Combination Therapy Effective Against Microorganisms, Including Drug Resistant Microorganisms
20220233557 · 2022-07-28
Assignee
Inventors
- John Lee Pace (Burlington, NJ)
- Marc Edwin Wiles (Mason, OH)
- Stacy Michelle Adams (Cary, NC)
- Elizabeth Hussey (Chapel Hill, NC)
Cpc classification
A61K31/505
HUMAN NECESSITIES
A61K31/4178
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/665
HUMAN NECESSITIES
A61K31/44
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/4178
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K31/665
HUMAN NECESSITIES
International classification
A61K31/665
HUMAN NECESSITIES
A61K31/44
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Abstract
Novel antimicrobial compositions comprise at least a form of a fosfomycin (or suitable analogs, or derivatives thereof), and at least one sulfonamide (or suitable analogs, or derivatives thereof), and/or at least one diaminopyridine (or suitable analogs, or derivatives thereof). Said antimicrobial compositions generally include two or more of said components as a novel combination, provided in a single combination or formulation, or provided independently with overlapping exposures of the two or more components. These novel compositions, when provided as the two or more components, are effective against susceptible microorganisms, and are effective against susceptible drug-resistant microorganisms (e.g., bacteria, parasites), including multi-drug resistant bacteria. These novel compositions are also effective against microorganisms or strains thereof now susceptible to the combination even though said microorganisms or strains thereof were previously found or were considered resistant or intolerant to at least one of the components of the novel composition, when the at least one component was used alone at what was considered an effective dosing amount or concentration.
Claims
1. A method of inhibiting growth of a microorganism, or killing the microorganism, the method comprising: providing a course of antimicrobial compounds or agents for use against the microorganism, the course of antimicrobial compounds or agents comprising a combination of at least three antimicrobial compounds or agents comprising at least a fosfomycin, at least one sulfonamide, and at least one diaminopyridine, the course of antimicrobial compounds or agents for inhibiting growth of the microorganism, or for killing the microorganism by a synergistic activity of the at least three antimicrobial compounds or agents provided over the course, such that the fosfomycin, the at least one sulfonamide, and the at least one diaminopyridine are each provided as a pharmaceutically acceptable salt, or ester, in which the fosfomycin will have inhibitory activity on cell wall synthesis of the microorganism, wherein the course of antimicrobial compounds or agents in use display a broad spectrum activity against Gram-positive bacteria, Gram-negative bacteria, and parasites, and wherein the microorganism is selected from any one or more of a drug-resistant bacteria, a multi-drug resistant bacteria, a drug-resistant parasite, and a multi-drug resistant parasite.
2. The method of claim 1, wherein the at least one sulfonamide includes functional analogs, and functional derivatives thereof, any of which have inhibitory activity in synthesis of dihydrofolate, or is sulfamethoxazole.
3. The method of claim 1, wherein the at least one diaminopyridine includes a functional analog, or functional derivative thereof, any of which inhibit dihydrofolate reductase, or is one of the group comprising trimethoprim, pyrimethamine, and iclaprim.
4. (canceled)
5. The method of claim 1, wherein the microorganism is one or more of: (i) one suspected to cause a bacterial infection selected from the group consisting of a gastrointestinal infection, genitourinary infection, respiratory infection, skin infection, systemic infection, wound infection, and sexually transmitted infection; and (ii) considered resistant to one or more of the fosfomycin, the at least one sulfonamide, and the at least one diaminopyridine.
6. (canceled)
7. (canceled)
8. The method of claim 1, wherein the microorganism resides in or on a subject, and the method includes administering the course of the antimicrobial compounds or agents to the subject.
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. The method of claim 1, wherein the combination of the at least three antimicrobial compounds or agents includes a combination of the at least sulfonamide and the at least one diaminopyridine, in which the at least one sulfonamide and the at least one diaminopyridine are in a ratio of between about 3:1 and 6:1.
22. (canceled)
23. (canceled)
24. The method of claim 8, wherein the subject is any of a human, and an animal.
25. (canceled)
26. Antimicrobial compounds or agents provided over a course for use in inhibiting growth of a microorganism, or killing the microorganism comprising: at least three antimicrobial compounds or agents each provided daily over the course, the at least three antimicrobial compounds or agents comprising: a fosfomycin as a pharmaceutically acceptable salt or ester, any of which have inhibitory activity on cell wall synthesis of the microorganism; a sulfonamide as a pharmaceutically acceptable salt or ester any of which have inhibitory activity on folic acid metabolism of the microorganism; and a diaminopyridine as a pharmaceutically acceptable salt or ester any of which inhibit dihydrofolate reductase in the microorganism, such that the course of antimicrobial compounds or agents will inhibit growth of the microorganism or kill the microorganism by a synergistic activity of the at least three antimicrobial compounds or agents, wherein over the course, the at least three antimicrobial compounds or agents in use display a broad spectrum activity against Gram-positive bacteria, Gram-negative bacteria, and parasites, and wherein the microorganism is selected from any one or more of a drug-resistant bacteria, a multi-drug resistant bacteria, a drug-resistant parasite, and a multi-drug resistant parasite, any of which is suspected of causing an infection.
27. The antimicrobial compounds or agents of claim 26, wherein any one or more of the at least three antimicrobial compounds or agents further comprises an excipient.
28. The antimicrobial compounds or agents of claim 26, wherein the at least three antimicrobial compounds or agents includes a combination of the sulfonamide to the diaminopyridine ratio in a ratio of between about 3:1 and 6:1.
29. The antimicrobial compounds or agents of claim 26, wherein over the course the at least three antimicrobial compounds or agents are administered to a subject in need thereof.
30. The antimicrobial compounds or agents of claim 26, wherein over the course the at least three antimicrobial compounds or agents are administered to a subject in need thereof in addition to another agent selected from the group consisting of antibiotic, antiviral agent, antifungal agent, anti-parasitic agent, and combinations thereof.
31. (canceled)
32. (canceled)
33. (canceled)
34. The antimicrobial compounds or agents of claim 26, wherein over the course of the at least three antimicrobial compounds or agents, one or more of the at least three antimicrobial compounds or agents further comprises a carrier to increase AUC of the one or more of the at least three antimicrobial compounds or agents, as compared with AUC of the one or more of the at least three antimicrobial compounds or agents without the carrier.
35. The antimicrobial compounds or agents of claim 26, wherein over the course of the at least three antimicrobial compounds or agents, one or more of the at least three antimicrobial compounds or agents is in an amount that delivers an increased AUC upon administration.
36. The antimicrobial compounds or agents of claim 26, wherein the microorganism is resistant to at least one of the at least three antimicrobial compounds or agents.
37. The antimicrobial compounds or agents of claim 26, wherein the microorganism is a bacteria selected from at least one of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Bacillus anthracis, Enterobacteriaceae, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Stenotrophomonas maltophila, Burkholderia spp., Acinetobacter baumanii, Proteus mirabilis, Proteus vulgaris, Providencia sp., Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, Legionella pneumophila, and Yersinia pestis.
38. The antimicrobial compounds or agents of claim 26, wherein the infection is a complicated bacterial infection.
39. The antimicrobial compounds or agents of claim 26, wherein the therapeutically effective amount of the composition includes an amount of at least one of the components that is less than an amount of that component indicated as a therapeutically effective amount when used alone.
40. The antimicrobial compounds or agents of claim 26, wherein one or more of the at least three antimicrobial compounds or agents are suitable for any one or more of inhalation and parenteral injection.
41. An antimicrobial fosfomycin composition suitable for oral administration comprising at least three antimicrobial compounds or agents each provided over a course, the at least three antimicrobial compounds or agents comprising: a fosfomycin; a sulfonamide; and a diaminopyridine, such that at least one of the fosfomycin, the sulfonamide and the diaminopyridine is in a therapeutically effective amount, and when provided over the course, the at least three antimicrobial compounds or agents inhibit growth of a microorganism or kill the microorganism by a synergistic activity of the at least three antimicrobial compounds or agents, wherein over the course, the at least three antimicrobial compounds or agents in use display a broad spectrum activity against Gram-positive bacteria, Gram-negative bacteria, and parasites, and wherein the microorganism is selected from any one or more of a drug-resistant bacteria, a multi-drug resistant bacteria, a drug-resistant parasite, and a multi-drug resistant parasite, any of which is suspected of causing an infection, including a complicated bacterial infection, the fosfomycin being provided as a pharmaceutically acceptable salt or ester, any of which have inhibitory activity on cell wall synthesis of the microorganism, the at least three antimicrobial compounds or agents each being in a pharmaceutically acceptable form in which each of the pharmaceutically acceptable forms is separable or in a premix.
42. (canceled)
43. (canceled)
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45. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] For a more complete understanding of the description provided herein, and the advantages thereof, reference is now made to the brief description below, taken in connection with the accompanying drawing and detailed description.
