Compositions, uses and methods for attenuating anti-infective resistance

Abstract

A composition is disclosed that includes a chelating polymer including a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent, and an anti-infective agent. The composition can be used to treat a bacterial infection or to reduce bacterial resistance to the anti-infective agent in a subject.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 426,460, filed November 18, 2022, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION FIELD OF THE DISCLOSURE The present disclosure relates to compositions for attenuating resistance to anti-infective agents, and more particularly to compositions comprising chelating polymers. [Background technology]

[0003] Resistance to anti-infective agents, such as antibiotics, is a widespread and troublesome phenomenon and a major cause of mortality and morbidity in humans and animals infected with resistant organisms, such as bacteria. There are multiple mechanisms by which organisms, such as bacteria, adapt to adverse environmental conditions that allow them to survive. These inherent adaptation and survival mechanisms allow bacteria to overcome the effects of anti-infective agents.

[0004] Most antibiotics currently in use are of bacterial origin, and bacteria may have the inherent ability to resist any one or several antibiotics. On the other hand, clinically relevant antibiotic resistance is acquired when bacteria are exposed to antibiotics during the treatment of infection, or when antibiotics are present in the environment as pollutants or waste. Organisms can also develop resistance naturally when exposed to antibiotics for a period of time, either at inhibitory or subinhibitory concentrations.

[0005] A need exists to address one or more deficiencies in the art.

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art to the present disclosure. Summary of the Invention

[0007] The present disclosure relates to compositions for increasing the effectiveness of and / or reducing resistance of an organism to an anti-infective agent, the compositions comprising a large (greater than 1500 Da), water-soluble chelating polymer and an anti-infective agent, such as a fluoroquinolone antibiotic.

[0008] According to one aspect of the present disclosure, there is provided a composition, which may be used alone or in combination with any other aspect described herein, comprising: a transition metal chelate polymer; an anti-infective agent, The chelating polymer a reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent; The first monomer unit is represented by compound (I):

[0009] [ka] During the ceremony, R 1 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R 2 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R 3 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12, the second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene; the chelating polymer is soluble in an aqueous medium; Compositions are provided in which the chelating polymer has a molecular weight of at least about 1500 Da prior to chelation.

[0010] In some embodiments of the composition, R 1 is H, R 2 is methyl, R 3 is methyl, n is 1 to 6, and optionally n is 2.

[0011] In some embodiments of the composition, the first monomer unit is Compound (II):

[0012] [ka] is expressed by

[0013] In one aspect of the composition, the second monomer unit is 1-vinyl-2-pyrrolidone. In another aspect of the composition, the second monomer unit is N,N-dimethyl-acrylamide.

[0014] In some embodiments of the composition, the addition-fragmentation chain transfer agent is independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

[0015] In some embodiments of the composition, the transition metal is one or more of iron, manganese, cobalt, copper, or nickel.

[0016] In some aspects of the composition, the anti-infective agent is an antibiotic. In further aspects of the composition, the antibiotic is a fluoroquinolone antibiotic. In further aspects, the fluoroquinolone antibiotic is moxifloxacin or ciprofloxacin. In some aspects, the fluoroquinolone antibiotic is ciprofloxacin.

[0017] In some embodiments, the composition further comprises thiourea. Some compositions further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0018] In some aspects, the composition is for use in reducing resistance of an organism to an anti-infective agent. In further aspects, the organism is susceptible to the anti-infective agent prior to exposure to the composition. In some aspects of use, the organism is a bacterium. In further aspects, the bacterium is gram-positive. In yet further aspects, the bacterium is Staphylococcus aureus.

[0019] According to another aspect of the present disclosure, which may be used alone or in combination with any other aspect described herein, there is provided a method of treating a bacterial infection in a subject, the method comprising administering a composition disclosed herein.

[0020] According to another aspect of the present disclosure, which may be used alone or in combination with any other aspect described herein, there is provided a use of a composition disclosed herein for reducing bacterial resistance to an anti-infective agent in a subject, wherein the composition is for administration to a subject having the bacteria.

[0021] According to another aspect of the present disclosure, which may be used alone or in combination with any other aspect described herein, there is provided use of a composition described herein in the manufacture of a medicament for attenuating bacterial resistance to an anti-infective agent in a subject, wherein the composition is for administration to a subject having the bacteria.

[0022] In some embodiments, the bacterium is gram-positive. In further embodiments, the bacterium is Staphylococcus aureus.

[0023] According to another aspect of the present disclosure, which may be used alone or in combination with any other aspect described herein, there is provided a transition metal chelating polymer for use as an adjuvant in an antibiotic composition, comprising: The chelating polymer a reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent; The first monomer unit is represented by compound (I):

[0024] [ka] During the ceremony, R 1 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R 2 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R 3 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12, the second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene; the chelating polymer is soluble in an aqueous medium; Transition metal chelating polymers are provided, wherein the chelating polymer has a molecular weight of at least about 1500 Da prior to chelation.

[0025] In some embodiments of the transition metal chelate polymer, R 1 is H, R 2 is methyl, R 3 is methyl, n is 1 to 6, and optionally n is 2.

[0026] In some embodiments of the transition metal chelate polymer, the first monomer unit is Compound (II):

[0027] [ka] is expressed by

[0028] In some embodiments of the transition metal chelate polymer, the second monomer unit is 1-vinyl-2-pyrrolidone.

[0029] In some embodiments of the transition metal chelate polymer, the second monomer unit is N,N-dimethyl-acrylamide.

[0030] In some embodiments of the transition metal chelate polymer, the addition-fragmentation chain transfer agent is independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

[0031] In some embodiments of the transition metal chelating polymer, the antibiotic is a fluoroquinolone antibiotic.

[0032] In some embodiments of the transition metal chelating polymer, the fluoroquinolone antibiotic is moxifloxacin or ciprofloxacin.

[0033] In some embodiments of the transition metal chelating polymer, the fluoroquinolone antibiotic is ciprofloxacin.

[0034] According to one aspect of the present invention, there is provided a chelating composition soluble in an aqueous medium for chelating a transition metal element, which may be used alone or in combination with any other aspect described herein, comprising a carrier material and one or more suitable types of metal element-binding chemical groups fixed to or incorporated into the structure of the carrier material, wherein the one or more suitable types of metal element-binding chemical groups are one or more of carboxyl, hydroxyl, phenolate, catecholate, hydroxamate, hydroxypyridinone, and hydroxyphenyltriazole carboxyl types, the carrier material comprising vinylpyrrolidone, styrene, or styrene, and the chelating composition is capable of binding to a cell membrane of a living cell. Chelate compositions are provided that have a minimum molecular weight (nominally greater than 1500 Da) large enough to prevent normal uptake into intracellular aspects, are capable of binding transition metals, and the compositions are used to treat biological cells with anti-cellular agents, wherein the chelate composition binds to the transition metal and remains substantially soluble with its bound metal in the extracellular environment of the cells, thereby reducing uptake of the metal into intracellular aspects within the cellular membrane of the biological cell, such that the internal aspects of the biological cell underlying the cellular membrane of the biological cell lack a sufficient amount of the transition metal required for metal-requiring enzymes to resist the activity of the anti-cellular agent or to develop resistance to the anti-cellular agent.

[0035] According to another aspect of the present disclosure, there is provided a method for preparing a chelate composition described herein, which may be used alone or in combination with any other aspect described herein, comprising: prepared from at least a first monomeric unit and a second monomeric unit, wherein the first monomeric unit has incorporated therein or affixed thereto a suitable metal binding chemical group optionally independently selected from the group consisting of carboxyl, hydroxyl, phenolate, catecholate, hydroxamate, hydroxypyridinone, and hydroxyphenyltriazole; the first and second monomer units are polymerized by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent; Optionally, the first monomer unit is compound (I):

[0036] [ka] During the ceremony, R1 is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; R2 is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; R3 is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; n is 1 to 12, Optionally, a method is provided wherein the second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene.

[0037] In a further embodiment, the one or more suitable metal binding chemical groups is a hydroxypyridinone:

[0038] [ka] wherein X, Y and Z are independently N or C, such that: when X is N, Y and Z are C; when Y is N, X and Z are C; If Z is N, then X and Y are C.

[0039] In a further embodiment, compound (I) is represented by compound (Ia):

[0040] [ka] During the ceremony, R 1 is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; R 2is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; PG is a protecting group containing compound (Ia).

