2, 3
US8309531 B2	2012	Turner at al.	IFN-α-containing vector	Cancer therapeutic, treats or reduces infection	1, 3
US7875620 B2	2011	Warburton	Uric acid compounds	Bactericidal	1, 2, 3
US7771736 B2	2010	Abraham	Glyphosate (herbicide) and dicarboxylic acid	Inhibits growth of phylym Apicomplexa parasites	1, 2, 3
US7655759 B2	2010	Hamers et al.	Heavy chain immunoglobulin	Recognize and bind specific antigens	1, 2
US7838006 B2	2010	Jirathitikal et al.	Denatured hepatitis type B or C viral pathogen	Therapy or prophylaxis of pathogen induced infection	1, 2, 3
US7744681 B2	2010	Marcoon	Filtration article – facemask containing Oligodynamic metal/salt fibers	Immobilizes and neutralizes airborne pathogens	1, 3

*Previously, some of the patents have been filed with another country or region; however, their most recent versions are described above. 1 indicates anthrax, 2 botulism, 3 plague, and 4 nerve gas.

Only patents/applications with more than one indication have been included in this table.

Due to the references limit for this article, citations for each patent presented in this table have not been included in the list of references. Only references that have been cited within the manuscript text have been included in the list of references.

Beginning letters in patent number signifies which patent office approved this property: US: United States Patent and Trademark Office; EP: European Patent Office; CN: China Patent & Trademark Office; JP: Japan Patent Office; CA: Canadian Intellectual Property Office.

Table 2. Patents of countermeasures for nerve gas.

Patent number*	Publication year	Inventor	Molecule/Compound details	Properties
US9132135B2	2015	Albuquerque et al.	Galantamine	Allosteric potentiating ligand of nicotinic receptors
US9050331B2	2015	Asahara et al.	Paraoxonase	Degrades oxon, OP compounds, and aromatic carboxylic acids
US9162983B2	2015	Bauta et al.	Acetylcholinesterase reactivators	Inhibits OP
JP5730769B2	2015	Graham et al.	Morphinan compounds	Anticonvulsant; inhibits uptake of dopamine and serotonin
CN102491969B	2015	Verhoest et al.	Heteroaromatic compounds	Phosphodiesterase inhibitors
US8952023B1	2015	Wu et al.	TRPA1 channel antagonist	Alters intracellular free calcium concentration
EP2516664B1	2015	Yim et al.	Recombinant butyrylcholinesterases	Hydrolyzes OP
US8735124 B2	2014	Tawfik et al.	Isolated PON1 polypeptides	High catalytic efficiency for a VX type OP
EP2061458 B1	2014	Cloyd	Topiramate compositions	Anticonvulsant; neuroprotective
US8920824B2	2014	Rosenberg	Native or recombinant butyrylcholinesterase	Bioscavengers detoxify neurotoxins
CN101977658B	2013	Henry et al.	Diacetyl monoxime	OP neutralizer
US8404850B2	2013	Cabell et al.	Bis-quaternary pyridinium–aldoxime salt	Treats exposure to a phosphorous containing cholesterase inhibitor
US8557547B2	2013	Horne et al.	Phosphotriesterase from agrobacterium radiobacter P230	Enzymes hydrolyze OP molecules
US8299062B2	2012	Volvovitz	Huperzine compound	Acetylcholinesterase inhibitor; prophylactic or therapeutic
US8318728B2	2012	Chong	Acetamide compound	Modulates TRPA1 channel activity
US8252969B2	2012	Yokley et al.	Hypernucleophilic catalysts	Hydrolytic destruction of chemical agents
EP2142644B1	2011	Chabriere et al.	Mutated hyperthermophilic phosphotriesterases	OP degrading enzyme – prevents inactivation of acetylcholinesterase
US7947827B2	2011	Bailey et al.	Metaloporphyrin molecule	Counteracts the effects of toxic substances through chemical catalysis
US7741431B2	2010	Allon et al.	Novel targeting peptide incorporated into liposomes	Liposomes encapsulating cholinesterase genes
US7754461B2	2010	Doctor et al.	Human butyrylcholinesterase	OP nerve agent scavenger
US6566574B1	2010	Tadros et al.	Cationic surfactant hydrotrope and reactive compound (oxidizer)	Neutralizes chemical or biological weapons

*Previously, some of the patents have been filed with another country or region; however, their most recent versions are described above.

Due to the references limit for this article, citations for each patent presented in this table have not been included in the list of references. Only references that have been cited within the manuscript text have been included in the list of references.

Beginning letters in patent number signifies which patent office approved this property: US: United States Patent and Trademark Office; EP: European Patent Office; CN: China Patent & Trademark Office; JP: Japan Patent Office; CA: Canadian Intellectual Property Office.

OP: Organophosphate.

Table 3. Patents of countermeasures for botulism.

Patent number*	Year of publication	Inventor(s)	Molecule/Compound details	Properties
US8741945 B2	2015	Shure et al.	Deoxyactagardine compounds	Reestablishes natural balance of GI flora
US9161961 B2	2015	Tel-Ari	Tea tree oil	Topical antibacterial
CN102422158 B	2015	Wang et al.	a-SNAP-25 antibodies	Bind to cleavage site of BoNT A
US8697055 B2	2014	Farmer	Lactic-acid-producing bacteria	Reduces the rate of colonization and physiological effects of GI tract pathogens
US8809392 B2	2014	Li et al.	Sulfoperoxycarboxylic acids	Reduces surface microbial populations through oxidative properties
US8841281 B2	2014	Merola	Amino acid–transition metal complexes	Antibacterial against multidrug-resistant bacteria
US8575094 B2	2013	Wadman et al.	Type-B lantibiotic or derivatives and variants thereof	Prevents/Reduces GI infections (aids in correcting GI flora imbalance)
US8088422 B2	2012	Gu et al.	Purified pyrroloquinoline quinone	Inhibits microorganism growth through damage of DNA and RNA
US7999079 B2	2012	Marks et al.	BoNT A antibodies	Neutralizes BoNT A
EP2440533 B1	2012	Mulhbacher et al.	Guanine riboswitch binding compounds	Antibacterial; inhibits expression of microbial gene guaA

*Previously, some of the patents have been filed with another country or region; however, their most recent versions are described above.

Due to the references limit for this article, citations for each patent presented in this table have not been included in the list of references. Only references that have been cited within the manuscript text have been included in the list of references.

Beginning letters in patent number signifies which patent office approved this property: US: United States Patent and Trademark Office; EP: European Patent Office; CN: China Patent & Trademark Office; JP: Japan Patent Office; CA: Canadian Intellectual Property Office.

Table 4. Patents of countermeasures for anthrax.

