(12) United States Patent (10) Patent No.: US 9,629,875 B2 Lentini Et Al

US009629875B2

(12) United States Patent (10) Patent No.: US 9,629,875 B2 Lentini et al. (45) Date of Patent: Apr. 25, 2017

(54) CLOSTRIDIUM DIFFICILE SPORCIDAL (58) Field of Classification Search COMPOSITIONS None See application file for complete search history. (71) Applicant: Microdermis Corporation, Princeton, NJ (US) (56) References Cited (72) Inventors: Peter J. Lentini, Tarrytown, NY (US); U.S. PATENT DOCUMENTS Dawn Lembo, Leonia, NJ (US) 4,271,149 A 6, 1981 Winicov et al. 5,665,307 A 9, 1997 Kirschner et al. (73) Assignee: Microdermis Corporation, Princeton, 5,885,620 A 3, 1999 Foret NJ (US) 2009/0324737 A1* 12/2009 Walker ...... A61K 8,347 424,616 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U.S.C. 154(b) by 0 days. WO WO-00,054594 A1 9, 2000 (21) Appl. No.: 14/683,276 OTHER PUBLICATIONS Filed: Apr. 10, 2015 (22) Kelemen WO 2000/054594 A1, Sep. 2000.* Prior Publication Data Gershenfeld, L. and Witlin, B., Iodine as an antiseptic, Ann. NY (65) Acad. Sci., 53(1): 172-82 (1950). US 2016/0058,789 A1 Mar. 3, 2016 International Search Report for PCT/US2015/025259, 3 pages (Jun. 26, 2015). Related U.S. Application Data Written Opinion for PCT/US2015/025259, 8 pages (Jun. 26, 2015). (60) Provisional application No. 61/978,451, filed on Apr. * cited by examiner 11, 2014. Primary Examiner — Robert A Wax (51) Int. Cl. Assistant Examiner — Quanglong Truong A6 IK3I/74 (2006.01) (74) Attorney, Agent, or Firm — Choate, Hall & Stewart A6 IK 33/18 (2006.01) LLP: Brenda Herschbach Jarrell A6 IK3I/I94 (2006.01) AOIN 37/36 (2006.01) (57) ABSTRACT AOIN 59/2 (2006.01) Disclosed are sporicidal compositions, and methods of use AOIN 43/36 (2006.01) thereof. The sporicidal compositions contain water, an A6 IK 9/00 (2006.01) organic Solvent, and a spore-germinating agent containing A 6LX 3L/79 (2006.01) an iodide source, a citrate source or both. The compositions (52) U.S. C. have sporicidal activity against, e.g., Clostridium difficile CPC ...... A61K 33/18 (2013.01); A0IN 37/36 spores. The sporicidal compositions can be used alone, or (2013.01); A0IN 43/36 (2013.01); A0IN can form part of a disinfecting composition or an antiseptic 59/12 (2013.01); A61K 9/0014 (2013.01); composition. A61K 31/194 (2013.01); A61 K3I/79 (2013.01) 17 Claims, 2 Drawing Sheets U.S. Patent Apr. 25, 2017 Sheet 1 of 2 US 9,629,875 B2

------C. difficile Spore Count after Contact

8 CFU/mL (x10^5)

FG, U.S. Patent Apr. 25, 2017 Sheet 2 of 2 US 9,629,875 B2

1000000 g------

100000 - : : x contro 8& control 10000 :- ; : & FF-B. & FF-B. 1000 --~ w8w, FF.B.2 w8.FF.B.2 O ------

