Fibrinogen as an adjuvant for antimicrobial agents and therapies

Abstract

The present invention relates to a composition comprising fibrinogen for use in increasing the susceptibility of microorganisms to antimicrobial compounds. The invention can be used to alleviate antimicrobial resistance problems of microbial biofilms or aggregates (e.g. microbial biofilms) on medical devices, in particular indwelling devices, or in wound beds. The device or wound bed can be (pre-) coated with fibrinogen to increase antimicrobial susceptibility. The antimicrobial agent can be supplied separately or together with the fibrinogen.

Description

Technical Field

[0001] This invention relates to a method and therapy for increasing the sensitivity of microorganisms to antimicrobial agents, and in particular, the method can be used to reduce the resistance of biofilms to antimicrobial agents. Background Technology

[0002] Antimicrobial resistance in pathogens is a growing global concern. Often, pathogens manage to evade the effects of once-powerful antimicrobial compounds such as penicillin. Different strategies are being employed to overcome antimicrobial resistance: 1) new antimicrobials are being developed; 2) combination therapies using two or more existing antimicrobials are being used; and 3) therapeutic strategies and products used as adjuvants to combine with existing antimicrobials to reduce pathogen resistance to those antimicrobials. Antimicrobial adjuvant products and therapeutic strategies include drugs that combat resistance (such as β-lactamase inhibitors), antitoxic drugs (such as bacterial toxin inhibitors), and host-directed therapies (such as innate immune system agonists).

[0003] One mechanism by which microorganisms increase resistance to antimicrobial agents and therapies is the formation of aggregates, small colonies, or biofilms. Pathogens in biofilms or aggregates are known to be 10 to 1000 times more resistant to antimicrobial agents than free-floating pathogens in planktonic form. This is a serious problem in the field of medical devices when biofilms form on indwelling medical devices such as catheters, stents, heart valves, pacemakers, and joint replacements and account for up to 65% of all infections. Biofilm formation can lead to device failure and the spread of device-associated infections. The currently recommended approach is to remove the device followed by antibiotic treatment or a combination of both, and to replace the device after eradication of the infection. Alternatives to removal and replacement have been proposed. Kretlow et al. (2014) Plast Reconstr Surg. 2014, 133(l):28e-38e describe in situ treatment of left ventricular assist devices with antibiotic beads.

[0004] Another complicating factor is the presence of multimicrobial biofilms containing certain fungi and viruses or fungi and bacteria, leading to increased resistance to antiviral, antibacterial, and antifungal agents.

[0005] Since pathogen adhesion is essential for colonization and subsequent disease development, some research has focused on preventing pathogen adhesion to surfaces to prevent biofilm formation. Fibrinogen, one of the most abundant host plasma proteins, is considered an important host protein that promotes biofilm formation. Flores-Mireles et al. (J Urol 2016:416) described fibrinogen deposition as an accumulation surface for urinary pathogens. Kwiecinski et al. (J. Infect. Dis. 2016:213) described how plasmin prevents biofilm formation.

[0006] Fibrinogen is also thought to increase antibiotic resistance in biofilms. Bedran et al. (Biomed Research International vol. 2013, article ID 431465) reported in vitro results of fibrinogen-induced biofilm formation in *Streptococcus mutans*, with increased penicillin resistance. Jorgensen et al. (Microorganisms 2016:4) described how fibrinolytic enzymes that specifically degrade fibrinogen or fibrin significantly increase antibiotic susceptibility in bacterial biofilms in vitro. These publications demonstrate that, in vitro, the presence of fibrinogen stimulates biofilm formation, and fibrinolysis leading to fibrinogen depletion enhances the efficacy of antibiotics in the treatment of biofilm infections. Attached Figure Description

[0007] Figure 1 Adhesion of Staphylococcus aureus to different immobilized fibrinogen species.

[0008] Figure 2 Surviving Staphylococcus aureus bacteria associated with biofilms formed in vitro on surfaces pre-coated with different fibrinogen species.

[0009] Figure 3 During biofilm formation, viable Staphylococcus aureus bacteria are detected in biofilms formed in vitro on uncoated surfaces using plasma Fib or rhFibγ′ in bacterial culture medium.

[0010] Figure 4 Surviving Staphylococcus aureus bacteria were detected in the biofilm formed on PTFE cages pre-coated with plasma-derived Fib or rhFibγ′, implanted subcutaneously in mice, and infected with Staphylococcus aureus.

[0011] Figure 5 Surviving Staphylococcus aureus bacteria were detected in biofilms formed in vitro on surfaces pre-coated with different fibrinogen species and subsequently exposed to different doses of vancomycin or ampicillin.

[0012] Figure 6 shows viable Staphylococcus aureus bacteria detected in biofilms formed in vitro on uncoated surfaces in the presence of plasma Fib (6A and 6C) or rhFibγ′ (6B and 6D) in bacterial culture media; 6E) a 50 / 50 mixture of plasma Fib and rhFibγ′ subsequently exposed to vancomycin.

[0013] Figure 7 shows the viable Staphylococcus aureus bacteria detected in biofilms formed in mice on PTFE cages pre-coated with plasma Fib, rhFibWT, or rhFibγ′ and implanted under the skin after therapeutic (7A) and prophylactic (7B) daptomycin treatment. Detailed Implementation

[0014] This invention relates to the use of compositions comprising fibrinogen for the treatment or prevention of antimicrobial resistance in individuals.

[0015] The use according to the invention mitigates the problem of microbial resistance to antimicrobial agents. In particular, it increases susceptibility to antimicrobial agent therapy and is especially beneficial against microorganisms that are typically highly resistant to antimicrobial agent compounds, such as microorganisms in biofilms, aggregates, or small colonies. Biofilms (which are microbial communities) are known to be resistant to antimicrobial agents, meaning these biofilms escape the effects of once-powerful antimicrobial agents. The compositions according to the invention can be used together with existing antimicrobial agents as antimicrobial adjuvants to reduce the resistance of microbial biofilms or aggregates to these antimicrobial agents.

