Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/65—Tetracyclines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4196—1,2,4-Triazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics

Definitions

• the present application relates to the field of antifungals, particularly antifungals against Candida species. It was found that antimicrobials from the tetracycline family potentiate the antifungal effect of fluconazole against Candida. Thus, these combinations are provided as medicament, particularly against Candida infections.
• Regulatable promoters are available to the research community, to induce or repress gene expression in C. albicans. Most of them, such as the PCK1 and the MAL2 promoters, allow conditional expression/repression of the gene of interest upon incubation of cells in growth media containing the repressing or the activating carbon source (3, 28).
• the tetracycline direct and reverse systems which allow gene induction or repression by addition of doxycycline to the growth medium, have also been successfully used to regulate gene expression in C. albicans (38, 45).
• doxycycline and to a lower extent tetracycline, two licensed antimicrobials that prevent bacterial protein synthesis, potentiate the antifungal activity of fluconazole against Candida albicans in a dosage-dependent manner.
• Doxycycline was found to act synergistically with the antifungal fluconazole against Candida albicans. Combination with doxycycline converts fluconazole from fungistatic to fungicidal, prevents the onset of drug resistance, and is also effective against a clinical isolate characterized by elevated resistance to fluconazole.
• Doxycycline-mediated growth inhibition can be reversed by externally added iron, indicating that iron depletion may account for the synergism. Consistently, we confirmed old literature data about iron-chelating activity of doxycycline. Synergism of fluconazole with doxycycline does not appear to be mediated by calcineurin, since doxycycline further aggravates the susceptibility to fluconazole of mutants lacking the catalytic or the regulatory subunits of calcineurin. Growth in the presence of fluconazole and doxycycline is restored by elevated dosage of ERG11 in Saccharomyces cerevisiae but not in C.
• doxycycline appears to have a major impact on prevention of fluconazole tolerance, defined as incomplete growth inhibition at supra-MIC concentrations of fluconazole. This finding is consistent with the role of doxycycline in converting fluconazole to a fungicidal drug, and may also have implications in the prevention of drug resistance and the combination of doxycycline and fluconazole can be used in treatment of candidiasis. The fact that we never recovered spontaneous mutants resistant to the combination of fluconazole and doxycycline indicates that multiple genetic alterations may be necessary for such resistance to occur, and makes this treatment especially attractive.
• a combination of a tetracycline antibiotic and fluconazole is provided. It is particularly envisaged that the tetracycline antibiotic is doxycycline.
• the combination can be provided as one composition, or as a kit of parts.
• the combination of the tetracycline and fluconazole is provided for use as a medicament.
• the combination is provided for use in treatment of Candida infections.
• this is equivalent to providing the use of a combination of a tetracycline and fluconazole for the manufacture of a medicament for treatment of Candida infections.
• methods of treatment of Candida infections are provided for subjects in need thereof, comprising:
• said Candida is Candida albicans. Even more particularly, the Candida albicans is a fluconazole resistant isolate of Candida albicans. BRIEF DESCRIPTION OF THE FIGURES
• Fig.1 Doxycycline synergizes with fluconazole in a dosage-dependent and medium- and carbon source-independent manner. Doxycycline synergizes with fluconazole in a dosage-dependent and medium- and carbon source-independent manner.
• Cells of the C. albicans reference strain SC5314 were spotted onto plates containing different drug combinations. Where indicated, fluconazole (Flu) is present at 10 ⁇ g ml, whereas the concentration of doxycycline (Dox) is expressed in ⁇ g ml.
• Doxycycline converts fluconazole from fungistatic to fungicidal and prevents the onset of drug resistance.
• A viable cell counts after 48 h or 96 h of incubation in the presence of fluconazole alone, or with 50 or 100 mg/ml doxycycline added. Open symbols: viable cell counts after 48 h of incubation. Filled symbols: viable cell counts after 96 h of incubation. Circles: fluconazole alone; squares: fluconazole + 50 mg/ml doxycycline; triangles: fluconazole + 100 mg/ml doxycycline. Values falling below the detection limit of 10 1 CFU/ml were approximated to 0.5 x 10 1 CFU/ml. Mean values from three independent experiments are shown.