10 gr:rixx-x-xx-r FF-G2

... FF-G2 1 -: initia 2 s O

F.G. 2 US 9,629,875 B2 1. 2 CLOSTRIDIUM DIFFICILE SPORCIDAL FIG. 2 shows reduction in C. difficile spore count after COMPOSITIONS contact with provided compositions according to Example 32. BACKGROUND DETAILED DESCRIPTION OF CERTAIN Spore-forming Clostridium difficile-associated diseases EMBODIMENTS (CDAD) remain an important nosocomial infection associ ated with significant morbidity and mortality. In recent years, the incidence of CDAD has unfortunately increased Definitions and Usage and high rates of recurrent disease continue with currently Unless otherwise specified, the word “includes” (or any available treatment regimens. Typically, Clostridium difficile 10 variation thereon, e.g., “include”, “including, etc.) is is transmitted by the fecal-oral route. Spores that persist in intended to be open-ended. For example, “A includes 1, 2 the environment Survive the gastric acid barrier and germi and 3’ means that A includes but is not limited to 1, 2 and nate in the colon. Toxins released from vegetative C. difficile 3. cells are responsible for clinical CDAD. Unless otherwise specified, the word “about’, when used Vegetative C. difficile can only survive 15 minutes aero- 15 to modify a numeric quantity, meanst8% of the numeric bically, but the bacteria are nonetheless very difficult to value. Thus, “about 5” means 4.6-5.4, “about 70” means eradicate because they form spores. C. difficile spores can be 64-76, etc. For this purpose, percentage values are consid found as airborne particles, attached to inanimate Surfaces ered as their nominal quantities, e.g., 5% is treated as 5 Such as hard Surfaces and fabrics, and attached to Surfaces of rather than 0.05. Thus, “about 5% means 4.6%-5.4%, living organisims, such as skin and hair. Spores can be found 20 “about 70% means 64%-76%, etc. on a patient’s skin as well as on any surface in the room that As used herein, the term "sporicidal' has the meaning the infected patient occupied. During exams these spores ascribed to it in the section labeled “Sporicidal Composi can be transferred to the hands and body of healthcare tions', below. workers and thereby spread to Subsequent equipment and Clostridium difficile Spores areas they contact. 25 Clostridium difficile, also known as "CDF/cdf or “C. Hospital discharges for CDAD in the United States diff", a species of Gram-positive, spore-forming anaerobic doubled between 1996 and 2003. These nosocomial infec tions are extremely costly to hospitals at S1.28 to S9.55 bacillus, can lead to severe complications ranging from billion annually in the U.S. alone, mostly due to infected antibiotic-associated diarrhea (AAD) to severe life-threat patients requiring extended stays of 3.6 to 14.4 days. Com ening pseudomembranous colitis, a severe infection of the plications of CDAD include life-threatening diarrhea, 30 colon. In fact, C. difficile is the cause of approximately 25% pseudo-membranous colitis, toxic megacolon, sepsis, and of all cases of antibiotic-associated diarrhea. Most cases of death. Expenses related to treatment of these conditions C. difficile-associated disease (CDAD) occur in hospitals or ranges from S3,669 to S27,290 per patient. CDAD causes long-term care facilities causing more than 300,000 cases death in 1-2% of affected patients. per year in the United States alone. The total US hospital People are most often infected in hospitals, nursing 35 costs for CDAD management have been estimated to be $3.2 homes, or institutions, although C. difficile infection in the billion per year. community, outpatient setting is increasing. C. difficile Clostridia are motile bacteria that are ubiquitous in nature infection (CDI) can range in severity from asymptomatic to and are especially prevalent in soil. Under microscopy, severe and life-threatening, especially among the elderly. clostridia appear as long drumstick-like irregularly-shaped The rate of C. difficile acquisition is estimated to be 13% in 40 cells with a bulge at their terminal ends. Clostridium difficile patients with hospital stays of up to 2 weeks, and 50% in cells show optimum growth on blood agar at human body those with hospital stays longer than 4 weeks. temperatures in the absence of oxygen. When stressed, the While currently available antibiotics used for treatment of bacteria produce spores, which tolerate extreme conditions recurrent CDAD lead to symptomatic improvement, they are that the active bacteria cannot tolerate. essentially ineffective against C. difficile spores, the trans- as In Small numbers, C. difficile do not result in significant missible form of the disease. This causes a high risk of relapse occurring post-therapy as sporulated microorgan disease. The first step in development of C. difficile coloni isms begin to germinate. Therefore, controlling C. difficile zation is the disruption of the normal flora of the colon, infection requires limiting the spread of spores by good usually by antibiotics. Antibotic treatments, especially those hygiene practices, isolation and barrier precautions, and with a broad spectrum of activity, cause disruption, often environmental cleaning. 50 resulting from eradication of the normal intestinal flora by Because of the prevalence of C. difficile in hospitals, antibiotics of normal intestinal flora, leading to an over healthcare workers and researchers have an interest in growth of C. difficile. C. difficile is currently the most developing an agent that can kill C. difficile and its spores. common cause of nosocomial diarrhea with significant mor bidity and mortality. The C. difficile bacteria, which natu SUMMARY 55 rally reside in the human intestines, overpopulate and release toxins that can cause bloating, constipation, or The present invention encompasses the insight that both diarrhea with abdominal pain, which may become severe. citrate and iodide are C. difficile germinants, i.e., they Latent symptoms often mimic Some flu-like symptoms. facilitate germination of C. difficile spores to the vegetative Antibiotic treatment of C. difficile infections can be dif state, where the bacterium is more susceptible to being killed 60 ficult, due both to antibiotic resistance as well as physiologi by stressors, such as antimicrobial agents, heat, and air. cal factors of the bacteria itself. Because the organism forms acid- and heat-resistant spores, C. difficile spores can persist BRIEF DESCRIPTION OF THE DRAWING in the environment for years, and contamination by C. difficile is very common in hospital, clinical, long-term care FIG. 1 shows reduction in C. difficile spore count after 65 or nursing home environments. Often, it can be cultured contact with provided compositions according to Example from almost any surface in a hospital. Patient-to-patient 3.1. transmission of C. difficile spores occurs by sharing medical US 9,629,875 B2 3 4 equipment or facilities in hospitals, nursing homes and other (NH4)2SO4 (40 mg/L), FeSO4.7H2O (4 mg/L), CoCl2.6H2O extended-care facilities. Typically, C. difficile is transmitted (1 mg/L), NaHCO, (5000 mg/L) and taurocholic acid (1000 from person to person by the fecal-oral route. Ingested mg/L). However, the authors note that one limitation of their spores of C. difficile survive the gastric acid barrier and study is that it is not known which components of the germinate in the colon. Vegetative cells release two potent 5 Solution are essential to stimulate germination to a stage in toxins that ultimately mediate diarrhea and colitis. which enhanced killing by stressors such as UV-C are Given the pathogenesis of C. difficile, judicious use of possible. Among other things, the present invention encom antibiotics and strict infection control and environmental passes the recognition of the source of a problem with the measures are keys to the prevention of disease. The imple Nerandzic solution. To be practical, or economical, or both, mentation of antibiotic stewardship programs has been asso 10 a spore-germinating composition should contain fewer com ciated with decreased incidence of CDAD. To prevent ponents than the 18 amino acids, 10 minerals and one bile spread of spores, environmental cleaning and patient isola salt of the Nerandzic solution. tion are needed. Several disinfectants commonly used in In a similar vein, Hoffman (U.S. Patent Application hospitals may be ineffective against C. difficile spores, and Publication No. 2011/0135702) reports the following as C. may actually promote spore formation. 15 difficile spore-germinating agents: sodium taurocholate, gly C. difficile spores are resistant to killing by alcohol-based cocholate, cholate, glycine and combinations thereof. Hoff hand hygiene products and by antimicrobial Soaps that are man notes that based on the reported experiments, antimi commonly used in healthcare facilities. M. M. Nerandzic crobial agents alone or in combination with a surfactant had and C. J. Donskey (2010), “Triggering Germination Repre little effect on inactivating C. difficile spores, but that spori sents a Novel Strategy to Enhance Killing of Clostridium cidal efficacy was seen when in combination with one or difficile Spores”. PLoS ONE 5(8): e12285, 1-8 (“Nerandzic et more spore-germinating agents. In the context of the present al.”) (citing K. J. Popovich et al. (2009), Infect. Control invention, it is noteworthy that relatively small reductions Hosp. Epidemiol. 30: 959-63). C. difficile spores survive for were seen for the spore-germinating agents alone. months on Surfaces and are resistant to killing by many Walker (U.S. Pat. No. 7,192,601) reports a composition commonly used disinfectants. Nerandzic et al. (citing D. N. 25 having an antibacterial agent and a “spore coat opener”. Gerding et al. (2008), Clin. Infect. Dis. 46: S43–49). More Walker lists the following as an antibacterial agent: a qua over, low levels of Some disinfectants may actually promote ternary ammonium compound, a phenolic compound, a increased sporulation by C. difficile. Nerandzic et al. (citing monohydric alcohol, hydrogen peroxide, iodine, triclocar W. N. Fawley et al. (2007), Infect. Control Hosp. Epidemiol. ban, triclosan, and combinations of these. As a spore coat 28: 920–25: M. H. Wilcox and W. N. Fawley (2000), Lancet 30 opener Walker lists an amino acid, a metal chelation agent, 356: 1324). Sodium hypochlorite (bleach) is a disinfectant a reducing agent or an emulsifier, a surface-active agent, and with sporicidal activity, but it has several disadvantages, combinations of these, with examples such as "amino acid including being corrosive to many materials, irritating to mixture, L-alanine, calcium dipicolinate, disodium EDTA, Some patients and staff members, and dependent on correct quinone, proanthocyanidin and “reducing agent'. application by housekeepers. Nerandzic et al. (citing F. 35 Walker reports the results of various spore-inactivating Barbut Fet al. (2009), Infect. Control Hosp. Epidemiol. 30: compositions on Bacillus subtilis VS. exposure time. The 507-14.). There is a need to develop a disinfectant that is spore-coat opener (in the compositions that contain a spore nonreactive to untargeted materials and nonharmful to coat opener) is reported to be L-alanine. In addition to the humans and environment. sporicidal compositions, Walker also reports a colony-form Spore-Germinating Agents 40 ing unit (CFU) relative count for compositions containing Spore germination, the irreversible loss of spore-specific 0.1% L-alanine in the absence of sporicidal agents. In the properties, is an essential step required prior to outgrowth of context of the present invention, it is noteworthy that an vegetative cells. Because germinated spores become more increase in CFU count—to 116% after 4 hours—was Susceptible to killing by other stressors, induction of germi reported for the spore-coat opener alone. nation is a strategy to facilitate eradication of C. difficile 45 Notwithstanding these reports, Worthington (U.S. Patent spores. See, e.g., M. M. Nerandzic and C. J. Donskey Application Publication No. 2013/0142856) reports that this (2010), “Triggering Germination Represents a Novel Strat 'germinate/exterminate' approach has had limited Success egy to Enhance Killing of Clostridium difficile Spores'. in the clinical setting. It reports in particular that attempts PLoS ONE 5(8): e12285, 1-8 (“Nerandzic et al.”). Nerandzic have been made in the literature to design antibacterial et al. report testing triggering germination as a strategy to 50 Solutions which stimulate germination and which have an enhance UV-C-induced killing of C. difficile spores on antibacterial agent to attack the germinated pathogens, but Surfaces, thereby reducing the time and radiation dose that the antibacterial agents used in Such approaches have an necessary for disinfection of hospital rooms with the UV-C inhibitory effect on the germinating Solution. device. In addition, Nerandzic et al. report evaluating the Sporicidal Compositions potential for initiation of germination to enhance killing of 55 Provided are sporicidal compositions containing water, an C. difficile spores by heat, alcohol, and exposure to room air. organic solvent, and a spore-germinating agent, as further Nerandzic et al. report the following aqueous formulation defined herein. As used herein, the term “sporicidal refers as a C. difficile spore-germination solution: histidine (100 to the ability to reduce the number of spores in a given area. mg/L), tryptophan (100 mg/L), glycine (100 mg/L), tyrosine In this sense, it is irrelevant whether a provided composition (100 mg/L), arginine (200 mg/L), phenylalanine (200 60 is able to kill a bacterium in its spore form, or, as is believed, mg/L), methionine (200 mg/L), threonine (200 mg/L), ala it is able to facilitate germination of a spore to the vegetative nine (200 mg/L), lysine (300 mg/L), serine (300 mg/L), state where the bacterium is more susceptible to being killed valine (300 mg/L), isoleucine (300 mg/L), aspartic acid (300 by stressors (such as air, antimicrobial agents, etc.). Accord mg/L), leucine (400 mg/L), cysteine (500 mg/L), proline ingly, as used herein, the term "sporicidal' is meant to be (600 mg/L), glutamic acid (900 mg/L). KHPO (300 mg/L). 