[0016] In one embodiment, the composition according to the invention for use increases the sensitivity of bacteria to antimicrobial agent compounds. Antimicrobial compounds are compounds that stop bacterial growth (bacteriostasis) or kill bacteria (bactericidal), and include antibiotics, bacteriophages and their endolysins, antimicrobial or antimicrobial peptides, and antimicrobial antibodies. Those skilled in the art will understand that a bactericidal compound effective against some bacteria may be bacteriostatic against others, and vice versa. Preferably, the antimicrobial compound is a natural, synthetic, or semi-synthetic antibiotic. Suitable natural, synthetic, or semi-synthetic antibiotics include aminoglycosides, antimicrobial imidazoles, β-lactams, macrolides, peptide antibiotics, polyclonal or monoclonal antibodies, quinolones, sulfonamides, and tetracyclines and their derivatives.

[0017] Examples of aminoglycosides are gentamicin and tobramycin. Examples of antimicrobial imidazoles are miconazole and metronidazole; examples of β-lactams are penicillins, including natural penicillins, aminopenicillins, β-lactamase-resistant penicillins, carboxypenicillins such as amoxicillin, ampicillin, and penicillin; cephalosporins such as cephalexin; and carbapenems such as meropenem and doripenem. Examples of macrolides are ketolides and macrolide antibiotics such as clarithromycin, erythromycin, and telithromycin. Examples of peptide antibiotics are glycopeptide antibiotics such as vancomycin, and lipopeptide antibiotics such as dapoxetine. Examples of antibodies are antibodies against Staphylococcus aureus aggregation factor A, alpha toxin, lipoteichoic acid, and teichoic acid. Examples of quinolones are fluoroquinolones such as ciprofloxacin and levofloxacin. Examples of sulfonamides are sulfacetamide and sulfamethoxazole. Examples of tetracyclines are tetracycline and doxycycline. In one embodiment, the antimicrobial compound is penicillin or a peptide antibiotic. In another embodiment, the antimicrobial compound is carboxypenicillin, a glycopeptide antibiotic, or a lipopeptide antibiotic. In yet another embodiment, the antimicrobial compound is selected from the group consisting of ampicillin, vancomycin, daptomycin, and tobramycin.

[0018] Technicians will be familiar with the fact that some antimicrobial agents are used to combat several groups of microorganisms. For example, some antimicrobial peptides can be used to combat both bacterial infections and viral or fungal infections.

[0019] This composition can also be used to treat infections involving several types of microorganisms. It can increase the susceptibility of one or more types of microorganisms to antimicrobial agents. For example, it can make viruses or yeast in bacterial biofilms more susceptible to antiviral or antiyeast agents.

[0020] Infectious microorganisms in biofilms or aggregates can also be called pathogens. Notable pathogens of biofilm-associated infections that can be treated with the adjuvant method of this invention are bacteria such as Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA).

[0021] The compositions according to the invention for use increase the antimicrobial susceptibility of microorganisms. Microorganisms treated according to the invention are preferably in cluster form, such as aggregated or clustered together, or they may adhere to a surface, such as in a biofilm. Microorganisms in cluster form or in a biofilm are less susceptible to antimicrobial agents compared to freely floating microorganisms. In a biofilm, microorganisms are typically surrounded by an extracellular matrix of polymeric substances (EPS) (such as glycoproteins) secreted by the microorganism itself or recruited from the host. Biofilms are also referred to in the art as glycocalyxes.

[0022] Attachment surfaces include living surfaces, such as tissue, cell surfaces, and wound beds, as well as inert surfaces, such as medical devices, particularly indwelling medical devices or implants, including catheters, cardiac implantable electronic devices (CIEDs), heart valves, fixators, joint replacements, stents, tracheostomies, and wound drainage tubes. Examples of catheters are peripherally inserted central catheters (PICC lines), implantable ports, and urinary catheters. Examples of CIEDs are pacemakers, left ventricular assist devices (LVADs), and implantable defibrillators. Examples of fixators are pins, plates, rods, screws, and filaments. Examples of joint replacements are hip, knee, and shoulder prostheses. Devices may include metals, ceramics, or non-biodegradable synthetic polymers, or may be composed 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 that will remain in the body for days, weeks, months or years.

[0023] The composition for use according to the invention comprises fibrinogen. The fibrinogen in the composition may be plasma fibrinogen (plasma fib), which is a mixture of all different fibrinogen variants present in the blood of a healthy individual; or it may be a formulation 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 WT fibrinogen, human fibrinogen γ′, human fibrinogen α extended variant, or human fibrinogen α truncated variant. In one embodiment, the fibrinogen in the composition consists of human plasma fibrinogen, human WT fibrinogen, human fibrinogen γ′ variant, human fibrinogen α extended variant, or human fibrinogen α truncated variant. Human fibrinogen refers to fibrinogen having the amino acid sequence found in most humans.

[0024] Plasma fibrinogen refers to fibrinogen isolated from plasma, typically comprising a mixture of native fibrinogen variants resulting from selective splicing, proteolytic degradation, or post-translational modification. Plasma fibrinogen may originate from one individual or multiple individuals. Preferably, the plasma fibrinogen has high purity, i.e., it is included in a composition comprising at least 95% w / w, at least 97% w / w, or at least 98% w / w plasma fibrinogen. Purity can be determined by coagulability.