Patent number*	Year of publication	Inventor(s)	Molecule/Compound details	Properties
US8933122 B2	2015	Bonvila et al.	Cationic surfactants	Inhibits proliferation of bacteria, fungi and yeasts
EP2510946 B1	2015	Cottam et al.	Synthetic TLR agonists phospholipid conjugate	Immunostimulatory agent
US8933016 B2	2015	Cowan et al.	Metallodrugs – antimicrobial peptide with antibiotic covalently bound by a metallic binding domain	The metal moiety generates ROS causing oxidative stress/death in the target pathogenic microorganism
US9181340 B2	2015	Croix et al.	Tem8 antibody conjugates	Interacts with IL-5
EP2723722 B1	2015	Johnson	3,5-Diamino-6-chloro-N-(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino) ethoxy)phenyl)butyl)carbamimidoyl) pyrazine-2-carboxamide	Blocks sodium channels
US8926986 B2	2015	Kublier-Kielb et al.	Conjugates of 3 methyl-3-hydroxybutyrate and 3-hydroxybutyrate-substitute saccharides	Elicits immune response
US9089507 B2	2015	Moeck et al.	Oritavancin	Inhibits B. anthracis growth
US9012608 B2	2015	Thullier et al.	G immunoglobin	Works against protective antigen anthrax toxin
US8840899 B2	2014	Ahmed et al.	mTOR inhibitor	Enhances antigen specific T-cell immune response
US8853228 B2	2014	Anderson et al.	Pyrimidine derivative compounds	Inhibits DHFR
CA2621136C	2014	Bartlett et al.	IMiDs	Induces autophagy
US8685396 B2	2014	Chen et al.	Monoclonal antibodies	Neutralizes anthrax PA toxin
CN102732468 B	2014	Cui et al.	Entercoccus faecalis products	Antimicrobial
US8764701 B1	2014	Hicks	Wound treatment apparatus	Delivers reactive species to wound to kill bacteria
CA2574477C	2014	Kim et al.	Alpha-defensin	Binds/inhibits B. anthracis toxin
CN103550814 B	2014	Liang	Chitosan bio-film forming glue	Blocks sodium channels
US8741223 B2	2014	Mason et al.	Chlorine dioxide gas	Application eliminates surface contamination
US8685393 B2	2014	Parikh	Ang-2 antagonists compounds	Reduce or block Ang-2 signaling pathways
US8440646 B1	2013	Alekshun et al.	Substituted tetracycline compounds	Antibacterial; inhibits protein synthesis
US8853228 B2	2013	Anderson et al.	Heterocyclic analogs	Inhibits dihydrofolate reductase
US8343495 B2	2013	Brock et al.	Equine-derived antibody compositions	Bind to Bacillus anthracis protective antigen
US8617548 B2	2013	Casey	Anti-anthrax antibodies in conjunction with fluoroquinolone antibiotic	Neutralizes B. anthracis toxin
US8501182 B2	2013	Chen et al.	Monoclonal antibodies	Bind to poly-γ-d-glutamic acid
US8580553 B2	2013	Fischetti et al.	Phage-associated lytic enzymes	Detects and kills B. anthracis
EP2083858 B1	2013	Glennie et al.	CD27 agonistic antibodies	Promotes T-cell immunity
US8937099 B2	2013	Goldstein	NAC amide	Prevents and counteracts oxidative stress
US8420607 B2	2013	Carlson et al.	Isolated oligosaccharide antibody	Binds to β-Gal disaccharide
US8450300 B2	2013	Jones	Fusidic acid	Inhibits bacterial protein synthesis
CA2595889C	2013	Najafi et al.	N-halogenated amino acids, N, N-dihalogenated amino acids and derivatives	Releases halogen; antibacterial, -infective, -viral, -microbial, -fungal
US8524484 B2	2013	Sabbadini et al.	Immunogenic minicells	Induce immune response
US8414926 B1	2013	Turos et al.	Nanoparticle made of biologically active agent and surfactant covalently attached to polymer backbone	Drug delivery system for antibiotics or other biologically active agents
US8574608 B2	2013	Texter	Silver surfactant compounds	Topical antimicrobial; inhibits bacterial growth
US8389469 B2	2013	Yoong et al.	Bacteriophage lytic enyzmes	Bacteriolytic agent; antimicrobial
US8209131 B2	2012	Coombe et al.	Glycoaminoglycan molecules	Bactericidal
US8268825 B2	2012	Dreier et al.	2,4-Diaminopyrimidine compounds	Antibacterial; dihydrofolate reductase inhibitor
US8088395 B2	2012	Ghosh	Phytol derived immune-adjuvants; used with an antibiotic	Induces humoral and cytotoxic immune responses
US8114913 B1	2012	Guilford et al.	Glutathione and n-acetyl-cystein	Detoxification of anthrax toxin
US8211656 B2	2012	Hyde et al.	Modified red blood cells containing target binding units	Recognizes and targets bacteria in blood
US8242174 B2	2012	Johnson et al.	Hydroxamic acid/aniline derivatives	Inhibit lethal factor toxin
US8192720 B2	2012	Kozel et al.	Capsular polypeptide	CD40 agonist elicits immune response
US8198251 B2	2012	Vollmer et al.	CpG oligonucleotides	Elicits immune response
US8012961 B2	2011	Hubschwerlen et al.	Tricyclic antibiotics	Antibacterial agent
US7955596 B2	2011	Jones et al.	Furanone	Inhibits toxin production
EP1781708 B1	2011	Nair et al.	Novel d-glucan polysaccharide ‘RRI’	Th1-type immune response to antigen conjugate
US7906119 B1	2011	Rosen et al.	Monoclonal antibody	Binds to protective antigen of B. anthracis
EP1902719 B1	2011	Schischkova et al.	Bacteriolitic complex produced by Lystobacter sp.XL 1	Bactericidal to B. anthracis spore cells
US7901687 B2	2011	Werz et al.	Oligosaccharides conjugates	Vaccine
CA2487727C	2011	Xiong et al.	Sulfonamide compounds	Inhibits lethal factor component activity
US7838252 B2	2010	Cohen et al.	Antibodies or binding fragments specific for lipoprotein-related receptor protein 6	Inhibits cellular internalization of anthrax toxin
EP1482791 B1	2010	Ninkov	Organic phenolic compounds	Antimicrobial
US7718680 B2	2010	Pellecchia et al.	Heterocyclic compounds	Inhibits lethal factor protease activity

mTOR: Mammalian target of rapamycin; DHFR: dihydrofolate reductase; IMiDs: immunomodulatory compound.

*Previously, some of the patents have been filed with another country or region; however, their most recent versions are described above.

Due to the references limit for this article, citations for each patent presented in this table have not been included in the list of references. Only references that have been cited within the manuscript text have been included in the list of references.

Beginning letters in patent number signifies which patent office approved this property: US: United States Patent and Trademark Office; EP: European Patent Office; CN: China Patent & Trademark Office; JP: Japan Patent Office; CA: Canadian Intellectual Property Office.

Table 5. Patents for plague countermeasures (protectors, mitigators, and therapeutics/treatments).