65 understood by reference to an observable effect rather than NaHPO (1500 mg/L), NaCl (90 mg/L), CaCl2.H2O (26 to a mechanism of action. Such compositions are useful, for mg/L), MgCl2.6H2O (20 mg/L), MnCl2.4H2O (10 mg/L), example, for reducing the count of Clostridium spores, US 9,629,875 B2 5 6 particularly Clostridium difficile spores, on living tissue citrate, magnesium citrate tribasic or calcium citrate tribasic, Surfaces (e.g., skin) and/or on non-living tissue surfaces. In or a mixture of two or more of these. In some embodiments, Some embodiments, provided compositions reduce the citrate source is citric acid, Sodium citrate monobasic, Clostridium spore counts on or in an environmental site. Sodium citrate dibasic, sodium citrate tribasic, potassium Among other things, in Some embodiments, such composi citrate monobasic, potassium citrate dibasic or potassium tions reduce the likelihood of infection or the spread of citrate tribasic, or a mixture of two or more of these. In some infection from the spore-forming bacteria. embodiments, the citrate source is citric acid, sodium citrate As shown in Example 3.1, in some embodiments, pro monobasic, sodium citrate dibasic or sodium citrate tribasic, vided compositions exhibit sporicidal activity even in the or a mixture of two or more of these. In some embodiments, absence of antimicrobial agents. Without wishing to be 10 bound by theory, it is believed that the citrate and iodide the citrate Source is citric acid, Sodium citrate tribasic, or a Sources facilitate germination of C. difficile spores to the mixture thereof. In some embodiments, the citrate source is vegetative state, where the bacterium is more susceptible to Sodium citrate. In some embodiments, the citrate source is being killed by stressors, including air. As described in this citric acid. In some embodiments, the citrate source is silver Example, spore count reductions of about 88% to about 92% 15 dihydrogen citrate. were seen after 8 minutes with compositions of water, Because the provided compositions are aqueous, a pro propylene glycol and sodium citrate (A1 compositions) or vided composition containing a citrate source will contain potassium iodide (A2 compositions). Similarly, provided one or more citrate species in differing amounts according to compositions formulated such that they are suitable for various factors, such as other solvents present, other ions topical administration (PP compositions) showed spore present, temperature, etc. Therefore, as is customary in the count reductions of 78% to about 84% after 8 minutes. art, in some embodiments a provided composition contain As shown in Example 3.2, in some embodiments, pro ing a citrate source is described by reference to its constitu vided compositions containing antimicrobial agents simi ent components prior to being combined, e.g., “water, pro larly exhibit sporicidal activity. Without wishing to be bound pylene glycol, and sodium citrate', recognizing that sodium by theory, it is believed that the citrate and iodide sources 25 citrate in aqueous solution at equilibrium will exist as one or facilitate germination of C. difficile spores to the vegetative more citrate species. A weight percentage (“w/w') of Such a state, where the bacterium is more susceptible to being killed component refers to the amount of the component as mea by stressors, including antimicrobial agents. However, con Sured prior to combination. For example, a composition trary to the limited Success of this germinate/exterminate resulting from the combination of 79.50 g water, 20 g approach reported by Worthington, provided compositions 30 containing antimicrobial agents exhibit more pronounced propylene glycol and 0.50 g sodium citrate can be described sporicidal activity than those without. Thus, spore count as a composition containing “water (79.50% w/w), propyl reductions of about 99% to about 100% are seen after 1 ene glycol (20% w/w) and sodium citrate (0.50% w/w). minute, and remain at about 100% after 10 minutes, with In some embodiments, a provided composition containing compositions containing 10% PVP-I. 35 a citrate source is described as containing a percentage Spore-Germinating Agents: Citrate Source. “citrate'. In many such embodiments, the weight percentage In some embodiments, provided compositions include a “citrate” is determined without regard to the remainder of spore germinating agent that is or includes a citrate source. the citrate source, and considering the tribasic form of citric Because citric acid, C(OH)(CH-COH)2COH, is a triprotic acid, i.e., HOC(COO )(CHCOO). For example, sodium weak acid, in aqueous solution it exists as a mixture of 40 citrate is 64.3% w/w "citrate', because the molecular weight citrate species: HCH-O, HCHO,-, HCH-O, and of HOC(COONa)(CHCOONa).2H2O is 294.1 and the CHO,. As used herein, a "citrate source' is any chemical molecular weight of HOC(COO-)(CHCOO) is 189.1, species that produces or exists at least in part as citric acid which is 64.3% of 294.1. Accordingly, a provided compo and/or one or more citrate ions in aqueous solution. sition described as containing 0.25% w/w sodium citrate Examples of citrate sources include citric acid, sodium 45 could equally be described as containing 0.16% w/w citrate. citrate monobasic, Sodium citrate dibasic, sodium citrate Such descriptions are especially useful when more than one tribasic, potassium citrate monobasic, potassium citrate Source of citrate is used to make a provided composition. dibasic, potassium citrate tribasic, magnesium citrate, mag For example, a provided composition described as con nesium citrate tribasic, calcium citrate tribasic (tricalcium taining 0.16% w/w citrate could be prepared in any number dicitrate), silver citrate (citric acid trisilver salt) and silver 50 of various ways and/or through use of any of a variety of dihydrogen citrate, including anhydrous forms and hydrates citrate sources. The % citrate of some exemplary citrate (both of which are intended at every mention herein unless Sources is shown below. otherwise specified). As used herein, “sodium citrate” refers to HOC(COONa)(CHCOONa).2HO, and is used inter citrate source MW % citrate changeably with sodium citrate dihydrate, citric acid triso 55 dium salt dihydrate, sodium citrate tribasic dihydrate and sodium citrate tribasic dihydrate 2.94.1 64.3 trisodium citrate dihydrate. Sodium citrate monobasic (anh.) 214.1 88.3 Sodium citrate dibasic sesquihydrate 263.1 71.9 In some embodiments, the citrate source is citric acid, potassium citrate tribasic monohydrate 324.4 58.3 Sodium citrate monobasic, sodium citrate dibasic, sodium potassium citrate monobasic (anh.) 230.2 82.2 citrate tribasic, potassium citrate monobasic, potassium cit 60 magnesium citrate 214.4 88.2 rate dibasic, potassium citrate tribasic, magnesium citrate, magnesium citrate tribasic nonahydrate 61.3.3 61.7 magnesium citrate tribasic, calcium citrate tribasic, silver (contains 2 equivalents of citrate) (378.2/613.3)*100 citrate or silver dihydrogen citrate, or a mixture of two or more of these. In some embodiments, the citrate source is From Such values, one skilled in the art can easily citric acid, Sodium citrate monobasic, sodium citrate dibasic, 65 determine appropriate weight percentages of any given Sodium citrate tribasic, potassium citrate monobasic, potas citrate source(s). Weight percentages of exemplary citrate sium citrate dibasic, potassium citrate tribasic, magnesium sources equivalent to 0.16% w/w citrate are shown below. US 9,629,875 B2 8 example, a composition resulting from the combination of % ww citrate source % wiw citrate 79.50 g water, 20 g propylene glycol and 0.50g potassium citrate source (measured) (calculated) iodide can be described as a composition containing “water sodium citrate tribasic dihydrate O.25 O16 (79.50% w/w), propylene glycol (20% w/w) and potassium Sodium citrate monobasic (anh.) O.18 O16 iodide (0.50% w/w). Sodium citrate dibasic sesquihydrate O.22 O16 potassium citrate tribasic monohydrate 0.27 O16 In some embodiments, a provided composition containing potassium citrate monobasic (anh.) O.19 O16 an iodide source is described as containing a percentage magnesium citrate O.18 O16 “iodide'. In these embodiments, the weight percentage magnesium citrate tribasic nonahydrate O.26 O16 “iodide' is determined without regard to the remainder of 10 the iodide source. For example, potassium iodide is 76.4% Those skilled in the art can readily calculate the appropriate w/w “iodide', because the molecular (atomic) weight of KI amounts of citrate Source(s) to use in a composition to obtain is 166.0 and the molecular (atomic) weight of F is 126.9, the desired '% w/w citrate. Those skilled in the art will also which is 76.4% of 166.0. Accordingly, a provided compo recognize that solubility limits of different citrate sources 15 sition described as containing 0.25% w/w potassium iodide will set upper limits of weight percentages achievable in a could equally be described as containing 0.19% w/w iodide. composition. Such descriptions are especially useful when more than one Spore-Germinating Agents: Iodide Source. Source of iodide is used to make a provided composition. In some embodiments, provided compositions include a In a manner analogous to that described above for citrate spore germinating agent that is or includes an iodide source. Sources, those skilled in the art can easily determine appro Because iodide is the conjugate base of a strong acid (HI), priate weight percentages of any given iodide Source(s), and in aqueous Solution it exists essentially as the species I can readily calculate the appropriate amounts of iodide (iodide), but may also form more complex species. For the Source(s) to use in a composition to obtain the desired '% purposes of the present invention, the collective anionic w/w iodide. Those skilled in the art will also recognize that species in solution attributable to an iodide source will be 25 solubility limits of different iodide sources will set upper referred to as “iodide', recognizing both that other iodine limits of weight percentages achievable in a composition. containing species may be present and that they are custom Solvents arily not considered by those skilled in the art owing to their A provided composition contains an organic solvent. The extremely small concentration relative to I. As used herein, organic solvent can be a single organic liquid or a mixture an "iodide source' is any chemical species that produces or 30 of two or more organic liquids. Suitable organic solvents exists at least in part as iodide in aqueous solution. Examples include glycols, such as ethylene glycol (1.2-ethanediol). of iodide sources include potassium iodide (KI), sodium propylene glycols (1,2-propanediol ("propylene glycol); iodide (NaI), lithium iodide (Li), cesium iodide (CsI), Zinc iodide (ZnI), copper(I) iodide (CuI), calcium iodide (Cal.), 1,3-propanediol), butylene glycols, (1.2-butanediol (“buty barium iodide (Bal), magnesium iodide (Mg), ammonium 35 lene glycol); 1,3-butanediol: 1,4-butanediol: 2,3-butane iodide (NHI), tetrabutyl ammonium iodide diol), diethylene glycol (bis(2-hydroxyethyl) ether), and the ((CHCH2CHCH)NI) and boron triiodide (BI), includ like: glycerine; glycol ethers, such as ethylene glycol ing anhydrous forms and hydrates (both of which are monomethyl ether, ethylene glycol monoethyl ether, ethyl intended at every mention herein unless otherwise ene glycol monopropyl ether, ethylene glycol monoisopro specified). 40 pyl ether, ethylene glycol monobutyl ether, ethylene glycol In some embodiments, the iodide Source is potassium monophenyl ether, ethylene glycol monobenzyl ether, dieth iodide, Sodium iodide, lithium iodide, cesium iodide, Zinc ylene glycol monomethyl ether, diethylene glycol mono iodide, copper(I) iodide, calcium iodide, barium iodide, ethyl ether, diethylene glycol mono-n-butyl ether, ethylene magnesium iodide, ammonium iodide, tetrabutyl ammo glycol dimethyl ether, ethylene glycol diethyl ether, ethylene nium iodide or borontriiodide, or a mixture of two or more 45 glycol dibutyl ether, and the like; polyethylene glycols, such of these. In some embodiments, the iodide source is potas as PEG-200 and PEG-400; and dimethyl isosorbide. sium iodide, Sodium iodide, calcium iodide, magnesium In some embodiments, the organic solvent is glycerine, a iodide, ammonium iodide or tetrabutyl ammonium iodide, glycol, or a mixture thereof. In some embodiments, the or a mixture of two or more of these. In some embodiments, organic solvent is glycerine. In some embodiments, the the iodide source is potassium iodide, Sodium iodide, cal 50 organic solvent is a glycol. In some embodiments, the cium iodide or magnesium iodide, or a mixture of two or organic solvent is glycerine, propylene glycol, butylene more of these. In some embodiments, the iodide Source glycol, or a mixture of two or more of these. In some comprises is iodide, ammonium iodide or tetrabutyl ammo embodiments, the organic solvent is a mixture of glycerine nium iodide, or a mixture of two or more of these. In some and propylene glycol. In some embodiments, the organic embodiments, the iodide Source is potassium iodide. 55 Solvent is a mixture of glycerine and butylene glycol. In Because the provided compositions are aqueous, a pro Some embodiments, the organic solvent is a mixture of vided composition containing an iodide source will contain propylene glycol and butylene glycol. In some embodi iodide in an amount according to various factors, such as ments, the organic solvent is propylene glycol. In some other solvents present, other ions present, temperature, etc. embodiments, the organic solvent is butylene glycol. Therefore, as is customary in the art, in some embodiments 60 Additional Description of Certain Embodiments a provided composition containing an iodide Source is In some embodiments, a provided composition comprises described by reference to its constituent components prior to a spore-germinating agent that “consists essentially of one being combined, e.g., “water, propylene glycol, and potas or more recited components. Such language refers to a sium iodide', recognizing that potassium iodide in aqueous composition that can include, in addition to the specifically Solution at equilibrium will exist as iodide. A weight per 65 recited spore-germinating agent, a plurality of components centage (“w/w') of Such a component refers to the amount that do not significantly affect the spore-germinating activity of the component as measured prior to combination. For of the composition.