[0041] [ka] During the ceremony, n is 1 to 12, R 4 But -COCCH2R 3 or a protecting group, and polymerizes with compound (Ib), Next, R 4 Ga-COCCH2R 3 then removing PG to give compound (I), or R 4 is a protecting group, R 4 and reacting with a suitable acrylate source, followed by removal of PG to give compound (I).

[0042] In another embodiment, R 1 is H and R 2 is methyl and R 3 is methyl and n is 1 to 6, or optionally n is at least 2.

[0043] In another embodiment, the first monomer unit is compound (II):

[0044] [ka] is expressed by

[0045] In another embodiment, the second monomer unit is 1-vinyl-2-pyrrolidone.

[0046] In another embodiment, the second monomer unit is N,N-dimethyl-acrylamide.

[0047] In another embodiment, suitable addition-fragmentation chain transfer agents are independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

[0048] In another embodiment, residues of the addition-fragmentation chain transfer agent are wholly or partially removed from the chelating composition after polymerization.

[0049] In another embodiment, the chelating composition has a molecular weight range defined by a low molecular weight limit, as measured prior to iron binding, of about 1500 daltons so as not to be normally incorporated into intracellular contexts within the cell membrane of a living cell, and a high molecular weight limit that is sufficiently low to allow the composition to remain soluble in aqueous media.

[0050] In a further embodiment, the chelating composition comprises a metal binding chemical group of 3-hydroxy-pyridin-4-one incorporated into a support material comprised of pyrrolidone, acrylamide, imidazole, or styrene.

[0051] In a further aspect, the metal chelate composition is for use in treating a disease in a human or other animal by administration, together with another anti-cellular agent, to a human or other animal having a disease resulting from one or more biological cells or activity of one or more cells.

[0052] In a further embodiment, the transition metal is one or more of iron, manganese, cobalt, copper, or nickel.

[0053] In one embodiment, the disease-causing cells are one or more of a microorganism, a multicellular parasite, or a cancer.

[0054] In further embodiments, the other anti-cellular agent is one or more of an antibiotic, an anti-infective, an anti-parasitic, or an anti-cancer agent.

[0055] In one aspect, a pharmaceutical composition is provided comprising a chelating composition described herein and a pharmaceutically acceptable carrier, excipient, or diluent, wherein the amount of the composition and the frequency and route of administration of the pharmaceutical composition are adjusted to take into account the particular disease being treated and other anti-cellular agents being administered.

[0056] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the present disclosure. [Brief explanation of the drawings]

[0057] Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. [Figure 1] 1 is a synthetic scheme showing the synthesis of MAHMP-pyrrolidone copolymer. [Figure 2] 1 is a UV-vis spectrum showing the metal-to-ligand charge transfer band (MLCT) band for iron loading of DIBI. [Figure 3] This graph shows the effects of ciprofloxacin, DIBI, and thiourea on the killing and recovery growth of Staphylococcus aureus. ATCC 25923 was grown in Mueller-Hinton Broth (MHB), and standardized inocula were added to 10 mL flask cultures of the following: untreated Roswell Park Memorial Institute (RPMI) control (●), 50 μg / mL DIBI (■), 0.5 μg / mL CIP (▲), 10 mM thiourea (◆), 50 μg / mL DIBI combined with 0.5 μg / mL CIP (□), and 10 mM thiourea combined with 0.5 μg / mL CIP (◇) in RPMI. Samples obtained during exposure were plated on blood agar (BA) to determine CFU / mL. Data represent the mean + / - SEM of three independent experiments. [Figure 4]This graph shows the long-term effects of ciprofloxacin, DIBI, and thiourea on the killing and recovery growth of Staphylococcus aureus. ATCC 25923 was grown in MHB, and standardized inocula were added to 10 mL flask cultures of untreated RPMI control (●), 50 μg / mL DIBI (■), 0.5 μg / mL CIP (▲), 10 mM thiourea (◆), 50 μg / mL DIBI combined with 0.5 μg / mL CIP (□), 10 mM thiourea combined with 0.5 μg / mL CIP (◇), and RPMI supplemented with 50 μg / mL DIBI and 10 mM thiourea combined with 0.5 μg / mL CIP (Δ). Samples obtained during exposure were plated on BA, and CFU / mL was determined. Data represent the mean + / - SEM of two independent experiments. [Figure 5] This is a series of graphs showing the effects of ciprofloxacin and DIBI on killing of Staphylococcus aureus and recovery growth of small-colony variants. S. aureus ATCC 43300 (Figures 5A and 5C) and ATCC 25923 (Figures 5B and 5D) were grown in MHB, and standardized inocula were added to 10 mL flask cultures of untreated RPMI control (○) or RPMI supplemented with 50 μg / mL DIBI (□), 0.5 μg / mL CIP (Δ), or 50 μg / mL DIBI in combination with 0.5 μg / mL CIP (▽). Samples taken during exposure were plated on BA for enumeration of total CFU / mL (Figures 5A and 5B) and percentage of SCVs (Figures 5C and 5D) in the recovered population. Data represent the mean + / - SEM of two independent experiments. [Figure 6] 1 is a photograph of a plate showing the effect of ciprofloxacin and DIBI on Staphylococcus aureus small colony variant formation. DETAILED DESCRIPTION OF THE INVENTION

[0058] One or more exemplary embodiments have been described by way of example. Described herein are compositions, methods, and uses relating to metal chelating polymers for preventing anti-infective resistance in organisms. It will be understood that the embodiments and examples are provided for illustrative purposes intended for those skilled in the art and are not meant to be limiting in any way. All references to embodiments, examples, aspects, formulas, compounds, compositions, solutions, and the like are intended to be exemplary and non-limiting.

[0059] Iron is an essential trace nutrient metal required by all vertebrates because it provides the active catalytic center for a variety of important enzymes involved in DNA synthesis, metabolism, and cell defense, and, with few exceptions, cannot be replaced by alternative trace metals.

[0060] Furthermore, because iron is also required by pathogens to invade and infect the body, there is an active iron-suppressing defense that occurs early during infection with a reduction in the amount of circulating iron on transferrin, limiting access to the iron required by pathogens for proliferation. For a review of these infection-related aspects, see, e.g., "Iron-withdrawing anti-infectants for new host-directed therapies based on iron dependence, the Achilles' heel of antibiotic-resistant microbes." Bruce E. Holbein, M. Trisha C. Ang, David S. Allan, Wangxue Chen, and Christian Lehmann. Environ Chem Lett. 2021;19(4):2789-2808 (the entire contents of which are incorporated herein by reference).

[0061] Compositions for chelating and retaining iron in the extracellular environment of growing cells to limit the uptake of iron required for intracellular growth and thus affect cell growth are described, for example, in U.S. Pat. No. 10,709,784, entitled "Metal chelating compositions and methods for controlling the growth or activities of a living cell or organism," and U.S. Pat. No. 11,059,785, entitled "Polymeric metal chelating compositions and methods of preparing the same for controlling growth and activities of living cells," both of which are incorporated herein by reference in their entireties. Such compositions have been shown in various examples, such as those reviewed by Holbein et al. (2021), to assist vertebrates' natural iron-withdrawal defenses in combating diseases in which iron supply in the body promotes the growth of pathogens (i.e., either microbial invading pathogens or cancer cells with uncontrolled growth occurring within the animal's body).

[0062] Exposure of bacteria to antibiotics is known to induce the emergence of antibiotic-resistant persisters, which may represent a minor subpopulation of the initially present resistant phenotype that becomes positively enriched by antibiotic selection. Alternatively, antibiotic-resistant small colony variants (SCVs) may arise from accelerated mutations induced by DNA damage resulting from antibiotic exposure. SCV isolates obtained from clinical specimens after treatment of infected human patients with β-lactams and aminoglycosides often have menadione or hemin auxotrophy, whereas thymidine auxotrophy is typically seen in SCVs from patients treated with antifolates. Importantly, regardless of the initial drug treatment that induced the SCV phenotype, these variants often have cross-resistance to other unrelated antibiotics due to their altered metabolism and electron gradient deficiency.

[0063] The SCV response to antibiotics is considered part of the systemic SOS response observed from stress induced by various agents, including antibiotics. Interestingly, intracellular free iron plays a role in the generation of reactive oxygen species (ROS), but bacterial defense against ROS also requires catalase and superoxide dismutase, both of which are iron-dependent enzymes. Furthermore, DNA damage repair involves ribonucleotide reductase, another enzyme whose activity is iron-dependent. Thus, iron requirements and bacterial iron regulation may influence the development and expression of antibiotic resistance at several distinct physiological levels; these aspects have been reviewed previously.

