DESC:FIELD OF INVENTION
The current invention relates to methods and compositions for enhancing the efficacy of antibiotics and for overcoming antibiotic resistance in pathogenic bacteria. The invention specifically relates to the use of compositions comprising co-crystals of antibiotics with Nonsteroidal anti-inflammatory drugs such as celecoxib and meloxicam to increase the uptake of these antibiotics by pathogenic bacteria.
BACKGROUND
Drug –resistance by microbes is one of the biggest challenges currently in health and pharmaceutical sector. The increasing, and many times superfluous use of antibiotics has contributed to the current problem of bacterial resistance to antibiotics. Moreover, irrespective of how effective a new antibiotic is upon clinical introduction, there is invariably some drug resistance by microbes, which limits the effectiveness of a new drug. Thus, recent emergence of “superbugs,” clinically resistant to several existing antibiotics, combined with no new antibiotics being discovered in the recent years, is posing a great problem in treating bacterial infections.
There are several mechanisms by which antibiotic resistance develops in bacteria. Many bacteria use efflux pumps to pump out antibiotics, leading to antibiotic resistance (Poole, K; Clinic. Microbiol. Infec. 2004, 10, 12-26). Moreover, the second membrane in Gram negative bacteria provides a reduced influx of antibiotics into the cell. Hence, the antibiotic resistance in Gram negative bacteria is contributed by reduced influx (permeability) of cells to antibiotic, as well as increased efflux. The majority of ESKAPE pathogens (the term ESKAPE pathogens refers to the six bacterial species that collectively cause majority of the nosocomial infections, are largely resistant to existing repertoire of drugs, these species are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are Gram negative pathogens, and cause serious diseases ( Nikaido, H., Science, 1994, 264, 382-388). Numerous approaches have been reported for increasing antibiotic concentration in the cell cytoplasm of efflux pump expressing bacteria (Mahamoud, et al., J. Antimicrob. Chemoth. 2007, 59, 1223-1229). However, there are not many combination therapies available that can increase antibiotic efficacy and prevent resistance development by bacteria.
The development of new antibiotics is becoming increasingly sporadic, and is unable to keep pace with the rapid emergence of resistant pathogens. Therefore novel drugs and new approaches to combat resistant pathogens are urgently needed. Both de novo drug discovery and drug rescuing and repurposing have been used in the search for an effective antibiotic. Unlike the lengthy and costly process of de novo drug discovery, drug rescuing and repurposing can reduce the time, cost and risk associated with drug innovation. There is thus a need for new agents that can work to increase efficacy of already known antibiotics, and help to combat resistance to these antibiotics in bacteria. Thus, combination therapies that can enhance antibiotic efficacy as well as help to decrease antibiotic resistance in bacteria, is a highly effective economic strategy. According to FDA, identification of non-traditional treatment strategies using combination of two or more drugs containing antibiotics targeting bacteria and another drug as potentiator of antibiotic activity would help in overcoming drug resistance (Miró-Canturri A, Ayerbe-Algaba R and Smani Y (2019) Drug Repurposing for the Treatment of Bacterial and Fungal Infections. Front. Microbiol. 10:41. doi: 10.3389/fmicb.2019.00041).
Examples of combination drugs that are already being sold and are effective antimicrobials are amoxicillin and clavulanic acid, sold as AUGMENTIN® and piperacillin and tazobactam, sold as ZOSYN®.
The current invention discloses compositions and methods of enhancing antibiotic efficacy in treating bacterial infections, as well as decreasing antibiotic resistance in bacteria by co-administering antibiotics with Nonsteroidal anti-inflammatory drugs (NSAID as co-crystals. More specifically, the current invention discloses the increase in antibiotic efficacy by using co-crystals of the antibiotics with NSAID compounds such as celecoxib and Meloxicam, which increases antibiotic uptake into the bacterial cells and antibiotic efficacy in inhibiting bacterial growth.
SUMMARY
The current invention discloses compositions and methods of enhancing the antimicrobial activity of an antibiotic. In one embodiment, the invention encompasses a pharmaceutical composition comprising an antibiotic and an NSAID, wherein the antibiotic and the NSAID are in co-crystal form. In one embodiment, the NSAID in the pharmaceutical composition is Celecoxib or meloxicam. In one embodiment, the antibiotic is co-administered with a non-steroidal anti-inflammatory drug like celecoxib or meloxicam. In one embodiment, the co-administration of antibiotic and Celecoxib or Meloxicam increases the uptake of the antibiotic into the bacterial cell, by changing the cell membrane potential, or increasing permeability of the bacterial membrane, or by doing both. In one embodiment, the co-crystal of the antibiotic with Celecoxib or Meloxicam increases the uptake of the antibiotic by the bacterial cells to a greater extent than the increase that occurs by co-administration of the antibiotic and the NSAID as a physical mixture to the bacterial cells.
One embodiment of the invention is a co-crystal comprising at least one antibiotic and at least one NSAID.
In one embodiment, the NSAID has at least one functional group selected from the group consisting of alcohol, thiol, ester, carboxylic acid, primary amine, secondary amine, tertiary amine.
In one embodiment, when compared to the antibiotic alone, or to a physical mixture of the antibiotic and the NSAID, the co-crystal exhibits at least one of the following:
(a) increased efficacy
(b) increased dose response
(c) increased bioavailability
(d) increased solubility
In one embodiment, the antibiotic is selected from the group consisting of Ampicillin, Kanamycin, Chloramphenicol, Ciprofloxacillin, Oxacillin and Isoniazid.
In one embodiment, the antibiotic is an antibiotic that is used to treat tuberculosis.
In one embodiment, the NSAID is celecoxib or meloxicam, their enantiomers or salts thereof.
In one embodiment, the antibiotic is effective at inhibiting bacterial growth at less than the minimum inhibitory concentration (MIC) of the antibiotic.
In one embodiment, the antibiotic is effective at inhibiting bacterial growth at half the minimum inhibitory concentration of the antibiotic.
In one embodiment, the co-crystal exhibits increased permeability into the bacterial cells to a larger extent, as compared to antibiotic alone, or to a physical mixture of the antibiotic and the NSAID.