US 9,629,875 B2 11 12 {G18): (a) q is about 0.69% to about 0.84% w/w; (b) q. citrate dibasic or a hydrate thereof, or sodium citrate tribasic is about 0.58% to about 0.71% w/w: {G19}: (a) q is about or a hydrate thereof. In some embodiments, K6): a pro 0.73% to about 0.80% w/w; (b) q is about 0.61% to about vided sporicidal composition is as K1} or K2}, and the 0.68% w/w: {G20}: (a) q is about 0.76% w/w; (b) q is citrate source consists essentially of one or more of sodium about 0.64% w/w: {G21: (a) q is about 0.65% to about citrate tribasic dihydrate and citric acid. In some embodi 0.88% w/w; (b) q is about 0.73 to about 0.91% w/w: ments, {K7): a provided sporicidal composition is as {K1} {G22}: (a) q is about 0.69% to about 0.84% w/w; (b) q. or K2, and the citrate source consists essentially of one or is about 0.76% to about 0.88% w/w: {G23}: (a) q is about more of sodium citrate tribasic dihydrate. In some embodi 0.73% to about 0.80% w/w; (b) q is about 0.79% to about ments {K}: a provided sporicidal composition is as any one 10 of K1 to K7. 0.85% w/w: {G24}: (a) q is about 0.76% w/w; (b) q is Disinfecting Compositions about 0.82% w/w. In some embodiments {G*}: a provided In some embodiments, the invention provides a disinfect sporicidal composition is as any one of{G1 to G24}. ing composition, comprising a provided sporicidal compo In some embodiments, a provided sporicidal composition is as any one of {A*}, {B*}, {C*}, {D*}, {E*}, {F} or sition, and a disinfectant, wherein the provided sporicidal {G*}, and q is H1}: about 5% to about 50% w/w, H2}: 15 composition is as any one of {A*}, {B*}, {C}, {D}, about 10% to about 50% w/w, {H3}: about 5% to about 40% {E}, {F*}, {G*}, {H*}, {I*}, {J*} or {K*}. In some w/w, {H4}: about 10% to about 40% w/w, H5}: about 5% embodiments, the disinfectant comprises an alcohol disin to about 30% w/w, H6}: about 10% to about 30% w/w, fectant, an aldehyde disinfectant, an oxidizing disinfectant, {H7): about 10% to about 25% w/w, or H8): about 15% to a phenolic disinfectant, a quaternary ammonium disinfec about 25% w/w. In some embodiments H*}: a provided tant, chlorhexidine gluconate, polyhexamethylene biguanide sporicidal composition is as any one of {H1} to {H7. (polyhexanide; PHMB) or 4-chloro-3,5-dimethylphenol In some embodiments, I1}: a provided sporicidal com (chloroxylenol; p-chloro-m-xylenol; PCMX). position is as any one of {A*}, {B*}, {C}, {D*}, {E*}, In some embodiments, the disinfectant comprises an {F}, {G*} or {H}, and the organic solvent comprises alcohol disinfectant. In some embodiments, the alcohol glycerine, propylene glycol or butylene glycol. In some 25 disinfectant comprises ethanol or isopropanol. embodiments, {I2}: a provided sporicidal composition is as In some embodiments, the disinfectant comprises an any one of A*}, {B*}, {C}, {D*}, {E*}, {F}, {G*} or aldehyde disinfectant. In some embodiments, the aldehyde {H}, and the organic solvent consists essentially of glyc disinfectant comprises formaldehyde, glutaraldehyde or erine, propylene glycol, butylene glycol, or a mixture of two o-phthalaldehyde. or more of these. In some embodiments, I3}: a provided 30 In some embodiments, the disinfectant comprises an sporicidal composition is as any one of {A*}, {B*}, {C*}, oxidizing disinfectant. In some embodiments, the oxidizing {D*}, {E*}, {F*}, {G*} or {H*}, and the organic solvent disinfectant comprises sodium hypochlorite, calcium consists of glycerine, propylene glycol, butylene glycol, or hypochlorite, chloramine, chloramine-T, chlorine dioxide, a mixture of two or more of these. In some embodiments Sodium chlorite, Sodium chlorate, potassium chlorate, {1}: a provided sporicidal composition is as any one of I1} 35 hydrogen peroxide, iodine, peracetic acid, performic acid, to 13. potassium permanganate or potassium peroxymonosulfate. i. an embodiments, J1}: a provided sporicidal com In some embodiments, the disinfectant comprises a phe position is as any one of {A*}, {B*}, {C}, {D*}, {E*}, nolic disinfectant. In some embodiments, the phenolic dis {F}, {G*}, {H*} or {1}, and the spore-germinating agent infectant comprises phenol, o-phenylphenol, chloroxylenol, consists essentially of an iodide source or a combination of 40 hexachlorophene, thymol, amylmetacresol. 2,4-dichlo an iodide Source and a citrate source. In some embodiments, robenzyl alcohol or phenoxyethanol (Rose ether). {J2}: a provided sporicidal composition is as J1}, and the In some embodiments, the disinfectant comprises a qua spore-germinating agent consists essentially of an iodide ternary ammonium disinfectant. In some embodiments, the source. In some embodiments, J3}: a provided sporicidal quaternary ammonium disinfectant comprises benzalko composition is as J1} or J2}, and the iodide source 45 nium chloride, benzethonium chloride, methylbenzetho comprises potassium iodide. In some embodiments, J4): a nium chloride, cetalkonium chloride, cetylpyridinium chlo provided sporicidal composition is as J1} or (J2), and the ride, cetrimonium, cetrimide, dofanium chloride, iodide Source consists essentially of potassium iodide. In tetraethylammonium bromide, didecyldimethylammonium some embodiments J*}: a provided sporicidal composition chloride or domiphen bromide. is as any one of J1} to J4}. 50 In some embodiments, the disinfectant comprises chlo In some embodiments, K1}: a provided sporicidal com rhexidine gluconate. In some embodiments, the disinfectant position is as any one of {A*}, {B*}, {C}, {D*}, {E*}, comprises polyhexamethylene biguanide. In some embodi {F}, {G*}, {H*} or {1}, and the spore-germinating agent ments, the disinfectant comprises 4-chloro-3,5-dimethyl consists essentially of a citrate Source or a combination of a phenol. citrate Source and an iodide source. In some embodiments, {K2}: a provided sporicidal composition is as K1, and the 55 Antiseptic Compositions spore-germinating agent consists essentially of a citrate In some embodiments, the invention provides an antisep source. In some embodiments, K3}: a provided sporicidal tic composition, comprising a provided sporicidal compo composition is as {K1 or {K2}, and the citrate source sition, and an antiseptic, wherein the provided sporicidal comprises citric acid, sodium citrate monobasic or a hydrate composition is as any one of {A*}, {B*}, {C}, {D}, thereof, sodium citrate dibasic or a hydrate thereof, or 60 {E}, {F*}, {G*}, {H*}, {I*}, {J*} or {K*}. In some sodium citrate tribasic or a hydrate thereof. In some embodi embodiments, the antiseptic comprises an alcohol antiseptic, ments, {K4}: a provided sporicidal composition is as {K1} a quaternary ammonium antiseptic, chlorhexidine gluconate, or K2, and the citrate source comprises sodium citrate hydrogen peroxide, polyhexamethylene biguanide (poly tribasic dihydrate. In some embodiments, K5}: a provided hexanide; PHMB), 4-chloro-3,5-dimethylphenol (chlorox sporicidal composition is as {K1} or K2, and the citrate 65 ylenol; p-chloro-m-xylenol; PCMX), iodine, octenidine Source consists essentially of one or more of citric acid, dihydrochloride, sodium hypochlorite, calcium hypochlorite Sodium citrate monobasic or a hydrate thereof, sodium or Sodium bicarbonate. US 9,629,875 B2 13 14 In some embodiments, the antiseptic comprises an alcohol ments, a provided composition comprises about 10% to antiseptic. In some embodiments, the antiseptic comprises about 12% w/w PVP-I. In some embodiments, a provided chlorhexidine gluconate. In some embodiments, the antisep composition comprises about 10% to about 11.5% w/w tic comprises hydrogen peroxide. In some embodiments, the PVP-I. In some embodiments, a provided composition com prises about 10% to about 11% w/w PVP-I. In some embodi antiseptic comprises polyhexamethylene biguanide. In some ments, a provided composition comprises about 10% to embodiments, the antiseptic comprises 4-chloro-3,5-dimeth about 10.5% WFw PVP-I. ylphenol. In some embodiments, a provided composition comprises In some embodiments, the antiseptic comprises iodine. In about 5% to about 12% w/w PVP-I. In some embodiments, Some embodiments, the iodine is at least in part in the form a provided composition comprises about 5% to about 11% of an iodophor. In some embodiments, the iodophor com 10 w/w PVP-I. In some embodiments, a provided composition prises povidone-iodine (“PVP-I). Povidone-iodine is a comprises about 5% to about 10.5% w/w PVP-I. In some complex of polyvinylpyrrolidone (“PVP) and elemental embodiments, a provided composition comprises about 6% iodine. See, e.g., International Specialty Products, PVP to about 10.5% w/w PVP-I. In some embodiments, a pro Iodine. Povidone Iodine Antiseptic Agent (2004). vided composition comprises about 7% to about 10.5% w/w In some embodiments, a provided composition comprises 15 PVP-I. In some embodiments, a provided composition com about 8% to about 12% w/w PVP-I. In some embodiments, prises about 8% to about 10.5% w/w PVP-I. In some a provided composition comprises about 9% to about 11% embodiments, a provided composition comprises about 9% w/w PVP-I. In some embodiments, a provided composition to about 10.5% W/w PVP-I. comprises about 9.5% to about 10.5% w/w PVP-I. In some In some embodiments, an antiseptic composition is Suit embodiments, a provided composition comprises about able for topical administration. In some embodiments, a 9.8% to about 10.2% w/w PVP-I. In some embodiments, a topical antiseptic composition is a composition according to provided composition comprises about 10% w/w PVP-I. Example 2.3, 2.4 or 2.5. Examples of products suitable for In some embodiments, a provided composition comprises the components of these compositions are shown in the table about 10% to about 12.5% w/w PVP-I. In Some embodi below.