[0064] Bactericidal antibiotics such as CIP can cause oxidative stress that can damage iron-sulfur clusters. Substantial evidence that CIP induces resistance-conferring mutations via ROS- and iron-dependent mechanisms has been obtained in Escherichia coli; both Δfur and ΔsodAB knockout mutants, which are associated with loss of iron homeostasis control or loss of superoxide dismutase activity, respectively, leading to intracellular iron accumulation, had significantly higher rates of CIP-resistant mutations after exposure compared with wild-type. CIP and related structures are also known to bind iron and may therefore transport iron to or translocate iron within the bacterial cytoplasm.

[0065] CIP has been shown to be mutagenic in Staphylococcus aureus (S. aureus), increasing both the mutation rate and DNA recombination through activation of RecA, which functions directly in DNA repair, and through self-cleavage of the repressor molecule LexA, allowing induction of the SOS response, which involves the error-prone UmuC-type polymerase. Topoisomerase IV (GrlA) is the primary target of CIP in Gram-positive bacteria and functions (along with DNA gyrase, a secondary target of CIP) to assist DNA replication. Therefore, interference with GrlA results in DNA damage requiring repair. RecA repairs DNA by forming stabilized nucleoprotein filaments, which then enable repair; this process is enabled in Gram-positive bacteria by AddAB, a nuclease that acts through an essential iron-sulfur cluster.

[0066] All of the above mechanisms leading to antibiotic resistance require DNA synthesis / repair and growth, as well as metabolic and macromolecular synthesis activities, and therefore require an adequate supply of key iron-dependent enzymes. Therefore, sufficient iron sequestration to limit the adequate supply of these critical iron-dependent enzymes can be expected to inhibit bacterial repair and recovery from antibiotic-induced damage and prevent the development of resistance mechanisms. Exploiting this iron-dependence of antibiotic resistance with novel iron-sequestering therapeutics offers a potential new means for addressing microbial antibiotic resistance.

[0067] Apo-transferrin, an iron sequestrant synthesized by the host during infection, has been reported to suppress the formation of CIP-resistant persister clones in Klebsiella pneumoniae. The synthetic iron chelator DIBI has previously been shown to enhance CIP activity against S. aureus through synergistic killing.

[0068] composition As used herein, the term "composition" is intended to encompass dosage forms containing a specified amount of a chelating polymer and an anti-infective agent, as well as any dosage form resulting directly or indirectly from the combination of a specified amount of a specified compound. Such a term is intended to encompass dosage forms containing a chelating polymer and an anti-infective agent, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients. Thus, a composition of the present disclosure can encompass any composition made by mixing a compound of the present disclosure with one or more pharmaceutically acceptable carriers or excipients. "Pharmaceutically acceptable" means that the carrier or excipient is compatible with the compounds disclosed herein and the other components of the composition.

[0069] The compositions described herein, when used with other anti-cellular agents, including anti-infective antibiotics, can not only enhance the activity of the antibiotic, but also inhibit the development and emergence of resistance to the anti-cellular or anti-infective agents. The inhibition of resistance development and emergence of resistance to anti-cellular or anti-infective agents by the compositions is disclosed herein.

[0070] The compositions described herein include a transition metal chelate polymer and an anti-infective agent. The transition metal chelate polymer comprises the reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent.

[0071] The first monomer unit is represented by compound (I):

[0072] [ka] During the ceremony, R 1 are independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 2 are independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 3are independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12, The second monomer units are independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene.

[0073] The iron chelating polymer is water-soluble. The iron chelating polymer has a molecular weight of at least about 1500 Da before chelation. The molecular weight can be adjusted during synthesis by varying the relative amounts of reagents, such as the ratio of monomers or polymerization agents. In some cases, the polymer can have a molecular weight of greater than 100 kDa before chelation.

[0074] The polymers can be prepared by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent. Suitable addition-fragmentation chain transfer agents can be independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid. During preparation of the polymers, residues of the addition-fragmentation chain transfer agent can be wholly or partially removed from the chelating composition after polymerization. The polymers can be prepared using techniques and reagents known in the art, such as those disclosed in U.S. Pat. No. 11,059,785 and Ang, M, et al. DIBI, "A 3-hydroxypyridine-4-one chelator iron-binding polymer with enhanced antimicrobial activity." Medchemcomm. 2018;9(7):1206-1212 (incorporated herein by reference).

[0075] The protecting group is in compound (Ia):

[0076] [ka] In the synthesis or preparation of compound (Ia):

[0077] [ka] Compound (Ib)

[0078] [ka] It may be used during synthesis by polymerizing with During the ceremony, R 1 is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; R 2 is independently selected from the group consisting of H, alkyl, and alkyl substituted with O, N, or S; PG is a protecting group; n is 1 to 12, R 4 But -COCCH2R 3 or a protecting group, and polymerizes with compound (Ib), R 4 COCCH2R 3 then removing PG to give compound (I), or R 4 is a protecting group, R 4 and reacting with a suitable acrylate source, followed by removal of PG to give compound (I).

[0079] Suitable protecting groups (PG) include any protecting group known in the art that is suitable for protecting a hydroxyl group on a pyridinone. For example, phenylmethyl or silyl ethers, such as trimethylsilyl ethers, may be used.

[0080] The polymer comprises a first monomer unit represented by compound (II) and

[0081] [ka] and a second monomer unit comprising 1-vinyl-2-pyrrolidone or N,N-dimethyl-acrylamide.

[0082] In some embodiments, the polymer is a reaction product of a first monomeric unit and a second monomeric unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent, wherein the first monomeric unit is represented by compound (II):

[0083] [ka] The second monomer unit is 1-vinyl-2-pyrrolidone.

[0084] The compositions described herein include an anti-infective agent. An anti-infective agent can be any substance or composition that inhibits the growth of an organism, such as an infectious organism. In some cases, the anti-infective agent is an antibiotic, an antifungal agent, an antiviral agent, and / or an antiparasitic agent. When the anti-infective agent is an antibiotic, the antibiotic can be effective against gram-positive or gram-negative bacteria. The antibacterial agent may be from the quinolone or fluoroquinolone antibacterial family, such as moxifloxacin, ciprofloxacin, levofloxacin, flumequine, fleroxacin, balofloxacin, grepafloxacin, pazufloxacin, sparfloxacin, temafloxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, oxolinic acid, losoxacin, clinafloxacin, gatifloxacin, sitafloxacin, prulifloxacin, besifloxacin, and / or delafloxacin.

[0085] Methods and Uses of the Composition The polymers described herein can be used as adjuvants. The compositions described herein can be used to increase or enhance the effectiveness of anti-infective agents. For example, the compositions can be more effective at inhibiting the growth of organisms such as bacteria than the anti-infective agent alone. In some cases, the compositions described herein can reduce the minimum inhibitory concentration (MIC) of the anti-infective agent.

[0086] Resistance of an organism to an anti-infective agent can be understood as a lack of inhibition of growth when exposed to the anti-infective agent. For example, if an organism such as E. coli or S. aureus is resistant to an antibiotic, the antibiotic may have a relatively higher minimum inhibitory concentration (MIC) than a corresponding susceptible strain of the same organism. Resistance can be measured over short (e.g., less than 12 hours) or long (e.g., 96 hours or more) exposure periods. In some cases, a resistant population may have unchanged or higher colony forming units per mL (CFU / mL) than a susceptible population at the same exposure time and concentration of anti-infective. Attenuation of resistance can be understood as substantially preventing this resistance. For example, a composition containing ciprofloxacin and DIBI may have a relatively similar MIC to the anti-infective agent alone after delayed exposure (e.g., up to 96 hours or more). In some cases, the MIC decreases due to increased effectiveness of the composition compared to the anti-infective agent alone.

[0087] In some cases, the MIC or CFU / mL may vary by ±0.1 to 50% or any value therebetween (optionally rounded to the nearest 0.1), or any subrange spanning between any two of these values, e.g., ±1 to 5%, is contemplated. For example, ±0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24. 5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, and 50% are considered.

[0088] The compositions described herein can be used to attenuate resistance to anti-infective agents by organisms. Attenuation of resistance can occur through preventing or substantially reducing the formation of anti-infective resistant subpopulations of the organism. In some cases, these resistant subpopulations may harbor genetic mutations that confer resistance to anti-infective agents, and the compositions described herein can substantially reduce the occurrence or spread of these mutations. The compositions described herein can suppress the formation of small colony variants (SCVs) of organisms such as S. aureus that are resistant to at least one anti-infective agent, such as a fluoroquinolone. Attenuation of resistance to anti-infective agents can be measured by genetic or phenotypic testing of the organism population to determine the concentration of resistant SCVs. One possible factor for determining whether resistance is at least partially attenuated is if the resistant SCVs are present at a relatively lower concentration than when the anti-infective agent is used alone.