In one embodiment, the uptake of antibiotic by the bacterial cells is greater for the co-crystal, as compared to antibiotic alone, or to a physical mixture of the antibiotic and the NSAID.
In one embodiment, the bacteria are antibiotic -resistant bacteria.
In one embodiment, the celecoxib concentration is 10 µM.
One embodiment of the invention is, a pharmaceutical composition comprising the co-crystal disclosed herein, wherein the co-crystal is present at a therapeutically effective amount of the co-crystal in a physiologically acceptable medium.
In one embodiment, the molar ratio between the antibiotic and the NSAID in the co-crystal is 2:1.
One embodiment of the invention is, a method of making the co-crystal, the method comprising the steps of:
(a) weighing desired amounts of antibiotic and NSAID so as to achieve molar ratio of 1:1, 1:2, 1:3 1:4, 2:1, 3:1 or 4:1 between antibiotic and NSAID;
(b) grinding the antibiotic and NSAID from step (a) together;
(c) dissolving the ground mixture of antibiotic and NSAID from step (b) in a suitable organic solvent; and
(d) Slow Evaporating the solvent from step (c) to form the co-crystals over a period of several days.
In one embodiment, the method disclosed herein, wherein the organic solvent in step (c) is methanol or ethanol.
In one embodiment, the co-crystals formed by the method disclosed herein.
In one embodiment, the molar ratio between the antibiotic and the NSAID is 2:1 in the method disclosed herein. In one embodiment, the NSAID is celecoxib or meloxicam, their enantiomers or salts thereof.

BRIEF DESCRIPTION OF FIGURES:
Fig 1: shows Co-crystal studies of ampicillin and celecoxib,
1A. Graph showing cell viability of S aureus in presence of Ampicillin and celecoxib cocrystals at various concentrations of the co-crystal. The different ratios of ampicillin and celecoxib are denoted as AC01 (only celecoxib), AC10 (only ampicillin), AC11, AC12, AC13, AC14, AC21, AC31 (molar ratios of ampicillin to celecoxib 1:1, 1:2, 1:3, 1:4, 2:1, 3:1 ).
1B. Graph showing efficacy of co-crystal AC21 against growth of S. aureus when compared to ampicillin alone (AC10) at different concentrations.
1C. PXRD (Powder X ray diffraction) analysis of the crystals AC10 (ampicillin alone) (a), AC10 (celecoxib alone) (b), and co-crystal AC21 (c). The PXRD analysis of the cocrystals showed that there were weak interactions between ampicillin and celecoxib, which is evidenced from the new peaks observed and the disappearance of existing peaks in drug mixture crystals when compared with crystals made from pure compounds (AC01 and AC10).
1D. Merged chromatograms of the PXRD.
1E. DSC (Differential Scanning Calorimeter) analysis of the crystals AC10 (ampicillin alone) (a), AC10 (celecoxib alone) (b), and co-crystal AC21 (c). DSC analysis of cocrystals showed that the crystalline nature of celecoxib was preserved in all combinations, but there was a phase transition in ampicillin in combination with celecoxib in AC21 when compared with the amorphous form of the pure compound.
1F. Graph showing the absorbance of ampicillin at 259 nm in membrane pellets and supernatant fractions of membrane ghosts treated with AC10 (Ampicillin only), AC01 (Celecoxib only), AC21 and physical mixture (PM) of ampicillin and celecoxib.
Fig. 2: The graphs showing the number of CFUs (colony forming units) of MRSA (methicillin resistant Staphylococcus aureus) treated with or without antibiotic and/or celecoxib physical mixtures or co-crystals (A, C, E, G) and the graph showing the absorbance of antibiotic in membrane pellets and supernatant fractions of MRSA membrane ghosts treated with or without antibiotic and/or celecoxib physical mixtures or co-crystals, showing increasing partitioning of the antibiotic into the pellet fraction when the cocrystal is used, which shows increased uptake into bacterial cells. The wavelengths for absorbance studies are different
(B, D, F, H). All antibiotics are used at half their MIC for these studies, which shows a increased dose response for the cocrystals.
Fig. 2A shows cocrystal studies for Celecoxib and Kanamycin: Celecoxib alone (10µM) ; Kanamycin (5µg/ml); K+C (PM with 10µM celecoxib and 5µg/ml kanamycin); KC21 (cocrystal 5µg/ml concentration) . Kanamycin is used at half of its MIC (MIC is 10µg/ml ) for these studies.
Fig. 2B shows increasing partitioning of Kanamycin into the pellet fraction when the cocrystal is used, compared to antibiotic alone and the physical mixture, which shows increased uptake into bacterial cells.
Fig. 2C shows cocrystal studies for Celecoxib and Chloramphenicol: Celecoxib alone (10µM) ; Chloramphenicol (2.5µg/ml); Ch+C (PM with 10µM celecoxib and 2.5µg/ml Chloramphenicol); ChC21 (cocrystal 5µg/ml concentration) . Chloramphenicol is used at half of its MIC (MIC is 5µg/ml) for these studies.
Fig. 2D shows increasing partitioning of Chloramphenicol into the pellet fraction when the cocrystal is used, compared to antibiotic alone and the physical mixture, which shows increased uptake into bacterial cells.
Fig. 2E shows cocrystal studies for Celecoxib and Ciprofloxacin: Celecoxib alone (10µM) ; Ciprofloxacin (0.125µg/ml); Ci+C (PM with 10µM celecoxib and 0.125µg/ml Ciprofloxacin); CiC21 (cocrystal 5µg/ml concentration) . Ciprofloxacin is used at half of its MIC (MIC is 0.25µg/ml ) for these studies.
Fig. 2F shows increasing partitioning of Ciprofloxacin into the pellet fraction when the cocrystal is used, compared to antibiotic alone and the physical mixture, which shows increased uptake into bacterial cells.
Fig. 2G shows cocrystal studies for Celecoxib and Oxacillin: Celecoxib alone (10µM) ; Oxacillin (25µg/ml); O+C (PM with 10µM celecoxib and 25µg/ml Oxacillin); OC21 (cocrystal 5µg/ml concentration) . Oxacillin is used at half of its MIC (MIC is 50 µg/ml ) for these studies.
Fig. 2F shows increasing partitioning of Oxacillin into the pellet fraction when the cocrystal is used, compared to antibiotic alone and the physical mixture, which shows increased uptake into bacterial cells.