hydroxyethylcellulose Natrosol (R) CS, Natrosol (R) 250 HHR, Natrosol (R) 250 HR, Natrosol (R) 250 HX, Natrosol (R) 250 M, Natrosol (R) 250 MR, Sclerotium gum Amigel (R), Vegetensor (R), Actigum TM CS 6 benzalkonium chloride benzalkonium chloride (e.g., Spectrum Chemical, TCI America), Stepanguat (R) 50 NF, Stepanguat (R) 65 NF, Swanol TM CA-101 stearic acid Stearic acid (e.g., Jeen International Corp. (Stearic Acid NF), Protameen Chemicals, Parchem; grades with lower iodine number preferred), Bergazid TM C1835, Bergazid TM C1852, Bergazid TM, C1892, Bergazid TM C1895, Emersol (R) 7036 NF, Pristerine TM 4900, Pristerine TM 4911, Pristerine TM 9559 cetyl alcohol? cetyl alcohol (e.g., Jeen International Corp. (Cetyl Alcohol NF), Protameen Chemicals; grades with lower iodine number preferred), Crodacol TM C-70, Crodacol TM C90, Crodacol TM C90 EP, Crodacol TM C95, Kalcol TM 6098, Lanette (R) 16, Lipocol (R) C, Tego (R) Alkanol 16 polyquaternium-7 Cetarol TM PQ7, Conditioneze (R) 7, Dehyguart (R) 701/NA, Dehyguart (R) CC7 BZ, Mackernium TM 007, Mackernium TM 007S, Merquat (R) 550, Merquat (R) S, Mirapol (R) 550, Salcare (R) Super 7, Thorcocquat TM 550, Zetesoft TM PQ7 distearyldimonium chloride' Genamin (R) DSAC, Genamin (R). DSAC 75, Genamin (R). DSAP, Nikkol TM CA-3475, Protaquat (R) 2HT-75, Swanol TM CA-3475, Varisoft (R) TA100 glycerol monolaurate glycerol monolaurate (e.g., Jeen International Corp., Mosselman Oleochemicals), Lauricidin (R) PEG-20 methyl glucose Glucamate TM SSE-20, Glukosa TM SSE-20 sesquistearate Emulsifying Wax" Emulsifying Wax NF, PolawaxTM NF PEG-7 glyceryl cocoate Acconon (R) CO-7, Cetarol TM HE 7, Cetiol (R) HE, Cremophor (R) GC-7, GlyceroxTM HE, Hetoxide TMGC-7, Jeechem TM GC-7, Liponate (R) GMC-7, Protachem TM GC-7, Saboderm TM HE, Sympatems TM GMC 070 ammonium nonoxynol-4 Alipal TM CO 436, Rhodapex (R) CO-436 sulfate octoxynol 99 Igepal (R) CA-9, Protachem TM OP-9, Triton TMX-100 nonoxyno1-9' Jeechem TM NP-9, Protachem TM NP-9, Tergitol TMNP-9 4-hydroxyacetophenone 4-hydroxyacetophenone (e.g., Sigma-Aldrich, Spectrum Chemical), Sym Save (R) H 'n-octadecanoic acid 'hexadecan-1-ol copolymer of acrylamide and diallyldimethylammonium chloride 'dimethyldioctadecylammonium chloride 2,3-dihydroxypropyl dodecanoate poly(oxy-1,2-ethanediyl). O-hydro-0)-hydroxy-, ether with methyl 3-d-glucopyranoside (4:1), octadecanoate (2:3) (20 mol EO average molar ratio) 'cetearyl alcohol, PEG-150 stearate, polysorbate 60, steareth-20 poly(oxy-1 2-ethanediyl), C.C.O."-1,2,3-propanetriyltris-()-hydroxy-, monococonut acid ester (7 molEO average molar ratio) 'poly(oxy-1,2-ethanediyl). O-sulfo-0)-(nonylphenoxy)-, ammonium salt "poly(oxy-1,2-ethanediyl). O-4-(1,1,3,3-tetramethylbutyl)phenyl-(1)-hydroxy-(9 mol EO average molar ratio) '2-2-[2-(2-[2-(2-[2-(2-[2-(4-nonylphenoxy)ethoxyethoxyethoxyethoxylethoxy-ethoxyethoxylethoxylethanol US 9,629,875 B2 15 16 EXAMPLES 1. At 45° C., add the spore-germinating agent to the batch. Mix until uniform and make Sure no lumps are present. Example 1 2. If an additional spore-germinating agent is present in the composition, add it to the batch. Mix until uniform and Preparation of Sporicidal Compositions make Sure no lumps are present. Repeat this step for each additional spore-germinating agent, if any, present in the Example 1.1 composition. Phase VII. Topical Antiseptic Compositions Containing PVP-I 1. Begin cooling batch to 40° C. 10 2. In a separate vessel, add the following ingredients one at Topical antiseptic compositions containing PVP-I can be a time with propeller mixing: glycerine (if present in the prepared according to the following general procedure. composition), ammonium nonoxynol-4 Sulfate, octoxy Phase I. nol-9 and nonoxynol-9 1. Add DI Water (deionized water) to suitably sized vessel 3. Once the above ingredients are uniform, slowly sprinkle with propeller agitation. 15 povidone-iodine (PVP-I) into premixed liquids, using 2. Slowly sprinkle hydroxyethylcellulose into room tem propeller type mixing. Mix until uniform and no lumps perature water with propeller agitation. are present. 3. After addition complete, let Phase mix until hydroxyeth 4. Once this premixed phase is uniform and lump free, add ylcellulose is dissolved. entire Pre Mix into Batch in Main Kettle and mix until 4. If Sclerotium gum is also present in formula, slowly uniform with propeller type mixing. Make sure batch is sprinkle Sclerotium gum into room temperature water completely uniform. with propeller agitation. After addition complete, let 5. Once batch is completely uniform, change to a Sweep Phase mix until Sclerotium gum is dissolved. blade and continue to mix and cool until batch is at room 5. Once Sclerotium gum is dissolved, begin heating batch to 25 temperature (25° C.). 830-860 C. 6. Once temperature is reached, make Sure a viscous, clear Example 1.2 gel is formed. Product should be lump-free and uniform before adding next phase. Topical PVP-I Placebo Compositions . Add benzalkonium chloride. Mix until uniform. 30 . Add stearic acid. Mix until uniform and melted. Let this Placebo compositions PPA1.L3 and PPA2. L3 were pre phase mix for an additional 5-10 minutes. pared according to the general procedure described in Phase II. Example 1.1, but with the following differences. . Add cetyl alcohol. Mix until uniform and melted. 1. In Phase I, polycuaternium-10 is added and mixed fol . Add polycuaternium-7. Mix until uniform. 35 lowing addition of DI water and before addition of : . Add distearyldimonium chloride slowly to batch. Mix hydroxyethylcellulose. well until batch is uniform and no lumps are present. 2. Phase IV was conducted at 70° C. rather than 65° C. 4. Once batch is lump free, add triethanolamine to batch. 3. Phase VI was conducted at 50° C. rather than 45° C. After addition, mix batch for 15 minutes. 4. Phase VII was conducted at 50° C. rather than 40° C. 5. Make sure batch temperature remains at 83°-86°C. before 40 continuing to next phase. Phase III. Example 2 1. Maintain constant temperature of the batch at 83°-86° C. during the following additions. Constitution of Sporicidal Compositions 2. Add glycerol monolaurate to the batch. Mix until melted 45 and uniform with propeller mixing. Example 2.1 3. Add PEG-20 methylglucose sesquistearate to the batch. Mix until dispersed and uniform with propeller mixing. Sporicidal Compositions Lacking Antiseptic 4. Add Emulsifying Wax NF to the batch. Mix until melted and uniform with propeller mixing. Mix batch for addi 50 The indicated sporicidal compositions were composed as tional 5 minutes after addition is complete. shown in the following table. Phase IV. 1. Begin cooling the batch to 65° C. 2. At 65° C., add PEG-7 glyceryl cocoate to batch with sodium citrate potassium iodide propeller mixing. Mix until uniform. 55 identifier solvent (% w/w) (% w/w) (% w/w) Phase V. A1.L.1 DI HO 79.75 O.25 1. Cool batch to 60° C. propylene glycol 20 2. At 60° C., add disodium ethylenediamine tetraacetate to A1.L2 DI HO 79.50 OSO batch. Mix until completely uniform with propeller mix propylene glycol 20 A1.L3 DI HO 79.25 0.75 ing. If hydroxyacetophenone is present in the composi 60 propylene glycol 20 tion, pre-mix with 3% glycerine, add to batch and mix A2.L.1 DI HO 79.75 O.25 until completely uniform with propeller mixing. Make propylene glycol 20 A2.L2 DI HO 79.50 O.SO sure batch is lump free. propylene glycol 20 3. Begin slowly cooling batch to 45° C. A2.L3 DI HO 79.25 0.75 Phase VI. The spore-germinating agent can vary. In some 65 propylene glycol 20 formulations, it is potassium iodide, Sodium citrate, citric acid, a combination of two of these, or a mixture of all three. US 9,629,875 B2 17 18 Example 2.2 Component FF-B.1 FF-B.2

