Antibiotic combination therapy

Abstract

The present invention provides an antibiotic combination therapy for use in the treatment of an Acinetobacter baumannii (A.baumannii) infection in a subject. In particular, these combination therapies include members of antibiotics of the class of rifabutin and polymyxin, such as commercially available natural product antibiotics, e.g., polymyxin B and polymyxin E (colistin and its prodrug colistin mesylate, CMS), as well as synthetic or semi-synthetic derivatives and analogs, e.g., SPR206, MRX-8 and QPX9003, and polymyxin-like antibiotics, e.g., Pol7306.

Description

Technical Field

[0001] The present invention generally relates to rifabutin combination therapies for treating Acinetobacter baumannii (A. baumannii) infections. Background Art

[0002] The emergence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) bacterial strains has made bacterial infections an increasingly serious public health problem over the past few decades. A bacterial species that poses a major threat to health is Acinetobacter baumannii, which may cause pneumonia, meningitis, and infections of the blood, urinary tract, and skin. Because Acinetobacter baumannii cells can survive for a long period of time on artificial surfaces, the bacteria are easily spread in hospital environments, and most Acinetobacter baumannii infections are hospital-acquired. For example, many soldiers in the Middle East were infected with Acinetobacter baumannii when they were treated for injuries during combat, and multidrug-resistant strains of the bacteria represent serious complications in the rehabilitation process of injured soldiers.

[0003] Treating A. baumannii infections is challenging. Using a variety of strategies, strains of A. baumannii have developed resistance to several different classes of antibiotics, including aminoglycosides, aminocyclines, tetracyclines, chloramphenicol, and carbapenems. Polymyxins such as colistin are often used as a last resort due to their severe side effects, but some strains of Acinetobacter baumannii are also resistant to colistin (Zubair et al., 2015, Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance, Clin Infect Dis. 60(9): 1295-303; Trebosc et al., Dissecting Colistin Resistance Mechanisms in Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates, Therapeutics and Prevention, Vol. 10, No. 4, July / August 2019, e01083-19; the contents of each of these documents are incorporated herein by reference). Therefore, the existing tools for treating and preventing diseases caused by this bacteria are not enough for many patients. In order to find a solution for treating these nosocomial pathogens, significant efforts have been made, and one of these solutions is combination therapy (Levin et al., 1999; Wood et al., 2003). The combination of two antibiotics shows different effects on each other, and in many cases, the effect is synergistic or enhanced, but in some cases, antagonism is observed (Montero et al., 2004; Tripodi et al., 2007). The unexpected activity of rifabutin, an antibiotic belonging to the rifamycin class, for Acinetobacter baumannii has been recently reported (US20210077471A1, the content of the document is incorporated herein by reference in its entirety).In addition, rifabutin has recently been reported to have a synergistic effect with polymyxin antibiotics, which include commercially available natural product antibiotics such as polymyxin B and polymyxin E (colistin and its prodrug colistin methane sulfonate (CMS)), as well as synthetic or semi-synthetic derivatives and analogs currently in clinical development, such as SPR206, MRX-8 (Vaara, 2019, Polymyxins and Their Potential Next Generation as Therapeutic Antibiotics, Front. Microbiol. 2019; 10: 1689, the contents of which are incorporated herein by reference in their entirety) and QPX9003 (Roberts et al., 2019, the contents of which are incorporated herein by reference in their entirety) (US20210077472A1, the contents of which are incorporated herein by reference in their entirety). Summary of the invention

[0004] The present invention provides a combination therapy including rifabutin and polymyxin (e.g., colistin, colistin mesylate (CMS), polymyxin B, polymyxin derivatives and analogs, as exemplified by SPR206, MRX-8 and QPX9003) for treating Acinetobacter baumannii infection. The present invention is based on the discovery that rifabutin synergizes with these antibiotics to inhibit the growth of Acinetobacter baumannii cells, so that the individual dose of each drug in the combination therapy can be lower than the therapeutic dose (if given alone). By allowing the use of a lower dose of a potentially toxic drug (e.g., polymyxin) at its standard therapeutic dose, it can be used in more cases and in early intervention, providing another tool for Acinetobacter baumannii infection, which is a major concern for drug resistance.