[0045]
[0046]
[0047]
[0048]
[0049]
DETAILED DESCRIPTION
[0050] Although making and using various embodiments are discussed in detail below, it should be appreciated that as described herein are provided many inventive concepts that may be embodied in a wide variety of contexts. Embodiments discussed herein are merely representative, and do not limit the scope of the invention.
[0051] The novel compositions described herein includes a combination of compounds or agents. Individually, each compound or agent on its own may provide only mild or moderate antimicrobial activity, and/or effectiveness. In combination, the grouping of said compounds or agents in the unique arrangements, and formulations described herein unexpectedly allow said compounds to behave synergistically. Surprisingly, the synergistic effect is greater than predicted.
[0052] The combination of the described composition includes at least a first compound or agent. The first compound or agent is or includes a form of a fosfomycin. In one or more forms, the fosfomycin has a structure as represented by formula I, or formula II, in which formula II is a fosfomycin salt depicted as a disodium salt, or formula III, which is a monobasic hydrosoluble fosfomycin salt depicted as fosfomycin tromethamine (fosfomycin-trometamol). The fosfomycin includes any of said formulas, suitable analogues, derivatives, salts, esters, or prodrugs thereof. The salts may include, chemically, (−)(cis-1,2-epoxy-propyl)phosphonic acid. Said salts may include mono-salts, and di-salts (e.g., sodium, potassium, calcium, magnesium), as well as salts formed with amines, such as α-phenethylamine, quinine, lysine, procaine, etc., which can be mono- or di-salts. The first compound or agent may also comprise any analogues, and derivatives, and salts thereof of the representative compounds as described, and illustrated but not limited to those of formula I, II, and III.
##STR00001##
[0053] In some embodiments, a fosfomycin calcium salt form may be more suitable, such as when other salts are not suitable, or are too large. The first compound or agent may include analogues, and/or derivatives thereof having inhibitory activity of cell wall synthesis (inhibits MurA or peptidoglycan synthesis).
[0054] The first compound or agent has activity against Gram-positive bacteria, and Gram-negative bacteria, and may be therapeutically useful in the treatment of infections caused thereby. These bacteria will include but are not limited to Staphylococcus aureus, Klebsiella spp., Streptococcus pyogenes, Enterobacter spp., Streptococcus pneumoniae, Serratia marcescens, Enterococcus faecalis, Pseudomonas aeruginosa, Neisseria meningitidis, Salmonella spp., Neisseria gonorrhoeae, Shigella spp., Haemophilus influenzae, Campylobacter jejuni, Legionella pneumophila, Yersinia enterocolitica, Escherichia coli, Acinetobacter calcoaceticus, Acinetobacter baumanii, Indole (−) Proteus spp., Vibrio cholerae, Indole (+) Proteus spp., Aeromonas spp., Peptococcus spp., Fusobacterium spp., Peptostreptococcus spp., and Clostridium spp. The first compound or agent in some forms has a history of use, such as being provided orally, and by IV, for use with urinary tract infections, and respiratory tract infections, said use providing a long safety record, as is available to one of ordinary skill in this field.
[0055] A second compound of the described compositions may be or may include a sulfonamide or at least one sulfonamide having a functional group represented in formula IV.
##STR00002##
[0056] The sulfonamide may also include a N.sup.1-heterocyclic substituted sulfonamide, such as a 5- or 6-membered heterocycle (e.g. a pyrimidine, pyrazine, pyridazine, oxazole, isoxazole, thiazole or thiadiazole ring). Specific examples include but are not limited to sulfadiazine, sulfamethoxazole, sulfatroxazole, sulfamerazine, sulfadoxine, sulfadimethoxine, sulfamethazine, sulfapyrazole, sulfaquinoxaline, sulfachloropyridazine, sulfaguanidine, sulfalene, sulfametin, sulfamethoxine, sulfamethoxy-pyridazine, sulfamethylphenazole, sulfaphenazole, sulfamoxole, sulfapyrazine, sulfapyridazine, sulfapyridine, sulfasymazine, sulfathiozole, sulfametrole, and sulfixoazole. The second compound may include analogues, derivatives, and/or prodrugs thereof having inhibitory activity in the synthesis of dihydrofolate, as depicted in
[0057] In one or more embodiments, the sulfonamide may be represented by sulfamethoxazole (SMX) represented by formula V, or may be its analogue, derivative, prodrug, salt, or thereof.
##STR00003##
[0058] The second compound or agent in the described compositions may also include or may further comprise or may be replaced by at least one diaminopyridine, including those analogues, derivatives, and/or prodrugs that inhibit dihydrofolate reductase, as also depicted in
[0059] In one or more embodiments, the diaminopyridine may be represented by a formula VI, which is pyrimethamine, or may be any analogue, derivative, prodrug, salt, or ester thereof.
##STR00004##
[0060] In one or more embodiments, the diaminopyridine may be represented by a formula VII, which is trimethoprim (TMP), or may be any analogue, derivative, prodrug, salt, or ester thereof.
##STR00005##
[0061] The second compound may be one or more of a sulfonamide (SM), and/or a diaminopyridine (DP) having inhibitory activity in the dihydrofolate pathway (as described above).
[0062] Interestingly, resistance to the first compound or agent, the fosfomycin (a phosphonic acid derivative, cis-1,2-epoxypropyl phosphonic acid, also provided as a monobasic hydrosoluble fosfomycin salt, or fosfomycin-trometamol) has occurred, sometimes frequently, in many clinically relevant bacteria (both Gram negative, and Gram positive). As an anti-infective, it is only approved for use in the U.S. in a salt form as fosfomycin tromethamine ((1R,2S)-(1,2-epoxypropyl)phosphonic acid, and 2-amino-2-(hydroxymethyl)-1,3-propanediol (1:1)) for treatment of mild (uncomplicated) urinary tract infection (acute cystitis) in women due to susceptible strains of Escherichia coli (Gram negative), and Enterococcus faecalis (Gram positive). The approved treatment is administration of a single oral dose, in which the active component is 3 g, given in the single dose. The minimum inhibitory concentration (MIC) of fosfomycin is 64 μg/μl or less against said bacteria (i.e., 32-128 μg/μl against E. faecalis, and 0.5-2 μg/μl against E. coli). There is use of fosfomycin outside the U.S. some more serious infections. In general, fosfomycin, and its salt forms are considered to have mild or moderate activity against Gram-positive, and/or Gram-negative microorganisms.
[0063] In some embodiments, a combination of a diaminopyridine (DP), such as a trimethoprim (2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine or TMP) with a sulfonamide (SM), such as sulfamethoxazole (N1-(5-methyl-3-isoxazolyl)sulfanilamide or SMX) may form the second compound. A specific combination of trimethoprim (TMP), in combination with sulfamethoxazole (SMX) has been used in a combination approach, commonly referred to as TMP-SMX or co-trimoxazole. The two agents (TMP and SMX) act along a same pathway, in which one inhibits synthesis of a compound one-step ahead of the other in a single metabolic path, which is the typical approach when using a combination of agents. This may also be referred to as sequential targeting depicted as pathway III in
[0064] While activity of co-trimoxazole can be considered synergistic, resistance to co-trimoxazole also occurs with some frequency in both Gram-positive, and Gram-negative microorganisms. Resistance is also common against TMP, which can be used alone. Co-trimoxazole was approved for use in the U.S. in 1973; TMP alone was approved for use in the U.S. in 1980.
[0065] Co-trimoxazole, like fosfomycin, is generally not considered for sustained activity against severe infections. As an anti-infective, co-trimoxazole is generally used against mild or moderate (uncomplicated) infections, including, for example, infections of the urinary tract (e.g., due to susceptible strains of at least the following organisms: Escherichia coli, Klebsiella species, Enterobacter species, Morganella morganii, Proteus mirabilis and Proteus vulgaris), for bronchitis (e.g., due to susceptible strains of Streptococcus pneumoniae, and Haemophilus influenzae), and otitis media (e.g., for acute infections due to susceptible strains of S. pneumoniae or H. influenzae), skin (Staphylococcus aureus), with some prophylactic use against opportunistic infections (e.g., traveler's diarrhea due to susceptible strains of enterotoxigenic E. coli), and Pneumocystitis jiroveci. TMP with SMX (co-trimoxazole) has a history of use, such as providing orally, and by IV, for use including with urinary tract infections; said use providing a long safety record. Administration of co-trimoxazole in the U.S. is usually once or twice a day (every 12 hours) for up to 14 or 21 days, in which a daily dose may include 80 or 160 mg of TMP, and 400 or 800 mg of SMX, respectively (for adults). The MIC of co-trimoxazole against organisms such as Enterobacter species, and H. influenzae is also less than 64 μg/μl (e.g., ≤2 μg/μl for TMP, and ≤38 μg/μl for SMX against E. faecalis, and ≤0.5 μg/μl for TMP, and ≤9.5 μg/μl for SMX against H. influenzae).