Topical PVP-I Placebo Compositions deinonized water 27.95 27.95 potassium iodide 0.75 O.SO PVP-I placebo compositions were composed as shown in 5 citric acid O.25 O.25 the following table (numbers are expressed in percentage glycerine O.OO S.OO butylene glycol 2O.OO 1S.OO w/w). powidone-iodine 10.00 1O.OO Sclerotium gum 41.05 41.30 benzalkonium chloride (50% solution) (total) (total) Component PPA1.L3 PPA2.L3 10 Stearic acid cetyl alcohol deinonized water 45.10 45.10 polyguaternium-7 sodium citrate 0.75 O.OO distearyldimonium chloride potassium iodide O.OO 0.75 Eriethanolamine propylene glycol 1S.OO 1S.OO glycerol monolaurate polycuaternium-10 39.15 39.15 PEG-20 methyl glucose sesquistearate hydroxyethylcellulose (total) (total) 15 Emulsifying Wax benzalkonium chloride (50% solution) PEG-7 glyceryl cocoate Stearic acid ethylenedia mine tetraacetate cetyl alcohol ammonium nonoxynol-4 Sulfate distearyldimonium chloride Octoxynol-9 triethanolamine nonoxynol-9 glycerol monolaurate PEG-20 methyl glucose sesquistearate Emulsifying Wax caprylicicapric triglycerides Example 2.5 ethylenediamine tetraacetate ammonium nonoxynol-4 Sulfate octoxynol-9 25 Topical Antiseptic Compositions Containing nonoxynol-9 Glycerine The compositions were prepared according to the proce Example 2.3 dure of Example 1.1, but with the following differences: 30 Phase IV was conducted at 70° C., and Phase VI was Topical Antiseptic Compositions Containing conducted at 50° C. They were composed as shown in the Propylene Glycol following table (numbers are expressed in percentage w/w).

