Fibrinogen as an antimicrobial agent and adjuvant for antimicrobial therapy.

Abstract

The present invention relates to a composition for increasing the antimicrobial susceptibility of microorganisms, comprising fibrinogen. The present invention can be used to alleviate the problem of antimicrobial resistance of microbial biofilms or aggregates, for example, on medical devices, particularly indwelling devices, or on wound beds. The device or wound bed is (pre)coated with fibrinogen to increase its antimicrobial susceptibility. The antimicrobial agent may be provided separately from the fibrinogen or together with the fibrinogen.

Description

[Technical Field]

[0001] The present invention relates to methods for increasing the susceptibility of microorganisms to antimicrobial agents and antimicrobial treatments, and in particular to methods used to reduce the resistance of biofilms to antimicrobial agents. [Background technology]

[0002] Antimicrobial resistance among pathogenic bacteria is a growing concern worldwide. Often, pathogenic bacteria manage to evade the effects of previously powerful antimicrobial compounds, such as penicillin. Various strategies are being used to overcome the problem of antimicrobial resistance: 1) new antimicrobial drugs are being developed; 2) combination therapy using two or more existing antimicrobial drugs is being used; and 3) treatment strategies and therapeutic agents that act as adjuvants to existing antimicrobial drugs are used in combination with these antimicrobial drugs to reduce pathogen resistance to these drugs. Antimicrobial adjuvant formulations and treatment strategies include anti-resistance drugs (e.g., β-lactamase inhibitors), anti-virulence drugs (e.g., bacterial toxin inhibitors), and host-targeted therapies (e.g., innate immune system agonists).

[0003] One mechanism used by microorganisms to increase resistance to antimicrobial agents and antimicrobial treatments is the formation of aggregates, microcolonies, or biofilms. Pathogens in biofilms or aggregates are known to be 10 to 1,000 times more resistant to antimicrobial agents than planktonic pathogens. Biofilm formation on indwelling medical devices, such as catheters, stents, heart valves, pacemakers, and artificial joints, is a serious problem in the medical device field, where it accounts for 65% of all infections. Biofilm formation can lead to device failure and the spread of device-related infections. Device replacement after removal or simultaneous antimicrobial treatment and decontamination is currently recommended. Alternatives to removal and replacement have been proposed. Kretlow et al. (2014) (Plast Reconstr Surg. 2014, 133(1):28e-38e) reported in situ treatment of left ventricular assist devices with antimicrobial beads.

[0004] A further complication is the presence of polymicrobial biofilms containing certain fungi and viruses or fungi and bacteria, resulting in increased resistance to antiviral, antibacterial and antifungal agents.

[0005] Because pathogen adhesion is necessary for colonization and subsequent disease progression, some have focused on preventing pathogen adhesion to surfaces to prevent biofilm formation. Fibrinogen, one of the most abundant host plasma proteins, is thought to be a key host protein that promotes biofilm formation. Flores-Mireles et al. (J Urol 2016: 416) reported fibrinogen deposition as an accumulation surface for uropathogenic bacteria. Kwiecinski et al. (J. Infect. Dis. 2016: 213) reported how fibrinolytic enzymes prevent biofilm formation. Summary of the Invention

[0006] Fibrinogen is also thought to increase the antimicrobial resistance of biofilms. Bedran et al. (Biomed Research International vol. 2013, article ID 431465) reported in vitro results showing that fibrinogen induces biofilm formation by Streptococcus mutans and increases penicillin resistance. Jorgensen et al. (Microorganisms 2016: 4) reported in vitro how fibrinolytic enzymes that specifically degrade fibrinogen or fibrin significantly increase the antimicrobial susceptibility of bacterial biofilms. These reports demonstrate that the presence of fibrinogen stimulates biofilm formation in vitro and that fibrinolysis, resulting in fibrinogen depletion, improves the effectiveness of antimicrobial agents in treating biofilm infections. [Brief explanation of the drawings]

[0007] [Figure 1] Adhesion of Staphylococcus aureus to different immobilized fibrinogen species [Figure 2] Viable counts of Staphylococcus aureus associated with biofilms formed on surfaces precoated with different fibrinogen species in vitro [Figure 3] In vitro viable counts of Staphylococcus aureus detected in biofilms formed on non-precoated surfaces using plasma Fib or rhFibγ´ in bacterial culture medium during biofilm formation [Figure 4] In vivo viable counts of S. aureus detected in biofilms formed on PTFE cages pre-coated with plasma Fib or rhFibγ´, implanted subcutaneously in mice, and infected with S. aureus. [Figure 5] Viable counts of Staphylococcus aureus detected in biofilms formed on surfaces pre-coated with different fibrinogen species and exposed to different doses of vancomycin or ampicillin in vitro [Figure 6A] In vitro viable counts of S. aureus detected in biofilms formed on non-precoated surfaces in the presence of plasma Fib (6A and 6C) or rhFibγ´ (6B and 6D) in bacterial cultures; and in 6E) a 50 / 50 mixture of plasma Fib and rhFibγ´, subsequently exposed to vancomycin. [Figure 6B] In vitro viable counts of S. aureus detected in biofilms formed on non-precoated surfaces in the presence of plasma Fib (6A and 6C) or rhFibγ´ (6B and 6D) in bacterial cultures; and in 6E) a 50 / 50 mixture of plasma Fib and rhFibγ´, subsequently exposed to vancomycin. [Figure 6C] In vitro viable counts of S. aureus detected in biofilms formed on non-precoated surfaces in the presence of plasma Fib (6A and 6C) or rhFibγ´ (6B and 6D) in bacterial cultures; and in 6E) a 50 / 50 mixture of plasma Fib and rhFibγ´, subsequently exposed to vancomycin. [Figure 6D] In vitro viable counts of S. aureus detected in biofilms formed on non-precoated surfaces in the presence of plasma Fib (6A and 6C) or rhFibγ´ (6B and 6D) in bacterial cultures; and in 6E) a 50 / 50 mixture of plasma Fib and rhFibγ´, subsequently exposed to vancomycin. [Figure 6E] In vitro viable counts of S. aureus detected in biofilms formed on non-precoated surfaces in the presence of plasma Fib (6A and 6C) or rhFibγ´ (6B and 6D) in bacterial cultures; and in 6E) a 50 / 50 mixture of plasma Fib and rhFibγ´, subsequently exposed to vancomycin. [Figure 7A]In vivo viable counts of S. aureus detected in biofilms formed on PTFE cages pre-coated with plasma Fib, rhFibWT, or rhFibγ´, implanted subcutaneously in mice, and treated with therapeutic (7A) and prophylactic (7B) daptomycin. [Figure 7B] In vivo viable counts of S. aureus detected in biofilms formed on PTFE cages pre-coated with plasma Fib, rhFibWT, or rhFibγ´, implanted subcutaneously in mice, and treated with therapeutic (7A) and prophylactic (7B) daptomycin. DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention relates to the use of a composition comprising fibrinogen for the treatment or prevention of antimicrobial resistance in an individual.