DESCRIPTION OF THE INVENTION
The current invention discloses compositions and methods of enhancing the antimicrobial activity of an antibiotic. The current invention provides pharmaceutical compositions comprising effective amount of an antibiotic and an NSAID, the NSAID being either celecoxib or Meloxicam.
In one embodiment, the antibiotic is co-administered with the non-steroidal anti-inflammatory drug like celecoxib or meloxicam. In one embodiment, the co-administration is done by making a physical mixture of the antibiotic with the celecoxib or meloxicam. In one embodiment the co-administration is done by first making co-crystal of the antibiotic with and NSAID. In one embodiment, the NSAID is celecoxib or meloxicam. In one embodiment there drug-NSAID interaction is different in the co-crystal than in the physical mixture of the two components. In one embodiment, the co-crystal exhibits increased uptake/ permeability of the antibiotic into the bacterial cells of more than the physical mixture of the antibiotic and the NSAID does.
Definitions:
The terms “antibiotic” and “antimicrobial” are used interchangeably herein, and refer to an antibacterial agent, and may mean any compound that either inhibits or completely arrests or prevents microbial growth or kills the microbe.
As used herein, the “antimicrobial activity” of an antimicrobial agent is defined as the property of a substance to inhibit the growth and reproduction of a microbial organism or to kill it. Common terms generally applied to antibiotic action towards bacteria are bacteriostatic (stops growth) and bactericidal (kills bacteria). Depending on the concentrations applied, microbial growth can be slowed or stopped by the action of an antimicrobial agent.
“Active agents” are APIs which show a pharmaceutical effect and thus can be identified as being pharmaceutically active. In a more narrow sense this definition is encompassing all APIs being marketed or under clinical trial for the treatment of diseases. “Active agents with antimicrobial activity” are APIs (Active Pharmaceutical Ingredients) which show efficacy as antimicrobial agents.
ESKAPE, an acronym for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, pathogens are the global emerging cause of nosocomial infections and are multidrug resistant (MDR). The currently available antibiotics are not effective against some highly resistant ESKAPE pathogens such as Acinetobacter species, MDR- P. aeruginosa, Klebsiella species, and E. coli which led to the use of old antibiotics like colistin that are associated with severe toxicity
The term co-crystal refers to : Cocrystals are multicomponent systems in which two components, an active pharmaceutical ingredient and a coformer were present in stoichiometric ratio and bonded together with non-covalent interactions in the crystal lattice. The components in a co-crystal are held together by weak non-covalent interactions. Examples of weak interactions includes, but are not limited to,: hydrogen bonds, van der Waals forces, and p-p interactions (Ref 1, incorporated herein by reference).
“Co-crystal former” as use herein is defined as a molecule being an active agent selected from NSAIDs, and with which the antibiotic is able to form co-crystals.
In one embodiment, the co-crystal former itself can be an active pharmaceutical ingredient.

NSAIDs or Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used medications in the world because of their demonstrated efficacy in reducing pain and inflammation. They consist of a group of drugs that are used in fever, pain, and inflammation because these drugs possess antipyretic, analgesic, and anti-inflammatory properties. Clinically, they are useful in relieving pain in many conditions, ranging from menstrual and postoperative pain to arthritic pain. These drugs are well-known anti-inflammatory agents, and they exert their effects through the inhibition of prostaglandin synthesis by blocking the enzyme cyclooxygenase (COX).
“Mixture” or “physical mixture” of antibiotic and the corresponding cocrystal former” is defined as a mixture of the antibiotic and the NSAID/s which is only a physical mixture without any coupling forces between the compounds.
In one embodiment the NSAID used herein is a Coxib, a selective COX-2 inhibitor.. Examples of Coxibs include, but are not limited to: celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, valdecoxib, and cimicoxib.
Celecoxib: Celecoxib is a Cyclooxygenase-2 (COX-2) specific inhibitor approved by FDA for the treatment of rheumatoid arthritis. COX-2 is induced by inflammatory stimuli and is responsible for the prostaglandins (PGE2) production at the site of inflammation.
In one embodiment, Celecoxib is used for cocrystal formation with any antibiotics that can be used for treating human subjects.
In one embodiment, Meloxicam is used for cocrystal formation with any antibiotics that can be used for treating veterinary subjects.
In one embodiment, the NSAID itself does not show any antimicrobial activity.
Permeation/ permeability of a drug / compound through a membrane is an important parameter affecting its efficacy and bioavailability. The uptake of an antimicrobial compound by bacterial cells is also a limiting factor which leads to drug resistance in bacteria. The increased permeability of a drug into membranes can affect uptake of the drug by the bacterial cells, as well its permeating through the GI (gastrointestinal) cells to get absorbed into the blood circulation.
Intestinal permeability refers to the flow of a substance across the organ, and one of the quantitative ways to define it is how deep can a substance/ drug penetrate into the intestinal wall per time unit.
Drug lipophilicity is a very important descriptor governing permeation across a biological membrane. Lipophilicity plays a crucial role in determining ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties and the overall suitability of drug candidates. Control of physicochemical properties such as lipophilicity, can improve compound quality and the likelihood of therapeutic success.
Most drugs need to pass through at least one cellular membrane to reach their intended target. Although tight binding of a drug molecule to its intended target is important for potency, poor membrane permeability often translates into poor or non-existent in vivo efficacy. In eukaryotic systems, two possible transport modes are available for a molecule to passthrough a membrane: active and passive. Active transport involves a transport protein that uses energy (e.g., ATP hydrolysis) to shuttle a molecule across a membrane. In contrast, passive transport, which is the most common mode of drug passage through membranes, involves diffusion of a molecule through the membrane with no outside assistance or energy input. The rate of passive diffusion across a membrane is proportional to the partition coefficient of the compound between the membrane (lipophilic environment) and the external medium (aqueous milieu), the diffusion coefficient of the compound through the membrane, and the compound’s concentration gradient across the membrane.
Increase in efficacy refers to increase in the action of a drug. For an antibiotic increase in efficacy refers to increase in its antimicrobial activity at the same concentration, or decrease in its effective concentration. Increase in efficacy of an antibiotic may be measured by any means, for example , inhibition of bacterial growth or bactericidal activity, to a greater extent than the control at the same or decreased doses than the reference or control , or to the same extent at a lower concentration than used in control.