[0005] The combination of antibiotics described herein shows synergy for a wide range of Acinetobacter baumannii strains. This synergy has significantly increased the susceptibility of Acinetobacter baumannii cells to rifabutin and polymyxin, and when there is a polymyxin antibiotic of sub-inhibitory concentration / sub-therapeutic exposure, the MIC90 of rifabutin (the maximum minimum inhibition concentration of the growth of 90% of the bacterial strain in the test group) is reduced by up to 32 times. Unexpectedly, in time killing experiments, the existence of the sub-inhibitory concentration / sub-therapeutic exposure of polymyxin antibiotics makes rifabutin sterilize faster, and prevents regeneration (preventing the selection of drug-resistant colonies).

[0006] Aspects of the invention may include methods for treating an Acinetobacter baumannii infection in a subject, wherein the method may comprise providing a therapeutic or subtherapeutic dose of rifabutin and a subtherapeutic dose of polymyxin to a subject infected with Acinetobacter baumannii. A therapeutic dose of rifabutin is described as a dose that achieves a Cmax of about 2 mg / L and a 24-hour exposure (AUC)>10 mg*hour / liter. A subtherapeutic dose of rifabutin may be defined as a dose that determines a Cmax of about 2 mg / L. max Any dose <2 mg / L and 24-hour AUC ≤10 mg*hour / L. Subtherapeutic doses of rifabutin administered intravenously may be about two-thirds or less of the standard therapeutic dose.

[0007] In certain embodiments, a subtherapeutic dose of a polymyxin antibiotic is defined as a mean fC required to achieve clinical efficacy if given alone. max , fAUC and fC trough dose, and the fCt of polymyxin rough Included in its MIC5 and its MIC 50 In certain embodiments, the subtherapeutic dose of polymyxin is a concentration that reaches a therapeutic dose of fC trough Half or less of fC trough In certain embodiments, a subtherapeutic dose of polymyxin is a dose that is less than or equal to the therapeutic dose. trough One quarter or less of fC trough The desired dose. In some embodiments, the therapeutic dose of intravenous rifabutin can be about 600 mg / day. In some embodiments, the subtherapeutic dose of intravenous rifabutin can be about 550 mg / day or less. The subtherapeutic dose of intravenous rifabutin can be about 400 mg / day or less. In various embodiments, the subtherapeutic dose of intravenous rifabutin can be about 250 mg / day to about 400 mg / day.

[0008] Rifabutin and polymyxin may be provided in a single formulation suitable for intravenous administration, wherein the polymyxin may be polymyxin B and the subtherapeutic dose of the polymyxin may be about 60 mg / day or less, or the polymyxin may be colistin methanesulfonate (CMS) and the subtherapeutic dose of the polymyxin may be about 90 mg / day or less.

[0009] In certain embodiments, rifabutin and polymyxin may be provided separately, wherein the polymyxin may be polymyxin B and the subtherapeutic dose of the polymyxin may be about 0.5 mg / kg / day to about 0.8 mg / kg / day, or the polymyxin may be colistin methanesulfonate (CMS) and the subtherapeutic dose of the polymyxin may be about 0.8 mg / kg / day to about 1.6 mg / kg / day.

[0010] In various embodiments, rifabutin can be administered intravenously or orally or by inhalation. Acinetobacter baumannii may include carbapenem-resistant Acinetobacter baumannii (CRAB). The polymyxin may be SPR206 or QPX9003 or MRX-8 or any other synthetic or semi-synthetic polymyxin or polymyxin-like antibiotic (e.g., Pol7306), and the subtherapeutic dose of the polymyxin is about one-third of the standard therapeutic dose.