[0066] Fosfomycin is considered bactericidal (bacterial killing). SMX alone, and TMP alone are each considered bacteriostatic (bacteria inhibiting, by inhibiting metabolism of folic acid).
[0067] Unexpectedly, the combination described herein of the first compound or agent (a form of fosfomycin, its analogue, derivative, prodrug, etc., which may also be referred to herein as fosfomycin) with the second compound (comprising one or both DP, and/or SM or as a form of co-trimoxazole) has a very robust, and unexpected synergistic effect, greater than the activity of each, which is either the form of fosfomycin when used alone or is, for example, DP, and/or SM when used. The synergistic effect contradicts some literature suggesting that rapidly bactericidal agents, such as fosfomycin, producing by-products, and/or toxins that appear directly related to morbidity, may be antagonized by bacteriostatic agents. The magnitude of synergism as has been found with the described combination of a first bactericidal antimicrobial or antibiotic agent (some form of fosfomycin, its analogue, derivative, prodrug, etc.), and a second antimicrobial or antibiotic agent (one or both DP, and/or SM or as a form of co-trimoxazole) was not predicted.
[0068] The metabolic pathway affected by DP, and SM, which reversibly inhibits successive steps in folate metabolism, as depicted in
[0069] The above, however, does not explain additional unexpected findings in which the combination described herein, containing the fosfomycin, the sulfonamide, and/or the diaminopyridine, has also been found to have superior activity against drug resistant bacteria, including bacterial strains resistant to one or both fosfomycin, and co-trimoxazole or its components individually. In view of the findings described herein, it is believed that in a resistant organism only partial inhibition of biochemical synthesis occurs with introduction of a first inhibitor (such as the first compound or agent described herein) even though bacterial growth is not inhibited. When a second inhibitor (such as the second compound described herein), having convergent inhibitory effect, is introduced, there is concomitant partial inhibition of bacterial growth that, together, disrupts at least one essential metabolite synthesis. This is generally depicted in pathway IV or
[0070] As used herein, the described composition, containing the fosfomycin, with the sulfonamide, and/or the diaminopyridine, may also include any comparable analogue, derivative or synthetic (such as so-called equivalents) of any one or more of the fosfomycin, the sulfonamide, and/or the diaminopyridine. The compositions in any of the combinations described herein may also comprise a co-administration of any one or more of the described first, and/or described second compounds or agents. The co-administration may be provided sequentially or concomitantly. The co-administration may also comprise a pre-mix of the some or all of the components as described herein. The composition may comprise a co-formulation of any one or more of the described first, and/or second compounds or agents.
[0071] In use, the described composition, containing at least two of the fosfomycin, the sulfonamide, and the diaminopyridine will, in some embodiments, be provided at dosages that differ from their independent use. For example, in the U.S., independently fosfomycin on its own is generally delivered once, in a dose that is generally 3 g (i.e., for uncomplicated urinary tract infections). At this dose, fosfomycin is only weakly bactericidal. Co-trimoxazole on its own is generally delivered twice daily, and up to four times daily, in a ratio of TMP to SMX that is 1:5, a dosing regimen that often includes a duration of at least about seven days and a maximum of about 21 days. At these current regimens, co-trimoxazole also behaves like a weak bactericidal agent. On the other hand, with the combinations described herein, the combination is strongly bactericidal, with better MIC (lower MIC for all components). With the combinations described herein, the dosing amount, and/or schedule of dosing have changed due to the synergistic effect, and significantly lower MICs when provided as the described combination. For example, at least one of the compounds when provided in the unique combination described herein (fosfomycin plus one or more of the sulfonamide, and/or the diaminopyridine) will include a lower amount of either or both the first compound (fosfomycin), and/or the second compound (sulfonamide, and/or diaminopyridine). Any of said combinations as described herein should be effective in inhibiting growth and or treating an infection caused by one or more susceptible microorganisms, including serious infections, due to an ability of the combinations described herein to achieve adequate concentrations at a site of infection. The combination as described herein should remain above the MIC of the susceptible microorganism (e.g., at the site of infection) beyond a time necessary to achieve optimal pharmacodynamic effect.
[0072] A dosing regimen of the described novel combination may include, for example, a once daily, twice daily, or up to six times per day of the described composition, in which fosfomycin is in an amount from between about 1-8 g per dose, and DP and/or SM (or as co-trimoxazole) is in an amount of between about 40 mg to 800 mg per dose. Because the combination of agents described herein has been found to have such high activity (often far greater than the compounds when used individually), the actual doses of each agent when administered to a subject in need does not need to be increased from, which avoids toxicity, and reduces adverse events. In some embodiments, the actual does of each agent when administered to a subject in need will be less than current dosing of said agents when use alone. For example, there may be a once daily, twice daily, or up to six times per day of the described composition, in which fosfomycin is in an amount from between about 1-8 g per dose, and DP and/or SM (or as co-trimoxazole) is in an amount of between about 40 mg to 800 mg per dose.
[0073] In some embodiments, a dosing regimen of the described novel combination may include one or more doses of the first compound or agent, and/or the second compound or agent currently used clinically. Said doses can also be optimized by pharmacodynamics, and pharmacokinetic data, thereby achieving a better clinical outcome in vivo as compared with outcome of said first compound or agent or second compound or agent when used independently. Said doses may be optimized to increase the AUC of the novel combination or provide an improved AUC for the novel combination, such as upon administration (oral, IV, inhalation, as examples), as compared to a same mode of administration with one of the components used individually. Said doses may be optimized to increase the AUC of the novel combination or provide an improved AUC for the novel combination, upon administration (oral, inhalation, or IV as examples), with inclusion of an effective amount of a carrier, as compared to administration using a same mode with one of the components individually, or as compared with administration using the same mode for the novel combination without said carrier. The carrier may include one or more of an effective linear chain polysaccharide, such as a cellulose, or microcrystalline cellulose, or hydroxymethyl cellulose, or hydroxypropylcellulose, or a polyvinylpyrrolidone, the carrier included in an amount that is at or less than 50% of the novel composition, or at or less than 40% of the novel composition, or at or less than 30% of the novel composition. The carrier may interact (physically, chemically, and/or transiently) with one or more of the components (first compound, and/or second compound) of the novel compositions described herein. Said dosing regimen when including current clinically useful doses for one or both of the first compound or agent, and second compounds or agent may, in many embodiments, result in an improved outcome (therapeutically, and/or clinically) as compared with outcome when only the first compound or agent or only the second compound or agent are provided independently at said (same) clinically useful dose. Further, in some embodiments, said dosing regimen when including current clinically useful doses for one or both of the independent first, and second compounds or agents may be provided to a subject having resistance or exhibiting clinical resistance to one or both of the first compound or agent, and the second compound or agent, thereby providing an improved outcome (therapeutically, and/or clinically) as compared with the outcome when only the first compound or agent or only the second compound or agent were provided independently at said (same) current clinically useful dose.
[0074] Evaluations of the described novel combination were performed in vitro against S. aureus, E. coli, K. pneumoniae, and P. aeruginosa strains, as provided in the tables below.
[0075] The described novel combination was tested against both reference, and clinically resistant strains of S. aureus, E. coli, K. pneumoniae, and P. aeruginosa. Reference and clinical isolates were obtained from the American Type Culture Collection (ATCC, Manasas, Va.) and International Health Management Associates, Inc. (Schaumberg, Ill.). Bacteria were cultured in trypticase soy broth, concentrated 10-fold by centrifugation, and cryopreserved in the same medium containing 20% glycerol by holding at −80° C. Every two weeks a small amount of frozen culture was removed aseptically, and streaked on fresh trypticase soy agar plates to act as a working inoculum. Following overnight growth at about 37° C., the plates were wrapped in parafilm, and held at about 4° C. Bacteria (4-5 colonies) were then aseptically transferred into broth growth medium for assays as needed.
[0076] Minimal inhibitory concentration (MIC) assays were performed in a manner as described for the CLSI Agar dilution or broth microdilution assay guideline (see, Clinical Laboratory Standards Institute, 2009, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, Eighth Edition, M07-A8, Vol. 29, No. 2). For example, known antibiotics or the compositions described herein were dissolved, serially diluted, and then added to tempered molten Mueller-Hinton Agar before pouring of plates; or were dissolved, and then serially diluted in 100 μL cation-adjusted Mueller-Hinton broth across the assay plate in the classical 2-fold geometric format. D-glucose-6-phosphate was included as a medium supplement in both agar, and broth at a concentration of 50 mg/L. Antibiotic concentrations for agar assay were at the final concentration. Antibiotic concentrations for broth microdilution in each well at this point were double what the final assay concentration would be. Bacterial inocula were prepared by swabbing several colonies from a fresh agar plate, and re-suspending the bacteria to a density that was the 0.5 McFarland turbidity standard, considered close or equivalent to 1×10.sup.8CFU/mL. For agar dilution, a steers replicator was utilized to deliver 1×10.sup.4 CFU/spot. For broth microdilution, the bacterial inocula were diluted in the cation-adjusted Mueller-Hinton broth to 1×10.sup.6 CFU/mL, and 100 μL was overlayered onto the wells containing a diluted antibiotic or a composition described. Following incubation as per the guideline, results were recorded as growth or no growth, and the lowest concentration of the antibiotic or the composition described where no growth was observed was declared the MIC.