• B emergence of fluconazole resistant strains on plates containing 128 mg/ml fluconazole, without (top) or with (bottom) 50 mg/ml doxycycline. Plates were incubated 4 days before being scanned.
• the fluconazole+doxycycline combination is also effective on a clinical isolate of C. albicans characterized by elevated resistance to fluconazole.
• A Cells of the C. albicans reference strain SC5314, and of the clinical isolate FH5 were spotted on SD plates containing doxycycline, without (mid panel) or with fluconazole (right panel). Concentrations are expressed in mg/ml. Plates were incubated 2 days before being scanned.
• B E-test measurement of fluconazole MIC of strain FH5 on RPMI-glucose plates. Left: medium without doxycycline; center: medium with 50 mg/ml doxycycline; right: medium with 200 mg/ml doxycycline.
• A Transformants of the S. cerevisiae reference strain BY4742 with plasmid pAFC88 (ERG11) or with an empty plasmid (control) were spotted on SD plates containing combinations of fluconazole and doxycycline, at the indicated concentrations (mg/ml). Plates were incubated 4 days before being scanned.
• B Top panel: cells of the C. albicans strains AFA60 (control) and AFA59b (ACT1p-CaERG11) were spotted on SD plates containing the indicated concentrations of fluconazole+doxycycline.
• cerevisiae reference strain BY4742 with plasmid pAFC99a (GAL1, 10p-CaERG11) or with an empty plasmid (control) were spotted on SGal plates containing combinations of fluconazole and doxycycline, at the indicated concentrations (mg/ml). Plates were incubated 5 days before being scanned
• Fig.6 Doxycycline synergizes with fluconazole via iron sequestration.
• A cells of C. albicans SC5314 were spotted on SD plates containing combinations of fluconazole and doxycycline at standard concentrations, with and without ferric citrate or ferric chloride added at the indicated concentrations. Plates were incubated 2 days before being scanned.
• B cells of S. cerevisiae BY4742 were similarly spotted on SD plates. Plate scans were taken after 2 days.
• C Colorimetric assessment of iron chelating activity of doxycycline, tetracycline and fluconazole. Iron chelation is expressed as a reduction of absorbance at 630 nm. Filled circles: doxycycline, open circles: tetracycline, filled squares: fluconazole, open squares: water.
• D cells of C. albicans SC5314 were spotted on SD plates containing combinations of fluconazole (10 mg/ml) and BPS (20 mg/ml). Plates were incubated 4 days before being scanned.
• tetracycline antibiotic or "a tetracycline” as used herein refers to the broad- spectrum polyketide antibiotic produced by the Streptomyces genus of Actinobacteria, indicated for use against many bacterial infections, or semisynthetic derivatives or related substances with antimicrobial activity, characterized by containing the same four-ring system. They can also be indicated as 'a subclass of polyketides having an octahydrotetracene-2- carboxamide skeleton' or as 'derivatives of polycyclic naphthacene carboxamide'.
• tetracyclines include, but are not limited to: tetracycline, chlortetracycline, oxytetracycline, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, tigecycline, penimepicycline and pipacycline.
• the tetracycline antibiotic is doxycycline.
• the combination of the tetracycline and fluconazole is provided for use as a medicament.
• the combination is provided for use in treatment of Candida infections.
• Candida is a genus of yeasts, and common Candida species that may cause an infection include, but are not limited to Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Candida parapsilosis.
• said Candida is Candida albicans. Even more particularly, the Candida albicans is a fluconazole resistant isolate of Candida albicans.
• the combination can be provided as one composition, or as a kit of parts.
• compositions containing a combination of a tetracycline antibiotic and fluconazole can be utilized to achieve the desired pharmacological effect by administration to a patient (or subject) in need thereof.
• a patient for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease.
• the patient (or subject) is immunocompromised.
• the present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a tetracycline antibiotic (e.g. doxycycline) and fluconazole, or salt thereof.
• a pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient.