[0011] Aspects of the invention may include combination therapies comprising therapeutically effective amounts of rifabutin and polymyxin to treat an Acinetobacter baumannii infection in a subject, wherein the rifabutin and polymyxin are present in amounts that would be subtherapeutic if provided alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is shown that in the presence of sub-MIC (sub-fC trough The accumulation index and MIC50 and MIC90 of rifabutin in the case of members of the polymyxin family antibiotics at 1% to 2% concentration (pH 7.0).

[0013] Figure 2 It is shown that in the presence of sub-MIC (sub-fC trough MIC distribution of rifabutin in the context of members of the polymyxin family of antibiotics at 1.5% to 2.5% concentration.

[0014] Figure 3 Kill curve results for the first A. baumannii strain are shown.

[0015] Figure 4 Kill curve results for a second A. baumannii strain are shown. DETAILED DESCRIPTION

[0016] The present invention provides a combination therapy for treating an Acinetobacter baumannii infection in a subject. The combination therapy is based on the discovery that rifabutin synergizes with polymyxin antibiotics such as polymyxin B and polymyxin E (colistin) and synthetic or semisynthetic derivatives and analogs currently in clinical development such as SPR206, MRX-8, and QPX9003 to inhibit the growth of Acinetobacter baumannii cells, even at subtherapeutic doses of individual antibiotics if given alone. Therefore, while polymyxins such as colistin are antibiotics of last resort due to potential toxicity and adverse events, the ability to reduce dosage when used in combination with rifabutin may open up new applications, providing another tool to combat the growing number of drug-resistant infections.

[0017] The activity of rifabutin against Acinetobacter baumannii (including multidrug resistance (MDR) and carbapenem-resistant Acinetobacter baumannii (CRAB)) has recently been reported in US20210077471A1, while the intravenous dosage form of rifabutin has been reported in US20210077470A1, the contents of each of which are incorporated herein by reference in their entirety. Additionally, the synergistic effect between rifabutin and polymyxin antibiotics has been reported in US20210077472A1, which is incorporated herein by reference in its entirety.

[0018] A surprising benefit of this synergistic effect is that patients affected by Acinetobacter baumannii infections such as CRAB can be administered low doses of members of the polymyxin class of antibiotics (as low as 1 / 4 of the normally prescribed dose) in combination with low doses of rifabutin (as low as 1 / 4 of the effective dose) as needed to achieve a Cmax of approximately 2 mg / L and / or an AUC of >10 mg*hr / L and <300 mg*hr / L.

[0019] Therefore, the methods and formulations of the present invention include a combination therapy of rifabutin at a therapeutic or subtherapeutic dose and a polymyxin at a dose that is considered to be subtherapeutic. In various embodiments, rifabutin can be administered in a single vial with a member of the polymyxin class of antibiotics (co-formulation) for reconstitution and dilution in a suitable diluent for infusion to a person. Alternatively, members of rifabutin and polymyxin class antibiotics can be contained in different vials, which are independently reconstituted and combined in a suitable diluent when infused to a person. In still other embodiments, members of rifabutin and polymyxin class antibiotics can be administered to a patient alone, and therefore can be administered in parallel at the same infusion site or at two different infusion sites. Rifabutin can also be administered orally, and members of polymyxin class antibiotics can be administered by infusion or any other suitable route of administration (e.g., inhalation). In fact, members of rifabutin and polymyxin class antibiotics can be administered together or both alone by inhalation.

[0020] The therapeutic dose of intravenous rifabutin is about 300 mg BID (i.e., 600 mg / day). When used in combination with polymyxins according to the methods of the present invention, the amount of rifabutin administered intravenously (IV) in the combination therapy may be less than about 600 mg / day (e.g., subtherapeutic if given alone). In some embodiments, the IV "subtherapeutic" rifabutin dose may be more than about 150 mg / day, and in preferred embodiments is about 250 to about 500 mg / day (125-250 mg BID).