Patent number*	Year of publication	Inventor(s)	Molecule/Compound details	Properties
US9062297 B2	2015	Curtiss et al.	Mutated recombinant Y. pestis	Vaccinates against bacteria with pCD1 or araC PBAD
US9109018 B2	2015	Emery et al.	Y. pestis polypeptide isolate	Decreases colonization by blocking attachment sites of gram negative bacteria
US8993627 B2	2015	Goldstein	N-acetylcysteine amide	Reduces oxidative stress
US9107906 B1	2015	Grossman et al.	Pooled human plasma immunoglobulins	Reduces incidence of infection in immunocompromised patients
US9138468 B2	2015	Lien et al.	LpxL-expressing Y. pestis	Administration increases the immune system’s ability to identify and protect against the unmodified Y. pestis
CA2757574C	2015	Moir et al.	Organic compound (multiple structures)	Inhibits type III secretion system; prevents secretion/translocation of toxin effectors
US9211327 B2	2015	Nilles	Antibody containing YscF fragment	Targets surface protein
US9028809 B2	2015	Stinchcomb et al.	Antigens or peptides associated with Y. pestis	Vaccine induces immune response against Y. pestis
US8946188 B2	2015	Tan et al.	5′-O-[N-(salicyl) sulfamoyl]-adenosine (salicyl-AMS)	Inhibits siderophore-producing salicylation enzymes
US8945580 B2	2015	Yusibov et al.	Vaccine comprised of at least two different Y. Pestis antigens fused to thermostable protein	Stimulates production of antigen specific antibodies or cellular immune response
US9045550 B2	2015	Zlotkin	Peptide-containing FNFDWY	Prevents microbial biofilm formation
US8722046 B2	2014	Amemiya et al.	Human monoclonal antibodies	Recognizes Y. pestis F1 or V antigen
US8747872 B2	2014	Baker et al.	Nanoemulsion compositions	Treats bacterial biofilms
US8715641 B2	2014	Filutowicz et al.	Amoebae spores	Amoebae kill or slow the growth of bacteria or fungus
US8709448 B2	2014	Gelder et al.	Immunostimulant microparticles	Activates innate antibacterial and -viral immune response
US8906868 B2	2014	Glinka et al.	Aminoglycosides	Antimicrobial
US8716283 B2	2014	Goguen et al.	Organic compounds	Inhibits Type III secretion system release of Yops into host cell
US8795677 B2	2014	Heath et al.	Single protein, composed of both F1 and V antigens	Elicits protective immune response
US8680136 B2	2014	Hirst et al.	1-cyclic-boronic acid	Inhibits beta-lactamase inhibitors
CA2589553C	2014	Mizel et al.	Flagellin adjuvant	Combined with Y. pestis antigen
US8795683 B2	2014	Oberreither et al.	Modified vaccinia Ankara virus containing mannitol	Mannitol improves the stability of vaccine agent to allow for better immune response
US8912314 B2	2015	Pier et al.	Monoclonal antibodies	Binds to poly-N-acetyl glucosamine
US8859760 B2	2014	Prestwich et al.	Porphyrin	Kills and prevents the growth of microbes
US8350061 B2	2013	Iyer et al.	Bacterial qurorum-sensing molecule analogs chemically linked to antibiotic	Allows for the direct delivery of antibiotic to pathogen
CA2366216C	2012	Alpar et al.	Polycationic carbohydrate	Stimulates immune system
US7947741 B2	2011	Bostian et al.	Pentamindine and analogous compositions coadministered with antimicrobial agents	Used as efflux pump inhibitors to treat drug resistant pathogens
US7815911 B1	2010	Straley et al.	YadC polypeptide	Induces immune response

*Previously, some of the patents have been filed with another country or region; however, their most recent versions are described above.

Due to the references limit for this article, citations for each patent presented in this table have not been included in the list of references. Only references that have been cited within the manuscript text have been included in the list of references.

Beginning letters in patent number signifies which patent office approved this property: US: United States Patent and Trademark Office; EP: European Patent Office; CN: China Patent & Trademark Office; JP: Japan Patent Office; CA: Canadian Intellectual Property Office.

Figure 1. Possible CBRN threats. Several CB threats have been identified for which there is need to develop medical countermeasures.

Figure 2. FDA-approved countermeasures for CB threats following the Animal Rule. In 2002, the FDA issued the Animal Rule to expedite the development of medical countermeasures against CBRN threats. In the past fourteen years, nine countermeasures have been approved by FDA following Animal Rule.

2. Countermeasures for chemical weapons

Chemical weapons use the toxic properties of their components to produce external or internal injuries. Some chemically based threats arise from nerve agents and sulfur mustard, which typically are liquids at ambient temperature. Nerve agents inhibit acetylcholinesterase, an essential molecule for signal transmission of neuronal tissues. This inhibition leads to a buildup of acetylcholine at neuronal synapses, producing an overstimulation of cholinergic receptors, ultimately leading to paralysis and often death. The organophosphorus nerve agents, tabun, sarin, soman, and cyclosarin, are among the most toxic chemical warfare agents known and grouped as G-series nerve agents [16]. At room temperature, G-series nerve agents are volatile liquids, making them serious exposure risks from inhalation. V-series agents, including O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate (also known as VX), are among the most toxic chemical warfare nerve agents. The V agents are more poisonous than sarin but have low volatility and are primarily detrimental after skin exposure [17].

G-series nerve agents display high volatility, which increases with rising temperatures; release of liquid G-type nerve agents in an enclosed environment may rapidly create lethal concentrations [6]. In a military or terrorist scenario, exposure to chemical G-series nerve agents will happen mainly via inhalation; nevertheless, percutaneous absorption is also feasible if there is exposure to the skin or to the eyes with the less volatile (than the G-series agents) V-series nerve agents.

2.1. Nerve gas

Nerve agents are considered to be the deadliest of the common chemical warfare agents. By binding to or inactivating acetylcholinesterase, nerve agents alter the cholinergic transmission which leads to a rapidly increasing cholinergic crisis [17]. This results in an uncontrolled and overwhelming stimulation of both muscarinic and nicotinic receptors leading to a wide variety of clinical symptoms, including death resulting from respiratory failure. Antidotal therapy largely consists of atropine, pralidoxime, and benzodiazepines, all of which must be administered immediately to limit the impact of nerve agents binding to the enzyme and to avoid the worsening of symptoms [18].

Patents for agents under development to treat nerve gas exposure are presented in Tables 1 and 2. These recent patents include several enzymes such as hyperthermophilic phosphotriesterases, butyrylcholinesterase, paraoxonase, and novel peptides. Pyridostigmine bromide has been approved by the FDA for pretreatment against the nerve agent soman. Apart from medical pretreatment, if cholinergic crisis is detected, pralidoxime (2-PAM) and atropine must be administered as soon as possible; the additional use of benzodiazepines (diazepam) is suggested [6]. Diazepam Chempack is currently stockpiled within the SNS, but will soon be substituted by the improved formulation, Midazolam, which is an intramuscular (im) injectable that acts faster than diazepam [19]. Atropine and 2-PAM are also available in SNS. Some new medical countermeasures utilize nerve agent scavengers; human butyrylcholinesterase is the principal stoichiometric bioscavenger and human paraoxonase 1 is the leading catalytic bioscavenger. The proof of concept for bioscavenger gene-delivery has already been established [20].

2.1.1. Pyridostigmine bromide

Soman triggers loss of muscle control and respiratory failure, leading to death. Results from studies in nonhuman primates (NHPs) and guinea pigs have provided support that pretreatment with pyridostigmine bromide, together with atropine and pralidoxime provided after exposure to soman, boosts rates of survival [21,22]. NHP-provided with mestinon (pyridostigmine) syrup-impregnated diet biscuits (40 mg/animal) displayed a reproducible inhibition of 40–50% whole blood cholinesterase activity for a period of 1–6 h. Pyridostigmine pretreatment was complemented by two doses of an antidotal combination (0.05 mg/kg atropine, 2.24 mg/kg trimedoxime bromide-4, and 0.4 mg/kg benactyzine) which ensured the survival of 5 of 6 animals following 3 separate exposures to soman concentrations of 10 LD₅₀. The protective period of oral pyridostigmine supported by antidotal combination therapy was between 30 min and 8 h prior to soman exposure. Oral pyridostigmine pretreatment in combination with atropine therapy (3 doses of 0.07 or 1.00 mg/kg, im) also protected NHPs exposed to 10 LD₅₀ soman. Oral pyridostigmine pretreatment was not able to protect NHPs from soman exposure, without the antidotal combination or atropine therapy [22]. Based on these results, the FDA approved pyridostigmine bromide on 5 February 2003 to enhance survival after exposure to soman. Pyridostigmine bromide was the first drug approved by the FDA under the Animal Rule [23]. The FDA approved pyridostigmine bromide specifically to be used prior to soman exposure and in combination with atropine and pralidoxime after exposure.