• a pharmaceutically effective amount of the combination is preferably that amount which produces a result or exerts an influence on the particular condition being treated, e.g. one which reduces Candida growth or reproduction.
• compositions of the present invention can be administered with pharmaceutically-acceptable carriers well known in the art using any effective conventional dosage unit forms, including immediate, slow and timed release preparations, orally, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.
• the compositions are foreseen for topical administration (as opposed to systemic administration). Nevertheless, according to alternative embodiments, the compositions are provided for systemic administration.
• compositions can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions.
• the solid unit dosage forms can be a capsule that can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.
• compositions of this invention may be tableted with conventional tablet bases such as lactose, sucrose and cornstarch in combination with binders such as acacia, corn starch or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, gum tragacanth, acacia, lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example talc, stearic acid, or magnesium, calcium or zinc stearate, dyes, coloring agents, and flavoring agents such as peppermint, oil of wintergreen, or cherry flavoring, intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient.
• binders such as acacia, corn starch or gelatin
• disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn star
• Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent or emulsifying agent.
• Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance tablets, pills or capsules may be coated with shellac, sugar or both.
• Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient (i.e. the tetracycline, the fluconazole or both) in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example those sweetening, flavoring and coloring agents described above, may also be present.
• the pharmaceutical compositions of this invention may also be in the form of oil-in-water emulsions.
• the oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils.
• Suitable emulsifying agents may be (1 ) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
• the emulsions may also contain sweetening and flavoring agents.
• Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil such as, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
• the oily suspensions may contain a thickening agent such as, for example, beeswax, hard paraffin, or cetyl alcohol.
• the suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.
• Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, and preservative, such as methyl and propyl parabens and flavoring and coloring agents.
• sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose.
• Such formulations may also contain a demulcent, and preservative, such as methyl and propyl parabens and flavoring and coloring agents.
• compositions of this invention may also be administered parenterally, that is, subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly, or intraperitoneal ⁇ , as injectable dosages of the composition or combination in preferably a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1 ,1 -dioxolane-4- methanol, ethers such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or, a fatty acid glyceride, or an acetylated fatty acid glyceride, with or without the addition of a pharmaceutical
• Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid.
• Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate.
• Suitable soaps include fatty acid alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; non-ionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and poly(oxyethylene- oxypropylene)s or ethylene oxide or propylene oxide copolymers; and amphoteric detergents, for example, alkyl-beta- aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, as well as mixtures.
• suitable detergents include cationic detergents, for example di
• compositions of this invention will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) preferably of from about 12 to about 17. The quantity of surfactant in such formulation preferably ranges from about 5% to about 15% by weight.
• the surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB.
• surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
• compositions may be in the form of sterile injectable aqueous suspensions.
• suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadeca-ethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from a
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
• Diluents and solvents that may be employed are, for example, water, Ringer's solution, isotonic sodium chloride solutions and isotonic glucose solutions.
• sterile fixed oils are conventionally employed as solvents or suspending media.
• any bland, fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid can be used in the preparation of injectables.
• composition of the invention may also be administered in the form of suppositories for rectal administration of the drug.
• These compositions can be prepared by mixing the drug with a suitable non-irritation excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• suitable non-irritation excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• suitable non-irritation excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• Such materials are, for example, cocoa butter and polyethylene glycol.
• transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the compositions of the present invention in controlled amounts.
• the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (see for example US 5,023,252). Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
• Controlled release formulations for parenteral administration include liposomal, polymeric microsphere and polymeric gel formulations that are known in the art. It may be desirable or necessary to introduce the pharmaceutical composition to the patient via a mechanical delivery device.
• the construction and use of mechanical delivery devices for the delivery of pharmaceutical agents is well known in the art.
• One such implantable delivery system, used for the transport of agents to specific anatomical regions of the body, is described in US 5,01 1 ,472.
• compositions of the invention can also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired.
• Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al., "Compendium of Excipients for Parenteral Formulations” PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-31 1 ; Strickley, R.G “Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1 " PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349 ; and Nema, S. et al., "Excipients and Their Use in Injectable Products” PDA Journal of Pharmaceutical Science & Technology 1997, 51 (4), 166-171.