[0021] Standard therapeutic polymyxin dosing is discussed in: Tsuji BT, Pogue JM, Zavascki AP, et al. International Consensus Guidelines for the Optimal Use of the Polymyxins: Endorsed by the America College of Clinical Pharmacology (ACCP), the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), the Infectious Diseases Society of America (TDSA), the International Society for Anti-infective Pharmacology (TSAP), the Society of Critical Care Medicine (SCCM), and the Society of Infectious Diseases Pharmacists (SIDP). Pharmacotherapy 2019;39(1):10-39, which is incorporated herein by reference in its entirety.

[0022] The standard therapeutic dose of polymyxin B may be about 1.5 to about 2.5 mg / kg / day. If administered separately from rifabutin, polymyxin B may be dosed in mg / kg and may be about 0.5 to about 0.8 mg / kg / day. If co-formulated with rifabutin, polymyxin B may be provided in a fixed dose, for example, at about 60 mg / day (30 mg BID).

[0023] The standard therapeutic dose of CMS can be about 2.5 to about 5.0 mg / kg / day. If administered separately from rifabutin, CMS can be dosed by mg / kg and can be about 0.8 to about 1.6 mg / kg / day. If co-formulated with rifabutin, CMS can be provided in a fixed dose, for example, at about 90 mg / day (45 mg BID).

[0024] In general, a subtherapeutic dose of a polymyxin antibiotic is considered to be a dose at which, if given alone, the mean fC required for efficacy would not be achieved. max , fAUC and fC trough , and / or the fC of the polymyxin trough Between its MIC5 and its MIC50 (ie, the concentration that inhibits the growth of 5% to 50% of the strains in a test panel of more than 100 recent clinical isolates of Acinetobacter baumannii). max It should be understood as the pharmacokinetic maximum free drug concentration achieved at steady state for a given dose. trough It should be understood as the pharmacokinetic minimum free drug concentration achieved between doses at steady state for a drug at a given dose.fAUC should be understood as the area under the pharmacokinetic free drug curve (i.e., total exposure typically over 24 hours) achieved by a drug at a given dose at steady state.

[0025] Combination therapy

[0026] The combination therapy of the present invention includes two antibiotics that act synergistically to inhibit the growth of Acinetobacter baumannii cells. The synergistic effect between antibiotics (such as rifabutin and polymyxin B or colistin or synthetic or semi-synthetic derivatives or analogs or polymyxin-like antibiotics in clinical development, such as SPR206, MRX-8, QPX9003 or Pol7306) can be determined by any suitable method.

[0027] Combination therapy of the present invention, one of these antibiotics is rifabutin. Combination therapy includes polymyxins, such as polymyxin B or colistin or synthetic or semi-synthetic derivatives or analogs or polymyxin-like antibiotics in clinical development, such as SPR206, MRX-8, QPX9003 or Pol7306 that act synergistically with rifabutin. Clinically, colistin can be provided in the form of colistin sodium methanesulfonate (colistimethate sodium) or colistin sulfate. In the case of multiple microbial infections, it is contemplated herein to include any additional antibiotics in the combination therapy, and this will be apparent to those of ordinary skill in the art, and 3, 4, 5 or more different antibiotics may be included. Exemplary additional antibiotics can include an aminocyclitol, an aminoglycoside, a β-lactam, a β-lactamase inhibitor, a carbapenem, a cephalosporin, a quinolone, a rifamycin, a sulfonamide, minocycline, eravacycline, sulbactam, or a tetracycline. Each antibiotic can independently be amikacin, trimethoprim-sulfamethoxazole, cefepime, cefiderocol, ceftazidime, chloramphenicol, ciprofloxacin, colistin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, penicillin, piperacillin, polymyxin B, rifampicin, tazobactam or tigecycline.