[0025] WT fibrinogen refers to the main mature form of human fibrinogen, accounting for 60-80% of the total circulating plasma fibrinogen. It has an Aα chain of 610 amino acids, a Bβ chain of 461 amino acids, and a γ chain of 411 amino acids. In the literature, this form of fibrinogen is also referred to as high molecular weight (HMW) fibrinogen or intact fibrinogen. The fibrinogen γ′ variant has two α polypeptide chains, two β polypeptide chains, and two γ polypeptide chains, at least one of which is a γ′ polypeptide chain. In humans, the γ′ polypeptide chain has 427 amino acids. If the fibrinogen molecule contains one WTγ polypeptide chain (γ411) and one γ′ polypeptide chain (γ427), this variant is referred to herein as fibrinogen γ′ heterodimer or Fibγ427 / 411. If both γ polypeptide chains are γ′ type (γ427), this variant is referred to as fibrinogen γ′ homodimer or Fibγ427 / 427. The composition for use according to the invention may include or consist of a mixture of γ′ variants (such as a mixture of fibrinogen γ′ heterodimer and homodimer), or it may include or consist of a type of γ′ variant.

[0026] The fibrinogen α-elongation variant has two α-peptide chains (at least one of which is an α-elongation polypeptide chain), two β-peptide chains, and two γ-peptide chains (at least one of which is a γ′ polypeptide chain). In humans, the α-elongation polypeptide chain has 847 amino acids. If the fibrinogen molecule comprises one WTα-peptide chain (Aα610) and one α-elongation polypeptide chain (Aα847), this variant is referred to herein as a fibrinogen α-elongation heterodimer or rhFib Aα847 / Aα610. If both α-peptide chains are α-elongation (Aα847), this variant is referred to as a fibrinogen α-elongation homodimer or Fib Aα847 / 847. Compositions for use according to the invention may comprise or consist of mixtures of α-elongation variants (such as mixtures of fibrinogen α-elongation heterodimers and homodimers), or may comprise or consist of one type of α-elongation variant.

[0027] Fibrinogen α-truncated variants have two α-polypeptide chains (where at least one of the α-polypeptide chains is truncated between the amino acid at position 251 and the carboxyl-terminal amino acid at position 610), two β-polypeptide chains, and two γ-polypeptide chains. If the fibrinogen molecule contains one WTα chain (Aα610) and one truncated α-polypeptide chain (Aα < 610 and > 251), this variant is referred to herein as fibrinogen α-truncated heterodimer or Fib Aα610 / truncated > 251 (also referred to in the literature as LMW fibrinogen when isolated from plasma). If both α-polypeptide chains are α-truncated (Aα < 610 and > 251), this variant is referred to as fibrinogen α-truncated homodimer or fibrinogen Aα-truncated (also referred to in the literature as LMW′ fibrinogen when isolated from plasma). The amino acid sequence of fibrinogen has been disclosed; see, for example, the sequences of the α, β, and γ chains and their variants in WO2010 / 004004.

[0028] The composition for use according to the invention may include or consist of a mixture of α-truncated variants (e.g., a mixture of fibrinogen α-truncated heterodimer and homodimer), or it may include or consist of a type of α-truncated variant.

[0029] Fibrinogen that binds an α-chain variant (extended or truncated) to a γ′ variant, including fibrinogen that is heterodimer for both variant polypeptide chains, heterodimer for either α or γ variant, and homodimer for both variant polypeptide chains, can be used in compositions for use according to the invention.

[0030] The fibrinogen in the composition can be obtained by methods known in the art. For example, plasma fibrinogen can be obtained by isolating from plasma, or it can be commercially available, for example, from Enzyme Research Labs in Swansea, UK, as FIB 3 or FIB 1. Preferably, purified plasma fibrinogen is used, such as fibrinogen depleted of at least 95% coagulating plasminogen. 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 a genome or cDNA, followed by transfection using host cells or cell culture systems such as mammalian or human cell culture systems. Recombinant production of proteins has many advantages over using plasma-derived materials. These advantages include its preferred safety characteristics, the possibility of producing variants in a pure manner, and the fact that there is an unlimited supply.

[0031] For economically viable production, high levels of complete, functional fibrinogen or its variants are required. Furthermore, appropriate post-translational modifications (e.g., glycosylation) are necessary for specific applications. Therefore, in one embodiment of the invention, for pharmaceutical standards, fibrinogen is preferably produced in mammalian cell culture systems, such as young hamster kidney (BHK) cells, NSO cells, Sp2 / O cells, PER.C6 cells, FIEK293 cells, insect cells, Chinese flamingo ovary (CFIO) cells, or COS cells derived from African green monkeys. In a preferred embodiment, the mammalian fibrinogen in the composition according to the invention for use is produced in CHO cells.

[0032] In a preferred embodiment, the fibrinogen in the composition for use according to the invention is composed of human fibrinogen γ′, which is preferably in its homodimer form and preferably produced by recombination.

[0033] In another preferred embodiment, the fibrinogen in the composition for use according to the invention comprises recombinant human fibrinogen α, preferably extended in its homodimeric form.

[0034] In another preferred embodiment, the fibrinogen in the composition for use according to the invention comprises recombinant human fibrinogen that binds a γ′ polypeptide chain to an α-extended polypeptide chain, preferably in homodimeric form for both the γ and α variant polypeptide chains.

[0035] The composition can be used to pre-coat a medical device intended for implantation before implantation, or to coat a wound surface before bandaging or closure. Pre-coating with the composition increases bacterial susceptibility to antibiotics in the body compared to an uncoated surface. The coating can be thin, such as a film, or thick, comprising several layers. The composition can be formulated as an aerosol, drops, dry powder, emulsion, foam, gel, ointment, paste, cream, semi-gel, spray, or suspension. Preferably, the composition is formulated as a spray, paste, or gel.

[0036] The coating can be continuous or discontinuous. Pre-coating of the medical device can be performed by incubating at 37°C with a fibrinogen solution in PBS containing 10-100 μg / mL fibrinogen for several hours, or by spraying a solution at a concentration of 100-1000 μg / mL at room temperature, optionally in combination with thrombin solution. In-situ coating of wounds or tissue surfaces can be performed by spraying a fibrinogen solution containing 1-50 mg / mL (optionally combined with thrombin solution). Typically, 0.1-1 mL of coating solution per square centimeter of surface area is sufficient. To ensure uniform efficacy of the final coating across the entire surface of the medical device or cellular tissue, the composition is preferably uniformly distributed across a given width of the medical device or tissue such that the fibrinogen concentration per unit area of ​​the surface to be coated varies by at most 50%, such as at most 25%, such as at most 10%. In one embodiment, the coating solution comprises 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.