• the effective dosage of the compositions of this invention can readily be determined for treatment of each desired indication.
• the amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular respective amounts of the combination, the tetracycline used, the dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.
• the total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 50 mg/kg body weight per day.
• Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing.
• "drug holidays" in which a patient is not dosed with a drug for a certain period of time may be beneficial to the overall balance between pharmacological effect and tolerability.
• a unit dosage may contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day.
• the average daily dosage for administration by injection will preferably be from 0.01 to 200 mg/kg of total body weight.
• the average daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
• the average daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
• the average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily.
• the transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg.
• the average daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight.
• compositions for inhalation are presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
• the particles of active combination suitably have diameters of less than 50 microns, preferably less than 10 microns, for example between 1 and 5 microns, such as between 2 and 5 microns.
• coated nanoparticles can be used, with a particle size between 30 and 500 nm.
• a favored inhaled dose will be in the range of 0.05 to 2 mg, for example 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2 mg.
• the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific combination employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like.
• the desired mode of treatment and number of doses of the combination of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests.
• compositions will usually be accompanied by written or printed directions for use in the medical treatment concerned.
• the tetracycline and fluconazole can be provided as one composition, or as different compositions which are intended for combination therapy (i.e. for simultaneous, concomitant or immediate subsequent administration). In the latter case, they will typically (although not exclusively be offered as kits.
• Kits typically will comprise at least a combination of a tetracycline antibiotic and fluconazole as described herein and at least a suitable buffer.
• the kits may contain any of the pharmaceutical compositions or agrochemical compositions described herein.
• the kit may additionally contain instructions for use.
• the kits will optionally contain devices for administration purposes (e.g. a syringe, a container with spraying nozzle).
• the use is provided of a combination of a tetracycline and fluconazole for the manufacture of a medicament for treatment of Candida infections.
• methods of treatment of Candida infections are provided for subjects in need thereof, comprising:
• the administration of the tetracycline (e.g. doxycycline) and fluconazole will result in treatment of the Candida infection.
• This can be by stopping or reducing growth of the Candida, by stopping or reducing reproduction of the Candida, by stopping or reducing biofilm formation by the Candida, and/or by killing the Candida.
• Administering the combination of a tetracycline and fluconazole may be simultaneous administration, concomitant administration, subsequent administration, but it is envisaged that both substances will be simultaneously present and active in the subject for at least an amount of time necessary to result in an effect on the Candida species.
• Minimal medium was supplemented with uridine when ura3D/ura3D strains of C. albicans were used.
• RPMI 1640 medium with L- glutamine without sodium bicarbonate (Sigma) was buffered with 0.165 M morpholinepropanesulfonic acid (MOPS).
• MOPS morpholinepropanesulfonic acid
• Glucose at the final concentration of 2% was added only to RPMI-agar medium.
• FBS medium consisted of 10% fetal bovine serum in sterile milliQ water (Millipore). Media were solidified with 2% agar.
• Sabouraud dextrose agar medium was bought from Fluka.
• Minimal medium for yeast experiments was supplemented with histidine, lysine, leucine and uracil at appropriate concentrations (59).
• Leucine and uracyl were omitted from media for experiments shown in Fig. 5A and 5D, respectively. All plate tests for C. albicans were carried out at 37°C; plate tests for S. cerevisiae were carried out at 30°C. The standard concentration of fluconazole on plates was 10 ⁇ g ml, whereas that of doxycycline was 50 ⁇ g ml. Indications are given in the text for experiments conducted with different drug concentrations.
• doxycycline which was dissolved in 50% ethanol, all above-mentioned chemicals were dissolved in milliQ water.
• RPS10 :pPCK1-GFP URA3-PCK1 vector
• ura3A0 MIC testing in broth microdilution assays Fluconazole MIC was determined according to the approved CLSI standard reference method for broth dilution antifungal susceptibility testing of yeasts M27-A3 (10).