[0009] The use of the present invention alleviates the problem of antimicrobial resistance in microorganisms. In particular, the present invention increases the susceptibility of microorganisms to antimicrobial treatment and is particularly advantageous for addressing microorganisms that typically have minimal susceptibility to antimicrobial compounds, such as microorganisms in biofilms, aggregates, or microcolonies. Biofilms, which are communities of microorganisms, are notorious for their resistance to antimicrobial agents, i.e., they escape the action of previously potent antimicrobial compounds. The compositions used in the present invention can act as adjuvants for antimicrobial agents used in combination with existing antimicrobial agents, reducing the resistance of microbial biofilms or aggregates to these antimicrobial agents.

[0010] In some embodiments, the compositions used in the present invention enhance the susceptibility of bacteria to antibacterial compounds. Antibacterial compounds prevent bacterial growth (bacteriostatic) or kill bacteria (bactericidal) and include antibacterial agents, bacteriophages and their endolysins, antibacterial or antimicrobial peptides, and antibacterial antibodies. Those skilled in the art will appreciate that a bactericidal compound against one bacterium may be bacteriostatic against another, and vice versa. Preferably, the antibacterial compound is a natural, synthetic, or semi-synthetic antibacterial agent. Suitable natural, synthetic, or semi-synthetic antibacterial agents include aminoglycosides, antibacterial imidazoles, beta-lactams, macrolides, peptide antibacterial agents, polyclonal or monoclonal antibodies, quinolones, sulfonamides, and tetracyclines, as well as their derivatives.

[0011] Examples of aminoglycosides include gentamicin and tobramycin. Examples of antibacterial imidazoles include miconazole and metronidazole; examples of beta-lactams include penicillins, including natural penicillins such as amoxicillin, ampicillin, and penicillin, aminopenicillins, beta-lactamase-resistant penicillins, and carboxypenicillins; cephalosporins such as cephalexin; and carbapenems such as meropenem and doripenem. Examples of macrolides include ketolide and macrolide antibacterial agents such as clarithromycin, erythromycin, and telithromycin. Examples of peptide antibacterial agents include glycopeptide antibacterial agents such as vancomycin and lipopeptide antibacterial agents such as daptomycin. Examples of antibodies include antibodies against Staphylococcus aureus clumping factor A, alpha toxin, lipoteichoic acid, and teichoic acid. Examples of quinolones include fluoroquinolones such as ciprofloxacin and levofloxacin. Examples of sulfonamides include sulfacetamide and sulfamethoxazole. Examples of tetracycline antibacterial agents include tetracycline and doxycycline. In one embodiment, the antibacterial compound is a penicillin or a peptide antibacterial agent. In another embodiment, the antibacterial compound is a carboxypenicillin, a glycopeptide antibacterial agent, or a lipopeptide antibacterial agent. In yet another embodiment, the antibacterial compound is selected from the group consisting of ampicillin, vancomycin, daptomycin, and tobramycin.

[0012] Those skilled in the art will be familiar with the fact that some antimicrobial agents are used against several groups of microorganisms, for example, some antimicrobial peptides can be used against both bacterial and viral or fungal infections.

[0013] The compositions can also be used to treat infections involving several microorganisms. The compositions can be used to increase the antimicrobial susceptibility of one or several microorganisms. For example, the compositions can increase the antiviral or antiyeast susceptibility of viruses or yeasts in bacterial biofilms.

[0014] The infectious microorganisms in the biofilm or aggregates are sometimes referred to as pathogens. A notorious pathogen in biofilm-associated infections that can be treated using the adjuvant methods of the present invention is Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA).

[0015] The compositions used in the present invention increase the antimicrobial susceptibility of microorganisms. Microorganisms treated by the present invention are preferably in clusters, such as aggregates or clumps, but may also be attached to a surface, such as in a biofilm. Microorganisms in clusters or biofilms tend to be less susceptible to antimicrobial agents than microorganisms in a planktonic state. In biofilms, microorganisms are typically surrounded by an extracellular matrix (EPS) derived from polymeric substances, such as glycoproteins, secreted by the microorganisms themselves or collected from the host. Biofilms are sometimes referred to in the art as glycocalyx.

[0016] Adhesion surfaces include biological surfaces such as tissues, cell surfaces, and wound beds, as well as inert surfaces such as medical devices, particularly indwelling medical devices or implants, including catheters, implantable cardiac electrical devices (CIEDs), heart valves, immobilizers, artificial joints, stents, tracheostomy tubes, and wound drains. Examples of catheters include peripherally inserted central catheters (PICC lines), implantable ports, and urinary catheters. Examples of CIEDs include pacemakers, left ventricular assist devices (LVADs), and implantable cardioverter-defibrillators. Examples of immobilizers include pins, plates, rods, screws, and wires. Examples of artificial joints include hip, knee, and shoulder prostheses. The devices may comprise or consist of metals, ceramics, or non-biodegradable synthetic polymers such as plastics or polytetrafluoroethylene (PTFE, Teflon®), or biodegradable polymers such as collagen, hyaluronic acid, polylactic acid, or polyurethane. The medical device is preferably an indwelling device or implant designed to remain in the body for days, weeks, months, or years.