[0077] To assay for synergy of antibacterial combinations, a recognized synergy checkerboard assay, adapted from the CLSI guideline microdilution assay, was utilized (see Eliopoulos, G. M., and Moellering, R. C, Jr., 1996. Antimicrobial Combinations, pg. 330-396. In: Lorian, V. (Ed.), Antibiotics in Laboratory Medicine, 4.sup.th Ed., Williams & Wilkins; Baltimore, Md.). In the case of the agar dilution method, each antibiotic alone, or in combination was added to agar prior to plate pouring. For broth microdilution, the first compound (or antibiotic or agent) was diluted in cation-adjusted Mueller-Hinton broth across the plate along the horizontal axis with each well in a row containing 50 μL of an identical concentration of compound (or antibiotic). Along the vertical axis a dilution series of the second compound (or antibiotic) was dispensed. At this point the drug concentrations are four-fold of the final desired working concentration. The bacterial inoculum is prepared as described above for the MIC assay, and 100 μL of culture is overlayered onto the combined compound (or antibiotic) matrix. Following incubation, wells with no growth are noted. The fractional inhibitory concentration (FIC), which provides the mathematical definition (as shown below) is calculated to determine whether there was synergy, or the MIC concentrations for fosfomycin, and trimethoprim for the combination were utilized for the calculation because the MIC for trimethoprim is commonly utilized to represent the value, and susceptibility of bacteria isolates to co-trimoxazole.
FIC=(MIC-A combination/MIC-A alone)+(MIC-B combination/MIC-B alone).
[0078] In the above, FIC: Synergy=FIC<0.5; Additive=FIC>0.5-1; Indifferent=FIC>1-2; Antagonism=FIC>2-4.
[0079] Synergy was also assayed by both agar, and broth microdilution methods. Additional information gathered about the synergistic effect of the described composition as a combination therapy was evaluated by determining bacterial killing effect. This was performed as described above for the checkerboard synergy assay with the exception that the bacterial count in an assay well was determined by plating at the beginning of the experiment, and identifying at the end of incubation for an assay well where growth was inhibited. This has been adapted from both the broth microdilution, and the bactericidal assays (Clinical Laboratory Standards Institute (1999) Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline M26-A, Vol. 19, No. 18). One additional difference from the guideline assay was that the starting inoculums were 5×10.sup.5 CFU/mL as per the MIC assay instead of 1×10.sup.6 CFU/mL in the guideline minimal bactericidal concentration assay. The value derived was the reduction in bacterial numbers (viability) for the compounds when alone versus the described composition comprising the combined antibiotics.
[0080] Synergism was found using the checkerboard microdilution assay, in which synergy was considered as a four-fold reduction in MIC. In fact, the synergistic effect of the described composition on all isolates was as much as an 8-fold reduction or as much as a 16-fold reduction. FIC values were <0.5.
[0081] Bactericidal synergy was demonstrated by determining the minimal bactericidal concentration, >3 log 10 reduction was observed for the combination ranging from 2-fold to as much as 266-fold reduction in concentrations of the individual agents.
[0082] Similarly, bactericidal synergy was greater than the defined value of ≥3 log.sub.10 change in viable count assessed by a time-kill assay after 24 hours.
[0083] The time kill assay was performed sequentially to the checkerboard synergy studies. Fifty milliliter Erlenmeyer flasks containing 10 mL of pre-warmed sterile cation-adjusted Mueller-Hinton Broth with or without antibiotic or the composition described herein were inoculated with bacteria to a starting level of 1×10.sup.6 CFU/mL, followed by incubation at 37° C. with shaking (.sup.˜200 rpm). At various time points, aliquots were withdrawn, serially diluted in antibiotic-free broth medium, and 0.1 mL aliquots of each dilution were spread plated on Trypticase soy agar plates. Enumeration was by counting colonies after 24-hours further incubation of the plates at 37° C., and calculating the reduction in viable bacteria.
[0084] The unique reduction in spontaneous mutational frequency, an indicator of the potential to experience lessened pre-existing antibiotic resistance from a bacterial population, was evaluated for the composition combination described herein, as well as for the fosfomycin and co-trimoxazole individually. To perform this study, a bacterial inoculum was prepared as described above for the minimal inhibitory concentration assay, and viable bacteria level confirmed by plate count, and 0.1 mL aliquots (1×10.sup.9 CFU/mL) of the bacteria were spread plated onto up to 500 Trypticase soy agar plates containing 4-fold MIC of the combination or 2-fold MIC or each antibiotic (fosfomycin or co-trimoxazole) alone providing a cumulative bacterial population of approximately 1×10.sup.10 in order to afford detection of a low resistance frequency. Following 24-hour incubation at 37° C., any colonies that grew up as suspect resistant mutants were streaked onto fresh agar plates of the same composition combination or antibiotic containing media to confirm resistance. MIC was further evaluated to confirm resistance, and the spontaneous mutational frequency was calculated based on the following equation:
[0085] The MIC (in μg/ml), and the fractional inhibitory concentration (FIC) of novel combination described herein against a wide variety of microorganisms, including Gram positive, and Gram negative bacteria presenting as clinically resistant, are provided in TABLE 1, demonstrating synergy, using a broth checkerboard assay, in which the novel composition described herein (Combo) was compared with each of the first compound or agent (as fosfomycin), and the second compound (as DP, and SM) when used alone. In TABLE 1, the second compound was a known DP and SM combination of trimethoprim and sulfamethoxazole or co-trimoxazole (in which data represents their independent MICs), and the Combo was the fosfomycin with the DP, and SM of the second compound. For FIC, synergy (S) was 0.5; additive (A) was >0.5-1; indifferent (I) was >1-2; and antagonistic (X) was >2. Bacterial strains resistant to antibiotics (*) as noted were based on reported CLSI susceptibility breakpoints: Fosfomycin, ≤64 μg/ml, S; co-trimoxazole, ≤2/38 μg/ml, S.
TABLE-US-00001 TABLE 1 Combo (μg/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound) Bacteria ((μg/ml) (μg/ml) (μg/ml) FIC Staphylococcus aureus 0.25/0.125/2.38 32 0.5/9.5 0.26 (ATCC 29213 - MSSA) Staphylococcus aureus 8/0.25/4.75 64 2/38 0.25 (ATCC 33591) Staphylococcus aureus 32/1/19 128* 4/76* 0.5 (MGH-04) Staphylococcus aureus 4/0.125/2.38 32 0.5/9.5 0.38 (918019 - MRSA) Staphylococcus aureus 2/0.25/4.75 16 2/38 0.25 (959797 - MRSA) Staphylococcus aureus 16/0.125/2.38 64 0.5/9.5 0.5 (USA-300 CDC- MRSA) Enterococcus faecalis 64/2/38 256* >8/152* <0.38 (MGH-01) (VanA) Enterococcus faecalis 32/0.5/9.5 128* 4/76* 0.38 (MGH-06) Enterococcus faecalis 16/0.25/4.75 64 1/19 0.5 (ATCC 29212) Escherichia coli 0.03/0.125/2.38 8 0.5/9.5 0.25 (ATCC 25922) Escherichia coli 16/2/38 64 8/152* 0.5 (854535) Escherichia coli 16/0.03/0.6 64 0.25/4.75 0.38 (928017) Pseudomonas aeruginosa 32/2/38 256* 4/76* 0.63 (924190) Pseudomonas aeruginosa 32/0.5/9.5 128* 2/38 0.5 (985543)
[0086] In view of TABLE 1, clinically relevant broad spectrum synergy was demonstrated for a composition comprising the first compound and the second compound, in which the described composition was effective against bacterial strains, including strains resistant to one or both of the trimethoprim, and the sulfamethoxazole. Additional synergy is identified in TABLE 2 (utilizing the broth checkerboard assay) and TABLE 3 (utilizing the agar checkerboard assay) for E. con; TABLE 4 (utilizing the broth checkerboard assay) and TABLE 5 (utilizing the agar checkerboard assay) for K. pneumonia; TABLE 6 (utilizing the broth checkerboard assay) and TABLE 7 (utilizing the agar checkerboard assay) for P. aeruginosa. In these analyses, for FIC, synergy (S) was ≤0.5; additive (A) was >0.5-1; indifferent (I) was >1-2; antagonistic (X) was >2; and certain bacterial strains (*) were resistant to antibiotics as noted based on reported CLSI susceptibility breakpoints: fosfomycin ≤64 μg/ml, 5; co-trimoxazole ≤2/38 μg/ml, S. MIC comparator data was performed using the following known antibiotics: Amoxicillin/clavulanic acid, Cefoxitin, Cefotaxime, Ceftazidime, Ceftriaxone, Piperacillin, Ampicillin, Merropenem, Polymixin B, Tobramycin, Amikacin, Ciprofloxacin (data not shown). The strains of E. coli in the comparator analyses included extended spectrum beta-lactamase (ESBL) producers based on phenotype (e.g., Amoxicillin/clavulanic acid resistance, or Cefoxitin resistance), as well as carbapenem resistant, or quinolone resistant (15 strains). Strains of K. pneumonia in the comparator analyses were highly resistant to many of the clinical comparators, a number of these strains express ESBLs based on phenotype, and several were carbapenem, and quinolone resistant. Strains of K. pneumonia in the comparator analyses were highly resistant to commonly utilized clinical comparators, including 3.sup.rd-generation cephalosporins, carbapenems, aminoglycosides, and quinolones.