[0037] The composition can be applied to a medical device or wound tissue using an applicator, dressing, wound packing, bandage, or medicated swab.

[0038] The method according to the invention enhances the sensitivity or susceptibility of microorganisms to antimicrobial compounds. This enhanced sensitivity, compared to a control without the composition, can be reflected in a variety of ways, such as through slower growth or cell death, which may be apparent, for example, through lower dry weight, a reduction in colony-forming units (CFU), or decreased metabolic activity. In one embodiment, the reduction in CFU is between 1 and 5 logarithms. In another embodiment, in mouse experiments, antibiotic sensitivity is significantly increased, decreasing from 6 logarithms or more to 0 CFU when the composition is used, compared to only a 3 logarithm reduction without the composition. The cure rate can be improved by at least 25% compared to a control group without fibrinogen. In another embodiment, the cure rate is increased by 5% to 100%, or 5% to 60%, or 60% to 100%.

[0039] Bacteria that may be more sensitive to antibiotics in this way are Gram-positive or Gram-negative, anaerobic, facultative anaerobic, or aerobic bacteria. Examples of suitable bacteria include those belonging to the genera Acinetobacter, Bacillus, Bacteroides, Burkholderia, Campylobacter, Clostridium, Enterococcus, Escherichia, Klebsiella, Listeria, Mycobacterium, Neisseria, Pseudomonas, Salmonella, Yersinia, Staphylococcus, and Streptococcus. In particular, those species with well-known antibiotic resistance and low susceptibility to antibiotics, such as Acinetobacter baumannii, Bacillus, Bacteroides, Burkholderia cepacia, Campylobacter, Clostridium difficile, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Listeria, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella, Yersinia, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Streptococcus pyogenes.

[0040] Preferably, the bacteria belong to the genera *Pseudomonas*, *Staphylococcus*, or *Streptococcus*. More preferably, the bacteria are strains of *Staphylococcus aureus*, 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*.

[0041] The compositions according to the invention can be advantageously used to treat or prevent any infection involving the invasion or proliferation of microorganisms that are abnormally present or present in abnormal amounts within an individual. The infection may be subclinical and asymptomatic, or clinical and symptomatic. Infections that can be treated or prevented include bacteremia, bone infections, chest infections, endocarditis, epidural abscesses, genital infections, meningitis, bone and joint infections, peritonitis, respiratory infections such as pneumonia; prosthetic joint infections; skin infections such as acne and rosacea; sepsis, septic thrombophlebitis, toxic shock syndrome, urinary tract infections, and wound infections.

[0042] The fibrinogen compositions according to the present invention may comprise a therapeutically effective amount or a preventatively effective amount of fibrinogen. A therapeutically effective amount refers to an effective amount that achieves the desired therapeutic effect within the necessary dose and time period. The therapeutically effective amount of variant fibrinogen or compositions thereof can be determined by those skilled in the art and can vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of mammalian fibrinogen or compositions to elicit the desired response in an individual. A therapeutically effective amount is also the amount by which any toxic or adverse effects of the variant fibrinogen or compositions of the present invention are exceeded by the therapeutically beneficial effects. A preventatively effective amount refers to an effective amount that achieves the desired preventative effect within the necessary dose and time period. Typically, because preventative doses are used in subjects before or in the early stages of disease, the preventatively effective amount will be lower than the therapeutically effective amount.

[0043] The fibrinogen content of the composition for use in a pre-coating device is preferably from 0.0001% w / w to 0.1% w / w, such as from 0.001% w / w to 0.05% w / w, 0.001% w / w to 0.1% w / w, or 0.005% w / w to 0.1% w / w, based on the weight of the composition. The fibrinogen content of the composition for coating wounds or tissue surfaces is preferably from 0.01% w / w to 10% w / w, such as from 0.1% w / w to 5% w / w, based on the weight of the composition.

[0044] In another embodiment of the invention, the fibrinogen composition according to the invention is a pharmaceutical composition or formulation, preferably sterile. Therefore, compositions according to the invention for use may further include pharmaceutically acceptable excipients, such as anti-adhesion agents, antioxidants, binders, fillers, carriers, colorants, disintegrants, diluents, fillers, flavoring agents, lubricants, preservatives, solvents, surfactants, sweeteners, transporters, or humectants. Excipients should not adversely affect the stability of the composition in the formulation. The term "pharmaceutically acceptable" means suitable for preparing pharmaceutical compositions, such as those generally considered safe and non-toxic.

[0045] Some examples of materials that can be used as pharmaceutically acceptable excipients include buffers such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions; and other non-toxic excipients such as sodium dodecyl sulfate and magnesium stearate, as well as preservatives and antioxidants, which may also be present in the composition. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences (Meade Publishing co., Easton, Pa.). Preferably, the carrier, transporter, or diluent comprises sterile water, sterile isotonic 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, isotonic saline, ethyl oleate, glycerol, hydroxylated castor oil, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent in a systemic composition can be from 80% to about 99.95% w / w or w / v.

[0046] The compositions according to the invention for use may further include an activator, such as thrombin or thrombin-like enzymes, such as snake venom thrombin or batroxobin, or a non-enzymatic activator, such as a fibrinogen-binding peptide. Thrombin and thrombin-like enzymes convert fibrinogen monomers into fibrin polymers by cleaving fibrin peptide A or B from the α-chain and β-chain, respectively. The cleaved fibrin(ogen) monomers spontaneously polymerize into double-chain fibrils, which can then grow and form fibrin polymers constituting a three-dimensional fibrin network. Preferably, the activator is human thrombin isolated or recombined from plasma, most preferably in a purified form.