2.2. Cyanide poisoning

Hydrogen cyanide is one of the two cyanide chemical warfare agents, the other is cyanogen chloride. Cyanide is a fast acting, lethal agent when used in enclosed spaces where high concentrations can be attained easily. Because of the widespread use of cyanide in US industrial processes, this agent presents a credible threat for terrorist use. Cyanide poisoning uncouples mitochondrial oxidative phosphorylation and inhibits essential and life-sustaining cellular respiration; cyanide poisoning can be fatal despite the extent of oxygen available to the body, and without treatment, exposure to a high dose of cyanide may result in death within minutes [24]. There are now two types of cyanide antidotes available, Cyanide Antidote kit and Cyanokit. Cyanide Antidote kit was the first available and contains inhalant amyl nitrite and injectable sodium nitrite and sodium thiosulfate. There is another version of this kit, Nithiodote, which contains injectable sodium nitrite and sodium thiosulfate. The second antidote, cyanide countermeasure (Cyanokit) is discussed below.

2.2.1. Cyanokit

Cyanokit is a cyanide antidote that includes hydroxocobalamin, a form of vitamin B₁₂ which was approved by FDA on 15 December 2006 following the Animal Rule [25]. Hydroxocobalamin binds to cyanide, allowing critical cellular respiration to resume. The ability of hydroxocobalamin for acute cyanide poisoning was compared with that of saline vehicle in canines [26]. Adult canines were injected with potassium cyanide (0.4 mg/kg/min, intravenously [iv]) and then treated after 3 min following the onset of apnea. The treatments, consisting of hydroxocobalamin (75 or 150 mg/kg, iv) or the saline vehicle (as a control),were then infused over a 7.5-min period while animals were ventilated with 100% oxygen for 5 min. Cyanide exposure reduced vital physiological functions that ended in a moribund state requiring euthanasia (within 4 h) of 59% of vehicle-treated animals; cyanide exposure also caused neurological deficits which required euthanasia in an additional 23% of vehicle-treated animals (within 2–4 days postexposure), resulting in an overall mortality rate of 82%. By contrast, those subjects administered with 75-mg/kg hydroxocobalamin had a mortality rate of 21% at 14 days postexposure, whereas those administered 150-mg/kg hydroxocobalamin had a 0% mortality (all animals survived), suggesting that hydroxocobalamin reversed cyanide toxicity and reduced mortality in the canine model [26].

2.3. Other important chemical threat

Sulfur mustard agents constitute another group of chemical threats and are often called blister agents since their injuries usually look like burns or blisters. Several cellular level mechanisms of action have been suggested for sulfur mustard [27,28]. Current therapy for mustard gas consists of treating the inflammation and where necessary, removing the dead eschar to expedite healing. Therapy for sulfur mustard poisoning is still a challenge although several promising methods are under investigation. Special attention is being paid to ameliorating the damage using low molecular weight antioxidants. Melatonin, epigallocatechin gallate, and flavone derivatives, in particular, have been studied recently [29].

3. Countermeasures for biological threats – bioterrorism

At large, there are a select group of biological agents (infectious and/or toxic constituent) which can be weaponized using relatively easy means (i.e. inexpensive production and storage) by individuals with criminal intents. Aerosolization is believed to be the most likely, successful dispersal method of bioweapons [30]. Furthermore, the injury produced by these weapons can spread well outside the initial delivery area, leading to dreadful results. These characteristics instill fear and incite public panic.

The Centers for Disease Control (CDC) and Prevention categorizes bioweapons into Categories A, B, and C, based on their infectious nature, mortality rate, public health impact, and ability to incite panic, and social disruption [31]. These threat agents are as follows: category A consists of: anthrax – Bacillus anthracis, botulism – Clostridium botulinum toxin, plague – Yersinia pestis, smallpox – variola major, tularemia – Francisella tularensis, viral hemorrhagic fevers – filoviruses – Ebola and Marburg, arenaviruses – Lassa and Machupo; category B: brucellosis – Brucella species, epsilon toxin – Clostridium perfringens, food safety threats, glanders – Burkholderia mallei, melioidosis – Burkholderia pseudomallei, psittacosis – Chlamydia psittaci, Q fever – Coxiella burnetii, ricin toxin – Ricinus communis, staphylococcal enterotoxin B (SEB), typhus fever – Rickettsia prowazekii, viral encephalitis – alphaviruses, and water safety threats; and category C emerging infectious diseases such as nipah virus and hantavirus. There are large numbers of agents identified as countermeasures for biological threats (Tables 1, 3–5); a few of these have obtained FDA approval as a result of the Animal Rule and are discussed below. The foremost therapeutic developments in countering exposures to given biologic agents focus on vaccine development. In addition to antibiotics, a few countering biologics have been approved; several of such agents are under development [32].

3.1. Botulism

Botulinum toxin is an exotoxin produced by the gram-positive bacteria Clostridium botulinum. Botulinum toxin-producing bacteria are divided into six groups: C. botulinum groups I–IV, as well as some strains of Clostridium baratii and Clostridium butyricum [33]. There are seven well-known serotypes of the botulism toxin, labeled A through G; almost all human cases of botulism are caused by types A, B, and E, with type E being the most prevalent [34]. It is the most powerful toxin known and has been projected (per unit mass) to be ~100,000 times more lethal than sarin gas [35]. The three main clinical presentations of botulism are as follows: foodborne botulism, infant botulism, and wound botulism; the main symptom of exposure is flat muscle paralysis. Though botulism is rare, it yields a life-threatening neuroparalytic syndrome. Patents for agents to treat botulism have been listed in Tables 1 and 3. The majority of listed patented countering agents are antimicrobial by nature and do not directly neutralize the botulism toxin. The only therapeutic countermeasure for the exotoxicemic effects of botulism, namely heptavalent botulinum antitoxin (HBAT), has been approved by the FDA using the Animal Rule and is discussed below.

3.1.1. HBAT

The C. botulinum toxin blocks acetylcholine released from motor neurons in a dose-dependent manner, which results in acute symmetric diplopia, dysphagia, dysarthria, dysphonia, and possible neurologic sequelae. Botulism is characterized by symmetrical descending paralysis that may develop to respiratory arrest. HBAT contains equine-derived, heptavalent antibody to the seven known botulinum toxin types (A–G) and is comprised of <2% intact immunoglobulin G (IgG) and >90% Fab and F(ab′)₂ immunoglobulin fragments; these fragments are created by the enzymatic cleavage and removal of Fc components [36]. In March 2010, the US CDC released HBAT as an Investigational New Drug (IND), for use as the preferred treatment for food-borne botulism, including type E, which had not been covered by the previously offered bivalent antitoxin. On 22 March 2013, the FDA approved the botulism antitoxin for use in neutralizing all seven known botulinum nerve toxin serotypes [37]. This product’s efficacy was studied utilizing guinea pig and NHP animal models under Good Laboratory Practice. In the guinea pig model, treatment with the vaccine resulted in a statistically significant improvement in the survival rate of animals across all serotypes tested at a dose of 1.5 LD₅₀ dose for guinea pigs injected im into their right hind limbs. In a controlled therapeutic NHP efficacy study, treatment with the antitoxin resulted in a statistical improvement in survival in animals challenged with 1.7 LD₅₀ dose of the toxin. These results provided substantial evidence that the antitoxin is likely to help humans with botulism, allowing for the FDA’s approval.