TABLE-US-00002 TABLE 2 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy Escherichia coli (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 846439 8/0.25/4.75 32 >4/76* <0.28 S 846446 0.25/0.06/1.25 2 >4/76* <0.13 S 846450 4/2/38 16 >4/76* <0.5 S 857021 8/2/38 32 >4/76* <0.5 S 863686 8/2/38 32 >4/76* <0.5 S
TABLE-US-00003 TABLE 3 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy Escherichia coli (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 846439 16/0.5/9.5 64 8/152* 0.31 S 846446 1/0.125/2.38 4 8/152* 0.26 S 846450 8/2/38 32 8/152* 0.5 S 846455 4/1/19 128* 4/76* 0.28 S 847149 16/1/19 128* 4/76* 0.38 S 854535 64/1/19 128* 4/76* 0.75 A 854566 4/1/19 128* 4/76* 0.28 S 857021 16/2/38 64 8/152* 0.5 S 863078 4/1/19 128* 4/76* 0.28 S 863686 16/2/38 64 16/304* 0.38 S ATCC 25922 0.12/0.25/4.75 16 1/19 0.26 S
TABLE-US-00004 TABLE 4 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy K. pneumoniae (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 848788 ND >64* >4/76* ND ND 857147 4/0.06/1.19 16 >4/76* ≤0.26 S 857187 32/0.5/9.5 64 1/19 1 I 868968 32/4/76; 64/.05/9.5 >64* >4/76* ND ND 868972 32/4/76 64 >4/76* ND ND 869028 ND >64* >4/76* ND ND 875638 16/2/38 32 >4/76* ≤0.75 A 892483 ND >64* >4/76* ND ND 926439 32/0.125/2.38 64 0.25/4.75 0.75 A
TABLE-US-00005 TABLE 5 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy K. pneumonia (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 848788 64/1/19 256* 16/304* 0.31 S 857147 8/0.125/2.38 32 16/304* 0.26 S 857187 32/1/19 128* 2/38 0.75 A 868968 64/2/38 256* 16/304* 0.38 S 868972 64/2/38 128* 16/304* 0.63 A 869028 64/2/38 256* 16/304* 0.38 S 870964 128/1/19 256* 4/76* 0.75 A 875638 16/2/38 64 16/304* 0.38 S 892483 64/2/38 256* 16/304* 0.38 S 926439 32/0.125/2.38 128* 0.5/9.5 0.5 S
TABLE-US-00006 TABLE 6 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy P. aeruginosa (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 826882 16/0.25/4.75 64 >4/76* <0.28 S 880238 16/1/19 64 >4/76* <0.38 S 906939 8/2/38 64 >4/76* <0.38 S 926474 8/1/19 32 >4/76* <0.38 S 945543 16/0.125/1.19 32 0.125/1.19 1.5 1 945545 8/2/38 64 >4/76* <0.38 S 945748 16/0.5/9.5 64 >4/76* <0.31 S
TABLE-US-00007 TABLE 7 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy P. aeruginosa (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 826882 32/0.5/9.5 128* 16/304* 0.28 S 880238 32/1/19 128* 16/304* 0.31 S 889058 4/1/19 128* 4/76* 0.28 S 906939 16/2/38 128* 16/304* 0.25 S 926474 16/2/38 64 16/304* 0.38 S 914881 4/1/19 128* 4/76* 0.28 S 924190 64/2/38 256* 8/152* 0.5 S 945543 4/1/19 128* 4/76* 0.28 S 945545 16/2/38 128* 16/304* 0.25 S 945748 32/0.5/9.5 128* 8/152* 0.31 S PAO1 4/0.5/9.5 32 2/38 0.38 S
[0087] Further synergy is identified in TABLE 8 (utilizing the broth checkerboard assay) against uUTI species. In these analyses, for FIC, synergy (S) was 0.5; additive (A) was >0.5-1; indifferent (I) was >1-2; antagonistic (X) was >2; and certain bacterial strains (*) were resistant to antibiotics as noted based on reported CLSI susceptibility breakpoints: fosfomycin ≤64 μg/ml, 5; co-trimoxazole ≥2/38 μg/ml, S. Synergy was demonstrated with the novel combination described herein including synergistic activity against a preponderance of Proteus mirabilis, Staphylococcus saprophyticus, and Enterococcus faecalis strains. MIC comparator data was performed using the following known antibiotics: Nitrofurantoin, Fosfomycin, Co-trimoxazole, and Levofloxacin.
TABLE-US-00008 TABLE 8 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X P. mirabilis 1128681 1/0.0075/0.15 4 0.06/1.19 0.38 S 1124256 16/0.5/9.5 64 2/38 0.5 S 1184042 1/0.015/03 4 0.06/1.19 0.5 S 1071120 0.25/0.06/1.19 1 0.25/4.75 0.5 S S. saprophyticus 1124324 8/0.125/2.38 32 0.5/9.5 0.5 S 1119421 8/0.125/2.38 32 0.5/9.5 0.5 S 1136894 16/0.125/2.38 64 0.25/4.75 ≤0.75 A 32/≤0.06/1.19 E. faecalis 10864321 0.125/0.5/9.5 32 >8/152* ≤0.04 S 1175598 8/2/38 32 >8/152* ≤0.38 S 1120278 8/0.25/4.75 64 0.5/9.5 0.63 A 1082155 8/2/38 32 >8/152* 0.38 S 1129099 1/0.125/2.38 4 0.25/4.75 ≤0.75 A 2/≤0.06/1.19
[0088] Synergy is also identified in TABLE 9 (utilizing the agar checkerboard assay) against N. gonorrhoeae. In these analyses, the agar dilution was performed utilizing GC agar supplemented with Isovitalex, and 50 mg/L of D-glucose-6-phosphate. For FIC, synergy (S) was ≤0.5; additive (A) was >0.5-1; indifferent (I) was >1-2; antagonistic (X) was >2; and certain bacterial strains (*) were resistant to antibiotics as noted based on reported CLSI susceptibility breakpoints: fosfomycin ≤64 μg/ml, 5; co-trimoxazole ≤2/38 μg/ml, S. Synergy was demonstrated as well as an additive effect with the novel combination described herein against gonococci. MIC comparator data was performed using the following known antibiotics: Ceftriaxone, Azithromycin, Cefixime, Ciprofloxacin, Tetracycline, Pristinamycin, Solithromycin, and Delafloxacin. Pristinamycin was purified from capsules obtained in France; Solithromycin, and Delafloxacin were synthesized internally for comparison.
TABLE-US-00009 TABLE 9 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy N. gonorrhoeae (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 127781 4/0.5/9.5 16 1/19 0.75 A 127782 4/0.25/4.75 16 1/19 0.5 S 127783 4/0.5/9.5 16 1/19 0.75 A 127786 4/0.25/4.75 16 0.5/9.5 0.75 A 1117522 4/0.25/4.75 16 0.5/9.5 0.75 A 1117523 4/0.125/2.38 16 1/19 0.38 S 1117524 2/0.25/4.75 8 1/19 0.5 S 1117525 4/0.25/4.75 8 0.5/9.5 1 A 1117527 2/0.5/9.5 8 2/38 0.5 S
[0089] Synergy was further demonstration using the broth checkerboard assay in TABLE 10 showing MIC for MRSA strains of S. aureus, many of which are associated with skin and skin structure infection (SSSI), and acute bacterial SSSI (ABSSSI). The breadth of the synergy against MRSA with the novel combination described herein is supportive of an ABSSSI indication. In these analyses, for FIC, synergy (S) was 0.5; additive (A) was >0.5-1; indifferent (I) was >1-2; antagonistic (X) was >2; and certain bacterial strains (*) were resistant to antibiotics as noted based on reported CLSI susceptibility breakpoints: fosfomycin ≤64 μg/ml, 5; co-trimoxazole ≤2/38 μg/ml, S.