[0047] Non-enzymatic activators are substances that include at least two fibrinogen binding sites and introduce a polymeric form of fibrinogen with properties similar to thrombin-induced fibrin by binding to two different fibrinogen molecules (which have two activator binding sites because fibrinogen is a symmetrical dimer).

[0048] The composition for use according to the invention can be used as an adjuvant to an antimicrobial agent and can be used in combination with an antimicrobial agent to reduce pathogen resistance to such antimicrobial agent. The antimicrobial agent can be administered separately or together with the composition comprising fibrinogen. If administered separately, the antimicrobial agent and fibrinogen can be administered simultaneously, or before or after each other. If the antimicrobial agent and fibrinogen are administered together, they can be present in separate compositions or combined in one composition. Therefore, the composition for use according to the invention may further include one or more antimicrobial agents, including antibiotics, antifungals, antiyeasts, and antivirals. In one embodiment, the composition for use according to the method of the invention is supplied separately or in the same composition as an antibiotic.

[0049] This composition can be used to increase the antimicrobial susceptibility of any individual in need, such as humans and mammals including alpacas, bison, buffalo, cats, cattle, deer, dogs, donkeys, horses, mice, pigs, rabbits, and sheep. For animal treatment, animal equivalents of human fibrinogen are typically preferred.

[0050] On the other hand, the present invention relates to a medical device coated with fibrinogen, particularly an indwelling device or implant that will remain in the body for days, weeks, months, or years. Examples of medical devices are as described above, and include catheters, endotracheal tubes, feeding tubes, heart valves, joint replacements, stents, tracheostomies, and wound drainage tubes. The device may be supplied or commercially available and pre-coated, or it may be pre-coated before use (e.g., hours, minutes, or seconds before use).

[0051] In another aspect, the present invention relates to a method for increasing the susceptibility of microorganisms to antimicrobial agents. In one embodiment, the method includes providing a human or animal individual with a composition comprising fibrinogen to prevent or treat microbial infection in the individual, optionally combined with an antimicrobial agent to reduce microbial resistance to the antimicrobial agent. In another embodiment, the method includes applying the fibrinogen composition to a medical device or wound bed. In one embodiment, the fibrinogen composition is applied ex vivo to the medical device prior to implantation or introduction into a human or animal individual. The composition comprising fibrinogen can be any composition available for use according to the invention as described above.

[0052] Those skilled in the art will understand that the embodiments and preferred embodiments mentioned above for using the composition can also be applied to medical devices and methods for increasing susceptibility to antimicrobial agents in microorganisms, and vice versa.

[0053] Example

[0054] Materials and methods

[0055] bacteria

[0056] Methicillin-resistant Staphylococcus aureus (MRSA) strains USA 300 and USA 43300 were obtained from ATTCC and were routinely grown on trypsin-soy broth (TSB) or trypsin-soy agar (TSA) plates at 37°C.

[0057] fibrinogen

[0058] Purified plasma fibrinogen (plasma Fib) was obtained from Enzyme Research Labs in Swansea, UK. rhFib WT, RhFibγ′, RhFibα extension, and RhFibγ′ / α extension were prepared using standard recombinant DNA protein production techniques. cDNA sequences encoding the fibrinogen Aα610 (WT), Aα847 (α extension), Bβ, γ411 (γ), and γ427 (γ′) chains were cloned into pcDNA 3.1 plasmids (Invitrogen, Carlsbad, California, USA) to construct expression vectors for different fibrinogen chains. Different fully assembled recombinant human strains were produced using combinations of expression plasmids containing cDNA encoding the Aα, Bβ, and γ chains, either transiently expressed in HEK 293 cells (Life Technologies EXPI293 system) according to the manufacturer's instructions or stably expressed in CHO cells. CHO cells were grown in ProCH05 cell culture medium (Westburg, Ruesden, Netherlands) and transfected by electroporation using an Amaxa device (Westburg, Ruesden, Netherlands) according to the manufacturer's instructions. Clones producing fibrinogen were selected using a previously described ELISA method with blastomycin and bleomycin as selection markers (Hoegee-de Nobel E. et al., Thrombosis Haemostasis 1988 Dec22;60(3):415-8). Selected CHO clones were grown in CD FortiCHO cell culture medium (Life Technologies Europe, Braiswick, Netherlands) at 37°C and 5% CO2 for 7 to 10 days. Cell supernatants were collected by centrifugation or filtration, and recombinant fibrinogen variants were purified on a GPRP column using affinity purification, essentially as described by Kuyas C et al. (Thrombosis Haemostasis 1990 Jun 28; 63(3):439-444), and prepared in phosphate-buffered saline (PBS) at pH 7.4 at concentrations of 2-40 mg / ml.

[0059] Blade Sky Blue Technique

[0060] The azadirachtin method for measuring cellular metabolic activity was performed as described in Lee A. and Jain E. Tip Biosystems Application Note AN016 Rev. 1.0 Mar 2017.

[0061] In summary, the biofilm sensitivity analysis based on rezamidoides was performed as follows. Containing 1×10 8 Working stock of USA300 Staphylococcus aureus cfu / mL (OD) 600 Approximately 0.1 AU was prepared by culturing bacteria in TSB. Fibrinogen dilution was prepared in PBS at pH 7.4. 90 μL of TSB and 90 μL of fibrinogen dilution were added to a 96-well tissue culture microtiter plate. Subsequently, 20 μL of Staphylococcus aureus working stock solution was added to each well, and the plate was 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 cationic-adjusted Mueller Hinton broth (CAMHB) with different concentrations of antibiotics (0–100 μg / mL) was added to each well, and the plate was incubated at 37°C for 24 hours. 10 μL (10% of the sample volume) of Alamar Blue solution was added to each well, gently shaken, and incubated at 37°C for 1–4 hours. The absorbance at 570 nm and 600 nm was measured to determine cell viability.