3.2. Anthrax

Since the anthrax (B. anthracis) spore incidents in the fall of 2001 involving its use in the US mail, the threat of weaponized biological agents being used by terrorists has been of the paramount concern to national security [30]. Anthrax is one of the seven globally ignored zoonotic and endemic diseases, according to the World Health Organization [38]. Anthrax is the potentially fatal disease triggered by infection of gram-positive B. anthracis; this disease can present in one of four distinct clinical patterns depending on the route of infection (cutaneous, gastrointestinal, pneumonic, or injectional). Once the bacterial spore gains entry to a mammalian host, it is taken up by phagocytic cells and transported to the draining lymph nodes; the host cells undergo apoptosis and the bacteria germinate into vegetative cells able to produce the tri-partite complex of exotoxins. Pulmonary anthrax arises after the inhalation of small, ~2–5 µm, aerosolized spores; spores of this size are able to bypass the mucociliary system in the sinonasal tract and reach deep within the lung into the alveolar spaces [39,40]. The incubation time can be up to 100 days in the mediastinal lymph nodes, eventually leading to toxin-mediated edema, hemorrhage, and necrosis [41,42]. Inhalational symptoms include fevers, chills, sore throat, malaise and fatigue, nausea and vomiting, nonproductive cough, and respiratory distress. Ongoing attempts to produce more effective countermeasures involve three basic approaches: (1) improving the quality of existing vaccines as well as developing new and improved vaccines; (2) developing and testing more efficient antimicrobials; and (3) developing arrays of antitoxins. The process of producing higher quality and defined therapeutics is based on a growing understanding of the complex pathogenesis of anthrax infection; it is insufficient to simply reduce the bacteremia with antibiotic therapy, since beyond a specific point, the toxemia associated with anthrax infection is often fatal. Five agents have received FDA approval for human use following the Animal Rule to counteract exposure to anthrax; several more countermeasures have been identified and patented (Tables 1 and 4). Comparatively, more countermeasures for anthrax have been developed as an indirect result of the public concern and pressure following the 2001 anthrax incident that resulted directly in more scientific interest, coupled to increased government funding. In aggregate, these listed patents clearly cover all three of the R&D approaches mentioned earlier; however, the vast majority of them (~75%) represent new types/approaches for antimicrobial therapies, while the remaining patents are for new types of vaccines and antitoxin therapies. Useful prophylactic strategies as well as postexposure therapies remain under development [43]. The agents that have been approved by the FDA under the Animal Rule are specifically identified and discussed below.

3.2.1. Anthrasil

Anthrasil (or more commonly referred to as Anthrax ImmunoGlobulin) is a preparation of purified polyclonal human IgG for iv administration, with specificity to the anthrax toxin; these antibodies are present in the plasma of individuals vaccinated against anthrax. It is recommended for the treatment of inhalation anthrax in adult and pediatric patients in conjunction with antibacterial drugs. Anthrasil efficacy studies were accomplished in rabbit and NHP models [44]. In one study, NHPs were exposed to lethal doses of aerosolized B. anthracis spores, treated with Anthrasil or a placebo, and then assessed for survival. The survival of NHPs treated with Anthrasil (7.5, 15, or 30 mg/kg, started after animal became toxemic) ranged from 36% to 70% compared to 0% in the control group; survival rates enhanced as the treatment doses of Anthrasil were increased. Rabbits treated with Anthrasil after infection exhibited 26% survival compared to 2% survival in the control group. Another rabbit study demonstrated that a combination of Anthrasil and antibiotics (one of the fluoroquilones) resulted in 71% survival compared to 25% survival of those animals treated with antibiotics alone [44]. Simultaneous administration of Anthrasil with levofloxacin or ciprofloxacin in anthrax-exposed rabbits and anthrax-exposed NHPs, respectively, did not decrease the effectiveness of antibacterial therapy. This report also supported the concept that Anthrasil essentially alleviates the toxemic effects initiated by anthrax infection when administered in combination with antibiotics. These results offered sufficient support that Anthrasil is expected to benefit humans with inhalation anthrax. The safety of Anthrasil was examined in 74 healthy human volunteers [45]; the most frequently observed side effects were back pain, headache, nausea, and infusion site pain and swelling [44]. On 24 March 2015, Anthrasil was approved by the FDA following the Animal Rule [45].

3.2.2. Raxibacumab

Raxibacumab (ABthrax) is an anthrax-specific antitoxin, composed of human IgG1λ monoclonal antibody directed against the B. anthracis Protective Antigen. Therapy with Raxibacumab prevents the progression of the disease by binding the protective antigen and eventually inhibiting both, lethal factor and edema factor from entering the host cells [46]. Raxibacumab received fast track designation from the FDA and then approval for its indication on 14 December 2012 [47]; it was the first biologic product to be approved for adult and pediatric patients through the FDA Animal Rule [48].

A number of animal models have been used to determine Raxibacumab efficacy in both preexposure and postexposure situations [47]. Due to the unpredictable nature of potential anthrax releases, Raxibacumab may need to be administered to individuals in high-risk groups even before exposure events; therefore, the efficacy of prophylactic therapy is significant. Postexposure studies were vital in determining the therapeutic window in which the drug could be administered and still remain effective.

Raxibacumab’s efficacy for inhalation anthrax was shown in NHP and rabbit models [49,50]. All animals were administered aerosolized B. anthracis spores and received different doses of Raxibacumab, placebo, or antibiotics normally used to treat anthrax. In experiments treating anthrax-exposed animals (185 LD₅₀ spore dose for NHP and 202.5 LD₅₀ for rabbit) with a treatment dose of 40 mg/kg Raxibacumab, 64% of the NHPs and 44% of the rabbits survived, compared to 0% survival in the control groups of NHPs and rabbits; all surviving animals produced toxin-neutralizing antibodies. Another study with rabbits demonstrated that 82% of animals treated with a combination of antibiotics and Raxibacumab survived exposure compared with 65% of animals receiving antibiotic treatment alone [46,47]. To investigate the therapeutic window of treatment with Raxibacumab, rabbits were exposed to 100 LD₅₀ of B. anthracis spores and administered Raxibacumab at a single dose of 40 mg/kg at the time of exposure, 12, 24, or 36 h after exposure. Survival was 100% in animals treated at the time of exposure or 12 h after exposure, but decreased to 50% and 42% in animals treated at 24 and 36 h after exposure, respectively [51]. Raxibacumab has undergone human safety trials (326 healthy volunteers); common side effects included rash, extremity pain, itching, and drowsiness [46,52].

3.2.3. Levaquin

Levaquin (levofloxacin), a constituent of the fluoroquinolone family, is a synthetic broad-spectrum antibacterial agent for oral and iv administration. It is the l-isomer that provides the primary antibacterial activity of racemic ofloxacin, a quinolone antimicrobial agent [53]. Levofloxacin and other fluoroquinolone antimicrobials inhibit bacterial topoisomerase IV and DNA gyrase, enzymes needed for DNA replication, transcription, repair, and recombination [54].

Levaquin is indicated for inhalation anthrax to inhibit the occurrence or advancement of disease following exposure to aerosolized B. anthracis spores [55]. An animal study using NHPs exposed to an inhaled average dose of 49 LD₅₀ (~2.7 × 10⁶ spores) of B. anthracis was conducted; NHPs were administered a 30-day regimen of oral Levaquin beginning 24-h postexposure. The minimal inhibitory concentration of Levaquin for the anthrax strain used in this study was 0.125 μg/ml. Mortality of animals that received Levaquin (1/10) was significantly lower, compared to the control group (9/10). The one levofloxacin-treated animal that died of anthrax did so following the 30-day drug administration period.