“Dose response” is defined as the response of a subject to a drug.
“Increased dose response” refers to obtaining the same or increased desired (intended) response in a subject at lower concentration than the previously known effective concentration. The previously known effective concentration may the minimal inhibitory concentration (MIC). Thus increased dose response would refer to obtaining greater or same level of desired response (biologic activity) in a subject at lower than MIC, which means the drug has higher potency. Increased dose response may also refer to obtaining higher level of desired effect (biologic activity) at the same concentration, which means it has higher efficacy.
Solubility
As used herein, the “minimal inhibitory concentration” or “MIC” is the lowest concentration of any agent having antimicrobial activity that inhibits the growth of a microorganism as judged by visual inspection.
“Bioavailability” refers to the percentage of the weight of a therapeutic agent that is delivered into the general circulation of the animal or human being studied. The total exposure of a drug when administered intravenously is usually defined as 100% bioavailable. “Intranasal bioavailability” refers to the extent to which a therapeutic agent is absorbed into the general circulation when the pharmaceutical composition is taken intranasally as compared to intravenous injection.
Bioavailability is affected by multiple factors , including the rate and extent of drug absorption from the gastrointestinal (GI) tract which in turn is very complex and affected by many factors. These include physicochemical factors, physiological factors, and factors related to the dosage form. The fundamental key parameters controlling oral drug absorption are the permeability of the drug through the GI membrane and the solubility/dissolution of the drug dose in the GI milieu.
A “pharmaceutically effective” amount of an antibiotic is an amount sufficient to provide the intended treatment in the patient being treated. The “pharmaceutically effective” amount being administered should relieve to some extent one or more of the symptoms of the disease, i.e., infection, being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the disease, i.e., infection, that the subject being treated has or is at risk of developing.
A pharmaceutically effective amount of an antibiotic may also result in undesirable side effects, such as itching, stomach upset, swelling, inflammation etc.
As used herein, a “pharmaceutical composition” encompasses a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational and the like.
“Pharmaceutically acceptable salt” refers to those salts that retain the biological effectiveness and optionally other properties of the free bases and that are obtained by reaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.
A “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and/or adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used in the specification and claims includes one and more such excipients, diluents, carriers, and adjuvants.
As used herein, the term “subject,” or “patient,” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which compounds of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
As used herein, the term “administration” includes giving or administering a drugs such as an antibiotic or an NSAID, or any of the pharmaceutical compositions described herein, for testing or therapeutic purposes. Administering also includes adding antibiotics or any composition disclosed herein to a bacterial culture, a bacterial cell, for studies disclosed herein. The term administration also includes giving drugs/ pharmaceutical compositions to any subjects or patients for therapeutic or testing purposes. Administration can be by any means possible, and does not exclude any means of giving/ providing with a pharmaceutical composition.
As used herein, “co-administration” refers to more than one drug/ compound being given together. It includes all mechanisms by which the drugs/ compounds can be administered together, such as just mixing the two compounds to administer them together (physical mixture), or making co-crystals, as it has been shown in the current disclosure elsewhere.
A combination of the antimicrobial agent and the NSAID compound can be administered, or , in other words, can be co-administered, by any route, protocol, or means, that may be appropriate for its administration and embodiments of the current invention are not limited to any particular route, protocol, means, etc. of administration. For example, co-administration of the antibiotic and NSAID to the microbe can be done by by contacting the microbe in culture or in solution or by applying the antibiotic and the NSAID combination to a material, such as the surface of a material, in or on which the microbe resides. Administration can also be done to a subject/ patient having a microbial infection and such administration to the subject results in administration to the microbe. For example, the subject can be a plant or an animal. In certain embodiments, the subject can be a mammal. In certain embodiments, the mammal subject can be a human having and suffering from a microbial infection. In certain embodiments, a combination of an antibiotic and the NSAID as disclosed herein is administered in an effective amount.

The term “gram positive antibiotic” refers to an antibacterial agent active against gram-positive bacteria.
The term “gram negative antibiotic” refers to an antibacterial agent active against gram-negative bacteria.
The term “nosocomial infection”, “hospital acquired infection” and “health-care associated infections” are used interchangeably herein.
An “effective amount” is that amount, the administration of which to a subject (also referred to as a patient), either in a single dose or as part of a series, is effective for treatment. For example, and effective amount can be an amount that is sufficient to reduce the severity of a microbial infection (or one or more symptoms thereof), ameliorate one or more symptoms of an infection, prevent the advancement of the infection, cause regression of infection, or enhance or improve the therapeutic effect(s) of another therapy. In some embodiments, the effective amount cannot be specified in advance and can be determined by a caregiver, for example, by a physician or other healthcare provider, using various means, for example, dose titration. Appropriate therapeutically effective amounts can also be determined by routine experimentation using, for example, animal models.
As used herein, “biofilms” refer to biological films that develop and persist at interfaces in aqueous environments, especially along the inner walls of conduit material in industrial facilities, in household plumbing systems, on medical implants, or as foci of chronic infections. These biological films are composed of microorganisms embedded in an organic gelatinous structure composed of one or more matrix polymers that are secreted by the resident microorganisms. Biofilms can develop into macroscopic structures several millimeters or centimeters in thickness and can cover large surface areas. These biological formations can play a role in restricting or entirely blocking flow in plumbing systems and often decrease the lifespan or longevity of materials through corrosive action mediated by the embedded bacteria. Biofilms are also capable of trapping nutrients and particulates that can contribute to their enhanced development and stability. Biofilms can also prevent penetration of antimicrobial agents and therefore, make bacteria within biofilms drug resistant, which leads to persistent infection. Embodiments of the present disclosure can be used to inhibit the growth of a biofilm, where inhibits includes one or more of the following: stopping the growth of the biofilm, killing the biofilm, reducing the size of the biofilm, and the like.