[0062] Example 1: In vitro adhesion of Staphylococcus aureus to different fibrinogen species

[0063] The combined experiments were basically carried out as described above (Flick M. et al., Blood 2013 Mar 7; 121(10):1783-94). In short, the combined measurements were performed as follows:

[0064] 96-well microtiter plates were coated with fibrinogen solution in phosphate-buffered saline (PBS) (0-25 μg / ml) after overnight incubation at 37°C. Following washing and blocking, the wells were incubated with a Staphylococcus aureus USA 300 suspension in PBS (OD 600 nm approximately 0.4) for 2 hours at 37°C. After washing, the plates were incubated with 0.1% crystal violet solution for 30 minutes, and after washing, the bound crystal violet was dissolved in 10% acetic acid, and the absorbance at 570 nm was measured to quantify bacterial adhesion.

[0065] Figure 1The study showed that *Staphylococcus aureus* bacteria bound to purified plasma fibrinogen, rhFib WT, and rhFib α extension, but not to rhFib γ′ or rhFib α extension / γ′. This indicates that *Staphylococcus aureus* adhesion to fibrinogen depends on the presence of the C-terminus of the fibrinogen γ chain (γ411) containing the AGDV motif. Different α chain lengths (Aα610 vs Aα847) had no effect on the binding of *Staphylococcus aureus* to fibrinogen. These results are consistent with literature data.

[0066] Example 2: In vitro biofilm formation on a surface pre-coated with different fibrinogen species.

[0067] In vitro biofilm formation on pre-coated polystyrene surfaces has been previously described (Flarrison JJ et al. 2010 Nature Protocols 5, 1236-1254) and was performed using the Innovotech (Edmonton, Alberta, Canada) MBEC assay according to the manufacturer’s protocol.

[0068] In summary, the analysis was performed as follows. Polystyrene caps on 96-well microtiter plates containing 96 pegs were pre-coated by immersing the pegs in 0.25 ml of a solution containing different fibrinogen species at concentrations of 10 or 100 μL / ml dissolved in PBS buffer at 37°C for 24 hours. After washing, the pre-coated pegs (and the uncoated control) were incubated with a bacterial suspension of approximately 10E6 CFU / ml in TSB + 1% glucose at 35°C for 24 hours to allow biofilm formation. After washing the pegs to remove airborne bacteria, the biofilm-bearing pegs were incubated with CAMFIB at 37°C for 24 hours. After washing, viable bacteria were separated from the surface by gentle sonication, and the number of viable bacteria was determined by plating onto TSA plates. Colony counts were performed to determine the CFU / peg surface area. Results are as follows. Figure 2 As shown.

[0069] Compared with pegs coated with rhFib WT or RhFibα extension, a small reduction of approximately one log number of surviving bacteria was observed on uncoated controls and pegs pre-coated with RhFibγ′.

[0070] These results are surprising given the very significant differences in binding observed in Example 1 (and the literature), which demonstrate that binding strongly depends on the availability of the carboxyl terminus of fibrinogen containing the AGDV binding motif. The results indicate that the number of surviving bacteria bound to the biofilm depends only slightly on the interaction with the pre-coated fibrinogen.

[0071] In summary, pre-coating surfaces with fibrinogen in vitro does not significantly increase or decrease the number of surviving bacteria in biofilms formed on such surfaces.

[0072] Example 3: In vitro biofilm formation on an uncoated surface in the presence of different fibrinogen species in a bacterial culture medium.

[0073] The effects of different fibrinogen species present in the solution during surface biofilm formation were determined using the above-described resazu assay (compared to a pre-coated surface before biofilm formation).

[0074] Purified plasma fibrinogen (Fib) or rhFibγ′ at concentrations between 0 and 250 μg / ml was added to TSB, followed by bacterial inoculation of the mixture and incubation at 37°C for 24 hours to allow simultaneous fibrinogen coating and biofilm formation on the polystyrene surface. The number of surviving bacteria in the biofilms formed under different conditions was compared by measuring the OD 570 nm after the addition of resazurin.

[0075] The results show Figure 3 The results showed that the number of surviving bacteria in the biofilm did not differ significantly under all conditions. However, a slight increase in surviving bacteria was observed in the biofilm formed in the presence of plasma fibrinogen compared to the absence of fibrinogen. These results are very similar to those obtained in Example 2 using a surface pre-coated with fibrinogen, and confirm that the number of surviving bacteria associated with the biofilm is largely independent of fibrinogen.

[0076] Example 4: Formation of in vivo biofilm on pre-coated surfaces

[0077] The in vivo effects of pre-coating with different fibrinogen species on the formation of Staphylococcus aureus USA43300 biofilms were carried out essentially as described previously (Nowakowska J et al., Antibiotics 2014, 3, 378-397). In short, the study was conducted as follows.

[0078] Polytetrafluoroethylene (PTFE) cages and uncoated controls were implanted subcutaneously into C57BI mice. The PTFE cages were pre-coated by incubation at 37°C for 24 hours in a fibrinogen solution containing 10 μg / ml or 100 μg / ml rhFib WT or RhFibγ′. The cages were infected with approximately 400 CFU of Staphylococcus aureus per cage. Two days after implantation, the cages were removed, and bacteria were gently sonicated to separate them from the surface. The bacteria were then plated onto TSA plates to measure the adhering viable bacteria, and colonies were counted to determine the number of CFUs in each cage.

[0079] Figure 4 The results showed no difference in the number of CFUs between cages that were not pre-coated and those pre-coated with rhFib WT. A small, clinically insignificant reduction (approximately 1 log) in the number of CFUs was observed in cages coated with rhFib γ′ compared to cages that were not pre-coated and those pre-coated with rhFib WT.

[0080] These results indicate that, in vivo, the number of adhesive bacteria in biofilms formed on uncoated cages and on cages precoated with rhFib WT is virtually the same, while cages precoated with rhFib γ′ contain a slightly lower number of adhesive bacteria (approximately 1 log).