These compositions can be prepared according to the Component FF-G.1 FF-G.2 procedure of Example 1.2, and were composed as shown in 35 the following table (numbers are expressed in percentage deinonized water 36.20 35.20 potassium iodide 1.00 1.OO w/w). citric acid O.SO O.SO Sodium citrate O.SO O.SO glycerine 10.00 1O.OO Component FF-10.1 FF-10.2 FF-10.3 40 powidone-iodine 10.00 1O.OO hydroxyethylcellulose 418O 428O deinonized water 35.10 35.10 35.10 benzalkonium chloride (50% solution) (total) (total) Sodium citrate 0.75 0.75 Stearic acid potassium iodide O.OO 0.75 0.75 cetyl alcohol propylene glycol 1S.OO 1S.OO 1S.OO polyguaternium-7 powidone-iodine 10.00 1O.OO 10.00 45 distearyldimonium chloride polycuaternium-10 39.15 39.15 38.40 Eriethanolamine hydroxyethylcellulose (total) (total) (total) glycerol monolaurate benzalkonium chloride (50% solution) PEG-20 methyl glucose sesquistearate Stearic acid Emulsifying Wax cetyl alcohol PEG-7 glyceryl cocoate distearyldimonium chloride ethylenedia mine tetraacetate triethanolamine 50 ammonium nonoxynol-4 Sulfate glycerol monolaurate Octoxynol-9 PEG-20 methyl glucose sesquistearate nonoxynol-9 Emulsifying Wax hydroxyacetophenone caprylicicapric triglycerides ethylenedia mine tetraacetate ammonium nonoxynol-4 Sulfate 55 octoxynol-9 Example 3 nonoxynol-9 Activity of Sporicidal Compositions

Example 2.4 60 Example 3.1 Topical Antiseptic Compositions Containing Sporicidal Compositions Lacking Antiseptic Butylene Glycol Test Samples. The formulations were prepared according to the proce 65 Compositions according to Examples 2.1 (sporicidal com dure of Example 1.1, and were composed as shown in the positions lacking antiseptic) and 2.2 (topical PVP-I placebo following table (numbers are expressed in percentage w/w). compositions) were prepared. US 9,629,875 B2 19 20 Test Parameters. microorganism: C. difficile ATCC 43598 exposure temp: room temp. (endospores) Subculture no.: na type of sample: Solvent in dilution growth medium: C. difficile agar neutralize used: Dey/Engley (DfE) broth, 20 mL. culture dilution phosphate-buffered no. of replicates: Duplicate medium: saline (PBS) inoculum volume: 0.01 mL plate incubation: 36.0 + 1° C. (anaerobic conditions) contact times: 30 minutes incubation time: ~72 hours elapsed heat shock: ~8 minutes heat shock temp.: 70 + 2 C.

Test Procedure. 15 Balance A0.990 mL volume of each test sample (for PP 1.00+0.01 Test Procedure. g weighed and tested) was inoculated with 0.01 mL of a C. Preparation of the Spore Suspension. difficile endospore suspension, Vortex mixed, and the vessel Clostridium difficile was grown with AA anaerobically at was allowed to react for 30 minutes. After the 30-minute 36°C.-38°C. for 20 days. Spores were aseptically harvested contact time, the inoculated test Substances were subjected with sterile 80% Isopropyl Alcohol (“IPA') and collected in to 70° C. for 8 minutes. Each substance was then neutral a sterile vessel. The spore Suspension was then centrifuged ized with 20 mLDey/Engley (D/E) broth, and enumerated for 20 minutes at 5000 rpm and the pellets re-suspended in using spread plate techniques. sterile 80% IPA to achieve a final concentration between Results. Spore count reductions are shown below and in FIG. 1. 10x10' and 5.0x10" viable spores/mL. Plate counting was 25 performed to verify that spores count is appropriate. Inoculation of the Test Material and Enumeration of the Reduction from Organism. sample replicate CFU/mL (x10) Control, 8 min. (%)" 1 gram aliquot of the test material was transferred to a sterile glass jar. 104 aliquots of the prepared test organism time = 0 1S.OO were inoculated into the test material to achieve a concen 2 15.50 30 Control? 15.50 tration between 1.0x10 and 5.0x10 spores/mL. Thejar was 2 17.00 mixed immediately. After the appropriate contact time, 99 A1.L.1 1.63 89.5 mL of the neutralization broth was added into each jar and 2 1.83 89.2 A1.L2 1.39 91.0 thoroughly mixed. Recovery was performed by plate count 2 1.39 91.8 35 as follows: A1.L3 1.39 91.0 10 mL (to give a dilution of 10' plated with AA. 2 1.57 90.8 1 mL (to give a dilution of 10° plated with AA. A2.L.1 1.79 88.5 2 1.36 92.0 1 mL to 9.0 mL of saline followed by ten-fold serial A2.L2 1.53 90.1 dilution to achieve 10 to 10 dilutions and plating 2 1.18 93.1 40 1.0 mL aliquots onto AA. A2.L3 1.55 90.0 The plates were then incubated anaerobically at 36°C.1 2 1.90 88.8 PPA1.L3 3.38 78.2 C. for 4 to 5 days. At the end of the incubation periods, the 2 2.96 82.6 plates were counted using a Quebec colony counter and the PPA2.L3 2.63 83.9 number of viable organisms was determined. 2 2.92 82.8 45 Numbers Control. % Reduction = 100 x (B - A)/B 1.0 mL aliquot of sterile saline was transferred to a sterile A = number of surviving bacteria (spores) in sample after the contact time glass jar. 104 aliquots of the prepared test organism were B = number of bacteria (spores) in the untreated sample immediately after inoculation inoculated into the test material to achieve a concentration and or the contact time *Saline between 1.0x10 and 5.0x10 spores/mL. The jar was mixed 50 immediately. After the appropriate contact time, 99 mL of the neutralization broth was added into each jar and thor Example 3.2 oughly mixed. Ten-fold serial dilutions were made thereafter Topical Antiseptic Compositions Containing into 9.0 mL of neutralization broth. 2x1.0 mL from each Butylene Glycol and/or Glycerine dilution tube was plated into sterile Petri dishes and poured, 55 plated, incubated and counted as above. Test Parameters. Log Reduction and % Reduction Calculation. Microorganism: Clostridium difficile ATCC 43,603 logo reduction=logo initial bacterial population (spores) (Numbers Control)-logo number of Survivors Time Intervals: 0-Time (numbers control) 1 min, 2 min, 3 min, 5 min, 10 min 60 Media and Equipment: Neutralization Broth (Letheen initial bacterial population (Numbers Control) - Broth with 0.05% NaSO) number survivors (Test Sample) % reduction = - x 100 Anaerobic Agar (“AA) initial bacterial population (Numbers Control) Saline, 0.85% 36° C.1° C. Incubator 65 Calibrate Timer Results. Miscellaneous Spore count reductions are shown below and in FIG. 2. US 9,629,875 B2 21 22