• Inocula of C. albicans SC5314 and FH5 were prepared from cultures grown overnight on Sabouraud medium, adjusted to obtain final cell suspensions of 0.5 x 10 3 to 2.5 x 10 3 CFU/ml. Viable counts of the inocula were verified by plating serial dilutions on YPD plates. MIC plates were incubated at 35°C for 48 h for the reference strain SC5314, and for 24 h for strain FH5.
• MIC endpoints were defined as the lowest concentration of fluconazole causing a > 50% decrease in optical density (prominent decrease in turbidity, or score 2, according to CLSI guidelines), and a > 90% decrease in viability as compared to the drug-free control.
• Optical densities were recorded at 540 nm using a Molecular Devices SpectraMax Plus384 Absorbance Microplate Reader. Viable counts were measured plating 100 ml of 10 "1 to 10 "5 serial dilutions on YPD plates. For viable count checks of optically clear wells, 100 ml of undiluted wells content were also plated. Where appropriate, comparison of results obtained from plating 10 "1 /10 "2 dilutions and from 100 ml of undiluted wells content did not evidence a significant influence of drugs carry over in our experimental conditions. Experiments were repeated for a minimum of three times.
• the MIC-0 for fluconazole and doxycycline which resulted > 32 ⁇ g/ml and > 200 ⁇ g/ml, respectively, were considered to be 64 and 400 ⁇ g/ml. Experiments were repeated for a minimum of three times.
• Iron chelation assay Iron chelation was assayed using the colorimetric SideroTec Assay kit from Emergen Bio (Maynooth, Ireland), following instructions from the manufacturer. Briefly, fluconazole, tetracycline and doxycycline at the concentrations of 25, 50, 100, 200 and 400 ⁇ g ml were mixed with the provided reagents in flat-bottom 96 well plates, and incubated at room temperature for 15 minutes. Plates were read at 630 nm on the above-mentioned plate reader. Decreases in absorbance are inversely proportional to iron chelation activity. milliQ water was substituted for the chemicals in the negative control.
• Genomic screen for multicopy suppressors of susceptibility to fluconazole + doxycycline We transformed the S. cerevisiae genomic library in vector pFL44 constructed in the lab of F. Lacroute (60) in the yeast strain BY4742 by the lithium acetate procedure. One aliquot of each transformation was plated on SD medium containing histidine, leucine and lysine for estimation of the transformation efficiency. The remaining transformation mixture was outgrown 4 hours in liquid YPD before being spread on plates same as above containing 10 ⁇ g ml fluconazole and 50 ⁇ g ml doxycycline.
• True resistant transformants were separated from false positives by further re-streaking them in selective conditions, and by co-segregation analysis of resistance to fluconazole+doxycycline with the presence of a transforming plasmid.
• interesting plasmids were extracted from yeast, transformed in Escherichia coli and retransformed into BY4742 for confirmation of their suppressor activity.
• Genomic inserts of suppressor plasmids were sequenced using oligonucleotides M13F (5'- GTAAAACGACGGCCAG-3') (SEQ ID NO:1 ) and M13R (5'-CAG GAAACAG CTATGAC-3 ' ) (SEQ ID NO:2), identifying a -5.4 kb fragment containing incomplete SOD2, YHR007, ERG11 and incomplete STP2. Reintroduction of pAFC88 (see further) into BY4742 and subsequent suppression of the susceptibility to fluconazole+doxycycline of the resulting transformants confirmed ERG11 as the suppressor gene.
• Plasmids used in this study A Hindlll-Smal fragment, containing ERG11 surrounded by -730 nucleotides at its 5' and -670 nucleotides at its 3', was excised from the Lacroute library suppressor plasmid, and cloned into Hindlll-Smal YEplac181 (17), creating plasmid pAFC88.
• Hindlll-Smal YEplac181 Hindlll-Smal YEplac181
• CaERG11 was amplified from genomic DNA of strain SC5314 using primers CaERGH s (5'-CCCAAGCTTATGGCTATTGTTGAAACTGTCA- 3') (SEQ ID NO:3) and CaERG1 1 as (5'-GCGGCTAGCTGAATCGAAAGAAAGTTGCCG-3') (SEQ ID NO:4).