The estimated effective human dose of Levaquin is based on the mean plasma concentrations of Levaquin combined with a statistically significant enhancement in survival over control in the NHP model of inhalation anthrax. Plasma concentrations serve as a surrogate end point that has a realistic likelihood of predicting clinical advantage. Levaquin plasma concentrations reach or exceed those determined in NHP experiments in adult and pediatric patients receiving the recommended oral and iv dosage regimens [56]. Levaquin received FDA approval for this indication following the Animal Rule on the 24 November 2004 [56].

3.2.4. BioThrax

Anthrax Vaccine Adsorbed was originally licensed on 10 November 1970 and re-licensed to Emergent as BioThrax on 12 November 1998. On 23 November 2015, the FDA approved the supplement requesting a new indication for BioThrax: postexposure prophylaxis of suspected or confirmed Bacillus anthracis exposure, when combined with the recommended course of antimicrobial therapy. This was the first approval of a vaccine for any indication based on the Animal Rule [57]. BioThrax produces antibodies against protective antigen, ultimately neutralizing the activities of the cytotoxic lethal toxin and edema toxin of B. anthracis. Although other B. anthracis proteins besides those against protective antigen may be present in BioThrax, their role in protection against anthrax exposure has not been established.

Pivotal animal efficacy studies were conducted in rabbits and NHPs; animals received two im vaccinations 4 weeks apart with serial dilutions of BioThrax and were subjected to lethal quantities of aerosolized B. anthracis spores on study day 70 [58]. Logistic regression analysis established that an antibody 50% neutralization factor level of 0.56 in rabbits and 0.29 in NHPs was linked with 70% probability of survival.

The ability of BioThrax to increase rates of survival when the treatments were supplemented by antimicrobial treatment was examined in two postexposure animal model studies. In these studies, rabbits were challenged with aerosolized B. anthracis spores and then treated with levofloxacin, administered via oral gavage once daily for 7 days starting at 6–12 h postexposure, with or without two im injections of BioThrax 1 week apart. Rates of survival among animals receiving both levofloxacin and BioThrax ranged from 70% to 100% and incrementally increased as dose of the vaccine increased. In contrast, only 44% and 23% survival were noted among animals that received only antimicrobial treatment [58].

3.2.5. Ciprofloxacin hydrochloride

Ciprofloxacin hydrochloride (Cipro), a fluoroquinolone, is the monohydrochloride monohydrate salt of 1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid. The FDA approved Cipro in 2015 to decrease the chance of developing anthrax infection following exposure to the anthrax spores [59]. A study using NHPs exposed to an inhaled mean dose of 11 LD₅₀ (~5.5 × 10⁵ spores; range 5–30 LD₅₀) of B. anthracis was conducted. The minimal inhibitory concentration of ciprofloxacin for the anthrax strain used in this study was 0.08 µg/ml [60,61]. NHPs received a 30-day regimen of oral ciprofloxacin, initiated 24 h postexposure; the mortality of ciprofloxacin-treated NHPs was significantly lower (1/9), compared to the control group (9/10) [61]. The mean serum concentrations of ciprofloxacin associated with a statistically significant enhancement in survival in the NHP model of inhalation anthrax are attained or exceeded in adult and pediatric patients receiving oral and iv regimens [62,63].

3.3. Plague

Y. pestis is a gram-negative coccobacillus that is the causal agent of a rare but possibly fatal bacterial infection known as plague. It is classified by the CDC as a category A bioterrorism agent with the three most common forms being: bubonic plague (infection of the lymph nodes), septicemic plague (infection of the blood), and pneumonic plague (infection of the lungs) [30]. Y. pestis has been used as a bio warfare weapon in the past [64]. Agents that have been identified and patented for the treatment of plague are listed in Tables 1 and 5. The majority of these newly patented agents fall into three major groups: (1) vaccines against antigens that are predicted to confer protection to host; (2) new antimicrobials; and (3) new approaches to enhance specific immunity. Discussed below are three countermeasures with indications to treat plague that have been approved by the FDA utilizing the Animal Rule.

3.3.1. Avelox

Avelox (moxifloxacin hydrochloride) is a fluoroquinolone antibiotic, effective in treating experimental pneumonic plague in an animal infection model [65]. The FDA approved Avelox on 8 May 2015 to treat patients with pneumonic and septicemic plague [66]. In the NHP model of pneumonic plague, the mortality of Avelox-treated NHPs was significantly lower compared to the control group. Avelox’s safety was characterized in clinical studies and post-marketing information for the drug’s existing clinical uses. Common side effects are diarrhea, nausea, headache, and dizziness.

3.3.2. Levaquin

On 27 April 2012, the FDA approved Levaquin (levofloxacin) for the prophylactic use and treatment of patients with plague, including pneumonic and septicemic plague, caused by Y. pestis [47]. Approval for this indication is based on an efficacy study conducted in NHPs exposed to an inhaled mean dose of 65 LD₅₀ (range 3–145 LD₅₀) of Y. pestis [55]. The minimal inhibitory concentration of Levaquin for the Y. pestis strain used in this study was 0.03 μg/ml. Mean plasma concentrations of Levaquin attained at the end of a single 30-min iv infusion ranged from 2.84 to 3.50 μg/ml in NHPs. The mortality in the Levaquin-treated group was significantly lower (1/17) compared to the control group (7/7).

3.3.3. Cipro

The FDA approved Cipro on 2 February 2015 for treatment and prophylaxis of plague due to Y. pestis in adults and pediatric patients from birth to 17 years of age [67]. Studies of Cipro for use in the treatment of plague were conducted in NHPs [68]. Ciprofloxacin was significantly efficacious in the treatment of pneumonic plague in NHPs (10-day regimen, 15 mg/kg iv administered over 60 min twice a day, mimics the human regimen of 400 mg, iv twice a day).

3.4. Other biological agents

In addition to the infectious agents discussed above, there are several other infectious agents and toxins that pose significant health threats when weaponized. Some of these agents include tularemia, Q fever, smallpox, viral hemorrhagic fever, SEB (Staphylococcal aureus enterotoxin B), ricin, trichothecene mycotoxins including T-2 mycotoxin, saxotoxin, and tetrodotoxins [69]. Tularemia is a zoonotic disease caused by infection with the gram-negative, facultative intracellular bacterium Francisella tularensis. A live, attenuated vaccine is currently available with IND and is effective against aerosol infection [70]. Q fever is a zoonosis triggered by rickettsia-like organism, Coxiella burnetii, an obligate gram-negative intracellular bacterium. To treat Q fever, a 2-week course of doxycycline is recommended for adults and children aged ≥8 years. A Q fever vaccine is licensed in Australia but not in the United States [71].

Despite the eradication of naturally occurring smallpox and the availability of a vaccine, the potential weaponization of the variola virus continues to pose a significant health threat to the military and civilian populations, alike. A Vaccinia vaccine is an effective vaccine for preexposure prophylaxis against smallpox [72]. IMVAMUNE (also known as IMVANEX), which is based on a live, attenuated, modified Vaccinia Ankara virus that lacks replicative capacity in human hosts, is being developed for the prevention of smallpox infection, particularly in patients contraindicated to the traditional smallpox vaccine, such as immunosuppressed individuals. This vaccine has been approved by European Medical Agency in 2013 and may get FDA approval in the near future [73].