[0089] Suitable organisms for treatment or exposure to the compositions include bacteria, such as Staphylococcus aureus. Other suitable organisms may be any organism that is sensitive to anti-infective agents and develops natural resistance after prolonged exposure to the anti-infective agent. The compositions described herein may prevent the formation of resistant colonies after exposure to the anti-infective agent. For example, the compositions described herein may be used to attenuate or otherwise prevent the formation or spread of resistance to fluoroquinolone antibiotics.

[0090] The compounds disclosed herein and pharmaceutically acceptable carriers or excipients can be formulated into dosage forms adapted for administration to a subject by a desired route of administration. For example, dosage forms can include those adapted for (1) oral administration, such as tablets, capsules, caplets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets, and (2) parenteral administration, such as sterile solutions, suspensions, and powders for reconstitution.

[0091] Suitable pharmaceutically acceptable carriers or excipients vary depending on the specific dosage form selected. Furthermore, suitable pharmaceutically acceptable carriers or excipients can be selected for the specific function they may perform in the composition. For example, certain pharmaceutically acceptable carriers or excipients can be selected for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable carriers or excipients can be selected for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable carriers or excipients can be selected for their ability to facilitate the transport or transportation of the compounds disclosed herein from one organ or part of the body to another organ or part of the body after administration to a subject. Certain pharmaceutically acceptable carriers or excipients can be selected for their ability to enhance patient compliance.

[0092] Suitable pharmaceutically acceptable excipients may include the following types of excipients: diluents, lubricants, binders, disintegrants, fillers, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, thickening agents, antioxidants, preservatives, stabilizers, surfactants, and buffers.

[0093] Those skilled in the art have the knowledge and skill in the art to select suitable pharmaceutically acceptable carriers and excipients in appropriate amounts for use in the compositions of the present disclosure. Furthermore, there are many sources of information available to those skilled in the art that describe pharmaceutically acceptable carriers and excipients and may be useful in selecting suitable pharmaceutically acceptable carriers and excipients. Examples include REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company), THE HANDBOOK OF PHARMACEUTICAL ADDITIVES (Gower Publishing Limited), and THE HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (the American Pharmaceutical Association and the Pharmaceutical Press). The compositions of the present disclosure are prepared using techniques and methods known to those skilled in the art. Some methods commonly used in the art are described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company).

[0094] Any suitable dosage or delivery form may be used. In one embodiment, the present disclosure is directed to a solid oral dosage form such as a tablet or capsule, or a pharmaceutically acceptable salt of the foregoing, and a diluent or filler.

[0095] Suitable diluents and fillers may include lactose, sucrose, dextrose, mannitol, sorbitol, starches (e.g., corn starch, potato starch, and pregelatinized starch), cellulose and its derivatives (e.g., microcrystalline cellulose), calcium sulfate, and calcium hydrogen phosphate.

[0096] The solid oral dosage form may further comprise a binder.Suitable binders may include starch (e.g., corn starch, potato starch, and pregelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose).The solid oral dosage form may further comprise a disintegrant.

[0097] Suitable disintegrants may include crospovidone, sodium starch glycolate, croscarmellose, alginic acid, and sodium carboxymethyl cellulose. The solid oral dosage form may further comprise a lubricant.

[0098] Suitable lubricants may include stearic acid, magnesium stearate, calcium stearate, and talc.

[0099] Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The compositions can also be prepared so as to prolong or sustain release, for example, by coating or embedding particulate material in polymers, waxes, etc.

[0100] The compounds disclosed herein can also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethylene oxide polylysine substituted with palmitoyl residues. Furthermore, the compounds disclosed herein can be coupled to biodegradable polymer classes useful for achieving controlled drug release, such as polylactic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0101] In one embodiment, the present disclosure is directed to a liquid oral dosage form.

[0102] Oral liquids, such as solutions, syrups, and elixirs, can be prepared in dosage unit form, with each dose containing a predetermined amount of the compound disclosed herein or its pharmaceutically acceptable salt.Syrups can be prepared by dissolving the compound disclosed herein in a suitable flavored aqueous solution, and elixirs can be prepared by using a non-toxic alcoholic vehicle.Suspensions can be prepared by dispersing the compound disclosed herein in a non-toxic vehicle.Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservatives, flavor additives, such as peppermint oil, or other natural sweeteners or saccharin or other artificial sweeteners, can also be added.

[0103] In one embodiment, the present disclosure is directed to compositions for parenteral administration. Compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0104] The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0105] The present subject matter is further illustrated in the following examples. [Example]

[0106] Example 1: Synthesis of a soluble copolymer chelating composition containing an active pyridinone metal binding group in the form of MAHMP copolymerized with vinylpyrrolidone using the RAFT procedure (US Pat. No. 11,059,785).

[0107] Example 1A: General synthetic procedure The composition of this example (Batch No. IS09865-025) represents generally optimized synthesis conditions with respect to obtaining high yield conversion of monomer to copolymer product, and with reference to the synthesis scheme in FIG.

[0108] In a 50 mL two-neck round-bottom flask equipped with a magnetic stirrer and reflux condenser, N-vinyl-2-pyrrolidone (B) (3.5 g, 0.0315 mol) and MAHMP (A) (0.25 g, 0.00106 mol) were dissolved in 4 mL of deionized water (E), and the mixture was stirred under N for 20 min. To this mixture was added RAFT agent (0.136 g, 0.000652 mol), 2-ethoxythiocarbonylsulfanyl-2-methylpropionic acid (ETSPA) (F), and TMEDA (0.227 g, 0.00195 mol) (promoter (D)). The entire mixture was degassed by purging with N for 20 min. After degassing, tert-butyl hydroperoxide (0.35 g, 0.00388 mol) (initiator (C)) was added to the mixture. The mixture was then heated to 40°C, and the reaction was continued at the same temperature for 18 hours under N2. After 18 hours, the reaction mass was placed in a rotary evaporator and water was removed under reduced pressure. The crude mass was dissolved in methanol and filtered to remove insoluble impurities, and the filtrate was concentrated to an approximately 50 wt% polymer solution. This solution was then slowly precipitated with a 50% volume excess of MTBE under constant stirring. The precipitate was then filtered and dried on a rotary evaporator at 50°C under 35-50 mmHg for 4-6 hours until a constant weight of the product copolymer (Batch ISO9865-025) was observed. The mass yield of the MAHMP-pyrrolidone copolymer was 80% based on the mass of the monomers (A+B) initially fed to the polymerization reaction, and the copolymer was 1 It was found to contain 5.7% MAHMP content as determined by H NMR spectroscopy. The metal chelate copolymer was found to have a molecular weight (Mw) of approximately 7.3 kDa with a PDI (Mw / Mn) of 1.7 using GPC analysis.

[0109] Example 1B: Optimization of MAHMP:NVP Monomer Ratio With reference to Figure 1 and Example 1A above, a series of test polymerizations were conducted using the procedure of Example A (above), except that the ratio of the metal chelate monomer MAHMP (A) to the comonomer NVP (B) was varied while maintaining the total monomer (A + B) content at 50 equivalents for the polymerization reaction and keeping the other chemical components (C), (D), (E), and (F) the same for each test. The results in Table 1 below show that as the amount of MAHMP (A) fed to the polymerization increased relative to NVP (B), the overall copolymer yield decreased, but the percentage of MAHMP content in the product copolymer increased. Ideally, a copolymer MAHMP content of 10-20% may be preferred for chelating copolymer compositions with high metal binding capacity, but maintaining high copolymer yield is also very important for efficient monomer usage. RAFT-mediated polymerization using RAFT agent 4, 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid (ETSPA), was found to provide fairly narrow molecular weight distributions for the copolymer products, with the average Mw of the compositions being somewhat similar across different MAHMP:NVP conditions. Based on these results, an MAHMP:NVP ratio between 1:10 and 1:15 appeared to provide the best overall MAHMP content (10-15%) with a polydispersity index (PDI) of approximately 1.4-2.0, while maintaining a product yield of approximately 80%. The composition prepared by this synthetic procedure is referred to as DIBI for ease of reference with other examples provided.

[0110] [Table 1]

[0111] From these test syntheses, sample batch ISO9865-044 provided a relatively high copolymer yield (approximately 80%), allowing for approximately 15 mol% incorporation of MAHMP into the copolymer composition, and provided a relative (i.e., relative to a linear polystyrene calibration standard using gel exclusion chromatography) product mw of 3600 Da. Separate true mw determination of this composition (designated DIBI) by laser techniques indicated an actual true molecular weight (MW) of approximately 9 kDa.

