WO1993008806A1 - Procede de diminution de la resistance a la tetracycline dans des cellules vivantes - Google Patents
Procede de diminution de la resistance a la tetracycline dans des cellules vivantes Download PDFInfo
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- WO1993008806A1 WO1993008806A1 PCT/US1992/008965 US9208965W WO9308806A1 WO 1993008806 A1 WO1993008806 A1 WO 1993008806A1 US 9208965 W US9208965 W US 9208965W WO 9308806 A1 WO9308806 A1 WO 9308806A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
- C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
- C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/24—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a ring other than a six-membered aromatic ring of the carbon skeleton
- C07C237/26—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a ring other than a six-membered aromatic ring of the carbon skeleton of a ring being part of a condensed ring system formed by at least four rings, e.g. tetracycline
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
- C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/29—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
- C07C2601/08—Systems containing only non-condensed rings with a five-membered ring the ring being saturated
Definitions
- the research for the present invention was supported by funds obtained through Tufts University.
- the present invention concerns therapeutic tetracycline treatment of living cells, and is particularly directed to methods and materials for altering and overcoming resistance to tetracycline within microorganisms such as bacteria, fungi, rickettsia, and the like.
- microorganisms capable of producing bacteriocidal and/or bacteriostatic compositions.
- the first of these novel compounds was introduced in 1948 under the name chlortetracycline. Two years later oxytetracycline became available. The detailed elucidation of the chemical structure of these agents confirmed their similarity and furnished the analytical basis for the production of a third member of this group in 1952,
- compositions characterized chemically by the absence of the ring-attached CH 3 group present in the earlier compositions was prepared and became publicly available in 1959 under the official name demeclocycline. Subsequently, methacycline, a derivative of oxytetracycline, was introduced in 1966;
- tetracyclines became known as "broad spectrum" antibiotics. With the subsequent establishment of their in-vitro antimicrobial activity, effectiveness in experimental infections, and pharmacological properties, the tetracyclines as a class rapidly became widely used for therapeutic purposes. However, this widespread use of tetracyclines for both major and minor illnesses and diseases led directly to the emergence of resistance to these antibiotics even among highly susceptible bacterial species both commensal and pathogenic - as for example pneumococci and Salmonella.
- the principal site of action for tetracyclines is the bacterial ribosome; at least two different processes appear to be required for tetracyclines to gain access to the cytoplasm and the ribos ⁇ mes of bacteria.
- the first process is a passive diffusion of the tetracycline through hydrcphilic pores located in the outer cell membrane. One of these structures is the major outer membrane protein, Omp F in E. coli.
- the second process involves an energy-dependent active transport system that pumps all tetracyclines through the inner cytoplasmic membrane into the cytoplasm of the cell.
- TET inner membrane protein
- Tetracycline resistance is often regulated - that is, inducible by tetracycline. Investigations of active
- tetracycline resistance for cells is known and in effect.
- This resistance mechanism involves a cytoplasmic protein which protects the intracellular ribos ⁇ mes from the inhibitory action of tetracyclines.
- This form of tetracycline resistance is described within Burdett, V., J. Bact. 165:564-569 (1986); and Levy, S.B., J. Antimicrob.
- the present invention provides methods and compositions for therapeutically treating a tetracycline-resistant cell and also provides a method for altering a cell from a tetracycline- resistant state into a tetracycline-sensitive state.
- this method comprises the steps of:
- the cell is allowed to preferentially react with the blocking agent.
- the unique methodology is able to alter and to convert tetracycline-resistant cells or microorganisms into
- compositions which may be aditdissered alone against, for example, a sensitive or resistant strain of gram positive bacteria such as S. aureus and E. faecalis.
- the present invention represents a unique methodology by which to overcome the increasing resistance of many different varieties of cells and micrcorganisms to the antibiotic activity of tetracyclines, their analogues and derivatives.
- the present invention takes into account and acts upon the existence of specific DNA. sequences, which are typically found on plasmids and transposons, and which specify proteins for
- dete ⁇ ninants act via an active efflux system which maintains an intracellular tetracycline concentration below those levels able to inhibit protein synthesis within the microorganism such as described in above-mentioned U.S. Patent 4,806,529.
- Other determinants act by protecting the ribosome from tetracycline's inhibitory activity, e.g. by binding with tetracycline.
- the present invention represents improvement in efficacious and reliable techniques for overcoming tetracycline resistance in living cells and thus for reestablishing tetracyclines as an antibiotic of choice in the treatment of infectious diseases caused by the ever-increasing variety and diversity of disease agents.
- the invention relies on the action of a blocking agent which is capable of interacting with a product of at least one tetracycline resistance determinant which acts by protecting the cell from tetracycline's inhibitory activity.
- the determinant is capable of making a product, such as a cytoplasmic protein, which interacts with the ribosomes to make them tetracycline resistant or a membrane protein which keeps tetracycline out of the cell.