initial count 1 min 2 min 5 min 10 min Sample CFU/mL x 10 CFU/mL x 10 CFU/mL x 10 CFU/mL x 10 CFU/mL x 10 Control 2O. 115 121 118 190 24 12O. 117 117 120 og reduction: O.O163 O.O108 O.O163 -0.104 % reduction: 3.69 2.46 3.69 -27.1 FF-B.1 OO. O.130 O O O O.140 O O O og reduction: 2.88. NA NA NA % reduction: 99.9 100. 1OO. 1OO. FF-B.2 OO. O.O400 O.O2OO O.04OO O O.O400 O.OOSO O.O2OO O og reduction: 3.66 3.90 3.52 NA % reduction: 99.9 99.9 99.9 1OO. FF-G.1 O6 O.18O O O O O.12S O O O og reduction: 2.85 NA NA NA % reduction: 99.9 100. 1OO. 1OO. FF-G.2 OO. O O O O O O O O og reduction: NA NA NA NA % reduction: 1OO. 100. 1OO. 1OO.

Example 3.3 Results.

25 Topical Antiseptic Compositions on Hands LGM* Standard p-value Sample CFU 10 mL. deviation (vs. control) Test Composition. 1 (control) 4.36 O.17 Compositions according to Example 2.5 (topical antisep 2 (rinse) 3.31 O.30 <0.001 tic compositions containing glycerine). 30 3 (wash) 2.46 O.29 <0.001 4 3.04 O.22 <0.001 Experimental Samples. 5 4.38 O16 O681 6 2.82 0.17 <0.001

Test loggeometric mean (logo x) Sample composition? Test condition 35 Additionally, no skin irritation was noted after any of the 1 (control) No Spore contamination, and no intervention. experiments. 2 (rinse) No Spore contamination followed by a 30 s rinse 3 (wash) No Spore contamination followed by a 15 s hand What is claimed is: wash with a bactericidal soap and a 15 s rinse 1. A sporicidal composition, comprising: 4 Yes Spore contamination followed by application of water; test composition 5 Yes Application of test composition followed by " an amount q. of organic solvent, wherein q is about 5% spore contamination to about 60% w/w; and 6 Yes Application of test composition followed by an amount qi of spore-germinating agent, wherein q is spore application followed by reapplication of about 0.05% to about 5% w/w: test composition wherein: 45 the organic solvent comprises a glycol, glycerine, a Test Procedure. glycol ether, a polyethylene glycol or dimethyl isosorbide; and Preparation. the spore-germinating agent comprises: Five volunteers (10 hands) were recruited. A 60s scrub an amount q. of iodide Source, or with non-bactericidal soap followed by a 60 s rinse was 50 an amount q. of citrate source, or performed prior to each experiment. a combination thereof; Contamination. wherein: Hands were contaminated with 1x10 CFU/10 mL non 4ged;1+4.1; and toxigenic C. difficile spores (ATCC 700057). A modification of the standard hand wash test method, 55 at least one of q and q is at least about 0.05% ASTM E1174-06, was used to sample hands. For experi wfw. ments 4-6, a 30 second rinse after rubbing in the test 2. The sporicidal composition of claim 1, composition for 30 seconds was performed prior to hand wherein: sampling. the spore-germinating agent consists essentially of 60 an amount q. of iodide Source, or Evaluation of Viable C. difficile Spores. an amount q. of citrate source, or Neutralizing buffer was used to sample hands and inac a combination thereof; tivatetest composition. Quantitative cultures were per wherein: formed in quadruplicate on BHI agar with horse blood and taurocholate (Anaerobe Systems, Morgan Hill, Calif.). Log 65 4ged;1+4.1; and geometric mean (LGM) of viable C. difficile spores from at least one of q and q is at least about 0.05% each assay condition was calculated. wfw. US 9,629,875 B2 23 24 3. The sporicidal composition of claim 2, 8. The sporicidal composition of claim 5, wherein: wherein: q is about 0.06% to about 1.80% w/w; and one or both of (a) and (b): (114; 141. (a) q is at least about 0.08% w/w: 4. The sporicidal composition of claim 1, wherein: (b) q is at least about 0.06% w/w. q is about 0.10% to about 2.3% w/w; and 9. The sporicidal composition of claim 1, wherein the at least one of q and q is at least about 0.10% w/w. organic solvent comprises glycerine, propylene glycol or 5. A sporicidal composition, comprising: butylene glycol. water; 10. The sporicidal composition of claim 1, wherein the an amount q organic solvent, wherein q is about 5% to 10 spore-germinating agent consists essentially of an iodide about 60% w/w; and Source or a combination of an iodide source and a citrate an amount q spore-germinating agent, wherein q2 is SOUC. about 0.03% to about 3.8% w/w: 11. The sporicidal composition of claim 1, wherein the wherein: spore-germinating agent consists essentially of a citrate the organic solvent comprises a glycol, glycerine, a 15 glycol ether, a polyethylene glycol or dimethyl Source or a combination of a citrate source and an iodide isosorbide; and SOUC. the spore-germinating agent comprises: 12. The sporicidal composition of claim 10, wherein the an amount q. of iodide, or iodide Source comprises potassium iodide. an amount q. of citrate, or 13. The sporicidal composition of claim 11, wherein the or a combination thereof; citrate source comprises citric acid, sodium citrate monoba wherein: sic or a hydrate thereof, sodium citrate dibasic or a hydrate thereof, or sodium citrate tribasic or a hydrate thereof. 4,224:2+42; and 14. A disinfecting composition, comprising: one or both of (a) and (b): the sporicidal composition of claim 1; and (a) q is about 0.04% to about 3.8% w/w: 25 a disinfectant. (b) q is about 0.03% to about 3.2% w/w. 15. An antiseptic composition, comprising: 6. The sporicidal composition of claim 5, the sporicidal composition of claim 1; and wherein: an antiseptic. the spore-germinating agent consists essentially of 16. A method of reducing the bacterial spore count on a an amount q. of iodide, or 30 non-living tissue surface, comprising: an amount q. of citrate, or applying an effective amount of the composition of claim or a combination thereof; 1 to the surface; wherein: wherein the spore bacteria comprise bacteria of the genus Clostridium. 4.224;2+4.2; and 35 17. A method of reducing the bacterial spore count on a one or both of (a) and (b): living tissue surface, comprising: (a) q is about 0.04% to about 3.8% w/w: applying an effective amount of the composition of claim (b) q is about 0.03% to about 3.2% w/w. 1 to the surface; 7. The sporicidal composition of claim 6, wherein the spore bacteria comprise bacteria of the genus wherein: 40 Clostridium. 424,242.