• the amplified fragment was subcloned Hindlll-Nhel, replacing the Luciferase gene, in plasmid Cip10::ACT1 p-gLUC59 (15), and in plasmid pPCK1 -GFP (5), replacing GFP.
• the resulting plasmids are named pAFC89b ⁇ ACT1p-CaERG11) and pAFC92a ⁇ PCK1p- CaERG11). Cloned CaERG11 was sequenced to verify the absence of mutations.
• CaERG11 was extracted Hindlll-Nhel from pAFC89b, blunted with the Klenow enzyme, and subcloned into Smal pBEVY-GL (35), creating plasmid pAFC99a.
• Quantitative real-time PCR Cells of the AFA59b, AFA60a and AFA63a strains were grown to mid-log phase in minimal glucose medium before their RNA was extracted. For AFA63a, cells were collected, split, and each half was resuspended in either SD or SCAA media, and outgrown for 4 hours before RNA extraction. Complementary DNA was prepared from DNase- treated RNA samples with the Reverse Transcription kit A3500 (Promega). Quantitative PCR was performed on a StepOnePlus realtime PCR system (Applied Biosystems) using the Kapa SYBR Fast kit (Kapabiosystems). The fold regulation of each target gene was calculated using the DDCi method, using expression of the TEF1 to normalize data.
• the Ct value of TEF1 was subtracted from that of CaERG11 to calculate the DCf.
• the DCf value of AFA60a was used as a reference, and was therefore subtracted from the DCf value of the other samples, to obtain DDCi values.
• Gene expression levels relative to the reference were expressed as 2 "DDCf .
• Primers used are: CaERG1 1 up (5'-TTACCTCATTATTGGAGACGTGATG-3') (SEQ ID NO:5), CaERG1 1 down (5'-CACGTTCTCTTCTCAGTTTAATTTCTTTC-3') (SEQ ID NO:6), TEF1 a-fw (5'-CCACTGAAGTCAAGTCCGTTGA-3') (SEQ ID NO:7), and TEF1 a-rv (5'- CACCTTCAGCCAATTGTTCGT-3') (SEQ ID NO:8).
• Example 1 Doxycycline acts synergistically with fluconazole in a dosage-dependent manner.
• Example 2 Tetracycline acts synergistically with fluconazole with minor efficiency.
• Tetracyclines are antibacterial drugs that prevent the association of acylated tRNAs with the ribosome, therefore inhibiting translation (9). Since doxycycline is a derivative of tetracycline with improved pharmacokinetic properties, we decided to test whether amelioration of the antifungal properties of fluconazole was an exclusive feature of doxycycline or whether it could be extended to tetracycline.
• Tetracycline proved to be also synergistic with fluconazole, albeit with reduced potency (Fig. 1 C).
• an inhibitory effect of fluconazole plus tetracycline could be observed against C. albicans, this was less robust than that observed with doxycycline, given that a higher concentration of tetracycline was required to prevent fungal growth.
• the 200- ⁇ g ml tetracycline concentration that appeared to completely prevent growth of C. albicans on solid medium was considerably less efficient in liquid medium (our unpublished observations).
• Gentamicin and neomycin two unrelated antibiotics that also prevent bacterial translation, showed no effect in combination with fluconazole against C. albicans (Fig. 1 C).
• Example 3 Doxycycline eliminates fluconazole tolerance.
• CLSI standard reference method M27-A3 for broth dilution antifungal susceptibility testing of yeasts (10)
• the MIC for fluconazole of standard wild-type strain SC5314 was 0.5 ⁇ g ml
• the MIC of doxycycline alone was >200 ⁇ g ml.
• Doxycycline reduced the MIC for fluconazole 1 -fold (0.25 ⁇ g ml) when added at 200 ⁇ g ml (not shown) and led to no detectable changes at lower concentrations (described further below).
• calculation of the fractional inhibitory concentration index produced values of >0.5 for concentrations of doxycycline that were ⁇ 200 ⁇ g ml, failing to indicate synergism with fluconazole (22, 41 ).