[0112] Example 2: Demonstration of iron binding capacity and iron formation constant of the composition referenced as DIBI in Example 1B (Ang et al., 2018 and Gumbau-Brisa et al., 2020).

[0113] Example 2A: Spectrophotometric characterization of Fe chelation by DIBI The dried DIBI composition (30.3 mg, 3.4 μmol) prepared in Example 1B was dissolved in 25 mL of MOPS buffer. 1 mL aliquots of this solution were placed in vials, and an 8.6 mM aqueous Fe(NO) 9H O solution was added to a series of vials. A corresponding amount of MOPS buffer was then added to each vial to reach a final reaction volume of 1.1 mL for each vial.

[0114] The solution was gently shaken in a shaker plate for 24 h, at which point the absorbance of 300 µL of each vial was recorded from 350 to 800 nm. The UV-vis spectrum results showing the MLCT band for iron loading of DIBI are shown in Figure 2.

[0115] Plotting the absorption maximum at 460 nm against iron(III) concentration yielded a plateau at 371 μM iron(III). As a result, the nine MAHMP residues present on average on each molecule of DIBI, i.e., H 1Separate measurements by nuclear magnetic resonance spectroscopy (Ang et al., 2018) showed that iron tris-chelation (i.e., hexadentate binding of three iron atoms per molecule of DIBI with nine bidentate MAHMP residues, three MAHMP moieties for each bound iron atom) was fully available, resulting in a total iron-binding capacity of 338 μmol of iron per gram of DIBI. No precipitate was observed during the iron titration experiment, and the colored film obtained upon solution evaporation readily redissolved in water after drying for several weeks. These results indicate that both DIBI and iron-loaded DIBI are soluble in aqueous media.

[0116] Example 2B: Iron(III) Binding Affinity of DIBI and MAHMP To determine the binding affinity of DIBI to iron(III) and its component chelating ligand, MAHMP, acidic solutions of the corresponding 1:3 Fe:HPO complexes in a solution of constant ionic strength (I = 0.1 M) were titrated with NaOH. Titrations were performed under a humid nitrogen atmosphere to avoid concentration changes due to evaporation. Aliquots of each complex solution were periodically removed during the titration, and UV-vis spectra of the aliquots were collected as a batch once the titration was complete. The pH range tested spanned the equilibrium between bis- and tris-coordination for the ligand, as these are the only two species expected at physiological pH. For Fe3-DIBI, spectra were collected 24 h after collecting the batch of aliquots. Spectra were processed using HypSpec2014 to determine the iron(III) binding affinities of MAHMP, DIBI, and deferiprone (DFP) as a reference, as summarized in Table 2.

[0117] [Table 2] The value in parentheses is the standard deviation of the last digit.

[0118] MAHMP appeared similar to DFP, as might be expected given their close structural similarity, since both are bidentate chelators. That is, three chelator molecules are required for full hexadentate coordination of one Fe(III). DIBI, on the other hand, has multiple MAHMP chelating monomers (an average of nine per molecule) dispersed along its non-iron-binding polymer backbone. Thus, intramolecular tris complex formation could result from chelating MAHMP monomers in close spatial proximity along the polymer backbone backbone, or from structural loops that bring MAHMP residues into sufficient proximity with one another. DIBI's enhanced iron binding is reflected in higher Logβ2 and especially higher Logβ3 formation constants, with DIBI exhibiting up to 1000-fold higher Fe-binding affinity than either MAHMP or DFP.

[0119] Example 3: Initial testing of S. aureus, DIBI, and CIP Example 3A: Staphylococcus aureus accumulates excess medium iron Although MHB is considered the standard medium for MIC testing of antimicrobial agents, it is also known to contain a large excess of iron that exceeds the minimum microbial requirement for this growth-essential metal. This has been shown to affect the susceptibility of S. aureus to various iron chelators, particularly in the case of S. aureus, likely due to the ability of this bacterium to store excess iron provided in the growth medium.

[0120] We evaluated the cellular iron status of S. aureus grown in MHB, MHB substantially depleted of excess iron using FEC1 (FECMHB), and RPMI, a fully defined chemical medium containing only low amounts of inorganic iron sources. Because manganese has been shown to have similar electrochemical properties to iron and is involved in bacterial ROS regulation, the cellular concentration of manganese was also determined. Because zinc is not known to be involved in bacterial ROS regulation, zinc was also determined for comparison. Growth in MHB promoted cellular storage of excess iron and manganese, but in contrast, did not promote zinc, as shown in Table 3. Note that cellular iron and manganese concentrations were both approximately 10-fold higher in cultures in MHB compared to RPMI, indicating that RPMI provides sufficient iron for unlimited S. aureus growth. Growth in FECMHB resulted in a substantial reduction in the cellular load of both iron and manganese.

[0121] [Table 3]

[0122] Example 3B: Cellular iron status affects sensitivity to DIBI but not to CIP The susceptibility to both DIBI and CIP was tested by measuring the MICs for S. aureus ATCC 25923 and ATCC 43300 cultured in these three different media, and the results are shown in Table 4.

[0123] [Table 4]

[0124] Cellular iron status significantly affected susceptibility to DIBI. Bacteria grown in MHB had a 2000- to 4000-fold increase in MIC for DIBI compared with bacteria grown in FECMHB or RPMI. However, iron status did not significantly affect susceptibility to CIP, which remained similarly high (MIC = 0.25-5.0 μg / mL) whether the bacteria were loaded with excess iron or not.

[0125] It has previously been shown that the combination of a low, near-MIC concentration of DIBI (2.5 μg / mL (0.28 μM)) with 1×MIC CIP provided synergistic killing of FECMHB-grown S. aureus 43300 with a greater than 4 log CFU reduction by 24 hours of exposure compared to CIP-only treated cells, which grew substantially over 24 hours in the presence of this relatively low CIP concentration. Increasing CIP exposure to 2×MIC CIP resulted in rapid initial killing for both MHB- and FECMHB-cultured ATCC 25923 with an overall similar degree of killing by 24 hours, as shown in Table 5.

[0126] [Table 5]

[0127] Example 4: DIBI inhibits recovery growth of bacteria exposed to ciprofloxacin (CIP) In the test model, bacteria were exposed to CIP at twice its minimum inhibitory concentration (2xMIC) to provide substantial initial killing of the bacterium Staphylococcus aureus ATCC 25923, and the killed cultures were then incubated for 48 hours in the continued presence of CIP for periodic sampling of survivor growth. This antibiotic exposure resulted in a Log 10CIP was found to cause early killing of colony-forming units (CFU) / mL, and this initial killing phase by CIP was essentially complete by 12 hours of exposure, with no significant change in survivor numbers until 24 hours (Figure 3). The surviving population, while still exposed to CIP, showed substantial recovery growth after 24 hours, with survivor numbers increasing throughout the 48 hours of exposure.

[0128] Although both DIBI and thiourea, the compositions prepared in Example 1B, slowed the initial rate of kill by CIP, the overall extent of kill in both DIBI- and thiourea-treated CIP-exposed cultures was similar to that of CIP alone by 24 hours. For DIBI-treated CIP-exposed cultures, recovery growth after 24 hours was impaired, and bacterial populations at 48 hours were still below the initial inoculum CFU / mL. Thiourea appeared to significantly enhance recovery growth of CIP-exposed cultures after 24 hours.

[0129] Figure 3 shows the effects of ciprofloxacin, DIBI, and thiourea on the killing and recovery of Staphylococcus aureus. ATCC 25923 was grown in Mueller-Hinton Broth (MHB), and standardized inocula were added to 10 mL flask cultures of untreated RPMI control (●), 50 μg / mL DIBI (■), 0.5 μg / mL CIP (▲), 10 mM thiourea (◆), 50 μg / mL DIBI combined with 0.5 μg / mL CIP (□), and 10 mM thiourea combined with 0.5 μg / mL CIP (◇) in RPMI. Samples obtained during exposure were plated on BA, and CFU / mL was determined. Data represent the mean + / - SEM of three independent experiments.