[0028] Any additional antibiotic can be administered by any suitable route of administration. For example, and without limitation, each antibiotic can be independently administered intravenously, orally, parenterally, subcutaneously, by inhalation, by injection and / or by infusion.

[0029] Other antibiotics can be used according to the same dosage regimen.One or more antibiotics can be used according to different dosage regimens.Dosage regimens can include dosage, timetable or application or both.Dosage can be described by the absolute amount of the drug (e.g., mg) or by the relative amount of the drug relative to the experimenter (e.g., mg / kg).The timetable used can be described by the interval between dosages.For example and without limitation, the interval between dosages can be about one hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or longer.

[0030] Preparation

[0031] One or more of these antibiotics can be provided in the form of a single formulation. One or more antibiotics can be provided in the form of separate formulations. Each formulation can be prepared for delivery by a specific route of administration, such as intravenously, orally, parenterally, subcutaneously, by inhalation, by injection and / or by infusion.

[0032] These antibiotics can be provided in the form of pharmaceutically acceptable salts, such as non-toxic acid addition salts, which are salts of amino groups formed using inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or using organic acids such as, but not limited to, acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, glucuronic acid, malic acid, gluconic acid, lactic acid, aspartic acid or malonic acid.

[0033] The formulations can be administered by injection, infusion, implantation (intravenous, intramuscular, subcutaneous, etc.) or by inhalation in a dosage form, formulation or by a suitable delivery device or implant containing conventional non-toxic pharmaceutically acceptable carriers, solvents, diluents and adjuvants. The formulations and preparations of such compositions are well known to those skilled in the art of pharmaceutical formulations.

[0034] Formulations for parenteral use can be provided in unit dosage form (e.g., in single-dose ampoules and vials), in vials containing several doses and to which suitable preservatives (see below) may be added, in pre-filled syringes, or in pre-filled IV bags. Pharmaceutical compositions as described herein can be in a form suitable for sterile injection.

[0035] The formulation may include a solution containing rifabutin. Rifabutin solutions and methods for preparing rifabutin solutions are described in US20210077470A1, the contents of which are incorporated herein by reference in their entirety.

[0036] Depending on the needs of the patient and the clinical condition, administration of the composition by IV administration may be preferred over oral administration because IV administration allows for rapid introduction of the antibiotic into the systemic circulation, provides complete bioavailability, allows for better control of pharmacokinetic parameters that drive pharmacological efficacy, and avoids problems with stability and absorption in the gastrointestinal tract.

[0037] Treating Acinetobacter baumannii infections

[0038] The combination therapy of the present invention can be used to treat Acinetobacter baumannii infection in a subject. The subject can be a human. The subject can be a child, a neonate, an infant, a toddler, a child, a teenager, a young adult, an adult, or an elderly subject. The subject can be in emergency care, intensive care, neonatal intensive care, pediatric intensive care, coronary care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, operating room, ambulance, field hospital, or field environment outside the hospital.

[0039] The subject may have an Acinetobacter baumannii infection that is resistant to antibiotics or to multiple types of commercially available antibiotics (e.g., MDR, XDR, or PDR). Importantly, the combination therapy of the present invention can be used to treat infections caused by strains of Acinetobacter baumannii that have elevated MICs for rifabutin and / or resistance to polymyxin B or colistin or synthetic or semisynthetic derivatives or analogs in clinical development (e.g., SPR206, MRX-8, QPX9003, or Pol7306).

[0040] The antibiotics in the combination therapy of the present invention can be administered simultaneously or sequentially. Sequential administration or alternating administration can include providing each antibiotic exclusively for a certain period of time. Sequential administration can include overlapping periods during which both a formulation containing rifabutin and a formulation containing polymyxin are provided to the subject.