[0017] The composition used in the present invention contains fibrinogen. The fibrinogen in the composition may be plasma fibrinogen (plasma Fib), which is a mixture of all fibrinogen variants occurring in the blood of healthy individuals, or it may be a preparation consisting of one or more of these specific fibrinogen variants isolated from plasma (or plasma fibrinogen) or produced by recombinant protein production technology. The fibrinogen in the composition preferably comprises human plasma fibrinogen, human wild-type fibrinogen, human fibrinogen γ', human fibrinogen α-extension variant, or human fibrinogen α-truncated variant. In one embodiment, the fibrinogen in the composition consists of human plasma fibrinogen, human wild-type fibrinogen, human fibrinogen γ' variant, human fibrinogen α-extension variant, or human fibrinogen α-truncated variant. Human fibrinogen refers to fibrinogen having an amino acid sequence present in the majority of humans.

[0018] Plasma fibrinogen refers to fibrinogen isolated from plasma and typically contains a mixture of naturally occurring fibrinogen variants that are the result of alternative splicing, proteolysis, or post-translational modifications. Plasma fibrinogen may be derived from a single individual or pooled from multiple individuals. Preferably, the plasma fibrinogen is highly pure, i.e. constituted in a composition comprising at least 95% w / w, at least 97% w / w or at least 98% w / w plasma fibrinogen. Purity may be determined by its clotting capacity.

[0019] WT fibrinogen is the mature, dominant form of human fibrinogen, accounting for 60-80% of the total plasma fibrinogen in circulating blood. It has an Aα chain consisting of 610 amino acids, a Bβ chain consisting of 461 amino acids, and a γ chain consisting of 411 amino acids. Literally, this form of fibrinogen is also called high molecular weight (HMW) fibrinogen or intact fibrinogen.

[0020] A fibrinogen γ' variant has two α polypeptide chains, two β polypeptide chains, and two γ polypeptide chains, with at least one γ polypeptide chain being a γ' polypeptide chain. In humans, the γ' polypeptide chain has 427 amino acids. When a fibrinogen molecule contains one wild-type γ polypeptide chain (γ411) and one γ' polypeptide chain (γ427), the variant is referred to herein as a fibrinogen γ' heterodimer or Fibγ427 / 411. When both γ polypeptide chains are of the γ' type (γ427), the variant is referred to as a fibrinogen γ' homodimer or Fibγ427 / 427. The compositions used in the present invention may comprise or consist of a mixture of γ' variants, such as a mixture of fibrinogen γ' heterodimers and homodimers, or may comprise or consist of a single γ' variant.

[0021] A fibrinogen α-extension variant has two α polypeptide chains, two β polypeptide chains, and two γ polypeptide chains, with at least one α polypeptide chain being an extended polypeptide chain and at least one γ polypeptide chain being a γ' polypeptide chain. In humans, the α-extension variant has 847 amino acids. When a fibrinogen molecule contains one wild-type α polypeptide chain (Aα610) and one α-extension variant (Aα847), the variant is referred to herein as a fibrinogen α-extension heterodimer or rhFibAα847 / Aα610. When both α polypeptide chains are α-extension (Aα847), the variant is referred to as a fibrinogen α-extension homodimer or FibAα847 / 847. The compositions used in the present invention may comprise or consist of a mixture of α-extension variants, such as a mixture of fibrinogen α-extension heterodimers and homodimers, or may comprise or consist of a single α-extension variant.

[0022] Fibrinogen alpha-truncated variants have two alpha, two beta, and two gamma polypeptide chains, with at least one alpha chain truncated between amino acid 251 and the C-terminal amino acid 610. When a fibrinogen molecule contains one wild-type alpha chain (Aα) and one alpha-truncated polypeptide chain (Aα<610 and ≧251), the variant is referred to herein as fibrinogen alpha-truncated heterodimer or FibAα / truncated≧251 (also known literally as LMW fibrinogen when isolated from plasma). When both alpha polypeptide chains are alpha-truncated (Aα<610 and ≧251), the variant is referred to herein as fibrinogen alpha-truncated homodimer or FibAα truncated (also known literally as LMW' fibrinogen when isolated from plasma). The amino acid sequence of fibrinogen has been published, see for example WO2010 / 004004 for the sequences of the α, β and γ chains and variants thereof.

[0023] The composition used in the present invention may comprise or consist of a mixture of alpha-truncated variants, such as a mixture of fibrinogen alpha-truncated heterodimers and homodimers, or may comprise or consist of a single alpha-truncated variant.

[0024] Fibrinogen that combines an α chain mutant (extended or truncated) with a γ' mutant includes fibrinogen that is a heterodimer of both mutant polypeptide chains, a heterodimer of either the α or γ mutant, and a homodimer of both mutant polypeptide chains, and can be used as a composition for use in the present invention.

[0025] The fibrinogen in the composition can be obtained by methods known in the art. For example, plasma fibrinogen can be obtained by separation from plasma or commercially available, for example, as FIB3 or Fib1 from Enzyme Research Labs, Swansea, UK. Preferably, purified plasma fibrinogen is used, for example, as plasminogen-depleted fibrinogen with at least 95% clotting capacity. Specific fibrinogen variants can be obtained by purification from plasma or from commercially available plasma fibrinogen. Alternatively, specific fibrinogen variants can be obtained by recombinant production, for example, by cloning or chemically synthesizing the encoding gene or cDNA followed by transfection using a host cell or a cell culture system, such as a mammalian or human cell culture system. Recombinant production of proteins offers numerous advantages over the use of plasma-derived materials. These advantages include a favorable safety profile, the possibility of producing variants in a pure manner, and the fact that they are available in unlimited supply.

[0026] For economically viable production, high expression levels of intact and functional fibrinogen or its variants are required. Furthermore, for certain applications, appropriate post-translational modifications (e.g., glycosylation) are required. Therefore, in one embodiment of the present invention, to pharmaceutical standards, the fibrinogen is preferably produced in a mammalian cell culture system such as baby hamster kidney (BHK) cells, NSO cells, Sp2 / 0 cells, PER.C6 cells, HEK293 cells, insect cells, Chinese hamster ovary (CHO) cells, or African green monkey-derived COS cells. In a preferred embodiment, the mammalian fibrinogen in the composition used in the present invention is produced in CHO cells.

[0027] In a preferred embodiment, the fibrinogen in the composition used in the present invention is preferably in homodimeric form, and preferably consists of recombinantly produced human fibrinogen γ'.