TABLE-US-00010 TABLE 10 Combo (ug/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound Synergy S. aureus (μg/ml) (μg/ml) (μg/ml) FIC S/A/I/X 829025 1/0.015/0.3 4 0..06/1.19 0..5 S 829049 2/0.015/0.3 8 0..06/1.19 0.5 S 903887 2/0.015/0.3 8 0..06/1.19 0.5 S 920516 0.25/0.0078/0.15 2 0..06/1.19 0.5 S 920962 0.5/0.015/0.3 2 0.125/2.38 0.25 S 927596 2/0.03/0.6 8 0.125/2.38 0.38 S 938125 0.5/0.0078/0.15 4 0..06/1.19 0.5 S 953041 1/0.03/0.6 8 0.25/4.75 0.25 S 956270 0.5/0.015/0.3 28 0..06/1.19 0.5 S 956362 4/0.015/0.3 16 0..06/1.19 0.5 S 967287 2/0.0039/0.078 8 0..06/1.19 0.32 S 974980 2/0.03/0.6 8 0.125/2.38 0.5 S 979234 4/0.0078/0.15 16 0..06/1.19 0.38 S 1045190 0.5/0.015/0.3 2 0..06/1.19 0.5 S
[0090] With the novel combination described herein, the bactericidal (killing) synergy was demonstrated against all strains shown in TABLE 11. As there is no published definition for bactericidal synergy, MIC metric was utilized. For the minimal bactericidal concentration (MBC, μg/ml), TABLE 11 shows the lowest concentration providing a >3-log reduction within 24 hours of the first compound, the second compound, and the novel combination described herein.
TABLE-US-00011 TABLE 11 Combo (pg/ml) Fosfomycin (F) DP/SM (F/DP/SM) (first compound) (second compound) Bacteria (μg/ml) (μg/ml) (μg/ml) Staphylococcus aureus 1/0.5/9.5 128 2/38 (ATCC 29213 - MSSA) Staphylococcus aureus 32/1/19 256 8/152 (ATCC 33591) (MRSA) Staphylococcus aureus 128/4/76 256 16/304 (MGH-04) Staphylococcus aureus 16/0.5/9.5 128 2/38 (918019 - MRSA) Staphylococcus aureus 8/1/19 64 8/152 (959797 - MRSA) Staphylococcus aureus 16/0.5/9.5 256 2/38 (USA-300 CDC- MRSA) Enterococcus faecalis 128/4/76 ≥512 >16/304 (MGH-01) (VanA) Enterococcus faecalis 64/1/19 512 16/304 (MGH-06) Enterococcus faecalis 64/4/76 ≥512 >8/152 (ATCC 29212) Escherichia coli 0.12/0.5/9.5 32 2/38 (ATCC 25922) Escherichia coli 64/8/152 256 >8/152 (854535) Escherichia coli 64/0.25/4.75 256 2/38 (928017) Pseudomonas aeruginosa 64/4/76 >512 >8/152 (924190) Pseudomonas aeruginosa 128/2/38 512 8/152 (985543)
[0091] Bacericidal killing is demonstrated in
[0092] The described novel compositions (said combination of a first and second compounds or agents) were found to exhibit a synergy, and a growth inhibition well below breakpoint values. The unexpected values show synergy at unpredicted levels. Resistant breakpoints for the above first compound and second compound are provided below in TABLE 12, the data is based on CLSI MIC breakpoint data (i.e. from the Clinical Laboratories Standards Institute) in comparison with data obtained from the European Committee on Antimicrobial Susceptibility Testing (EUCAST).
TABLE-US-00012 TABLE 12 Resistance breakpoints (μg/ml) Fosfomycin DP with SM CLSI >256 >4/76 EUCAST >32 >4/76
[0093] Synergy with utilization of the composition described herein was found against both Gram negative bacteria, and Gram positive bacteria, which has not been found by other antimicrobial agents or combinations of antibiotic agents. In fact, the novel compositions described herein were effective against the most common Gram negative, and Gram positive bacteria independent of their resistance. In some embodiments, an FIC Index value was in the range of about 0.02 to 0.4 for E. coli, K. pneumoniae, and P. aeruginosa. The FIC Index value was in a range of about 0.07-0.25 for S. aureus. Synergy was found against S. aureus, including resistant strains of S. aureus, such as MRSA, which is a significant microorganism in hospital acquired bacterial pneumonia. Further, the novel compositions described herein were active against strains that are resistant to the independent compounds that make up the composition when used on their own. As such, the novel compositions described herein are able to overcome the resistance problems previously found with utilization of its constituent components.
[0094] While the susceptibility breakpoint is 64 for fosfomycin alone and is 4/76 for a trimethoprim/sulfamethoxazole combination (e.g., when provided as co-trimoxazole), with the combinations described herein and as exhibited in the tables, MICs are at least four- to eight-fold below these breakpoints. To provide sustained and/or acceptable exposures of the combination described herein, it may, in some embodiments, be that fosfomycin is provided in an amount so as to have a concentration above 64 μg/μl, at least for about 70% of the time, or for greater than 70% of the time. Similarly, it may be, in some embodiments, that the diaminopyridine (or its peak) is between about 5 and about 10 μg/μl, at least when administered, and/or for some time thereafter. An example, the diaminopyridine may be above the MIC for about or greater than about 50% of the time. Taking the above into account, in a first example, a dosing regimen of a combination described herein may include about 1 g of fosfomycin, given three times per day (about 1 g per dose), with at least one of the diaminopyridine provided three times per day (at about 110 mg per dose), and/or the sulfonamide provided three times per day (at about 550 mg per dose).
[0095] The described composition is able to reduce the MIC below the clinical breakpoints found for the individual components. This enables use of the novel compositions described herein against otherwise multi-drug resistant bacteria, including multi-drug resistant E. coli, multi-drug resistant P. aeruginosa, and multi-drug resistant S. aureus, as examples. Thus, with the described combinations and the low MIC values (low MIC within a “susceptible” range, and/or low MIC that is well below the accepted “susceptible” range) there will be better outcomes. With the number of strains, and types of strains showing susceptibility to the combinations described herein, lower doses (of each component, or of one or more of the components) will be possible with the combinations described herein (e.g., for treatment against, killing of, and/or inhibition of growth of the susceptible microorganisms). With the number of strains, and types of strains, showing susceptibility to the combinations described herein, shortened treatment duration (of each component, or of one or more of the components) will be possible with the combinations described herein (e.g., for treatment against, killing of, and/or inhibition of growth of the susceptible microorganisms). With the number of strains, and types of strains, showing susceptibility to the combinations described herein, a shorter median time to defervescence will be possible with the combinations described herein (when administered for treatment against, killing of, and/or inhibition of growth of the susceptible microorganisms).
[0096] With the described composition using the first compound and the second compound, such as ones identified in the tables, the frequency of spontaneous mutants resistant to said novel composition was representatively assessed, and found to be ≤1.8×10.sup.−10 as compared with 6.33×10.sup.−6 for fosfomycin alone or 2.67×10.sup.−9 for co-trimoxazole alone or 3.40×10.sup.−8 for nitrofurantoin. The spontaneous mutational frequency (SMF; resistance) was calculated as the number of resistant colonies observed divided by the total inoculum. The substantially lower SMF for the novel combination described herein indicates a lower potential for resistance evolution as compared with the individual components, or the nitrofurantoin comparator. Without being bound by theory, it is suggested that in addition to the substantially lower SMF and in view of the synergy described herein, susceptibility breakpoints with the described compositions may not have to change significantly over time. In some embodiments, susceptibility breakpoints with the described compositions will not have to change significantly.
[0097] With the described compositions, and without being bound by theory, but in view, in part, on the substantially lower SMF, and, in part, on the synergy, there will be an infrequent selection of resistant bacterial strains with the combinations described herein. The infrequent selection will also be infrequent at even low concentrations of the combinations described herein.
[0098] With the described novel compositions an MIC.sub.90≤16 μg/mL was identified for certain important Gram negative bacteria. For such strains, a representative dosing regimen of the novel composition against, for example, E. coli strains, such as those tested in the tables above may include 0.03-16 μg/mL of the first compound, and 0.61-38 μg/mL, and 0.03-2 μg/mL of the second compound, which comprises two individual components of DP, and SM.
[0099] With the described novel compositions MICs 64 μg/mL were identified for other, and most of the remaining important Gram negative bacteria. For such strains, a representative dosing regimen of the novel composition against, for example, P. aeruginosa strains, such as those tested in the tables above may include 2-32 μg/mL of the first compound, and 0.125-2 μg/mL, and 0.219-38 μg/mL of the second compound, which comprises two individual components of DP, and SM.
[0100] In view of prior safety analysis for components that may make up the compositions described herein, the combinations described herein will likely include amounts or doses of each of the components that are about or less than the amounts or doses of each of the components (when used individually) found to be safe when provided to a subject in need thereof.