- the present invention is intended for use with
- Gram-negative genera in particular Enterobacteriaceae, which harbor Class A-E tetracycline resistance determinants;
- Gram-positive genera including streptococci, staphylococci, and bacillus species which bear the Class K and L tetracycline resistance determinants; aerobic and anaerobic inicrcorganisms bearing the Class M, O or Q determinants represented by
- Streptococcus agalactiae Bacteroides. Enterococcus, Gardnerella and Neisseria species, Mycoplasma and Ureaplasma, and
- Clostridium Clostridium perfringens bearing the Class P tetracycline-resistant determinant.
- the present invention represents a major improvement over presently known methods for dealing with tetracycline resistance within disease-causing cells and organisms.
- the methodology requires only two essential steps: the administration to the tetracycline-resistant cell of a predetermined quantity of at least a first composition selected from the chemical group consisting of a blocking agent which is capable of interacting with a product of at least one
- tetracycline resistance determinant which is capable of protecting ribosomes in the cell from tetracycline , s inhibitory activity
- compositions which may be administered alone against, for example, a sensitive or resistant strain of gram positive bacteria such as S. aureus and E. faecalis.
- Tet M Tet O and Tet Q proteins for cytoplasmic protein products
- Tet A Tet B, Tet K and Tet L for membrane products.
- the resistance mechanism of the cell is allowed to
- the blocking agent preferentially react with the blocking agent so avoiding preferential reaction with the second administered composition which is the tetracycline, a tetracycline analogue or derivative composition.
- derivatives of methacycline are able to block the efflux protein and inhibit the resistance mechanism in both gram negative and gram positive cells, including different mechanisms of
- Patent No. 5,064,821 The present invention expands upon these earlier investigations in substantial degree. It also provides the user with novel blocking agents which unexpectedly have been found to show very high inhibition of the mechanisms for ribosome protection as well as efflux.
- the blocking agent is a tetracycline analogue which contains a sufficient part of tetracycline to interact with a product of at least one
- tetracycline resistance determinant capable of protecting cells from tetracycline's inhibitory activity.
- One specific class of blocking agents is the class of
- A is selected from the group consisting of hydrogen and a hydroxyl group
- B is selected from the group consisting of a hydrogen atom, a methylene group, and any linear, branched, or ring structure comprising from 1-6 carbon atoms and optionally including heteroatoms such as oxygen and nitrogen atoms;
- R is selected from the group consisting of organic entities comprising from 1-12 carbon atoms, with or without other heteroatoms including sulfur, oxygen, halogen, nitrogen, and the like, and takes form as linear, branched, or cyclic alkyl, aryl, or alkylaryl structures.
- preferred compound will have a lower K i than tetracycline (i.e., lower than about 4-8 ⁇ g/ml) .
- compositions having substitutions at the 13th carbon relate more to the size of the molecule than to the charge despite the presence of the sulfur atom.
- the longer chain length substitutions at the 13th carbon atom e.g., decyl and hexyl
- the shorter length substitutions e.g., butyl, propyl, and ethyl.
- the dihydroxypropyl derivative behaves more poorly in the blocking assay than the propyl or isopropyl derivative forms.
- a most preferred composition would be one having mercapto-substitutions on the 13th carbon atom in which the elipsoidal volume of the substituent joined to the sulfur atom is in the approximate size range of that provided by the butyl, benzyl or cyclopentyl derivatized structures.
- the blocking agent which can be employed in practicing the present invention is the class of C5 esters of tetracyclines of the formula (Formula II):
- R 1 and R 2 are selected from the group consisting of a methylene group, hydroxyl, hydrogen or a group consisting of organic entities comprising from 1-12 carbon atoms, with or without other heteroatoms including sulfur, oxygen, halogen, nitrogen, and the like, and takes form as linear, branched, or cyclic alkyl, aryl, or alkylaryl st-ructures; and A is selected from the group consisting of a hydrogen atom, a methylene group, and any linear, branched, or ring structure comprising from 1-6 carbon atoms and optionally including heteroatoms such as oxygen and nitrogen atoms.
- Certain C5 esters have been described by Bernardi et al. (II Farmaco, Ed. Sc. vol.
- hybrids of the above-described 6-deoxy-13 (substituted mercapto) and C5 ester may be employed as the blocking agent against resistant gram negative strains or alone against resistant gram positive strains.
- 13,5 derivatives may be accomplished by the anti-Mark ⁇ vnikov radical addition of alkyl or aryl thiols to the 6,13 exocyclic double bond of methacycline by the method of Blackwood et al., J. Am. Chem. Soc., 85:3943 (1963) the disclosure of which is incorporated by reference herein, followed by esterification with an appropriate
- the present invention requires that at least one other composition which is not chemically a blccking agent, such as the above-described 6-deoxy-13-(substituted
- mercapto)tetracycline or C5 ester be administered concurrently or simultaneously with the blocking agent to the cell.
- This additional adininistered composition is any "tetracycline-type antibiotic" (currently known which includes tetracycline itself; or any member of the tetracycline family including all analogues and derivatives which are NOT C5 ester derivatives nor 13-carbon substituted mercaptan compounds. Accordingly, the broadest definition for the additional tetracycline, analogue, or derivative to be administered concurrently is defined by FormulaIII below.