[0028] In another preferred embodiment, the fibrinogen in the composition used in the present invention consists of recombinant human alpha-extended fibrinogen, preferably in homodimeric form.

[0029] In another preferred embodiment, the fibrinogen in the composition used in the present invention consists of recombinant human fibrinogen in which a γ' polypeptide chain is combined with an α-extended polypeptide chain, preferably in which both the γ and α variant polypeptide chains are in homodimeric form.

[0030] The composition can be used to pre-coat implantable medical devices before implantation or to coat wound surfaces before dressing or suturing. Pre-coating with the composition increases the in vivo bacterial susceptibility to antimicrobial agents compared to uncoated surfaces. The coating can be thin, such as a film, or thick, comprising several layers. The composition can be formulated as an aerosol, drop, dry powder, emulsion, foam, gel, ointment, paste, salve, semi-gel, spray, or suspension. Preferably, the composition is formulated as a spray, paste, or gel.

[0031] The coating may be continuous or discontinuous. Pre-coating of medical devices can be accomplished by incubating with a fibrinogen solution containing 10-100 μg / ml fibrinogen in PBS for several hours at 37°C, optionally in combination with a thrombin solution, or by spraying a solution at a concentration of 100-1000 μg / ml at room temperature. In situ coating of wound or tissue surfaces can be accomplished by spraying a fibrinogen solution at 1-50 mg / ml, optionally in combination with a thrombin solution. Typically, a coating is applied to a surface of 1 cm2. 2 A coating solution of 0.1 to 1 ml per unit area is sufficient. To ensure uniform effectiveness of the final coating across the surface of the medical device or tissue, the composition is preferably spread evenly across a width of the medical device or tissue such that the fibrinogen concentration per unit area of ​​the coated surface varies by up to 50%, e.g., up to 25%, e.g., up to 10%. In certain embodiments, the coating solution contains at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 25 μg / ml, or at least 50 μg / ml of fibrinogen.

[0032] The composition may be applied to the medical device or injured tissue using an applicator, dressing, wound packing, bandage, or swab.

[0033] The antimicrobial susceptibility of microorganisms is increased by the methods of the present invention. Increased susceptibility can be reflected in several ways, such as reduced growth or cell death, and can be evidenced by a decrease in dry weight, colony-forming units (CFU), or metabolic activity compared to a control situation without the composition. In one embodiment, the decrease is between a 1-log and a 5-log decrease in CFU. In another embodiment, increased antimicrobial susceptibility is evidenced in mice experiments where the composition reduces CFU by 6 or more logs to 0 CFU, compared to a 3-log decrease without the composition. The cure rate is increased by at least 25% compared to a control group without fibrinogen. In another embodiment, the cure rate is increased from 5% to 100%, or from 5% to 60%, or from 60% to 100%.

[0034] Bacteria whose antimicrobial susceptibility can be improved by this method include gram-positive or gram-negative bacteria, anaerobes, facultative anaerobes, or aerobes. Suitable examples of bacteria include bacteria belonging to the genera Acinetobacter, Bacillus, Bacteroides, Burkholderia, Campylobacter, Clostridium, Enterococcus, Escherichia, Klebsiella, Listeria, Mycobacterium, Neisseria, Pseudomonas, Salmonella, Yersinia, Staphylococcus, and Streptococcus. In particular, examples of species notorious for antimicrobial resistance and low antimicrobial susceptibility include Acinetobacter baumannii, Bacillus spp., Bacteroides spp., Burkholderia cepacia, Campylobacter spp., Clostridium difficile, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Listeria spp., Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella spp., Yersinia spp., Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Streptococcus pyogenes.

[0035] Preferably, the bacterium belongs to the genus Pseudomonas, Staphylococcus or Streptococcus. More preferably, the bacterium is a Staphylococcus aureus strain, including ampicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus such as Staphylococcus aureus 300WT or Staphylococcus aureus 43300, oxacillin-resistant Staphylococcus aureus and multidrug-resistant Staphylococcus aureus.

[0036] The compositions used in the present invention can be advantageously used for the treatment or prevention of infection or proliferation of microorganisms that are not normally present or present in abnormal amounts in an individual. The infectious disease may be subclinical, i.e., asymptomatic, or clinical, i.e., with obvious symptoms. Treatable or preventable infectious diseases include bacteremia, bone infections, lung infections, endocarditis, epidural abscess, genital infections, respiratory infections such as meningitis, osteoarticular infections, peritonitis, and pneumonia, prosthetic joint infections, skin infections such as acne and rosacea, sepsis, septic thrombophlebitis, toxic shock syndrome, urinary tract infections, and wound infections.

[0037] The fibrinogen composition according to the present invention may contain a therapeutically effective amount of fibrinogen or a prophylactically effective amount of fibrinogen. A therapeutically effective amount refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic outcome. A therapeutically effective amount of a fibrinogen variant or composition thereof can be determined by one skilled in the art and may vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the mammalian fibrinogen or composition to elicit a desired response in the individual. A therapeutically effective amount is also an amount such that any toxic or adverse effects of the variant fibrinogen or composition of the present invention are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired prophylactic outcome. Typically, a prophylactic dose is used in patients at or before the early stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

[0038] The fibrinogen content of the compositions for pre-coating devices is preferably 0.0001% to 0.1% w / w, for example 0.001% to 0.05% w / w, 0.001% to 0.1% w / w, or 0.005% to 0.1% w / w, by weight of the composition. The fibrinogen content of the compositions for coating wounds or tissue surfaces is preferably 0.01% to 10% w / w, for example 0.1% to 5% w / w, by weight of the composition.

[0039] In another embodiment of the present invention, the fibrinogen composition according to the present invention is a pharmaceutical composition or formulation, preferably sterile. Therefore, the composition used in the present invention may further comprise pharmaceutically acceptable additives such as anti-adherents, antioxidants, binders, fillers, carriers, colorants, disintegrants, diluents, fillers, flavoring agents, lubricants, preservatives, solvents, surfactants, sweeteners, vehicles or wetting agents. The additives should not adversely affect the stability of the composition in the formulation. The term "pharmaceutically acceptable" refers to being suitable for the formulation of a pharmaceutical composition, such as being generally considered safe and non-toxic.