SEB is an exotoxin produced by Staphylococcus aureus. It is one of the toxins responsible for staphylococcal food poisoning in humans and has been produced previously as a biological weapon [74]. SEB is a superantigen; it acts by stimulating cytokine release and inflammation [75]. Ricin, a toxin derived from the castor bean, is a by-product of the process used to extract castor bean oil. Ricin acts against cells’ ribosomes, preventing the cells from producing proteins, thus leading to cell death, and possibly resulting in organ failure and death. Since there is no cure for ricin poisoning, treatment focuses on addressing the symptoms and, if possible, flushing the ricin out of the system [74]. Trichothecene is a type of mycotoxin produced by several toxic fungi such as Stachybotrys chartarum. Trichothecene is one of the most notorious mycotoxins because it is extremely toxic and difficult to destroy. There are 60 known types of trichothecene mycotoxins, though T-2 trichothecene mycotoxins are the only ones which have been used in biological weapons [74]. Saxitoxins and tetrodotoxins are a group of chemical agents produced by certain species of marine algae and fish. The lethal dose for a human is about 0.5–2.0 mg when entering the body orally (as in with food), and 0.05 mg if injected (as bite or sting) [76]. Saxitoxin and tetrodotoxin poisoning produces neurological symptoms such as muscle weakness, headaches, dysphonia, floating feeling, paralysis of the cranial and peripheral nerves, astigmatism, as well as paresthesia around the lips, tongue, gums, and distal segments of the limbs. There is no specific antidote for saxitoxins and tetrodotoxins, but supportive therapies are recommended [76].

3.5. Recent patents for CB threat countermeasures

A large number of agents have been patented as countermeasures for CB threats during the last few years. We have listed such agents in Tables 1–5 and have discussed them below.

3.5.1. Chemical/Nerve gas

Improvements in the therapeutic efficacies of the presently available treatment options for unwanted exposures to highly toxic, often lethal effects of organophosphorous nerve agents are needed. In this regard, galantamine (GAL-Hbr) has been recognized to be beneficial, at least experimentally, improving survival following exposures to various nerve agents [77,78]. Galantamine is a unique, dual mode-of-action agent. It is a reversible and weak competitive inhibitor of acetylcholinesterase and is one of a few drugs with activity as an allosteric modulator of nicotinic acetylcholine receptors [79]. Galantamine protects acetylcholinesterase from irreversible inhibition by organophosphate while preserving the scavenger capacity of plasma butyrylcholinesterase for organophosphates. Soman-induced inhibition of a major regulatory, neurotransmitter, GABA, was not observed in the hippocampus of galantamine-treated guinea pigs [79]. When administered instantly after VX exposure and in combination with two other well-known antidotes, atropine and pyridine-2-aldoxoime methochloride, GAL-Hbr effectively reduces the extent of diaphragmatic paralysis; this improves survival of the nerve agent-exposed guinea pigs [78,80]. Atropine, a current therapy for organophosphate poisoning and a muscarinic receptor antagonist, is administered postexposure. Galantamine can be administered multiple times per day for a protracted period of time. Another agent often discussed alongside galantamine as a potentially useful antidote is huperzine (HUP), a potent, naturally occurring, reversible inhibitor of acetylcholinesterase that can be successfully administered prior to or immediately following exposure to nerve agent, e.g. soman [81]. Despite HUP’s equivalent efficacy to that of physostigmine in mice (i.e. a pyridiostigmine-like related drug), the drug seems to be devoid of serious deleterious effects in healthy subjects [82].

3.5.2. Botulism

Several patents have recently been accepted as effective treatments for botulism (all seven known types of botulinum serotypes). These agents, currently under research and development, fall into several categories including biologics and nutraceuticals. Only one of the agents presented in Table 3, an oligoclonal (2–10 specificities) antibody of botulinum, has been tested for efficacy specifically against botulism. With prophylactic administration, the oligoclonal botulinum antibody binds solubilized toxin, preventing the toxin’s ability to disrupt natural levels of acetylcholine at synaptic neuromuscular junctions [83]. Experiments using an ex vivo mouse model suggest neuroparalysis can be significantly delayed by combining single chain Fv antibody fragments and either epitope 1 (S25) or 2 (C25) antibody fragments. This improved treatment modality may aid in improving the current treatment regime more effectively.

3.5.3. Anthrax

Patents for anthrax countermeasures have a wide range of drug-delivery schedules, including before or after exposure to B. anthracis. Activated C protein (Xigris) has shown efficacy in NHPs and mice when administered before exposure [84]. Untreated, control mice presented anthrax dose-dependent lethality whereas Xigris-treated mice were protected. Angiopoietins, members of vascular growth factor family, were augmented during the first 48 h of exposure to the bacterium and declined after 48 h in the individuals that survived infection. Angiopoietins seem to be a useful biomarker for anthrax infection and survival prediction. Additionally, poly-γ-d-glutamic acid (γDPGA), which is present on the surface of B. anthracis, can be targeted by prophylactic treatments; monoclonal antibodies bound to this surface molecule can protect mice from anthrax infection [85]. A treatment protocol for humans was developed utilizing these monoclonal antibodies and may prove suitable in the clinical management of victims following exposure. Treatment with a high-affinity anti-Protective Antigen (anti-PA) monoclonal antibody after exposure to B. anthracis may also be helpful [86] as it has shown to provide exposure mitigation to NHPs. When combined with levofloxacin, NHPs and rabbits demonstrated greater protection than with antibiotic treatment alone. Anti-PA provided comparable pharmacokinetics and bioavailability when administered either through iv or im routes, with the im route being more suitable during a mass causality scenario.

3.5.4. Plague

Elucidation of the pathogenic mechanisms of a given infectious microbe is generally an essential step for developing effective treatments. A recently published patent concerning a new type of Y pestis vaccine suggests that by selectively modifying Y. pestis’s lipopolysaccharide (LPS) by in vitro culturing at 37°C (typical body temperature), microbial infectivity becomes limited, and thus can serve as the basis of an effective, noninfectious, whole-organism vaccine. Limiting the infectivity of Y pestis and the potency of its cell wall-derived endotoxin might well serve in as a treating the causative root for plague symptoms [87]. The LPS may be part of the therapy in some situations. Protollin, a proteasome-LPS adjuvant (combination of Neisseria meningitides and Shigella flexneri), significantly enhanced mouse survival when combined with Y. pestis antigens to provide protection from plague [88,89].

In two recently published patents, NHPs were used to study the efficacy of the therapies for Y. pestis infections. NHPs were protected from Y. pestis exposure after immunization with plant-produced (F1-LcrV-LicKM (thermostable enzyme lichenase from Clostridium thermocellum) double fusion protein) (novel expression system) antigens [90]. No Y. pestis bacteria were evident in surviving NHP organs. A second biologic, flagellin, and F1/V antigen enhanced an appropriate antibody response in mice, suggesting that it may protect against exposure to Y. pestis [91]. Mice treated with this biologic had a 93% survival rate compared to 7% in the control group after administration of 100 LD₅₀ plague bacteria. The antibody efficacy is B-cell mediated, as further control tests revealed 100% mortality in both groups after exposure to 150 LD₅₀ doses of Y pestis in a B-cell knock-out mouse strain. Similarly, using the host’s natural immune system may allow for better protection. A novel trivalent nanoemulsion-based vaccine administered in mice demonstrated an antibody pattern that mimics that of untreated individuals that survive the plague [92]. Mice administered with this trivalent nanoemulsion vaccine demonstrated 90% protection when exposed to 150 LD₅₀. This vaccine can be used effectively through intranasal or im routes.