[0041] Examples

[0042] Example 1

[0043] The MIC90 (maximum minimum inhibitory concentration that inhibits the growth of 90% of the strains in the test group) of rifabutin was 2 or 1 mg / L when tested against a panel of 293 carbapenem-resistant Acinetobacter baumannii (CRAB) in RPMI / FCS (for broth microdilution) or MHA / PIH (for agar dilution), respectively (Trebosc V et al. 2020. In vitro activity of rifabutin against 293 contemporary carbapenem-resistant Acinetobacter baumannii clinical isolates and characterization of rifabutin mode of action and resistance mechanisms. J Antimicrob Chemother. 75(12):3552-3562, incorporated herein by reference).

[0044] When there is about one quarter of fC trough The MICs of rifabutin in the presence of 0.125 mg / L polymyxin B, 0.125 mg / L colistin, and 0.03 mg / L SPR206 when tested by broth microdilution in CAMHB / PIH with various members of the polymyxin class of antibiotics (trough free drug concentration) 90 Significantly decreased to 0.06-0.25 mg / L. At these corresponding polymyxin concentrations, no strains, 3% of strains, and <5% of strains would be susceptible to the corresponding polymyxins. Figure 1 The accumulation index and MIC of rifabutin in the presence of members of the polymyxin family antibiotics at sub-MIC (sub-trough concentrations) are shown. 50 and MIC 90 , showing the synergistic effect discussed above.

[0045] In addition, when tested in MHA / PIH, the rifabutin MIC distribution showed three different subgroups, of which group 1 (MIC <0.125 mg / L) accounted for 72% of the strains, group 2 (MIC ≥0.125 to <16 mg / L) accounted for 27% of the strains, and group 3 (MIC ≥16 mg / L) accounted for 1% of the strains (Trebosc V et al., 2020. In vitro activity of rifabutin against 293 contemporary carbapenem-resistant Acinetobacter baumannii clinical isolates and characterization of rifabutin mode of action and resistance mechanism. Journal of Antimicrobial Agents and Chemotherapy. 75 (12): 3552-3562, incorporated herein by reference in its entirety). When tested in CAMHB / PIH by broth microdilution in the presence of various members of the polymyxin class of antibiotics at about one-quarter of the fCtrough (free drug concentration at the trough), the MIC distribution of rifabutin showed only one major strain population, of which 99% of the strains had a MIC ≤1 mg / L. This indicates that the combination is active against strains with elevated rifabutin MICs. Importantly, 45, 40, and 18 strains had MICs greater than 2 mg / L for colistin, polymyxin B, and SPR206, respectively, indicating that the combination is active against polymyxin-resistant strains. In summary, these results suggest that the combination is active against strains with elevated rifabutin MICs, polymyxin-resistant strains, or both. Figure 2 The MIC distribution of rifabutin in the presence of members of the polymyxin family antibiotics at sub-MIC (sub-trough concentrations) is shown, illustrating the synergistic effect discussed above.

[0046] Example 2

[0047] Based on the data from Example 1, the bactericidal activity and resistance development of rifabutin and fixed rifabutin / polymyxin concentrations were evaluated in a time-kill curve experiment. The rifabutin concentration was fixed at 0.25 mg / L (average rifabutin MIC 90 The concentrations of colistin and polymyxin B were fixed at 0.125 mg / L and 0.03 mg / L, respectively (the fC trough 1 / 4 or less), and the concentration of SPR206 was fixed at 0.03 mg / L (a concentration that inhibits the growth of less than 5% of the strain if given alone). Figure 3 and 4 Results showed that low-dose rifabutin plus a low-dose member of the polymyxin class was at least as bactericidal as either full-dose rifabutin or full-dose polymyxins alone and completely prevented the development of resistance. Figure 3 and Figure 4Kill curve experiments of two different Acinetobacter baumannii strains (Pop1 and Pop2) with very different RIFABUTI MICs are shown. As shown, RIFABUTI at 0.25 mg / L and colistin or polymyxin B at 0.125 or 0.03 mg / L or SPR206 at 0.03 mg / L either have poor bactericidal properties or allow regeneration of both populations. However, the combination of RIFABUTI and a polymyxin provided together at low doses has a high and rapid bactericidal effect, indicating that the combination has a synergistic effect and can prevent the development of resistance (bacterial regeneration).