[0130] The series of experiments was then repeated, and survivor growth was followed over an extended 96-hour exposure period. The results are shown in Figure 4. Overall kill by CIP at 24 hours was again similar for CIP alone and DIBI- and thiourea-treated CIP-exposed bacteria, despite an early decline in the initial rate of kill for both thiourea- and DIBI-treated cultures. Thiourea promoted increased recovery after 24 hours, and survivor counts increased to those of the untreated control and CIP alone by 96 hours. DIBI-treated CIP-exposed survivor counts remained low throughout the 96-hour exposure, with final CFU / mL remaining lower than the initial inoculum, as shown in Figure 4. The combination of DIBI and thiourea resulted in overall kill at 24 hours similar to DIBI alone, but DIBI-thiourea only provided moderate recovery growth, i.e., compared to thiourea alone, which allowed strong recovery growth similar to CIP alone by 96 hours. DIBI-thiourea-treated survivor counts at 96 hours of CIP exposure remained below the initial inoculum count.

[0131] Figure 4 shows the long-term effects of ciprofloxacin, DIBI, and thiourea on the killing and recovery growth of Staphylococcus aureus. ATCC 25923 was grown in MHB, and standardized inocula were added to 10 mL flask cultures of the following: untreated RPMI control (●), 50 μg / mL DIBI (■), 0.5 μg / mL CIP (▲), 10 mM thiourea (◆), 50 μg / mL DIBI combined with 0.5 μg / mL CIP (□), 10 mM thiourea combined with 0.5 μg / mL CIP (◇), and RPMI supplemented with 50 μg / mL DIBI and 10 mM thiourea combined with 0.5 μg / mL CIP (Δ). Samples obtained during exposure were plated on BA, and CFU / mL was determined. Data represent the mean + / - SEM of two independent experiments.

[0132] This example suggests that the anti-cytotoxic agent ciprofloxacin provides initial kill of the majority of the bacterial population, but allows the recovery growth of survivors in its presence when used alone.

[0133] Example 5. Chronic CIP exposure is selective for small colony variant phenotypes, and DIBI prevents their formation All recovery count plating for the CIP exposure experiments in Examples 3 and 4 was on BA, and it was observed that for CIP-exposed bacteria, a gradually increasing proportion of recovered colonies over the recovery period had pinpoint morphology, which required additional incubation for up to 48 hours for reliable enumeration. These small colony variants (SCVs) were not evident in any of the other treatment groups at 96 hours.

[0134] Given that recovery growth in the presence of 2xMIC CIP was substantially in the form of SCVs, the response of two strains, ATCC 25923 and ATCC 43300, was compared to this prolonged CIP exposure and the effect of DIBI on recovery growth and SCV formation. Both strains had similar overall responses to 2xMIC CIP and DIBI (Figure 5). CIP-exposed bacteria recovered to near-control numbers by 96 hours. DIBI, by itself, partially inhibited growth and significantly inhibited recovery growth for both strains after CIP exposure. SCVs were enumerated for these experiments, and the results are shown in Figure 3. SCVs became increasingly enriched in CIP-exposed cultures for both strains between 48 and 96 hours, with approximately 80% of the total viable population present as SCVs by 96 hours of exposure to CIP. No SCVs were present in cultures not exposed to CIP (including DIBI alone) (Figure 5). Interestingly, when DIBI was combined with CIP, it suppressed overall recovery growth, with no survivors recovered as SCVs over the 96-hour exposure period.

[0135] Figure 5 shows the effects of ciprofloxacin and DIBI on Staphylococcus aureus killing and recovery growth of small-colony variants. S. aureus ATCC 43300 (A and C) and ATCC 25923 (B and D) were grown in MHB, and standardized inocula were added to 10 mL flask cultures of untreated RPMI control (○) or RPMI supplemented with 50 μg / mL DIBI (□), 0.5 μg / mL CIP (Δ), or 50 μg / mL DIBI combined with 0.5 μg / mL CIP (▽). Samples taken during exposure were plated on BA for enumeration of total CFU / mL (A and B) and percentage of SCV (C and D) in the recovered population. Data represent the mean + / - SEM of two independent experiments.

[0136] The colony morphology of recovered SCVs for ATCC 25923 compared to non-SCVs from survivors grown in various treatments over the exposure period is shown in Figure 6. Progressive enrichment of SCVs over time in CIP-exposed cultures is observed, and colony morphology for all other treatment groups shows the typical size variability for spot plating. Similar results were obtained for ATCC 43300 (not shown).

[0137] Figure 6 shows the effect of ciprofloxacin and DIBI on Staphylococcus aureus small-colony variant formation. S. aureus ATCC 25923 was grown in MHB, and standardized inocula were added to 10 mL flask cultures of untreated RPMI control, 50 μg / mL, 0.5 μg / mL CIP, or 50 μg / mL DIBI in combination with 0.5 μg / mL CIP in RPMI. Samples taken during exposure were plated on BA for enumeration of total CFU / mL and percentage of SCV in the recovered population. All photographs are at the same final magnification and represent two independent experiments.

[0138] This example suggests that bacterial survivors that recovered from exposure to the anti-cellular agent ciprofloxacin acquired altered phenotypes in response to the anti-cellular agent, and that compositions of the invention may prevent their formation.

[0139] Example 6: CIP-induced persister SCVs display stable acquired resistance to quinolones and their formation is blocked by DIBI. Six isolated subculture clones from each of the various treatments at 96 hours were confirmed to be S. aureus by growth on mannitol salt agar (MSA) and then grown in RPMI. Their ability to grow in this simple medium, although slower, indicated that these SCVs were neither auxotrophic nor fastidious. Growth was slow on MSA, BA, and the same basal medium lacking blood (Trypticase Soy Agar), indicating that the slow growth was not related to the recovery medium. SCVs exhibited hemolysis on BA, but this was only evident in heavily linearized regions or in cultures incubated for at least 48 hours. Subcultured clones were grown and tested in RPMI for susceptibility to DIBI, CIP, MOX, GEN, MUP, and VAN; median 24-hour MIC results are shown in Table 6.

[0140] [Table 6] MIC; minimum inhibitory concentration, DIBI; iron chelating polymer, CIP; ciprofloxacin, MOX; moxifloxacin, GEN; gentamicin, MUP; mupirocin, VAN; vancomycin; a ATCC43300 and ATCC25923 were tested; b Only ATCC 43300 was tested; c Only ATCC 25923 was tested.

[0141] All CIP-induced SCV clones (12 / 12, 6 from each strain) were found to have stable colony morphology after repeated subculture on antibiotic-free medium, suggesting genetic changes as opposed to epigenetic adaptation. All of these SCV clones exhibited increased resistance to CIP, with a median MIC of 1.0 μg / ml (3 μM), ranging from 0.5 to 2.0 μg / ml, approximately 4-8-fold higher than that of untreated controls. Similar results of increased resistance were also found for MOX (ATCC 43300). However, representative survivor SCV clones remained as susceptible as controls to MUP (ATCC 43300), GEN, and VAN (ATCC 25923) (Table 3). DIBI exposure alone did not result in significant changes in CIP, MOX, or GEN MICs in the recovered populations. When DIBI was combined with CIP, survivor clones had normal colony morphology (Figure 6) and had similar CIP and MOX MICs to controls (Table 4). The DIBI sensitivity of clones recovered from all treatments was similar, with an MIC of 4 μg / ml (0.4 μM). Non-SCV CIP survivors were also tested, and these had identical MIC results (not shown) to the SCVs, specifically a median CIP MIC of 1.0 μg / ml (3 μM) (range 0.5–1.0 μg / ml). Because these were rare, it is unclear whether they represent double mutants that compensated for the slow-growth phenotype, or less common types of mutations that do not affect growth, or epigenetic adaptations.

[0142] The killing action of second- and later-generation quinolone antibiotics (e.g., CIP and MOX, respectively) is thought to involve at least two separate but interrelated pathways. The primary pathway involves direct inhibition of CIP targets, topoisomerase and DNA gyrase, which causes DNA strand breaks, leading to inhibition of bacterial replication and ultimately death. The secondary pathway involves an ROS-dependent mechanism of bacterial death induced by quinolone exposure. It is also thought that the accumulation of DNA strand breaks from the primary pathway ultimately leads to further ROS production, to the point where ROS-mediated bactericidal activity has been suggested as the primary cause of bacterial cell death during quinolone treatment. However, the full extent of ROS involvement in bacterial antibiotic killing remains somewhat unclear, for example, in the case of Streptococcus pneumoniae, which is highly susceptible to killing by bactericidal antibiotics but still lacks an electron transport system, a presumed major source of ROS, because ROS detection using fluorescent probes may be unreliable given non-ROS antibiotic-induced autofluorescence.