4. Conclusion

The availability of CBRN countermeasures has become a critical US homeland security concern since the terrorist attacks in 2001. The challenges facing medical countermeasure development and availability are complex. Generally, medical countermeasure development and approval must follow the FDA’s arduous product review process. Yet, when human efficacy data are limited, the scientific and regulatory obstacles may be greater than the challenges faced during regular drug development. Furthermore, medical product development is very costly in terms of financial and research resources; it is roughly approximated that the cost to develop a new medical product spans from ~$1 million to well over ~$1 billion and the likelihood of failure is considerable (i.e. >85%) [4,93]. Although the 2004 Project BioShield Act funded a government market to procure additional medical countermeasures, the available funds are small compared to the possible monetary return made by developing and marketing a successful drug intended for other indications [94]. These challenges and the absence of a commercial market for many CBRN countermeasures have left those successful drug companies most experienced at navigating the complex regulatory process hesitant to enter into the business of CBRN countermeasures development. Stepping up are smaller biotechnology companies that are often technically strong, but have little or no experience developing any drug through the FDA review and approval process. Therefore, it is important that the FDA provides more guidance to such companies throughout the development process. This approach is not uncommon to the FDA as there are a large number of countermeasures for various threats at different stages of development and several recent review articles cover this area of development [95–97].

The DHHS as well as the DoD are committed to develop medical countermeasures for CBRN threats. Interagency interaction, cooperation, and support are essential for long-term goals to be met successfully and efficiently within and across the civilian and military sectors. These efforts will be important to allocate and dispense medical countermeasures to those who will need them in order to prevent, mitigate, and/or treat the detrimental effects of CBRN injuries. A critical consideration for medical countermeasure development is the diversity of the civilian population. In contrast to the DoD programs, which largely focus on the needs of a young, healthy military population, the Biomedical Advanced Research and Development Authority’s (BARDA, DHHS) efforts account for the total civilian population, including infants, children, pregnant women, elderly, and immunocompromised individuals. The special medical needs of each of these subpopulations must be predicted and accounted. One of BARDA’s approaches is to repurpose drugs already approved by the FDA for other indications; this strategy decreases risk within the development pipeline because these drugs have already proven some measure of safety in these select populations and often have well-defined biochemical targets that are relevant to chemical threats.

5. Expert opinion

Several factors have contributed to the mounting threat of a major crisis which would result in enormous numbers of CB exposure-related casualties. Despite the robust International Health Regulations Treaty that gave exceptional authority to World Health Organization after the 2005 severe acute respiratory syndrome outbreak, cutbacks in funding rapidly reduced the preparedness capability of responding to such an incident, especially before the 2015 Ebola crisis. This situation emphasizes the need for additional funding so that multiple countermeasures against various threats can be developed, stockpiled, and efficiently deployed under such circumstances. The US government has taken appropriate steps to develop a procurement system for CBRN threat countermeasures to protect citizens in the event of a public health emergency. The government has created a market and encouraged developers of medical countermeasures to participate in programs such as Project BioShield, BARDA, and Pandemic and All Hazards Preparedness Act [98]. The continued success of these programs will depend largely upon whether they are funded appropriately. The DHHS and DoD are dedicated to developing and implementing medical countermeasures for both civilian and military personnel. It is encouraging to note that nine countermeasures have received US FDA approval for CB threats; five of these countermeasures have been approved for anthrax and Ciprofloxacin has approval for both anthrax and plague.

The mission of the Medical Countermeasure Initiative is to promote the development of countermeasures for CBRN threats and emerging infectious diseases. They do so by enhancing the FDA’s regulatory processes, fostering the establishment of clear regulatory pathways for medical countermeasures, and establishing effective regulatory policies and mechanisms to facilitate timely access to medical countermeasures. The current regulatory framework and the lack of regulatory science create both real and perceived barriers for developers who are keen to develop medical countermeasures. Among the most important challenges faced during medical countermeasure development assessment and approval, are the regulatory uncertainties, which are driven by gaps in scientific knowledge. Developers perceive a higher-than-typical risk for engaging in novel medical countermeasure development if they do not identify a clear development pathway. Reducing regulatory uncertainty is one of the most important challenges that the US government must successfully address.

The threat of an international anthrax outbreak, and the search for improvements to the anthrax prophylaxis tools currently available, will likely remain a subject of great interest within the realm of biodefense for the foreseeable future. Efforts to gain FDA approval for a second-generation recombinant, protective antigen-based anthrax vaccine are ongoing and significant progress has been made. Additional vaccine approaches utilizing multiple components of the B. anthracis as immune response targets are under active investigation. In brief, a multi-pronged approach targeting distinct aspects of the B. anthracis seems like a logical objective for future anthrax vaccine.

There are several encouraging medical countermeasures under advanced stages of development for CB threat agents; IMVAMUNE, the Vaccinia Ankara virus vaccine for small pox, is a live, attenuated, and modified virus that may get FDA approval in near future. It has already been approved by the European Medical Agency for human use. There are additional antivirals under development for smallpox [36,94].

After 11 September 2001, and subsequent anthrax attack, the DHHS initiated large-scale development of products with FDA approval for other indications and to some extent unapproved products, to be used in the event of a mass causality scenario. Prior to this action, the only option for marketing unapproved products was an IND protocol requiring Institutional Review Board approval, documented informed consent from the patients, substantial record keeping, and follow-up requirements, even in the event of an emergency. Although the IND mechanism was appropriate for a clinical study and an emergency situation for an individual, this was unsuitable for mass causality scenario, particularly when essential components of the biodefense armamentarium were either unapproved or approved for different indications at that time [36,98].

Project BioShield of 2004 established the Emergency Use Authorization (EUA) program which allows the FDA to approve the emergency use of drugs without previous approval or the off-label use of approved products in a well-defined emergency situation. The EUA provides physicians and public health officials with new tools for care under emergency conditions [99]. It is imperative to note that countermeasures showing promising results do not need FDA approval or licensure to be produced in large quantities and then stockpiled in SNS in preparation of a mass causality scenario. Several medical countermeasures were procured for the SNS under Pandemic and All Hazards Preparedness Act, when the agents had only IND status, long before receiving FDA approval [98].

Article highlights

• Nine countermeasures have been approved by the FDA against chemical and biological threats following the Animal Rule.

• Pyridostigmine bromide and Cyanokit (hydroxocobalamin) have received FDA approval to combat effects of exposure to nerve gas (Soman) and cyanide poisoning, respectively.The FDA has approved Heptavalent botulinum antitoxin (HBAT; equine immunoglobulin G fragments) to treat botulism resulting from Clostridium botulinum toxin.

• Five countermeasures, Anthrasil (human immunoglobulin G), Raxibacumab (monoclonal antibody), Levaquin (Levofloxacin), BioThrax (vaccine) and Cipro (ciprofloxacin hydrochloride) have FDA approval for use against Bacillus anthracis.

• Avelox (moxifloxacin, an antibacterial agent), Levaquin (a member of the fluoroquinolone family), and Cipro have received FDA approval for the treatment of Yersinia pestis, including pneumonic and septicemic plague.

• Several additional countermeasures against CB threats have been patented and some are under advance development using Animal Rule.This box summarizes key points contained in the article.

Declaration of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Acknowledgments

The opinions or assertions contained herein are the professional views of the authors and do not necessarily represent the Armed Forces Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, or the Department of Defense, USA. Authors are thankful to Victoria L Newman for editorial help and to Patricia L.P. Romaine for patent searches. Mention of specific therapeutic agents does not constitute endorsement by the U.S. Department of Defense, and trade names are used only for the purpose of clarification. We apologize to those having contributed substantially to the topics discussed herein that we were unable to cite due to space constraints.

Additional information

Funding

The authors gratefully acknowledge the research support from Congressionally Directed Medical Research Programs (W81XWH-15-C-0117, JW140032) of the US Department of Defense to VK Singh.