[0040] Some examples of materials that can serve as pharmaceutically acceptable additives include, but are not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; saline; Ringer's solution; buffers such as ethyl alcohol and phosphate buffers, as well as other non-toxic additives such as sodium lauryl sulfate and magnesium stearate, as well as preservatives and antioxidants, which may be present in the composition. Techniques and formulations can generally be found in "Remington's Pharmaceutical Sciences" (Meade Publishing Co., Easton, Pennsylvania). Preferably, the carrier, vehicle, or diluent includes sterile water, sterile saline, sterile Ringer's solution, or sterile lactate solution, the selection of which is well known to those skilled in the art. Suitable solvents include water, saline, ethyl oleate, glycerin, hydroxide castor oil, alcohols such as ethanol, and phosphate buffers. The amount of solvent in a systemic composition may vary from 80% to about 99.95% w / w or w / v.

[0041] The compositions used in the present invention may further comprise an activator, such as thrombin or a thrombin-like enzyme such as reptilase or batroxobin, or a non-enzymatic activator, such as a fibrinogen-binding peptide. The thrombin and thrombin-like enzymes convert fibrinogen monomers into fibrin polymers by cleaving fibrinopeptides A and B from the α and β chains, respectively. The cleaved fibrin (and fibrinogen) monomers spontaneously polymerize into two-stranded protofibrils that can then elongate to form fibrin polymers that make up the three-dimensional fibrin network. Preferably, the activator is human thrombin, most preferably isolated from plasma or recombinantly produced in purified form.

[0042] The non-enzymatic activator is a substance that contains at least two binding sites and binds to two different fibrinogen molecules (fibrinogen is a symmetric dimer and therefore has two activator binding sites), thereby introducing a polymeric structure of fibrinogen with properties similar to those of thrombin-induced fibrin.

[0043] The compositions used in the present invention act as adjuvants for antimicrobial agents and can be used in combination with antimicrobial agents to reduce pathogen resistance to the antimicrobial agents. The antimicrobial agent may be applied separately from or together with the fibrinogen-containing composition. When applied separately, the antimicrobial agent and fibrinogen may be applied simultaneously or one after the other. When applied together, the antimicrobial agent and fibrinogen may be included in separate compositions or may be combined in a single composition. Thus, the compositions used in the present invention may further contain one or more antimicrobial agents, including antibacterial agents, antifungal agents, antiyeast agents, and antiviral agents. In some embodiments, the compositions used in the methods of the present invention are provided together with the antibacterial agent, separately, or in the same composition.

[0044] The compositions are used to enhance antimicrobial susceptibility in any individual in need thereof, such as humans and mammals including alpacas, bison, buffalo, cats, cows, deer, dogs, donkeys, horses, rats, pigs, rabbits, and sheep. For animal treatment, an animal equivalent of human fibrinogen is typically and preferably used.

[0045] In another aspect, the present invention relates to fibrinogen-coated medical devices, particularly indwelling devices or implants designed to remain in the body for days, weeks, months, or years. Examples of medical devices include those mentioned above, as well as catheters, endotracheal tubes, enteral feeding tubes, heart valves, artificial joints, stents, permanent tracheostomies, and wound drains. The devices may be supplied or commercially available pre-coated, or may be pre-coated immediately before use, such as hours, minutes, or seconds before use.

[0046] In yet another aspect, the present invention relates to a method for increasing the antimicrobial susceptibility of a microorganism. In one embodiment, the method comprises providing a human or animal individual with a composition comprising fibrinogen to prevent or treat a microbial infection in the individual, optionally in combination with an antimicrobial agent to reduce antimicrobial resistance in the microorganism. In another embodiment, the method comprises applying the fibrinogen composition to a medical device or wound bed. In one embodiment, the fibrinogen composition is applied to the medical device ex vivo prior to implantation or introduction of the medical device into the human or animal individual. The fibrinogen-containing composition may be any of the previously described compositions for use in the present invention.

[0047] Those skilled in the art will understand that the embodiments and preferred embodiments mentioned for the use of the compositions may also be applied to the methods for increasing the antimicrobial susceptibility of the medical devices and microorganisms, and vice versa. [Example]

[0048] material and method bacteria Methicillin-resistant Staphylococcus aureus (MRSA) strains USA300 and USA43300 were obtained from the ATTCC and routinely cultured at 37°C in tryptic soy broth (TSB) or on tryptic soy agar (TSA) plates.

[0049] fibrinogen Purified plasma fibrinogen (plasma Fib) was obtained from Enzyme Research Labs, Swansea, UK. rhFibWT and rhFibγ´, as well as rhFibα-extended and rhFibγ´ / α-extended forms, were generated using standard recombinant DNA protein production techniques. cDNA sequences encoding the fibrinogen Aα610 (WT) chain, Aα847 (α-extended) chain, Bβ chain, γ411 (γ) chain, and γ427 (γ´) chain were cloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) to construct expression vectors for the different fibrinogen chains. Expression plasmids containing the cDNAs encoding the Aα, Bβ, and γ chains were used in combination to produce different fully assembled recombinant human fibrinogen species by transient expression in HEK293 cells (Life Technologies EXPI293 system) or stable expression in CHO cells according to the manufacturer's instructions. CHO cells were cultured in ProCHO5 cell culture medium (Westburg, Leusden, The Netherlands) and transfected by electroporation using an Amaxa device (Westburg, Leusden, The Netherlands) according to the manufacturer's instructions. Fibrinogen-producing clones were selected using blasticidin and zeocin as selection markers using an ELISA method as previously described (Hoegee-de Nobel E. et al. Thrombosis Haemostasis 1988 Dec 22; 60(3): 415-8). Selected CHO clones were cultured in CD FortiCHO cell culture medium (Life Technologies Europe, Bleiswijk, The Netherlands) for 7 to 10 days at 37°C under 5% CO2. Cell supernatants were collected by centrifugation or filtration, and recombinant fibrinogen variants were purified using affinity purification on GRRP columns essentially as described by Kuyas C et al. (Thrombosis Haemostasis 1990 Jun 28; 63 (3): 439-444) and formulated at concentrations of 2-40 mg / ml in phosphate buffered saline (PBS) pH 7.4.