[0101] As shown, the combination described herein provides excellent activity against Gram-positive bacteria, and Gram-negative bacteria, thereby offering a broadened spectrum, with synergistic potency. Further, the combination described herein provides efficacy against a wide variety of infectious organisms, including parasites. The combination may be selected to have a therapeutic effect based on the sulfonamide(s) and/or the diaminopyridine(s) that are in the combination, in which certain sulfonamides and/or diaminopyridines exhibit greater or lesser anti-parasitic activity in addition to their antibacterial activity. The combination described herein is effective against strains resistant to one or more of the components in the described composition. The combination described herein is effective against strains resistant to existing drugs or agents that are currently used with some effectiveness in certain individuals in need thereof. The spectrum of activity, as provided with the described composition, is supportive of use with many infections, such as those of the urinary tract, respiratory tract, and skin and skin structures, including acute bacterial skin and skin structure infections, and sexually transmitted infections, and related infections or diseases. The bactericidal activity as provided with the described composition is supportive of use at reduced concentrations as compared with current use of the independent components when used for current indications as an antibiotic for bacterial infections, including infections of the urinary tract, respiratory tract, and skin a skin structure, including acute bacterial skin, and skin structure infections, and certain sexually transmitted diseases, and related infections. The very low frequency of spontaneous mutational frequency with the combination described herein is highly indicative of a much reduced potential for resistance with the compositions described herein. In some embodiments, the described combination may be administered in a lower dose while still providing efficacy. In some embodiments, the described combination may be administered at a higher dose while remaining safe for use. In one or more embodiments, the described combination provides an improved therapeutic index of the combination while not enhancing general toxicity.
[0102] Without being bound by theory, the synergism found with the novel compositions described herein may be related to specific inhibition of peptidoglycan (cell wall) synthesis in addition to a synergistic inhibition of a pathway leading to essential metabolites necessary for peptidoglycan synthesis rendering bacteria or other microorganisms highly susceptible to the novel compositions disclosed herein. In view of said mechanism of action of said novel composition, said novel composition should be effective against many or most clinically relevant Gram negative bacteria as well as Gram positive bacteria independent of the bacterial resistance profiles. Further, it is expected that there will be a reduced risk of use with said novel compositions in view of their known safety profiles showing they are well tolerated, and have safe usage when provided individually. Moreover, with lower dosing levels, as may be provided with the novel compositions described herein, there will be fewer safety concerns. A favorable spectrum against other multidrug resistant Gram negative, and Gram positive bacteria is provided and expected to be due in part to the highly synergistic interaction found with the novel composition described herein.
[0103] Providing the combination as described above, such as sequentially, or concomitantly, to a subject in need will preferably include providing all the components of the composition so that there is overlapping exposure with regard to each of the components. Providing the combination as described will preferably include having each of the components in sufficient amounts at a similar schedule or an overlapping schedule (same or overlapping times), or at or near maximal amounts at a similar a schedule or an overlapping schedule (same or overlapping times), whether or not the components are in a same formulation.
[0104] In one or more embodiments, a formulation, or the combination as described herein, may be provided once a day. In some embodiments, a formulation, or the combination as described herein may be provided twice daily. In one or more embodiments, a formulation, or the combination as described herein may be provided three times a day. In one or more embodiments, a formulation, or the combination as described herein may be provided four times a day, or at least one of the components is provided up to four times per day. Fosfomycin, having a short half-life, may, in some embodiments, be provided more than once per day, or at least in divided doses, two times a day, or three time a day, or four times a day. In one or more embodiments, a total daily amount of the fosfomycin may be in an amount in a range of about 0.5 g (per day) to about 6 g (per day). In one or more embodiments, a total daily amount of the sulfonamide (e.g., sulfamethoxazole) may be in an amount in a range of about 400 mg (per day) to about 3200 mg (per day). In one or more embodiments, a total daily amount of the diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) may be in an amount in a range of about 80 mg (per day) to about 500 mg (per day).
[0105] Additional representative dosing examples are provided. For example, a dosing may include a twice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 750 mg (each dose, 2 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 400 mg (each dose, 2 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 80 mg (each dose, 2 times/day). In another example, a dosing may include a thrice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 333 mg (each dose, 3 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 267 mg (each dose, 3 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 53 mg (each dose, 3 times/day). In another example, a dosing may include once daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 4 g (one dose/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 1600 mg (one dose/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 250 mg (one dose/day). In a further example, a dosing may include a twice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 2 g (each dose, 2 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 800 mg (each dose, 2 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 125 mg (each dose, 2 times/day). In still another example, a dosing may include a thrice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 1.33 g (each dose, 3 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 400 mg (each dose, 3 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 75 mg (each dose, 3 times/day). In yet another example, a dosing may include a twice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 1.5 g (each dose, 2 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 400 mg (each dose, 2 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 80 mg (each dose, 2 times/day). In still another example, a dosing may include a thrice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 1 g (each dose, 3 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 167 mg (each dose, 3 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 54 mg (each dose, 3 times/day). In another example, a dosing may include once daily dosing of fosfomycin provided in an amount of up to about or about 3 g (one dose/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided twice daily in an amount of up to about or about 400 mg (two doses/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided twice daily in an amount of up to about or about 80 mg (two doses/day). In a further example, a dosing may include a thrice daily dosing provided to a subject in need, in which fosfomycin is provided in an amount of up to about or about 500 mg (each dose, 3 times/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided in an amount of up to about or about 400 mg (each dose, 3 times/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided in an amount of up to about or about 75 mg (each dose, 3 times/day). In another example, a dosing may include twice daily dosing of fosfomycin provided in an amount of up to about or about 1 g (two doses/day), and in which the sulfonamide (e.g., sulfamethoxazole) is provided twice daily in an amount of up to about or about 800 mg (two doses/day), and/or diaminopyridine (e.g., trimethoprim, and/or pyrimethamine) is provided twice daily in an amount of up to about or about 125 mg (two doses/day). Of course, additional dosing schedules are contemplated and/or suitable.
[0106] The duration of the combination (in a single formulation or when said components are provided so that there is overlapping exposure of the components) may be from five days to 10 days, or may be from 10 days to 14 days. The duration of the combination may be from 1 day and up to about 21 days, or any length therebetween, the length of time, depending in part on the severity of the infection.
[0107] The novel compositions described herein may be provided for a number of uses, including but not limited to topical, oral, and parenteral (IV) use or for inhalation. Dosage forms of the novel compositions include pharmaceutically acceptable forms that provide effective treatment against one or more bacterial infections. Said forms may include pharmaceutically acceptable salts, esters or prodrugs thereof. Said forms may further include a pharmaceutically acceptable base addition salt, which refers to those salts that retain the biological effectiveness, and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Said forms may also include a pharmaceutically acceptable acid addition salt, which refers to those salts that retain the biological effectiveness, and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids. Said dosage forms will include dosages with acceptable side effect profiles when administered to a subject in need thereof. In one or more embodiments, the novel compositions described herein are provided as a pharmaceutical composition, which refers to a formulation containing each component of the composition along with a medium generally accepted in the art for the delivery of the novel composition on its whole or in its parts. For effective use, the pharmaceutical composition will be a biologically active composition, delivered to a subject in need thereof, such as a mammal, for example, humans. The medium will include any pharmaceutically acceptable carrier(s), diluent(s), and/or excipient(s).
[0108] In many embodiments, the novel composition will be provided at a therapeutically effective amount, which refers to that amount of the novel composition on its whole which, when administered to a subject in need thereof, is sufficient to effect treatment of a symptom, a condition, or a disease of interest in the subject or as presented by the subject. The amount of an antibiotic composition of the invention which constitutes a “therapeutically effective amount” will vary depending on the antibiotic composition, the disease or condition, and its severity, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge, and to this disclosure. The therapeutically effective amount may be modified by pharmacokinetic, and pharmacodynamic analysis as is understood in the field. In some embodiments, the novel composition may be provided prophylactically or preventatively or protectively, which may, in some instances, be at or below a therapeutically effective amount. A representative example of pharmacokinetic parameters is provided in TABLE 13.
TABLE-US-00013 TABLE 13 diaminopyridine sulfonamide (e.g., (e.g., Fosfomycin trimethoprim) sulfamethoxazole) Dose 3000 mg 720 mg 3600 mg Tmax 2 hrs 2 hrs 4 hrs Cmax 26.1 μg/mL 8.5 μg/mL 120 μg/mL T ½ 5.7 hrs kel 0.122 hrs 0.08 hrs 0.08 to 1 hr (elimina- tion rate constant) Cmax/dose 0.0087 mg 0.012 mg 0.033 mg
[0109] Treating a subject may be treating a subject in need thereof or treating a subject preventatively or protectively. Generally, treating as described herein, and a treatment as described herein may include treatment of a symptom, or a condition, or a disease of interest, or an infection as presented by a subject, or is defending against a condition or a disease or a symptom. Treatment may include but is not limited to: (i) preventing the symptom or disease or condition from occurring in a subject (such as when such subject is predisposed to the disease or condition, and has not yet been diagnosed or is not yet symptomatic; (ii) inhibiting a symptom or disease or condition (e.g., arresting its development; (iii) relieving a symptom or disease or condition (e.g., causing regression of the symptom disease or condition; or (iv) stabilizing the symptom or disease or condition.