• FIG. 2A shows robust recovery of cells incubated for 48 h with fluconazole alone at all concentrations tested (bottom row of cells) and decreases in viability in the presence of doxycycline. Starting from inocula of 10 3 CFU/ml, viable counts indicated increases of 3 and 2 logs, respectively, for cells incubated in the absence or presence of 0.5 ⁇ g ml fluconazole (Fig. 2B).
• doxycycline further reduced growth in the presence of fluconazole by 1 log, and resulted in optically clear wells (Fig. 2B).
• Addition of 25 ⁇ g ml doxycycline stably reduced the viable counts by 1 log and was associated with a strong potentiation of the fungistatic power of fluconazole (Fig. 2B).
• Higher concentrations of doxycycline caused further reductions in viable counts at supra-MICs of fluconazole, to levels just above the detection limit of 10 1 CFU/ml (50 ⁇ g/ml doxycycline) (Fig. 2B) or below that (100 pg/ml doxycycline) (Fig. 2B).
• Fluconazole MIC testing using Etest strips has been reported to deliver results comparable to those obtained using the broth microdilution method, within a 2-dilution margin of discrepancy (48). Consistent with the results reported above for broth microdilution MIC determinations, 50 ⁇ g ml doxycycline did not affect the fluconazole MIC of strain SC5314 in Etest measurements (Fig. 2C). However, the robust growth of smaller colonies within the fluconazole inhibition ellipse that is normally observed in these assays (48) was completely eliminated in the presence of the antibacterial (Fig. 2C).
• Example 5 Doxycycline prevents the onset of resistance to fluconazole.
• Candida albicans resistance to fluconazole has been documented to arise via different mechanisms, including mutations in ERG1 • /(encoding the drug target protein lanosterol 14a- demethylase) or increased expression of multidrug transporters and efflux pump-encoding genes such as CDR1, CDR2, and MDR1 (reviewed in references 1 , 36, and 58).
• ERG1 • /(encoding the drug target protein lanosterol 14a- demethylase) or increased expression of multidrug transporters and efflux pump-encoding genes such as CDR1, CDR2, and MDR1 (reviewed in references 1 , 36, and 58).
• CDR1, CDR2, and MDR1 multidrug transporters and efflux pump-encoding genes
• the fluconazole-doxycycline combination is instrumental in the prevention of cellular resistance to fluconazole, a result that is likely to arise from the conversion of fluconazole to a fungicidal drug.
• cells of C. albicans may be killed before resistance-conferring mutations can be fixed by cell proliferation.
• Example 6 The fluconazole + doxycycline combination is also effective on a clinical isolate of Candida albicans highly resistant to fluconazole.
• Example 7 Genome-wide screen for suppressors of fluconazole+doxycycline susceptibility in Saccharomyces cerevisiae reveals suppression by increased dosage of ERG11.
• Saccharomyces cerevisiae and Candida albicans are estimated to have diverged ⁇ 800 million years ago (21 ).
• Baker's yeast offers a number of tools for genetic studies that cannot be performed with C. albicans, because of the diploid nature of the latter and the absence of a known sexual cycle. For this reason, a number of studies of pathogenic fungi, including susceptibility/resistance to azole antifungals, have been conducted using S. cerevisiae as a tool, with the aim of translating the obtained results to pathogenic species (36, 58).
• S. cerevisiae susceptibility/resistance to azole antifungals
• BY4742 was transformed with a yeast genomic library in a 2 ⁇ plasmid (60), and transformations were plated on minimal medium containing fluconazole plus doxycycline. Putative suppressors were separated from false positives by restreaking them on the same medium and by allowing them to lose the transforming plasmids upon growth under nonselective conditions. Strains for which uracil prototrophy cosegregated with resistance to fluconazole plus doxycycline were analyzed further: their plasmids were extracted, amplified in E. coli, and retransformed into yeast to confirm plasmid-linked suppression.
• Example 8 Overexpression of CaERG11 is not sufficient for resistance to fluconazole and doxycycline in C. albicans.