Embodiments:
The current invention discloses compositions and methods of enhancing the antimicrobial activity of an antibiotic. In one embodiment, the invention encompasses a pharmaceutical composition comprising an antibiotic and an NSAID, wherein the antibiotic and the NSAID are in co-crystal form. In one embodiment, the NSAID in the pharmaceutical composition is Celecoxib or meloxicam. In one embodiment, the antibiotic is co-administered with a non-steroidal anti-inflammatory drug like celecoxib or meloxicam. In one embodiment, the co-administration of antibiotic and Celecoxib or Meloxicam increases the uptake of the antibiotic into the bacterial cell, by changing the cell membrane potential, or increasing permeability of the bacterial membrane, or by doing both. In one embodiment, the co-crystal of the antibiotic with Celecoxib or Meloxicam increases the uptake of the antibiotic by the bacterial cells to a greater extent than the increase that occurs by co-administration of the antibiotic and the NSAID as a physical mixture to the bacterial cells.
One embodiment of the invention is is a co-crystal comprising at least one antibiotic and at least one NSAID.
In one embodiment, the NSAID has at least one functional group selected from the group consisting of alcohol, thiol, ester, carboxylic acid, primary amine, secondary amine, tertiary amine.
In one embodiment, when compared to the antibiotic alone, or to a physical mixture of the antibiotic and the NSAID, the co-crystal exhibits at least one of the following:
(e) increased efficacy
(f) increased dose response
(g) increased bioavailability
(h) increased solubility
In one embodiment, the antibiotic is selected from the group consisting of Ampicillin, Kanamycin, Chloremphenicol, Ciprofloxacillin, and Oxacillin.
In one embodiment, the NSAID is celecoxib or meloxicam, their enantiomers or salts thereof.
In one embodiment, the antibiotic is effective at inhibiting bacterial growth at less than the minimum inhibitory concentration of the antibiotic.
In one embodiment, the antibiotic is effective at inhibiting bacterial growth at half the minimum inhibitory concentration of the antibiotic.
In one embodiment, the co-crystal increases the permeability of the bacterial membranes to a larger extent, as compared to antibiotic alone, or to a physical mixture of the antibiotic and the NSAID.
In one embodiment, the bacteria are antibiotic -resistant bacteria.
In one embodiment, the celecoxib concentration is 10 µM.
In one embodiment, the uptake of antibiotic by the bacterial cells is greater for the co-crystal, as compared to antibiotic alone, or to a physical mixture of the antibiotic and the NSAID.
In one embodiment, In the presence of the NSAID, there is increased uptake of ampicillin by bacteria due to increased membrane permeability, and altered physical properties of the drugs. In one embodiment, the co-crystal treatment increases permeation and bioavailability.
In one embodiment, this effect of celecoxib in combination cannot be defined by synergism since celecoxib alone does not show any effect on the bacterial growth. In one embodiment, the combinatorial treatment with celecoxib and antibiotic is a better treatment strategy to combat S. aureus infections than the antibiotic treatment alone, or treatment with the physical mixture.
In one embodiment, celecoxib increases uptake of the antibiotic by modulating membrane permeability and potential.
One embodiment of the invention is, a pharmaceutical composition comprising the co-crystal disclosed herein, wherein the co-crystal is present at a therapeutically effective amount of the co-crystal in a physiologically acceptable medium.
In one embodiment, the molar ratio between the antibiotic and the NSAID in the co-crystal is 2:1.
One embodiment of the invention is, a method of making the co-crystal, the method comprising the steps of:
(e) weighing desired amounts of antibiotic and NSAID so as to achieve molar ratio of 1:1, 1:2, 1:3 1:4, 2:1, 3:1 or 4:1 between antibiotic and NSAID;
(f) grinding the antibiotic and NSAID from step (a) together;
(g) dissolving the ground mixture of antibiotic and NSAID from step (b) in a suitable organic solvent; and
(h) Slow Evaporating the solvent from step (c) to form the co-crystals over a period of several days.
In one embodiment, the method disclosed herein, wherein the organic solvent in step (c) is methanol.
In one embodiment, the co-crystals formed by the method disclosed herein.
In one embodiment, the molar ratio between the antibiotic and the NSAID is 2:1 in the method disclosed herein. In one embodiment, the NSAID is celecoxib or meloxicam, their enantiomers or salts thereof.
One embodiment of the current invention is a pharmaceutical composition comprising cocrystal of an antibiotic with an NSAID.
In an embodiment, the pharmaceutical composition comprises a broad spectrum antibiotic.
In one embodiment, the NSAID is a COX inhibitor.
In certain embodiments, the antibiotic is an antibiotic selected from the group consisting of ampicillin, kanamycin, tobramycin, erythromycin, rifampicin, and tetracycline. In certain embodiments, the antibiotic is an antibiotic selected from the group consisting of erythromycin, rifampicin, and tetracycline. In certain embodiments, the antibiotic is kanamycin. In certain embodiments, the antibiotic is tobramycin. In certain embodiments, the antibiotic is erythromycin. In certain embodiments, the antibiotic is rifampicin. In certain embodiments, the antibiotic is tetracycline.
In one embodiment, the co-crystal comprises at least one antibiotic that can be used to treat tuberculosis. In one embodiment, co-crystal comprises at least one antibiotic that can be used to treat drug-resistant tuberculosis.
In an embodiment, the pharmaceutical composition can be used to treat an infection caused by one or more bacteria selected from the group consisting of: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae.

In one embodiment, treatment of microbial pathogens with antibiotic and celecoxib or meloxicam together causes differential gene expression in the microbes , when compared to microbial pathogens treated with antibiotic only. In one embodiment, these differentially expressed genes may be essential genes or non-essential genes. In one embodiment, the differentially expressed genes in microbial pathogens treated with antibiotic –celecoxib or meloxicam combination are genes responsible for pathogen virulence.
In one embodiment, the differentially expressed genes in microbial pathogens treated with antibiotic-celecoxib/ meloxicam combination are genes responsible for pathogenesis, drug-resistance or upstream signal transduction genes.
In one embodiment, enterotoxin and hemolysin genes are differentially expressed in microbial pathogens treated with antibiotic-celecoxib/ meloxicam combination compared to pathogens treated with only antibiotic.
In one embodiment, administration of antibiotic-NSAID cocrystal exhibits increased bacterial cell permeability to antibiotic.
In one embodiment, administration of co-crystal of antibiotic with the NSAID leads to increase in antibiotic uptake by pathogenic bacteria, when compared to antibiotic treatment alone.