[0110] Embodiments described herein further include treating one or more conditions or symptoms (e.g., infections, and/or diseases associate with one or more infections caused by or considered to be associated with a microbe, including bacteria described previously). Treating may including when presenting with symptoms or prior to presenting with symptoms. Microbial infections include but are not limited to wound infections, respiratory infections, systemic infections, skin infection, sexually transmitted diseases, and related infections, gastrointestinal, urogenital infections, pneumonia, tuberculosis, gonorrhea, syphilis, sepsis, meningitis, cholera, and diarrhea associated with a microbial infection. Treating may include prophylaxis, therapy, and/or cure. Treating of a subject may involve reducing infection, preventing infection, and/or delaying or slowing the onset of an infection or an associated condition, disease, or disorder (e.g. the symptoms associated with the disease, condition, or disorder). In some embodiments, the infection may be associated with antibiotic resistant bacteria.
[0111] Formulations prepared with the described compositions may include those suitable for oral, nasal, topical (including buccal, and sublingual), intravenous, rectal, vaginal, and/or parenteral administration. The formulations may conveniently be presented in unit dosage form, and may be prepared by any methods well known in the pharmacy field. The amount of active ingredient which can be combined with additional components, and/or a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, and other factors. The amount of active ingredient that can be combined with or without a carrier material to produce a single dosage form will, in many embodiments, generally be that amount of the compound, and/or the composition which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient, or from about 5% to about 80%, or any amount therein.
[0112] The described compositions may be provided as an injectable or parenteral formulation. Such compositions will include an effective amount of an excipient. The excipient will have one or more of the following functionalities: soluble; stabilization (e.g. antioxidant); anti-microbial preservation; impart tonicity (with a goal of achieving isotonicity); bulking agent. In some embodiments, the described compositions will be available in a sterile, pyrogen-free solution or as a lyophilized powder for reconstitution. They may be provided as single-use vials or formulated in, e.g., USP water for injection, or as a lyophilized powder, such as in an amount that will be reconstituted with dextrose for intravenous administration.
[0113] For oral administration, the described composition may be for immediate or sustained release (e.g., provided as granules, and/or by layering, such as a multi-layered tablet or capsule having granules therein, in which there are different release profile for each layer and/or granule, such as fast release in one layer (or set of granules), and delayed release in the other(s) which can be modulated to enable the ultimate or desired bioavailability). A binary combination may be provided into one multilayered film-coated tablet and/or capsule (as well as other suitable forms). An oral formulation may include a plurality of granules or plurality of layers or plurality of components each having its own unique release characteristics in order to maximize release and/or to ensure sufficient overlap and exposure of the components at the site of infection. A carrier may also be included for increasing AUC of one or more layers and/or set of granules, as described above. Layering, hot-melt extrusion (HME), granulation, and/or lipid drug delivery are exemplary means used to provide the described composition for oral administration. The described compositions may further comprise one or more of the following: tablet and/or capsular coating, disintegrant, surfactant, lubricant, glidant, anti-adherent, and corn starch anti-sticking agent.
[0114] Also provided are kits including one or more pharmaceutical compositions described herein. A kit may include one or more additional agents, compounds with compositions described herein. The different components of the kit may be provided in different containers. The kit may be compartmentalized to receive the containers in close confinement. The kit may also contain instructions for using the described compositions with accompanying components. Illustrative examples of containers for said kits include, but are not limited to, small glass containers, plastic containers, composite containers, or strips of plastic or paper. Containers may be those that allow a worker to efficiently transfer reagents or components from one compartment to another compartment. Such containers may also one that will accept a compound or compositions described herein, and/or may accept a resuspending solution. Said compositions being in any one or more of a powder (e.g. a lyophilized powder), precipitate, granules, tablet, gel or liquid form, as examples. Compositions described herein or one or more of the components that make us the described compositions may be provided in the same or different forms in a single kit, and may be provided in the same or different containers.
[0115] In one or more embodiments, DP, and SM may be prepared as a blend, by mixing the requisite ingredients as is known in the art. An organic solvent which is miscible with water (e.g., glycofurol or a polyethyleneglycol) may also be added during the preparation. The mixture may further comprise fosfomycin or its precursor or requisite ingredients. Alternatively, the DP, and SM may remain independent, and be prepared independently from the fosfomycin, in which case said DP, and/or the SM, and the fosfomycin may be co-administered (sequentially, or concomitantly).
[0116] Said aqueous compositions (or the DP, and/or SM and the fosfomycin independently) may be dried using known techniques, including freeze-drying, spray-drying. Precipitates may also be prepared from the aqueous compositions using methods known in the field, and dried, such as under vacuum. The resulting dry compositions, which are also a part of what is disclosed herein, may be reconverted, when desired (with or without sterilization, such as after sterilization) into solutions (e.g. injection solutions) by the addition of water or other suitable liquid for dissolving said dry components.
[0117] The compositions described herein may be used, in addition to use as injectable solutions, by other means of administration in which said combination in solution form should be used.
[0118] The compositions described herein may be used by any means of administration in which said combination in solid form should be used.
[0119] In another embodiment, the components are blended in dry form, and granulated by means known in the art (such as with a binding agent, polymeric component, etc.) to form a tablet or a pressed composition.
[0120] The compositions described herein may also be sterilized by means of known techniques which are usual in the preparation of parenteral administration forms as, for example, heat sterilization or sterile filtration.
[0121] Embodiments described herein include treating subjects infected with, and/or subjects at risk of developing, a microbial infection, including a drug-resistant microbial infection. A subject may be a mammal, including an animal or other multicellular organism. A subject may be a human. A subject may be a pet or a farm animal.
[0122] Several methods for killing the microorganisms are included with the inventions described herein. One or more methods include directly killing the microorganisms. The microorganisms may include Gram-positive bacteria. The microorganisms may include parasites. The microorganisms may include microorganisms considered resistance to alternative antimicrobial agents. The microorganisms may include microorganisms considered resistant to one of more of the ingredients used in the compositions described herein (e.g., at least one first compound or agent, and at least one second compound or agent). One or more methods may include killing the microorganisms in a subject in need thereof, the method comprising providing at least one form of the composition described herein (having at least one first compound or agent, and at least one second compound or agent). The method further comprises administering the at least one form of the composition described herein to the subject in a dose sufficient to kill or directly kill the microorganism. The method may further comprise administering the at least one form of the composition described herein to the subject in a dose and for a duration sufficient to kill or directly kill the microorganism. In addition, or as an alternative, the method may comprise administering the at least one form of the composition described herein to the subject in a dose and for a duration sufficient to control infection caused by or attributed to the microorganism. In addition, or as an alternative, the method may comprise administering the at least one form of the composition described herein to the subject in a dose and for a duration sufficient to suppress an infection caused by or attributed to the microorganism.
[0123] One or more methods described herein may include killing or inhibiting growth of microorganisms present on a surface or within an object or a subject in need thereof, the method comprising contacting the microorganisms with a formulation comprising an effective amount of one of the compositions described herein, wherein contacting results in killing or in inhibiting growth of the microorganism, the formulation comprising at least a pharmaceutically acceptable fosfomycin, and one or more of a pharmaceutically acceptable sulfonamide and/or a pharmaceutically acceptable diaminopyridine. The microorganism may be a bacteria. The microorganism may be a parasite. The microorganism may be resistant to one or more of the components that make of the formulation. The fosfomycin may be in the form of a pharmaceutically acceptable salt, ester, or prodrug, or an analog or derivative thereof, any of which have inhibitory activity in cell wall synthesis of the microorganism. The sulfonamide may be one or more sulfonamide in the form of a pharmaceutically acceptable salt, ester, or prodrug, or an analog or derivative thereof, any of which have any of which have inhibitory activity in folic acid metabolism. The diaminopyridine may be one or more diaminopyridine in the form of a pharmaceutically acceptable salt, ester, or prodrug, or an analog or derivative thereof, any of inhibit dihydrofolate reductase. The effective amount may be an amount of at least one of the fosfomycin, the diaminopyridine, and/or the sulfonamide that is less than an amount indicated as a therapeutically effective amount when used alone.
[0124] As used herein, and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” or to “a composition” includes a plurality of such agents or compositions, and equivalents thereof known to those skilled in the art, and so forth. It is understood that the term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude embodiments wherein, for example, any composition of matter, composition, method, or process, or the like, described herein may “consist of” or “consist essentially of” the described features.
[0125] Although representative processes, and articles have been described in detail herein, those skilled in the art will recognize that various substitutions and modifications may be made without departing from the scope, and spirit of what is described, and defined by the appended claims.