[0143] Here, we used a relatively high concentration of CIP (2× the MIC) and tested the effect of thiourea, a known ROS scavenger, during continuous exposure to CIP. Thiourea slowed the initial rate of death, but the overall extent of death by 24 h was similar to that of CIP alone. Others have reported that thiourea, along with the iron chelator dipyridyl, inhibited the ROS-dependent death pathway in MOX-treated Escherichia coli. Thiourea quenches existing ROS, while dipyridyl likely reduces ROS production by sequestering Fenton-ROS-reactive iron. DIBI, a potent iron chelator highly effective against S. aureus, was also found to slow the initial rate of CIP death but not the overall extent of death over 24 h during CIP exposure. While thiourea in combination with DIBI did not further slow the initial death rate, DIBI prevented the recovery growth after 24 h of exposure observed with thiourea alone.

[0144] Thus, the results herein suggest only a relatively small component of ROS-related early killing by CIP in the model system. In this regard, it is important to note that we tested CIP with fully filled / excess cellular iron stores to ensure there was excess intracellular iron for possible involvement in ROS generation. Previous studies of DIBI in combination with CIP showed a >3 log reduction in S. aureus killing during exposure to lower CIP concentrations (1 MIC). 10 These results suggest that CIP activity is dependent on iron-dependent systems unrelated to ROS production. Interestingly, intracellular iron-sulfur clusters are required for the activity of bacterial DNA polymerases for the repair of antibiotic-induced DNA damage in Staphylococcus. Therefore, it seems plausible that DIBI may interfere with intracellular bacterial iron-sulfur cluster synthesis or other iron-containing targets as part of its mechanism of action.

[0145] CIP has been found to likely induce the formation of mutant SCV survivors that have stable acquired resistance to CIP and its related quinolone MOX but not to the unrelated aminoglycoside GEN or other antibiotics. This suggests that the slow-growth phenotype observed here may be the result of CIP target-specific alterations rather than general metabolic changes that affect bactericidal activity across antibiotic species, as has been seen with other SCV phenotypes. If the topoisomerase or gyrase targets of CIP spontaneously alter to reduce CIP activity, this could potentially alter growth rate due to their role in DNA replication.

[0146] Various antibiotics, including quinolones, are known to induce the formation of bacterial persisters resistant to the antibiotic. This response to CIP in S. aureus has been shown to occur, at least in part, through the induction of an SOS response via ROS generation and activation of the RecA system, which can be inhibited by the herb baicalein, which has also been shown to be an iron chelator and inhibitor of the Fenton reaction.

[0147] The ROS scavenger thiourea and the iron chelator DIBI could both suppress SCV viable somatic cell formation in response to CIP, which may indicate a role for ROS in causing genetic mutations leading to antibiotic resistance. These putative SCV mutants were no more sensitive to DIBI than untreated clones, supporting the notion that DIBI prevented their initial formation, possibly through the suppression of ROS-mediated DNA damage that could increase DNA mutations, since DIBI alone did not suppress S. aureus growth over the entire incubation period. This possibility is indirectly supported by studies using the cell-permeable iron chelator o-phenanthroline with CIP-treated E. coli. DIBI is expected to be impermeable to bacteria, but it may similarly affect respiration (and therefore ROS production) through iron sequestration.

[0148] The extracellular location of DIBI may be beneficial during treatment, as curcumin, a well-studied turmeric extract that enters mammalian cells and chelates iron, has been shown to have diverse results as an antibacterial agent. For example, curcumin inhibited Listeria monocytogenes and Shigella flexneri but enhanced infection of mammalian cells by several other intracellular bacteria, including Salmonella species, S. aureus, and Yersinia enterolytica. Furthermore, curcumin interfered with CIP treatment of Salmonella species by affecting host cell signaling and the respiratory burst. Although S. aureus is largely extracellular in vivo, naturally occurring SCV variants can invade host cells, providing a reservoir for evading host defenses and antibiotic treatment; therefore, preventing their formation during antibiotic treatment is ideal.

[0149] DIBI was found to substantially prevent the growth of relatively quinolone-resistant S. aureus SCV survivors in multiple independent experiments with unrelated strains, limiting overall recovery growth from CIP exposure. This finding is relevant in the context of bacterial evolution of antibiotic resistance and antibiotic failure, as it shows that resistance can be acquired in incremental steps. The resulting MICs of CIP survivors were typically 1.0 g / mL and as high as 2.0 g / mL, at least twice the 0.5 g / mL concentration encountered in the initial antibiotic challenge assay. The observed increase in resistance could be from only a single CIP exposure, and although these increased MICs were approximately 4–8 times higher than those of control-treated survivors, they did not exceed the accepted MIC threshold (>1 g / mL) for classification as CIP-resistant using clinical breakpoints.

[0150] No resistance to DIBI was observed during prolonged exposure, consistent with our previous findings that resistance did not develop during repeated subculture of S. aureus in the presence of sub-MIC amounts of DIBI. During experimental infection with a CIP-resistant, highly virulent clinical isolate, DIBI has been shown to enhance CIP efficacy against Acinetobacter baumannii. Taken together, this may indicate the potential of DIBI as an adjunct to CIP and other antibiotics to improve antibiotic efficacy and prevent the development of antibiotic resistance.

[0151] This example suggests that bacterial survivors recovered from exposure to the antimicrobial agent ciprofloxacin acquired resistance to the antimicrobial agent and that the compositions of the present invention can prevent this resistance development. The examples herein demonstrate that the iron chelator DIBI can improve the outcome of ciprofloxacin treatment of S. aureus strains, at least by inhibiting growth during the recovery phase of an in vitro killing assay. Most bacterial colonies plated after 72-96 hours of CIP exposure were found to be in the small colony variant morphology, which was not observed when DIBI was combined with CIP. When these SCV clones were regrown in the absence of antimicrobial agents, the slow-growth phenotype remained, and these stable mutants were found to have high MICs to fluoroquinolones. In contrast, S. aureus clones exposed to CIP in the presence of DIBI did not change their MICs. The data presented here support the hypothesis that iron chelation by DIBI in combination treatment may suppress mechanisms that result in spontaneous resistance to CIP and further provide a body of evidence that DIBI may be useful as an adjunct antibacterial agent.

[0152] Example 7: Materials and Methods Antibiotics, media, and bacterial strains Ciprofloxacin (CIP), moxifloxacin (MOX), and mupirocin (MUP) (Sigma-Aldrich, St. Louis, MO, USA) were prepared as 1 mg / mL stocks in deionized water, and gentamicin (GEN) and vancomycin (VAN) (Sigma-Aldrich) were prepared at 10 mg / mL in water. All antibiotics were stored at −80°C, thawed, and diluted in RPMI immediately before use. The iron chelator DIBI-R12 (DIBI) (provided by Fe-Pharmaceuticals Canada Inc., formerly Chelation Partners Inc., Halifax, NS, Canada) was dissolved in Roswell Park Memorial Institute Medium 1640 (RPMI, Sigma-Aldrich) containing L-glutamine, buffered with 0.165 M 3-(N-morpholino)-propanesulfonic acid (MOPS, Sigma-Aldrich), filter-sterilized, and stored at 4°C as 200 mg / mL or 20 mg / mL. Thiourea (Sigma-Aldrich) was dissolved in RPMI at 100 mM, filter-sterilized, and used fresh or from storage at 4°C. DIBI was diluted to a final concentration of 50 μg / mL in 10 mL of RPMI in a sterile glass flask, CIP was diluted to 0.5 μg / mL (equivalent to 2× the MIC previously determined to be 0.25 μg / mL), and thiourea was diluted to 10 mM. Mueller-Hinton Broth (MHB, Oxoid, Basingstoke, Hampshire, UK) was partially deferred with an insoluble form of DIBI called FEC-1 (Fe-Pharmaceuticals Canada Inc., Halifax, NS, Canada), followed by removal of FEC-Fe by filtration, as previously described, to yield FECMHB. The selective medium mannitol salt agar (Sigma-Aldrich) was used for confirmation of S. aureus.Staphylococcus aureus ATCC 43300, ATCC 6538, and ATCC 25923 clinical reference strains were obtained from the Nova Scotia Health Authority (Halifax, NS, Canada) and grown overnight at 35°C from 10% glycerol stocks at −80°C on blood agar (BA, Oxoid, tryptic soy agar (TSA) with 5% sheep blood).