In one embodiment, the co-crystal of antibiotic with the NSAID has increased antibiotic uptake by bacterial cells when compared to that of a physical mixture (PM) of antibiotic and the NSAID.
In one embodiment, the co-crystal of antibiotic with celecoxib or meloxicam has increased antibiotic uptake by bacterial cells when compared to that of a physical mixture (PM) of antibiotic and the celecoxib or meloxicam.
In one embodiment, the co-crystal of antibiotic with the NSAID has increased bioavailability when compared to that of a physical mixture (PM) of antibiotic and the NSAID.
In one embodiment, the co-crystal of antibiotic with the NSAID has increased bioavailability when compared to that of the antibiotic alone.
In one embodiment, the co-crystal displays increased aqueous solubility compared to a physical mixture of the antibiotic and NSAID.
In one embodiment, the co-crystal displays increased aqueous solubility compared to the antibiotic or NSAID alone.
In one embodiment, combinatorial treatment with celecoxib and antibiotic leads to downregulation of virulence factors in pathogenic bacteria.
Examples of virulence factors that are downregulated upon combinatorial treatment of pathogens, include, but are not limited to, enterotoxins, two-component signal transduction system and antibiotic resistance genes.
An embodiment of the present disclosure includes a method of inhibiting the growth of a biofilm or the growth of bacteria, among others, that includes: exposing a surface having a biofilm thereon or exposed to bacteria to a co-crystal composition disclosed herein.
India is leading in the global burden of Tuberculosis (TB) with 24% of total global TB burden according to Global TB report. The clinical management of tuberculosis and other mycobacterial diseases with antimycobacterial chemotherapy remains a difficult task. The classical treatment protocols are long lasting and the drugs reach mycobacteria infected macrophages in low amounts. Misuse and mismanagement of anti-TB drugs is one of the major causes among several reasons for the emergence of multidrug resistant (MDR), extremely drug resistant (XDR) and pan drug resistant (PDR) strains of TB. Recently, totally drug resistant (TDR) strains have been reported in India. Government of India has decided to declare India TB-free by 2025 and has launched several programs including new drug discovery, rescuing and repurposing of old drugs along with antimicrobial stewardship programs. In such a situation, the present invention would be very useful in treating TB effectively. Since TB is classified as one of the granulomatous inflammatory diseases, using NSAID (celecoxib)-anti TB drug cocrystal will help in reducing inflammation in host and the anti TB drug will limit the bacterial growth.
The treatment regimen for newly diagnosed TB consists of eight weeks of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E) in the initial phase. The continuation phase consists of the three drugs Isoniazid, Rifampicin and Ethambutol given for another sixteen weeks.
However, the resistant TB treatment regimen is not empirical and depends on the patient response to the drug. Of the 4 drugs used in TB treatment – isoniazid, rifampicin, pyrazinamide and ethambutanol or streptomycin, isoniazid is used for the treatment of both active and latent TB treatment. However, resistance to isoniazid is more common. In one embodiment, the cocrystals of NSAID and isoniazid or NSAID-rifampicin can be made.
In one embodiment, the co-crystal of NSAID with isoniazid or rifampicin is highly effective in treating drug-resistant TB. In one embodiment, the co-crystal of NSAID with isoniazid or rifampicin is more effective in treating drug-resistant TB than isoniazid or rifampicin alone.
EXAMPLES:
Example 1
Co-crystal studies for celecoxib and Ampicillin
Co-crystals of different proportions of ampicillin (Sigma Chemicals) and celecoxib (Aurobindo Pharma Ltd.) were developed using neat grinding method followed by slow solvent evaporation. The cocrystals were then used to determine the percent growth inhibition of bacteria by counting colony forming units (CFU) (S aureus) at various concentrations. The results clearly demonstrated an increased inhibition of growth by cocrystals. The co-crystal of proportion AC21 (ampicillin 2 parts and celecoxib 1 part) showed more potency (Fig. 1A and 1B; The different ratios of ampicillin and celecoxib are denoted as AC01 (only celecoxib), AC10 (only ampicillin), AC11, AC12, AC13, AC14, AC21, AC31 (molar ratios of ampicillin to celecoxib 1:1, 1:2, 1:3, 1:4, 2:1, 3:1). The powder XRD (powder X-ray diffraction/PXRD) analysis of the cocrystals showed that there are weak interactions between ampicillin and celecoxib, which could be seen from the new peaks observed/ disappearance of existing peaks in drug mixture crystals compared with crystals made form pure compounds. (Figs. 1C and 1D).
Differential Scanning Calorimetric (DSC) analysis of co-crystals showed that the crystalline nature of celecoxib was preserved in all combinations, but there was a phase transition in ampicillin in combination with celecoxib in AC21 when compared with amorphous form of pure compound (Fig. 1E).
Example 2: Antibiotic-celecoxib co-crystal development with other antibiotics
Antibiotic-Celecoxib co-crystals were developed by co-grinding followed by slow solvent evaporation method using 2:1 molar proportion of Kanamycin, Chloremphenicol, Ciprofloxacillin, Oxacillin and celecoxib respectively. The co-crystals were designated as KC21, ChC21, CiC & OC21 respectively
Antibiotic and celecoxib were weighed as per the ratio and were manually ground for 30 min using a sterile ceramic mortar and pestle. This co-ground mixture is then dissolved in hot ethanol (100%) and was left at room temperature (RT) covered with a perforated aluminium foil until the solvent evaporated leaving behind the co-crystals.
Antibiotic-celecoxib co-crystal development
Antibiotic-Celecoxib co-crystals were also developed by co-grinding followed by slow solvent evaporation method using 2:1 molar proportion of Gentamycin, Tetracycline, Oxacillin and celecoxib respectively. We decided to choose only one proportion because as demonstrated earlier in our laboratory, this proportion of antibiotics with celecoxib gives the best results. The co-crystals were designated as GC21, TC21 & OC21 (details of all cocrystals made are given in Table 1).
Antibiotic and celecoxib were weighed as per the ratio and were manually ground for 30 min using a sterile ceramic mortar and pestle. This co-ground mixture is then dissolved in hot ethanol (100%) and was left at room temperature (RT) covered with a perforated aluminium foil until the solvent evaporated leaving behind the co-crystals.