[0048] Figure 3 Shown is the killing curve of rifabutin against Acinetobacter baumannii Pop1 strain in the presence of a member of the polymyxin family antibiotics at sub-MIC (sub-trough concentration) (rifabutin MIC = 0.016 mg / L).

[0049] Figure 4 Shown is the killing curve of rifabutin against Acinetobacter baumannii Pop2 strain in the presence of members of the polymyxin family antibiotics at sub-MIC (sub-trough concentration) (rifabutin MIC = 1 mg / L).

[0050] method

[0051] Antimicrobial agents

[0052] Rifabutin (lot number BV-015-3219-001-03) was manufactured by Olon SpA and a 10 g / L stock solution was prepared in DMSO. A stock solution of rifampicin (Sigma R3501) was prepared in DMSO at 10 mg / mL. Stock solutions of colistin sulfate (Sigma C4461), polymyxin B sulfate (Sigma P4932), and SPR206 acetate (MedChemExpress, HY-128780B) were prepared in water at 10 mg / mL. The stock solutions were stored at -20°C until use.

[0053] Bacterial strains

[0054] The A. baumannii clinical isolates used in this study were from the BioVersys strain collection. These strains were stored as 20% (v / v) glycerol stock cultures at -80°C.

[0055] Antimicrobial Susceptibility and Synergy Testing

[0056] According to the CLSI guidelines, the combination of rifabutin MIC and a fixed concentration of a specific polymyxin was determined by broth microdilution. The culture medium used to determine the MIC was cation-regulated Muller Hinton broth (CAMHB), supplemented with 0.1 mM pyridoxal-isonicotinylhydrazone (PIH). As previously described by Trebosc et al. 2020, the rifabutin MIC in this culture medium may be affected by the skipped well phenomenon, in which wells grown at a concentration higher than the concentration of the first inhibited well do not reflect the appropriate MIC of the strain. Therefore, the MIC value is set to the first minimum rifabutin concentration that prevents growth, as assessed by visual inspection of the assay plate.

[0057] Time Kill Curve

[0058] Time killing kinetics were carried out in CAMHB supplemented with 0.1mM PIH culture medium. Rifabutin was tested at a concentration of 0.25mg / L (with or without a specified concentration of polymyxin medicament). Samples were taken at 0, 2, 4, 8 and 24 hours to determine colony forming units (cfu). Bactericidal activity was defined as a reduction of ≥3-log in cfu compared to the cfu present in the inoculum (0 hour). The synergistic effect of the combination tested was defined as a reduction of ≥2-log in cfu compared to the cfu of the most active medicament in the combination.

[0059] Incorporated by Reference

[0060] Other documents, such as patents, patent applications, patent publications, journals, books, papers, web page content, have been referenced and cited throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0061] Equivalent

[0062] Various modifications to the invention and many other embodiments of the invention in addition to the embodiments shown and described herein will become apparent to those skilled in the art based on the entire content of this document (including references to the scientific literature and patent literature cited herein). The subject matter of this article contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and its equivalents.

Claims

1. A method of treating an Acinetobacter baumannii infection in a subject, the method comprising providing a subtherapeutic dose of rifabutin and a subtherapeutic dose of polymyxin to a subject infected with Acinetobacter baumannii.

2. The method of claim 1, wherein the subtherapeutic dose of the polymyxin is an average fC required to achieve clinical efficacy if given alone. max , fAUC and fC trough dosage.

3. The method of claim 1, wherein the subtherapeutic dose of the polymyxin produces fC trough , in the fC trough The growth of about 5% to about 50% of the strains in a test panel of 100 or more recent clinical isolates of Acinetobacter baumannii was inhibited.

4. The method of claim 1, wherein the subtherapeutic dose of rifabutin is about two-thirds or less of the standard therapeutic dose.