[0153] Antibiotic Kill and Recovery Growth Test Isolated colonies were used to inoculate 50 mL of MHB liquid culture in glass flasks with overnight shaking at 220 rpm at 35°C. Optical density (600 nm) was measured and used to generate standardized inocula for addition to 10 mL of RPMI flasks containing treated or untreated medium. The size of the inoculum was confirmed by serial dilution and plate counting on BA plates. At predetermined time points, the optical density (OD) of each flask was measured at 600 nm by removing an aliquot to be measured spectrophotometrically. Colony counts were performed by serial dilution in tubes containing phosphate-buffered saline (PBS, Sigma-Aldrich) and then spot-plating 50 μL of the appropriate dilution onto BA plates without antibiotics. Plates were incubated at 35°C, and colonies were initially counted after 24 h of incubation. Colonies significantly smaller than normal (pinpoint size) were counted as small colony variants (SCVs) and their locations were marked. The count plates were returned to the incubator for an additional 24 hours, and the plates were recounted. Any new colonies not previously counted were counted as SCVs and tallied together or separately for consideration apart from the total CFU / mL.

[0154] Isolation and characterization of treatment survivors Bacterial survivors from the CIP exposure experiment were selected from the 96-hour time point in the four treatment groups and streaked onto fresh BA plates containing no antibiotic to assess the stability of the slow-growth phenotype; plates were incubated for 48 hours to allow the slow-growths to reach a more normal colony size, if necessary. Surviving colonies were also plated onto MSA to rule out contamination, and collected growth was maintained as frozen stocks in 10% glycerol.

[0155] antibiotic sensitivity Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method recommended by CLSI. Re-cultured treatment survivors were inoculated into RPMI liquid tube cultures and incubated overnight at 35°C and 220 rpm. The OD of the cultures was measured and used as a reference to create a standardized suspension of 0.1 OD / mL, which was then further diluted 1:10 in RPMI to generate the inoculum for MIC determination. 96-well microplates containing 100 μL of serial two-fold antibiotic dilutions containing antibiotic or DIBI were prepared in duplicate wells, to which 5 μL of bacterial inoculum was added. The loaded microplates were incubated at 35°C. MIC endpoints were visually read at 24 and 48 hours of incubation as the lowest antibiotic concentration allowing zero visible growth in the wells.

[0156] Trace element determination of iron, manganese, and zinc Three strains of S. aureus were grown on BA for 24 hours at 35°C, after which isolated colonies were inoculated into 50 mL of MHB, 50 mL of FEC-MHB, or four colonies into 100 mL of RPMI in a flask. Cultures were incubated overnight at 35°C with shaking at 220 rpm. Cultures were recovered in 15 mL acid-washed polypropylene tubes and centrifuged at 3345 rcf for 15 minutes, and the supernatant was decanted. Phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich) was added to one tube, and the pellet was resuspended and combined into a single tube per sample. Centrifugation was repeated for 10 minutes, followed by an additional wash with 10 mL of PBS and finally 5 mL of deionized water. The pellet was resuspended in 2 mL of deionized water. The washed samples were transferred to pre-weighed quartz pressure vials and centrifuged for 5 minutes. The supernatant was carefully removed with a micropipettor as little as possible to avoid disturbing the pellet. The sample vials were placed in a vacuum oven at 85°C for 48 hours. After cooling to room temperature, the samples were weighed six times to determine the average dry weight. The samples were then subjected to trace element quantification. For this purpose, microwave-assisted digestion was used to homogenize the bacterial samples using a Discover SP-D microwave digester (CEM Corporation, Matthews, NC, USA). To the samples in 10 mL quartz pressure vials, 600 μL of concentrated nitric acid (trace metals-grade nitric acid, Thermo Fisher Scientific, Waltham, MA, USA) and 450 μL of hydrogen peroxide were added. The digested samples were diluted with Milli-Q water to obtain a nitric acid concentration of approximately 2% before analysis. Iron, manganese, and zinc were measured using an iCAP Q inductively coupled plasma mass spectrometer (ICP-MS, Thermo Fisher Scientific, Waltham, MA, USA) coupled with an ESI SC-4DXS autosampler (Elemental Scientific, Omaha, NE, USA). All samples were run in kinetic energy discrimination mode using high-purity helium (99.999%) as the collision gas.

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[0205] All cited documents are incorporated herein by reference. In the event of conflicting statements and information between any reference to or incorporated herein and this disclosure, this disclosure serves as the guiding authority.

[0206] What has been described is merely illustrative of the application of the principles of the present disclosure, but it will be apparent to those skilled in the art that certain changes and modifications can be made without departing from the scope of the claims set forth below.

Claims

1. a transition metal chelate polymer; an anti-infective agent, The chelating polymer is a reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent; The first monomer unit is represented by compound (I): 【Chemistry 1】 During the ceremony, R １ is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R ２ is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R ３ is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12; the second monomer units are independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene; the chelating polymer is soluble in an aqueous medium; A composition wherein the chelating polymer has a molecular weight of at least about 1500 Da prior to chelation.

2. R １ is H, R ２ is methyl, R ３ is methyl, 2. The composition of claim 1, wherein n is 1 to 6, and optionally n is 2.

3. The first monomer unit is represented by compound (II): 【Chemistry 2】 The composition according to claim 1 or 2.

4. The composition of any one of claims 1 to 3, wherein the second monomer unit is 1-vinyl-2-pyrrolidone.

5. The composition of any one of claims 1 to 4, wherein the second monomer unit is N,N-dimethyl-acrylamide.

6. 6. The composition of any one of claims 1 to 5, wherein the addition-fragmentation chain transfer agents are independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

7. The composition of any one of claims 1 to 7, wherein the transition metal is one or more of iron, manganese, cobalt, copper, or nickel.

8. The composition of any one of claims 1 to 8, wherein the anti-infective agent is an antibiotic.

9. 10. The composition of claim 9, wherein the antibiotic is a fluoroquinolone antibiotic.

10. 11. The composition of claim 10, wherein the fluoroquinolone antibiotic is moxifloxacin or ciprofloxacin.

11. 12. The composition of claim 11, wherein the fluoroquinolone antibiotic is ciprofloxacin.

12. The composition of any one of claims 1 to 12, further comprising thiourea.

13. The composition of any one of claims 1 to 13, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

14. A composition according to any one of claims 1 to 14 for use in reducing the resistance of an organism to said anti-infective agent.

15. 16. The composition for use of claim 15, wherein the organism is susceptible to the anti-infective agent prior to exposure to the composition.

16. 17. The composition for use according to claim 15 or 16, wherein the organism is a bacterium.

17. 18. The composition for use according to claim 17, wherein the bacterium is gram-positive.

18. 19. The composition for use according to claim 18, wherein the bacterium is Staphylococcus aureus.

19. 15. A method of treating a bacterial infection in a subject, comprising administering to the subject a composition according to any one of claims 1 to 14.

20. 15. Use of a composition according to any one of claims 1 to 14 for reducing bacterial resistance to said anti-infective agent in a subject, wherein the composition according to any one of claims 1 to 14 is for administration to said subject having said bacteria.

21. 15. Use of a composition according to any one of claims 1 to 14 in the manufacture of a medicament for reducing bacterial resistance to said anti-infective agent in a subject, wherein the composition according to any one of claims 1 to 14 is for administration to said subject having said bacteria.

22. 22. The use according to claim 20 or 21, wherein the bacterium is gram-positive.

23. 23. The use according to claim 22, wherein the bacterium is Staphylococcus aureus.

24. 1. A transition metal chelating polymer for use as an adjuvant in an antibiotic composition, comprising: The chelating polymer is a reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using an addition-fragmentation chain transfer agent; The first monomer unit is represented by compound (I): 【Transformation 3】 During the ceremony, R １ is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R ２ is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R ３ is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12; the second monomer units are independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene; the chelating polymer is soluble in an aqueous medium; A transition metal chelating polymer, wherein said chelating polymer has a molecular weight of at least about 1500 Da prior to chelation.

25. R １ is H, R ２ is methyl, R ３ is methyl, 2. The composition of claim 1, wherein n is 1 to 6, and optionally n is 2.

26. The first monomer unit is represented by compound (II): 【Chemistry 4】 26. The composition of claim 24 or 25.

27. The composition of any one of claims 24 to 26, wherein the second monomer unit is 1-vinyl-2-pyrrolidone.

28. The composition of any one of claims 24 to 26, wherein the second monomer unit is N,N-dimethyl-acrylamide.

29. 29. The composition of any one of claims 24 to 28, wherein the addition-fragmentation chain transfer agents are independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

30. The use according to any one of claims 24 to 29, wherein the antibiotic is a fluoroquinolone antibiotic.

31. 31. The use of claim 30, wherein the fluoroquinolone antibiotic is moxifloxacin or ciprofloxacin.

32. 32. The use of claim 31 , wherein the fluoroquinolone antibiotic is ciprofloxacin.

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