The efficacy of the GC21, TC21 & OC21 in inhibiting the multidrug resistant Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) along with methicillin-sensitive Staphylococcus aureus (MSSA) was determined by counting the Colony forming units (CFUs).
Table 1: Co-crystals with different antibiotics
antibiotic nsaid Ratio of antibiotic to nsaid Abbreviated name
ampicillin celecoxib 2:1 AC21
Kanamycin, celecoxib 2:1 KC21
Chloremphenicol, celecoxib 2:1 ChC21
Ciprofloxacillin, celecoxib 2:1 CiC
Oxacillin celecoxib 2:1 OC21
Gentamycin, celecoxib 2:1 GC21
Tetracycline, celecoxib 2:1 TC21

Example 3:
Determination of Colony Forming Units (CFUs)
The efficacy of the KC21, ChC21, CiC & OC21 in methicillin-resistant Staphylococcus aureus (MRSA) was determined by counting the Colony forming units (CFUs) as described earlier (Ref 2, incorporated herein by reference;)
The efficacy of the physical mixtures of the antibiotic and celecoxib were also evaluated and compared with the co-crystals.
Results
The co-crystals were more efficient at 3 ug/ml concentration than the physical mixtures of the antibiotic and celecoxib at the same concentration. The physical mixtures of the antibiotic and celecoxib were significantly more effective at inhibiting bacterial growth than the antibiotic alone. (Fig. 2).
The fold change in the number of CFUs also showed that the co-crystals were more effective than the physical mixtures (Table 1).
Table 2: Fold Difference in The Number Of cfus Between Physical Mixture And Co-Crystal Of Antibiotic And Celecoxib
Physical mixture Co-crystal
Ampicillin 2.8 (A+C) 5.1 (AC21)
kanamycin 1.38 (K+C) 1.835 (KC21)
Chloramphenicol
1.6 (Ch+C) 1.94
Ciprofloxacin 1.27 (Ci +C) 1.48
Oxacillin 1.81 2.77

Example 4: Determining changes in solubility and permeability in co-crystal form
Method 1: Further , to determine the effect of celecoxib on increasing the permeation/ permeability, the absorbance of antibiotic in pellet and supernatant fractions of the bacterial cells was measured.
Bacterial ghost membrane preparation: Bacterial membrane ghosts were prepared as described previously (Amara AA, Salem-Bekhit MM, Alanazi FK. Sponge-like: a new protocol for preparing bacterial ghosts. Scientific world journal 2013; 2013: 545741). The ghost membranes were incubated with celecoxib (10µM), ampicillin (3 µg/mL) or both the drugs in PBS. The entry of drugs into the membrane ghosts was monitored by spectrophotometry at wavelengths of 239 and 259 nm for ampicillin and celecoxib, respectively.
The concentrations of celecoxib and ampicillin inside bacteria using empty bacterial membrane vesicles was determined. The results clearly indicated increased absorbance for ampicillin in the presence of celecoxib in the pellet fraction, suggesting increased entry of ampicillin.
The bacterial membrane ghosts were incubated with AC21 and physical mixture of both the drugs (without crystallization) and the absorbance of ampicillin and celecoxib at 239 nm and 259 nm respectively in the pellet (membrane) and supernatant fraction was measured (? 239 and ? 259 respectively). The co-crystal AC21 showed more absorbance at 239 nm corresponding to ampicillin in pellet fraction suggesting increased entry of the ampicillin (Fig. 1F) compared to the physical mixture.
The absorbance values were similarly measured for co-crystals made with other antibiotics too . Only antibiotic absorbance is considered as control with 100% and the % antibiotic in different conditions is calculated as % control (% antibiotic=absorbance in different conditions * 100 / absorbance of control).
More % in Co-crystal in pellet fraction and less in supernatant fraction compared to physical mixture indicates more entry of antibiotic in Co-crystal form. The results are shown in Figs. 2 and Table 3.
Table 3: % antibiotic in supernatant and pellet fractions:
Antibiotic /
antibiotic + celecoxib physical mixture/
co-crystal Supernatant Pellet
Kanamycin 100 100
K + C 74.2346939 197.058824
KC21 55.3571429 255.882353
Chloramphenicol 100 100
Ch + C 71.641791 156.880734
ChC21 60.0746269 184.40
Ciprofloxacin 100 100
Ci+ C 73.5099338 130.612245
CiC21 66.5562914 146.938776
Oxacillin 100 100
O+C 74.5704467 145.801527
OC21 64.9484536 170.229008
Ampicillin 100 100
A + C 79.09 111.25
AC21 58.06 181.875

Method 2: Partition coefficient (LogP) determination
Drug lipophilicity is a very important descriptor governing permeation across a biological membrane . Lipophilicity is generally expressed quantitatively as the log10 of the partitioning of a neutral drug species between n-octanol and water (logP) and is the most widely used predictor for drug permeation.
Most drugs need to pass through at least one cellular membrane to reach their intended target. Although tight binding of a drug molecule to its intended target is important for potency, poor membrane permeability often translates into poor or non-existent in vivo efficacy. Two possible transport modes are available for a molecule to passthrough a membrane: active and passive. Active transport involves a transport protein that uses energy (e.g., ATP hydrolysis) to shuttle a molecule across a membrane. In contrast, passive transport, which is the most common mode of drug passage through membranes, involves diffusion of a molecule through the membrane with no outside assistance or energy input. The rate of passive diffusion across a membrane is proportional to the partition coefficient of the compound between the membrane (lipophilic environment) and the external medium (aqueous milieu), the diffusion coefficient of the compound through the membrane, and the compound’s concentration gradient across the membrane.
The n-octanol-water partition coefficient of ampicillin and cocrystal AC21 was determined by shake flask method as described previously (Ref 3 ;incorporated herein by reference;). Briefly, equal volumes of n-octanol (Merck) (10 mL) and water (10 mL) were taken in a flask and were mutually saturated on a thermostat shaker at 100 rpm, 25°C for 24 h. After saturation, the two phases were allowed to separate on standing at room temperature. A calibration curve was prepared for serial dilutions of ampicillin in water and n-octanol phases by measuring the absorbance at 259 nm on a UV-Vis spectrophotometer (Shimadzu). The concentration of ampicillin of AC21 co-crystal in both the phases was determined using the calibration curve and the logP was calculated as log10 of the ratio of concentration of ampicillin in n-octanol phase to the concentration of ampicillin in water phase. The logP value is average of a minimum of three replicates + standard deviation.