5. The method of claim 1, wherein the subtherapeutic dose of the polymyxin is less than about half of a standard therapeutic dose.

6. The method of claim 1, wherein the subtherapeutic dose of rifabutin is about 550 mg / day or less.

7. The method of claim 6, wherein the subtherapeutic dose of rifabutin is from about 250 mg / day to about 400 mg / day.

8. The method of claim 1, wherein the rifabutin and the polymyxin are provided in a single formulation.

9. The method of claim 8, wherein the polymyxin is polymyxin B and the subtherapeutic dose of the polymyxin is about 60 mg / day or less.

10. The method of claim 8, wherein the polymyxin is colistin methane sulfonate (CMS) and the subtherapeutic dose of the polymyxin is about 90 mg / day or less.

11. The method of claim 1, wherein the rifabutin and the polymyxin are provided separately.

12. The method of claim 11, wherein the polymyxin is polymyxin B and the subtherapeutic dose of the polymyxin is from about 0.5 mg / kg / day to about 0.8 mg / kg / day.

13. The method of claim 11, wherein the polymyxin is colistin methanesulfonate (CMS) and the subtherapeutic dose of the polymyxin is from about 0.8 mg / kg / day to about 1.6 mg / kg / day.

14. The method of claim 1, wherein the polymyxin is SPR206, QPX9003, MRX-8, or Pol7306, and the subtherapeutic dose of the polymyxin produces an fCtrough at which the growth of about 5% to about 50% of the strains in a test panel of 100 or more recent clinical isolates of Acinetobacter baumannii is inhibited.

15. A combination therapy for treating an Acinetobacter baumannii infection in a subject, the combination therapy comprising therapeutically effective amounts of rifabutin and a polymyxin, wherein the rifabutin and the polymyxin are present in amounts that would be subtherapeutic if provided alone.

16. The combination therapy of claim 15, wherein the rifabutin is present in an amount of about two-thirds or less of the standard therapeutic dose or less if provided alone.

17. The combination therapy of claim 15, wherein the polymyxin is administered at an average fC required for clinical efficacy that would not be achieved if administered alone. max , fAUC and fC trough The amount exists.

18. The combination therapy of claim 15, wherein the subtherapeutic dose of the polymyxin produces fC trough , in the fC trough The growth of about 5% to about 50% of the strains in a test panel of 100 or more recent clinical isolates of Acinetobacter baumannii was inhibited.

19. The combination therapy of claim 15, wherein the polymyxin is present in an amount of about half or less of the standard therapeutic dose or less if provided alone.

20. The combination therapy of claim 15 comprising about 550 mg / day or less of rifabutin.

21. The combination therapy of claim 15 comprising about 250 mg / day to about 400 mg / day of rifabutin.

22. The combination therapy of claim 15, wherein the rifabutin and the polymyxin are provided in a single formulation.

23. The combination therapy of claim 22, wherein the polymyxin is polymyxin B and is present in an amount of about 60 mg / day or less.

24. The combination therapy of claim 22, wherein the polymyxin is colistin methanesulfonate (CMS) and is present in an amount of about 90 mg / day or less.

25. The combination therapy of claim 15, wherein the rifabutin and the polymyxin are provided separately.

26. The combination therapy of claim 25, wherein the polymyxin is polymyxin B and is present in an amount of about 0.5 mg / kg / day to about 0.8 mg / kg / day.

27. The combination therapy of claim 25, wherein the polymyxin is colistin methanesulfonate (CMS) and is present in an amount of about 0.8 mg / kg / day to about 1.6 mg / kg / day.

28. The combination therapy of claim 15, wherein the polymyxin is SPR206, QPX9003, MRX-8, or Pol7306, and the subtherapeutic dose of the polymyxin produces an fCtrough at which the growth of about 5% to about 50% of the strains in a test panel of 100 or more recent clinical isolates of Acinetobacter baumannii are inhibited.

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