[0081] These in vivo results are consistent with the in vitro results in Examples 2 and 3 and support the finding that the number of surviving adhesive bacteria in the biofilm is largely independent of the pre-coating of fibrinogen.

[0082] Example 5: In vitro analysis of antibiotic sensitivity of biofilms formed on pre-coated surfaces

[0083] To determine the effect of pre-coating surfaces with different fibrinogen species on the antibiotic susceptibility of bacteria in biofilms, in vitro experiments were performed as described in Example 2. Vancomycin or ampicillin at different concentrations (0 μg / ml to 100 μg / ml) were present during the incubation step with CAMBFI. After antibiotic incubation, the surface was gently sonicated to release bacteria from the surface, and the CFU associated with the surface were determined.

[0084] The results show Figure 5 In the study, Staphylococcus aureus USA300 biofilms formed on uncoated pegs or pegs pre-coated with rhFibγ′ or rhFibα extensions were shown to be more sensitive to vancomycin and ampicillin compared to biofilms formed on pegs pre-coated with rhFib WT.

[0085] These results indicate that biofilms formed on uncoated pegs or on pegs pre-coated with RhFibγ′ or RhFibα extensions are more sensitive to antibiotics than biofilms formed on pegs pre-coated with RhFib WT. In other words, in vitro, the antibiotic sensitivity of biofilms is increased by using RhFibγ′ and RhFibα extensions.

[0086] Example 6: In vitro analysis of antibiotic sensitivity of biofilms formed on uncoated surfaces

[0087] To determine the effect of the presence of different fibrinogen species and mixtures thereof in the solution during biofilm formation on the antibiotic sensitivity of bacteria in the biofilm, in vitro experiments were performed using the resazurin assay described above, as described in Example 3. The percentage of viable cells compared to a control group containing 0 μg / ml antibiotics was calculated from OD measurements at 570 nm and 600 nm.

[0088] For purified plasma Fib, Figure 6A The vancomycin results showed more surviving bacteria compared to the control group. This indicates that the presence of plasma fibrinogen (Fib) increases the resistance of biofilms to vancomycin compared to the control group. Similar results were observed for ampicillin. Figure 6C ).

[0089] For RhFibγ′, Figure 6B The vancomycin results showed a similar number of surviving bacteria compared to the control group. This indicates that the biofilm's resistance to vancomycin is not altered compared to the control group if RhFibγ′ is present. For ampicillin (… Figure 6D Compared to the control group, higher RhFibγ′ concentrations resulted in lower viable bacterial counts. This indicates that, in vitro, the use of RhFibγ′ can increase the susceptibility of ampicillin-resistant strains to ampicillin.

[0090] Figure 6E The results showed that, compared with surfaces pre-coated only with plasma fibrinogen, biofilms formed in vitro on uncoated surfaces using a 50 / 50 mixture of 1 mg / ml plasma fibrinogen / rhFibγ′ in bacterial culture and treated with vancomycin contained significantly fewer viable Staphylococcus aureus bacteria. This result indicates that RhFibγ′ can increase antibiotic susceptibility, even in the presence of WT fibrinogen, making it clinically relevant.

[0091] Example 7: In vivo analysis of antibiotic sensitivity of pre-coated surface biofilms

[0092] To determine the in vivo effects of surfaces pre-coated with different fibrinogen species on the antibiotic sensitivity of bacteria in biofilms, studies were conducted in mice as described in Example 4, with prophylactic and therapeutic daptomycin treatments added. PTFE cages, either uncoated or pre-coated with 10 μg / ml or 100 μg / ml purified plasma Fib, rhFib WT, or rhFibγ′, were implanted subcutaneously into C57BI mice.

[0093] Mice in the prevention 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 post-infection, the cages were removed, cleaned, and sonicated to release bacteria from the cage surface. CFU was used to determine the CFU associated with the cage.

[0094] Therapeutic treatment ( Figure 7A ) and preventative treatment ( Figure 7B The results clearly demonstrate that despite prophylactic or therapeutic treatment with daptomycin, all (100%) uncoated cages (controls) contained large numbers of surviving planktonic and adherent Staphylococcus aureus bacteria. This indicates that biofilms formed on uncoated surfaces in vivo are highly resistant to antibiotic treatment. This contrasts with the in vitro results of Example 5, which showed that biofilms formed in vitro on uncoated surfaces were sensitive to antibiotic treatment. Clearly, in vitro results have no predictive value for in vivo results.

[0095] To avoid being bound by theory, one possible explanation for this discrepancy is that, in in vivo experiments, host extracellular proteins (such as fibronectin) adhere to implanted, uncoated cages, leading to biofilms that make bacteria resistant to antibiotic treatment.

[0096] Therapeutic use of daptomycin ( Figure 7A ) or preventive ( Figure 7B After treatment, only 50-60% of the cages pre-coated with plasma fibrin (10 μg / ml and 100 μg / ml coatings) contained viable adhering Staphylococcus aureus bacteria. This indicates that the biofilm formed in vivo on the pre-coated plasma fibrin surface provides only partial resistance to antibiotic treatment. Pre-coated with rhFibr WT (10 μg / ml and 100 μg / ml) and treated with daptomycin (… Figure 7A Similar results were observed in cages, with 56-67% of cages containing surviving bacteria. These results appear to contradict the in vitro findings in Example 5, which may be due to synergy with in vivo host defense mechanisms.

[0097] Daptomycin was used for prophylactic treatment. Figure 7B ) or therapeutic ( Figure 7A After treatment, cages pre-coated with rhFibγ′ (10 μg / ml and 100 μg / ml) showed the strongest reduction in surviving bacteria. Figure 7BThe results showed that only 7% of cages pre-coated with rhFibγ′ and prophylactically treated with daptomycin contained viable bacteria (compared to 100% in uncoated cages). When treated with therapeutic daptomycin ( Figure 7A In cages pre-coated with rhFibγ′, 37% contained viable bacteria (compared to 100% in uncoated cages and 55-67% in cages coated with plasma Fib / rhFib WT). This surprisingly strong in vivo effect (a 5-6 log reduction) of RhFibγ′ pre-coating on the antibiotic treatment sensitivity of bacteria in biofilms appears consistent with in vitro results (Example 5), which showed increased antibiotic sensitivity in bacteria within biofilms formed in vitro on RhFibγ′ pre-coated surfaces. However, the in vivo effect was more pronounced than the in vitro effect.