• yeast cells overexpressing CaERG 11 were less susceptible to fluconazole and fluconazole plus doxycycline than control cells, demonstrating that increased dosage of CaErgl 1 can suppress fluconazole susceptibility in yeast but not in C. albicans, and, as a consequence, that the suppression activity is retained by t eCaERGH gene.
• ERG11 in the two yeast species underline the difference existing between them in terms of their responses to the antifungal fluconazole.
• Example 9 Doxycycline potentiates the action of fluconazole via iron chelation.
• Iron-chelating activity of tetracyclines was confirmed using a colorimetric assay. We could demonstrate that both tetracycline and doxycycline chelate iron, with doxycycline showing higher affinity than tetracycline (Fig. 6C), whereas fluconazole showed no intrinsic iron-binding activity. Notably, the stronger iron-chelating activity of doxycycline correlated with its stronger synergism with fluconazole.
• Example 10 Synergism between fluconazole and doxycycline is not mediated by Calcineurin.
• CsA and FK506 were reported to be synergistic with fluconazole against Candida albicans (31 , 33), converting fluconazole to a fungicidal drug and eliminating trailing growth (33).
• the synergistic action of fluconazole with CsA and FK506 is mediated by inhibition of the phosphatase activity of calcineurin by the immunosuppressants (13, 55).
• Calcineurin is inactive under normal conditions, being activated only in the presence of certain external cues (52).
• a strain of C. albicans in which calcineurin was permanently activated by removal of the self-inhibitory C-terminal domain of Cna1 was less susceptible to fluconazole (55). While we confirmed a moderately increased tolerance to fluconazole in this strain (our unpublished observations), we also found this strain to be as susceptible to the fluconazole- doxycycline combination as an isogenic control strain carrying a reintegrated copy of wild- type CNA 1 (Fig. 7), demonstrating that constitutively active calcineurin is incapable of rescuing the cells' susceptibility to fluconazole plus doxycycline. Taken together, these observations suggest that the synergism of doxycycline with fluconazole is not mediated by inhibition of calcineurin activity.
• doxycycline and to a lesser extent tetracycline, two licensed antimicrobials that prevent bacterial protein synthesis, potentiate the antifungal activity of fluconazole against Candida albicans in a dosage-dependent manner.
• Doxycycline converts the action of fluconazole from fungistatic to fungicidal and prevents the onset of drug resistance.
• Addition of doxycycline appears to have a major impact on prevention of fluconazole tolerance, defined as incomplete growth inhibition at supra-MICs of fluconazole. This finding is consistent with the role of doxycycline in converting fluconazole to a fungicidal drug and may also have implications in the prevention of drug resistance.
• Iron depletion by doxycycline may result in lower incorporation of heme in the Erg1 1 protein in yeast, and an increase in the copy number of Erg1 1 may help the protein compete for heme.
• iron deprivation has been demonstrated to result in downregulation of ERG1 1 in C. albicans (25, 50)
• ERG1 1 overexpression may simply restore gene expression to wild-type levels.
• the situation appears to be more complex in C. albicans.
• iron depletion as the major cause of growth inhibition has been demonstrated by reversal upon addition of ferric iron to the medium.
• Iron depletion has been proposed to decrease ergosterol content in C. albicans, leading to higher fluidity in cell membranes, with consequent increased passive diffusion of fluconazole (50).
• Some of the results reported in this paper appear similar to those obtained using BPS as the iron chelator: addition of 200 ⁇ BPS lowered the MIC of C. albicans for fluconazole (50), and so did that for 200 ⁇ g ml doxycycline.
• the iron chelation properties of the two chemicals at the indicated concentrations are similar (our unpublished observations). Minor differences could be due to different experimental conditions.
• Candida albicans a molecular revolution built on lessons from budding yeast. Nat. Rev. Genet. 3:918-930.
• Dapl p a heme-binding protein that regulates the cytochrome P450 protein Erg1 1 p/Cyp51 p in Saccharomyces cerevisiae. Mol. Cell. Biol.

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