[0050] Resazurin method The resazurin method for measuring cell metabolic activity was performed as described by Lee A. and Jain E. Tip Biosystems Application note AN016 Rev. 1.0 Mar 2017.

[0051] Briefly, the resazurin-based biofilm susceptibility assay was performed as follows: 1x10 8 CFU / mL (OD 600 A working stock of USA300 S. aureus at a concentration of approximately 0.1 AU was prepared by culturing the bacteria in TSB. Fibrinogen dilutions were prepared in PBS pH 7.4. 90 μL of TSB and 90 μL of the fibrinogen dilutions were added to a 96-well tissue culture microtiter plate. Subsequently, 20 μL of S. aureus working stock was added to each well and incubated at 37°C for 24 hours to allow biofilm formation. The plate was washed three times with 0.9% sterile saline, and 100 μL of cation-adjusted Mueller-Hinton agar (CAMHB) with different concentrations of antimicrobial agents (0–100 μg / mL) was added to the 96-well plate and incubated at 37°C for 24 hours. 10 μL (10% of the sample volume) of Alamar Blue (resazurin) solution was added to each well, gently rocked, and incubated at 37°C for 1–4 hours. To determine cell activity, absorbance was measured at 570 and 600 nm.

[0052] Example 1: Adhesion of Staphylococcus aureus to different fibrinogen species in vitro Binding experiments were performed essentially as previously described (Flick M. et al. Blood 2013 Mar 7; 121(10): 1783-94). Briefly, binding assays were performed as follows: 96-well microtiter plates were coated with fibrinogen solution (0-25 μg / ml) in phosphate-buffered saline (PBS) by overnight incubation at 37°C. After washing and blocking, a suspension of Staphylococcus aureus USA300 in PBS (OD600nm approximately 0.4) was added to the wells and incubated for 2 hours at 37°C. After washing, the plates were incubated with 0.1% crystal violet solution for 30 minutes. After washing, the bound crystal violet was solubilized with 10% acetic acid, and the bacterial adhesion was quantified by measuring the absorbance at 570 nm.

[0053] Figure 1 shows that S. aureus binds to purified plasma Fib, rhFibWT, and rhFibα-extended, but not rhFibγ' or rhFibα-extended / γ'. This indicates that S. aureus adherence to fibrinogen is dependent on the presence of the C-terminus of the fibrinogen γ-chain (γ411), which contains the AGDV motif. Differences in the length of the α-chain (Aa610 vs. Aa847) do not affect S. aureus binding to fibrinogen. These results are consistent with literature data.

[0054] Example 2: Biofilm formation on surfaces pre-coated with different fibrinogen species in vitro In vitro biofilm formation on pre-coated polystyrene surfaces was performed using the Innovotech (Edmonton, Alberta, Canada) MBEC assay as previously described (Harrison JJ. et al. 2010 Nature Protocols 5, 1236-1254) according to the manufacturer's protocol.

[0055] Briefly, the assay was performed as follows. Polystyrene lids of 96-well microtiter plates with 96 pegs were precoated by submerging the pegs in 0.25 ml of a solution containing different fibrinogen species dissolved in PBS buffer at concentrations of 10 or 100 μg / ml for 24 hours at 37°C. After washing, the precoated pegs (and non-precoated controls) were added with a bacterial suspension of ∼10E6 CFU / ml in TSB with 1% glucose and incubated for 24 hours at 35°C to allow biofilm formation. After washing the pegs to remove planktonic bacteria, CAMHB was added to the biofilm-bearing pegs and incubated for 24 hours at 37°C. After washing, the viable bacteria were detached from the surface by mild sonication and plated on TSA plates to determine viable bacterial counts. Colonies were counted to determine CFU / peg surface. The results are shown in Figure 2.

[0056] A small reduction in viable count, approximately 1 log, is observed on non-precoated control and rhFibγ′ precoated PEGs compared to PEGs coated with rhFibWT or rhFibα extended forms.

[0057] These results are surprising given the highly significant binding differences observed in Example 1 (and in the literature), which demonstrate that binding is strongly dependent on the presence or absence of the fibrinogen C-terminus containing the AGDV-binding motif. The present results suggest that the number of viable bacteria incorporated into biofilms is only weakly dependent on interactions with the pre-coated fibrinogen. In conclusion, pre-coating surfaces with fibrinogen in vitro does not significantly increase or decrease the number of viable bacteria in the biofilms formed on each surface.

[0058] Example 3: Biofilm formation on non-precoated surfaces in the presence of different fibrinogen species in bacterial cultures in vitro The resazurin assay described previously was used to determine the effect of different fibrinogen species in solution present during biofilm formation on surfaces (as opposed to pre-coated surfaces prior to biofilm formation).

[0059] Purified plasma Fib or rhFibγ´ was added to TSB at concentrations ranging from 0 to 250 μg / ml, and then the mixture was inoculated with bacteria and incubated at 37°C for 24 hours. Fibrinogen coating and biofilm formation occurred simultaneously on the polystyrene surface. After the addition of resazurin, the number of viable bacteria in biofilms formed under different conditions was compared by measuring OD 570 nm.

[0060] The results, shown in Figure 3, indicate that there was no significant difference in viable bacterial counts in biofilms under all conditions, although a small increase in viable bacterial count was observed in biofilms formed in the presence of plasma Fib compared to those without fibrinogen. These results are very similar to those obtained in Example 2, using surfaces pre-coated with fibrinogen, and confirm that the viable counts associated with biofilms are largely independent of fibrinogen.

[0061] Example 4: Biofilm formation on pre-coated surfaces in vivo The in vivo effect of pre-coating with different fibrinogen species on biofilm formation by S. aureus USA43300 was investigated essentially as previously described (Nowakowska J et al. Antibiotics 2014, 3, 378-397). Briefly, the experiment was performed as follows.

[0062] Polytetrafluoroethylene cages precoated with fibrinogen solutions in PBS containing 10 or 100 μg / ml of rhFibWT or rhFibγ´ by incubation for 24 hours at 37°C, and non-precoated controls, were implanted subcutaneously in C57B1 mice. The cages were infected with approximately 400 CFU / cage of Staphylococcus aureus. Two days after implantation, the cages were removed, the bacteria were detached from the surface using mild sonification, and adherent viable bacteria were counted. Colonies were then plated on TSA plates and enumerated to determine the number of CFU / cage.