Result:
The logP value indicates the solubility and permeation of a drug. A drug with logP between 1-3 shows moderate solubility and moderate permeation. We experimentally determined the logP value, indicating the permeation/solubility of the drug, for the cocrystal AC21 and ampicillin by shake flask method along with prediction of the logP using various software such as ALOGPS and Molinspiration (Ref 4, Molinspiration Cheminformatics free web services, https://www.molinspiration.com) by giving the SMILES of both ampicillin and celecoxib together as structural input in ampicillin-celecoxib and celecoxib-ampicillin format. The experimental logP value was determined to be 2.08 + 0.089 for AC21 and 1.16 + 0.12 for ampicillin. The ALOGPS predicted the logP of the cocrystal as 2.01 and the Molinspiration predicted it to be 2.3.
The logP value for ampicillin alone according to Pubchem database is 1.35 which is much lower than the AC21. Ampicillin logP is 1.35 and is less when compared to 2.08 in cocrystal AC21 indicating an increased permeation of ampicillin in presence of celecoxib, and also higher in the cocrystal form than as a physical mixture.
The simplified molecular-input line-entry system (SMILES) is a specification in the form of a line notation for describing the structure of chemical species using short ASCII strings. (Ref 5).
Example 6: Experiment to measure increased dose response of the co-crystal form
Preparation of co-crystal with half MIC (Minimum inhibitory concentration) dose of antibiotic + celecoxib in 2:1 ratio
Cocrystal development of ampicillin and celecoxib: Ampicillin and celecoxib cocrystals were prepared as described in Example 1, mainly by co-grinding followed by solvent (methanol) evaporation at room temperature as described earlier (Ref 3). Briefly, accurately weighed ampicillin and celecoxib in different molar ratios (1:1, 1:2, 1:3, 1:4, 2:1, 3:1 and 4:1) were co-ground together with a mortar and pestle for 30 min followed by dissolving the known amount of co-ground mixture 10 in methanol. The solvent was slowly evaporated at room temperature and cocrystals, formed in 5 days, were characterized by powder-X-Ray diffraction (PXRD) and differential scanning calorimetry (DSC) for stability. They were named AC01 (only celecoxib), AC10 (only ampicillin), AC11, AC12, AC13, AC14, AC21, AC31 and AC41.
Fig. 1B shows concentration-dependent dose efficacy of AC21 cocrystal
S. aureus was taken from glycerol stock and grown overnight in tryptic soy broth (TSB) medium at 37°C, with shaking at 180 rpm. For dose efficacy studies, 1% of overnight culture was inoculated into 50 mL of fresh TSB medium, with or without supplementation of different concentrations of AC21 Cocrystals from 0.122 ug/ml to 125 ug/ml concentration, and incubated at 37°C , with shaking at 180 rpm for 5h. Cell growth was estimated by plating the serially diluted culture on agar plates and counting the CFU after incubation of plates overnight. It shows clearly that the co-crystal decreases cfu to a larger extent than the physical mixture of antibiotic and celecoxib.
,CLAIMS:We Claim:
1. A co-crystal comprising at least one antibiotic and at least one NSAID.
2. The co-crystal of claim 1, wherein the NSAID has at least one functional group selected from the group consisting of alcohol, thiol, ester, carboxylic acid, primary amine, secondary amine, tertiary amine
3. The co-crystal of claim 1, wherein when compared to the antibiotic alone, or to a physical mixture of the antibiotic and the NSAID, the co-crystal exhibits at least one of the following:
a. increased efficacy
b. increased dose response
c. increased bioavailability
d. increased solubility
4. The co-crystal of claim 1, wherein the antibiotic is selected from the group consisting of Ampicillin, Kanamycin, Chloremphenicol, Ciprofloxacillin, and Oxacillin.
5. The co-crystal of claim 1, wherein the NSAID is celecoxib or meloxicam, their enantiomers or salts thereof.
6. The co-crystal of claim 1, wherein the antibiotic is effective at inhibiting bacterial growth at less than the minimum inhibitory concentration of the antibiotic.
7. The co-crystal of claim 6, wherein the antibiotic is effective at inhibiting bacterial growth at half the minimum inhibitory concentration of the antibiotic.
8. The co-crystal of claim 1, wherein it increases the permeability of the bacterial membranes to a larger extent, as compared to antibiotic alone, or to a physical mixture of the antibiotic and the NSAID.
9. The co-crystal of claim 1, wherein the uptake of antibiotic by the bacterial cells is greater for the co-crystal, as compared to antibiotic alone, or to a physical mixture of the antibiotic and the NSAID.

10. The co-crystal of claim 8, wherein the bacteria are antibiotic -resistant bacteria.
11. The co-crystal of claim 6, wherein the celecoxib concentration is 10 µM.
12. A pharmaceutical composition comprising the co-crystal of claim 1, wherein the co-crystal is present at a therapeutically effective amount of the co-crystal according to any of claims 1 to 13 in a physiologically acceptable medium.
13. A co-crystal according to claim 1, wherein the molar ratio between the antibiotic and the NSAID is 2:1
14. A method of making the co-crystal, the method comprising the steps of:
a. weighing desired amounts of antibiotic and NSAID so as to achieve molar ratio of 1:1, 1:2, 1:3 1:4, 2:1, 3:1 or 4:1 between antibiotic and NSAID;
b. grinding the antibiotic and NSAID from step (a) together;
c. dissolving the ground mixture of antibiotic and NSAID from step (b) in a suitable organic solvent; and
d. Slow Evaporating the solvent from step (c) to form the co-crystals over a period of several days.
15. The method of claim 14, wherein the organic solvent in step (c) is methanol.
16. The co-crystals formed by the method of claim 14.
17. The method of claim 14, wherein the molar ratio between the antibiotic and the NSAID is 2:1.
18. The method of claim 14, wherein the NSAID is celecoxib or meloxicam, their enantiomers or salts thereof.