[0098] Figure 7A and Figure 7B Data from the study indicate that pre-coating medical devices or tissue surfaces with fibrinogen increases the sensitivity of biofilm-associated bacteria to bactericidal treatments such as antibiotics. The results with rhFibγ′ are even more pronounced.

[0099] Example 8: In vivo analysis of antibiotic susceptibility of biofilms of different Staphylococcus species.

[0100] To determine the in vivo effects of pre-coated surfaces with different fibrinogen species on antibiotic susceptibility to biofilms formed by several Staphylococcus species, studies were conducted in mice as described in Example 7, with prophylactic addition of daptomycin. One of the species included is Staphylococcus epidermidis, which binds fibrinogen via its β-chain. The β-chains in rhFib WT and rhFibγ′ are identical.

[0101] C57BI mice were subcutaneously implanted with PTFE cages pre-coated with 10 μg / ml or 100 μg / ml purified plasma Fib, rhFib WT, or rhFibγ′. Uncoated 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. At 48 hours post-infection, the cages were removed, washed, and sonicated to release bacteria from the cage surface. CFU associated with the cages were determined. The results are shown in Table 1, demonstrating that pre-coating with any type of fibrinogen was more effective than not pre-coating.

[0102] Table 1

[0103]

[0104] The results showed that when the biofilm surface was pre-coated with fibrinogen, the bacteria in the biofilm exhibited increased susceptibility and therefore lower resistance to antibiotics compared to uncoated surfaces. Optimal results were obtained when the surface was pre-coated with rhFibγ′, also for Staphylococcus epidermidis biofilms.

[0105] Example 9: In vivo analysis of antibiotic susceptibility of Pseudomonas biofilms

[0106] Example 8 was repeated using *Pseudomonas aeruginosa* strain PA01 with a prophylactic addition of tobramycin to determine the in vivo effect of fibrinogen pre-coating of the surface on antibiotic susceptibility of Gram-negative bacterial biofilms. *Pseudomonas aeruginosa* is a Gram-negative bacterium without a known fibrinogen binding site. Tobramycin (50 mg / kg) was used instead of daptomycin because tobramycin is typically used as an antibiotic against Gram-negative bacteria. The results are shown in Table 2 and demonstrate that pre-coating with fibrinogen was more effective than not pre-coating. When the biofilm surface was pre-coated with fibrinogen, the susceptibility of Gram-negative bacteria in the biofilm increased compared to no coating.

[0107] Table 2

[0108] % of mice without surviving adherent Pseudomonas aeruginosa Tobramycin (50 mg / kg) Pre-coating none 50 rhFibγ′ 100

Claims

1. Use of a composition comprising fibrinogen in the preparation of a medicament for treating or preventing antimicrobial resistance of microorganisms in human or animal individuals, wherein the fibrinogen comprises rhFibγ′, wherein the antimicrobial agent is selected from the group consisting of ampicillin, daptomycin, and tobramycin, and wherein the microorganism comprises bacteria.

2. The use according to claim 1, wherein, The microorganisms are in the form of biofilms or aggregates, and the composition is used to increase the susceptibility of the biofilms or aggregates to antimicrobial agents.

3. The use according to any one of claims 1-2, wherein, The biofilm is located on the medical device or wound tissue.

4. The use according to any one of claims 1-2, wherein, The composition further comprises plasma fibrinogen, wild-type fibrinogen, fibrinogen γ′ other than rhFibγ′, fibrinogen α extended variant or fibrinogen α truncated variant, or the composition consists of fibrinogen γ′.

5. The use according to any one of claims 1-2, wherein, The fibrinogen is used to coat a medical device or implant before implantation, or to coat a wound before bandaging or sealing.

6. The use according to any one of claims 1-2, wherein the composition further comprises thrombin, batroxobin, or snake venom thrombin.

7. The use according to any one of claims 1-2, wherein, The treatment or prevention also includes the supply of antimicrobial agents.

8. The use according to any one of claims 1-2, wherein, The fibrinogen in the composition is produced by recombinant synthesis.

9. The use according to any one of claims 1-2, wherein, The microorganisms are either Gram-positive or Gram-negative bacteria.

10. The use according to claim 9, wherein, The bacteria are either Pseudomonas or Staphylococcus species.

11. The use according to claim 9, wherein, The bacteria are Staphylococcus aureus.

12. The use according to claim 9, wherein, The bacteria are ampicillin-resistant Staphylococcus aureus strains, vancomycin-resistant Staphylococcus aureus strains, methicillin-resistant Staphylococcus aureus strains, or multidrug-resistant Staphylococcus aureus strains.

13. The use according to any one of claims 1-2, wherein, The composition is a spray, paste, or gel.

14. A method for preparing a medical device for treating or preventing antimicrobial resistance of microorganisms in an individual, the method comprising ex vivo application of a fibrinogen composition of any one of claims 1 to 13 to the medical device, wherein the antimicrobial agent is selected from the group consisting of ampicillin, daptomycin, and tobramycin, and wherein the microorganism comprises bacteria.

15. The method according to claim 14, wherein, The fibrinogen composition further includes plasma fibrinogen, wild-type fibrinogen, fibrinogen γ′ other than rhFibγ′, fibrinogen α extended variant, or fibrinogen α truncated variant.

16. The method of claim 14, wherein, The medical device is a catheter, endotracheal tube, enteral feeding tube, heart valve, joint replacement, stent, tracheostomy, vascular access device, or wound drainage tube.

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