[0063] The results, shown in Figure 4, indicate that there was no difference in the number of CFUs between non-precoated cages and cages pre-coated with rhFibWT. There was a small, clinically insignificant decrease (approximately 1 log) in the amount of CFUs in rhFibγ´-coated cages compared to non-precoated and rhFibWT-precoated cages.

[0064] These results indicate that in vivo, the number of attached bacteria in biofilms formed on non-precoated cages and cages precoated with rhFibWT was essentially the same, while cages precoated with rhFibγ´ contained slightly lower numbers of attached bacteria (approximately 1 log).

[0065] These in vivo results are consistent with the in vitro results of Examples 2 and 3 and support the finding that the number of adherent viable bacteria in biofilms is largely independent of fibrinogen pre-coating.

[0066] Example 5: In vitro analysis of antimicrobial susceptibility of biofilms formed on pre-coated surfaces To determine the effect of pre-coating surfaces with different fibrinogen species on the antimicrobial susceptibility of the bacteria in biofilms, in vitro experiments were performed as described in Example 2. Different concentrations (0 to 100 μg / ml) of vancomycin or ampicillin were present during the incubation step after the addition of CAMHB. After the antimicrobial incubation, the surfaces were treated with mild sonification to release the bacteria, and the surface-associated CFUs were determined.

[0067] The results shown in Figure 5 indicate that S. aureus USA300 biofilms formed on non-precoated PEGs or PEGs precoated with rhFibγ´ or rhFibα extended forms were more susceptible to vancomycin and ampicillin exposure compared to biofilms formed on PEGs precoated with rhFibWT.

[0068] These results indicate that biofilms formed on non-precoated pegs or pegs precoated with rhFib γ' or rhFib α extended forms are more susceptible to antimicrobial agents than biofilms formed on pegs precoated with rhFibWT. In other words, the antimicrobial susceptibility of biofilms is enhanced in vitro by the use of rhFib γ' and rhFib α extended forms.

[0069] Example 6: In vitro analysis of antimicrobial susceptibility of biofilms formed on non-precoated surfaces To determine the effect of different fibrinogen species and their mixtures present in solution during biofilm formation on the antimicrobial susceptibility of the bacteria in biofilms, an in vitro experiment was performed using the previously described resazurin assay as described in Example 3. The percentage of viable cells was calculated from OD measurements at 570 and 600 nm compared to a control with an antimicrobial concentration of 0 μg / ml.

[0070] The vancomycin results shown in Figure 6A show that purified plasma Fib resulted in more viable bacteria remaining than the control, indicating that vancomycin resistance in biofilms is increased in the presence of plasma Fib compared to the control. Similar results were observed with ampicillin (Figure 6C).

[0071] The vancomycin results shown in Figure 6B indicate that the number of viable bacteria remained similar in rhFibγ´ compared to the control, indicating that the presence of rhFibγ´ did not alter vancomycin resistance in biofilms compared to the control. In the case of ampicillin (Figure 6D), a lower number of viable bacteria was observed at higher concentrations of rhFibγ´ compared to the control, indicating that the use of rhFibγ´ may enhance the ampicillin susceptibility of ampicillin-resistant strains in vitro.

[0072] The results shown in Figure 6E demonstrate that in vitro biofilms formed on non-precoated surfaces using a 50 / 50 mixture of plasma Fib / rhFibγ´ in bacterial culture medium at a concentration of 1 mg / ml and treated with vancomycin induced significantly lower viable S. aureus counts compared with surfaces precoated with plasma Fib alone. This finding is clinically relevant, demonstrating that rhFibγ´ can enhance antimicrobial susceptibility even in the presence of fibrinogen WT.

[0073] Example 7: In vivo analysis of antimicrobial susceptibility of biofilms on pre-coated surfaces To determine the in vivo effect of surfaces precoated with different fibrinogen species on the antimicrobial susceptibility of the bacteria in biofilms, experiments were performed in mice using the method described in Example 4 with the addition of prophylactic and therapeutic daptomycin treatment. Polytetrafluoroethylene cages, either non-precoated or precoated with 10 or 100 μg / ml of purified plasma Fib, rhFibWT, or rhFibγ′, were implanted subcutaneously in C57B1 mice.

[0074] Mice in the prophylactic group were treated with daptomycin (50 mg / kg) 30 minutes before implantation and infection. Mice in the treatment group were treated with daptomycin (50 mg / kg) 24 hours after implantation and infection. Mice were infected with Staphylococcus aureus 43300 at a dose of 400-600 CFU / cage. 48 hours after infection, the cages were removed, washed, and sonicated to release bacteria from the cage surface. CFU associated with the cages were determined.

[0075] The results of therapeutic (Figure 7A) and prophylactic (Figure 7B) treatments clearly show that despite prophylactic or therapeutic treatment with daptomycin, significant amounts of planktonic and attached viable S. aureus remained in all (100%) non-precoated cages (controls). This indicates that in vivo, biofilms formed on non-precoated surfaces are more resistant to antimicrobial treatment. This contrasts with the in vitro results in Example 5, where biofilms formed on non-precoated surfaces were sensitive to antimicrobial treatment. Clearly, in vitro results are not definitive of in vivo results.

[0076] Without being bound by theory, one explanation for this discrepancy is that in in vivo experiments, host extracellular proteins (e.g., fibronectin) adhere to the implanted non-precoated cages, resulting in the formation of a biofilm that renders the bacteria resistant to antimicrobial treatment.

[0077] Only 50–60% of cages precoated with plasma Fib (10 and 100 μg / ml coatings) retained viable adherent S. aureus after therapeutic (Figure 7A) or prophylactic (Figure 7B) treatment with daptomycin. This indicates that in vivo, biofilms formed on plasma Fib precoated surfaces are only partially resistant to antimicrobial treatment. Similar results, with 56–67% of cages retaining viable bacteria, were observed for cages precoated with rhFibWT (10 and 100 μg / ml) and therapeutically treated with daptomycin (Figure 7A). These results appear to contrast with the in vitro findings in Example 5, which may be due to synergistic effects with host defense mechanisms in vivo.

[0078] Cages precoated with rhFibγ´ (10 and 100 μg / ml) showed the most significant reduction in viable bacterial counts after prophylactic (Figure 7B) or therapeutic (Figure 7A) treatment with daptomycin. Figure 7B shows that only 7% of cages precoated with rhFibγ´ and prophylactically treated with daptomycin still contained viable bacteria (compared to 100% of non-precoated cages). After therapeutic treatment with daptomycin (Figure 7A), 37% of cages precoated with rhFibγ´ still contained viable bacteria (compared to 100% of non-precoated cages and 55–67% of plasma Fib or rhFibWT-coated cages). This surprisingly strong effect of rhFibγ' precoating in vivo on the susceptibility of the bacteria in biofilms to antimicrobial treatment (5-6 log reduction) appears to be consistent with in vitro results (Example 5) showing increased antimicrobial susceptibility of bacteria in biofilms formed on rhFibγ' precoated surfaces in vitro. However, the effect seen in vivo is much more pronounced compared to the in vitro effect.

[0079] The data shown in Figures 7A and B suggest that pre-coating a medical device or coating a tissue surface with fibrinogen increases the susceptibility of bacteria and associated biofilms to antiseptic treatments, such as antimicrobial treatment. The results for rhFibγ´ are even clearer.

[0080] Example 8: In vivo analysis of antimicrobial susceptibility of biofilms from different Staphylococcus species To determine the in vivo effect of surfaces pre-coated with different fibrinogen species on the antimicrobial susceptibility of biofilms formed by several species of Staphylococcus aureus, experiments were performed in mice using the method described in Example 7 with the addition of prophylactic daptomycin treatment. One of the species is Staphylococcus epidermidis, which binds fibrinogen through its β-chain. The β-chain in rhFibWT and rhFibγ´ is identical.

[0081] Polytetrafluoroethylene cages precoated with 10 or 100 μg / ml of purified plasma Fib, rhFibWT, or rhFibγ´, were implanted subcutaneously in C57B1 mice. Non-precoated cages served as controls. Mice were treated with daptomycin (50 mg / kg) 30 minutes before implantation and infection. The bacterial dose was 400–600 CFU / cage. 48 hours after infection, the cages were removed, washed, and sonicated to release bacteria from the cage surface. The cage-associated CFU were determined. The results, shown in Table 1, demonstrate that both types of fibrinogen precoating work better than no precoating. [Table 1]

[0082] The results show that when the biofilm surface is pre-coated with fibrinogen, the bacteria in the biofilm exhibit increased antimicrobial susceptibility and therefore lower antimicrobial resistance compared to the uncoated biofilm. The best results were also obtained for S. epidermidis biofilms when the surface was pre-coated with rhFibγ'.

[0083] Example 9: In vivo analysis of antimicrobial susceptibility of Pseudomonas biofilms Example 8 was repeated with P. aeruginosa strain PA01, with the addition of prophylactic tobramycin treatment, to determine the in vivo effect of fibrinogen-precoated surfaces on the antimicrobial susceptibility of Gram-negative biofilms. P. aeruginosa is a Gram-negative bacterium with no known fibrinogen-binding site. Tobramycin (50 mg / kg) was used instead of daptomycin, as tobramycin is typically used as an antimicrobial agent against Gram-negative bacteria. The results, shown in Table 2, demonstrate that fibrinogen precoating works better than no precoating. When the biofilm surface was precoated with fibrinogen, increased antimicrobial susceptibility of the Gram-negative bacteria in the biofilm was observed compared to the uncoated biofilm. [Table 2]

Claims

1. A composition comprising fibrinogen for use in a method for treating or preventing microbial antimicrobial resistance in a human or animal individual receiving prophylactic or therapeutic antimicrobial treatment.

2. The composition of claim 1 , wherein the antimicrobial agent is an antibacterial agent.

3. The composition of claim 1, wherein the antimicrobial agent is an antibiotic.

4. The composition according to any one of claims 1 to 3, wherein the microorganism is in a biofilm or aggregate, and the composition is intended to increase the antimicrobial susceptibility of the biofilm or aggregate.

5. The composition of claim 4 , wherein the biofilm is on a medical device or injured tissue.

6. The composition according to any one of claims 1 to 5, wherein the composition comprises or consists of plasma fibrinogen, wild type fibrinogen, fibrinogen gamma', fibrinogen alpha extension mutants or fibrinogen alpha truncation mutants.

7. The composition of any one of claims 1 to 6, wherein the fibrinogen is used to coat a medical device or implant prior to implantation, or to coat a wound prior to dressing or suturing.

8. The composition of any one of claims 1 to 7, further comprising thrombin, batroxobin or reptilase.

9. The composition of any one of claims 1 to 8, wherein the method further comprises providing an antimicrobial agent.

10. The composition of any one of claims 1 to 9, wherein the fibrinogen in the composition is recombinantly produced.

11. The composition according to any one of claims 1 to 10, wherein the microorganism is a gram-positive or gram-negative bacterium.

12. The composition of claim 11 , wherein the bacterium is of the genus Pseudomonas or Staphylococcus.

13. The composition described in claim 11, wherein the bacterium is Staphylococcus aureus.

14. The composition of claim 11, wherein the bacteria is an ampicillin-resistant Staphylococcus aureus, a vancomycin-resistant Staphylococcus aureus, a methicillin-resistant Staphylococcus aureus or a multidrug-resistant Staphylococcus aureus strain.

15. The composition of any one of claims 1 to 14, wherein the composition is a spray, a paste or a gel.

16. A method for treating or preventing microbial antimicrobial resistance in an individual, comprising applying ex vivo to a medical device a fibrinogen composition according to any one of claims 1 to 15.

17. 17. The method of claim 16, wherein the fibrinogen composition comprises plasma fibrinogen, wild type fibrinogen, fibrinogen gamma', fibrinogen alpha extension mutants or fibrinogen alpha truncation mutants.

18. 18. The method of claim 17, wherein the medical device is a catheter, an endotracheal tube, an enteral feeding tube, a heart valve, a prosthetic joint, a stent, a tracheostomy tube, a vascular access device, or a wound drain.

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