- R 1 -R 5 may be a hydrogen atom, a halogen atom, a hydroxyl group, or any other organic composition comprising from 1-8 carbon atoms and optionally include a heteroatom such as nitrogen, oxygen, in linear, branched, or cyclic structural formats.
- a heteroatom such as nitrogen, oxygen, in linear, branched, or cyclic structural formats.
- R 1 and R 2 are hydrogen or a hydroxyl group
- R 3 is a hydrogen or a methyl group
- R 4 is a hydrogen atom, a halogen, or a nitrogen containing entity
- R 5 is a hydrogen atom, or a nitrogen ⁇ nteining ring
- tetracycline analogues and derivatives including the following: oxytetracycline;
- chlortetracycline demeclocycline; doxycycline; chelocardin; minocycline; rolitetracycline; lymecycline; sancycline;
- methacycline apicycline; clomocycline; guamecycline;
- meglucycline mepyclycline
- penimepicycline pipacycline
- etamocycline etamocycline
- penimocycline etamocycline
- these specific tetracycline compositions may be employed as the alternative tetracycline-type composition which does not contain a C5 ester nor a 13-carbon substituted mercapto group as part of its formulation and chemical structure.
- 13-S-derivatives or Formula II, C5 esters, or the 13,5 derivative, and Formula III, alternative tetracycline compounds, can be administered concurrently, sequentially or simultaneously in any appropriate carrier for oral, topical or parenteral administration. It is also possible that the two discrete compositions could be linked covalently or otherwise joined to each other and/or to other ligands. These compositions can be introduced by any means that affects an infectious or disease state caused by tetracycline-resistant microorganisms in humans and/or animals. The specific route of administration, the choice of carrying materials, and the particular means for introducing each composition concomitantly to the
- tetracycdine-resistant cells are of no major importance or relevance.
- the 13-S-derivative, the C5 derivative composition or the 13,5 derivative and the other alternative tetracycline-type compound are to be applied topically, they can be individually or mutually admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, or a cream.
- topical carriers include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
- topical carriers are liquid petrolatum, iscprcpylpalmitate, polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% in water, sodium lauryl sulfate 5% in water, and the like.
- materials such as anti-oxidants, humectants, viscosity
- stabilizers may also be added if and when necessary.
- tetracycline-type composition are to be introduced concurrently, sequentially or simultaneously in parenteral form, each
- composition will be prepared individually or in combination in sterile form; in multiple or single dose formats; and be dispersed in a fluid carrier such as sterile physiological saline or 5% dextrose solutions commonly used with injectables.
- each of the two requisite compositions may be provided individually or in combination in the form of prepared capsules, cachets, or tablets each containing a predetermined quantity of the 13-S-derivative, the C5 ester composition or the 13,5 derivative and the tetracycline-type antibiotic.
- Their preparation may also take form as a powder or granules; or dissolved or suspended in a solution or suspension within an oil-in-water emulsion or conversely within a
- water-in-oil liquid emulsion for ingestion or for oral cavity lavage treatments.
- These solid or liquid formulations may generally include one or more carrier materials such as flavoring agents, binders, buffers, diluents, surface active agents, thickeners, lubricants, preservatives, and the like. It is deemed that all of these methods for formulating, preparing, and administering the requisite compositions are conventionally known.
- the C5 derivative composition or the 13,5 derivative and each embodiment of the alternative tetracycline composition will have individual specific pharmacological activity which can be represented and evaluated as the Minimal Inhibitory
- MIC Concentration
- MLC Minimal Lethal Concentration
- the degree of tetracycline resistance is known to very substantially among the different cell types, their delineated genera, and among the different species comprising a single genus; this varying degree of tetracycline resistance is without regard to whether the mechanism of resistance is based upon an active efflux system or a ribosome protection system intracellularly.
- each specific route of in vivo administration is conventionally recognized to require markedly different dose concentration of conventionally known tetracycline compounds; accordingly, in vivo therapeutic dosages will vary depending upon whether the tetracycline-type composition is given orally, parenterally, or topically. Each of these individual factors should be taken in consideration by the user when deciding the proper dosage or concentration for the 13-S-derivative, the C5 derivative composition or the 13,5 derivative and the other tetracycline antibiotic composition.
- the dosage and concentration of the 13-S-derivative, the C5 derivative composition (broadly defined by Formula I or Formula II, respectively) or the 13,5 derivative be administered in a subinhibitory quantity - that is, less than the minimum
- inhibitory quantity - that is, less than the minimum inhibitory concentration or the minimum lethal concentration for that specific composition when employed against a
- tetracycline analogue or tetracycline derivative meeting the broad definitional requirements of Formula III above be employed in at least a minimum inhibitory concentration; and preferably be administered at an effective dosage to provide a minimal lethal concentration in-situ. Accordingly, it is deemed that the concentrations for the two concomitantly administered compositions are conventionally known within the art; and can be pptimalized with a minimum of difficulty.
- the C5 derivative composition or the 13,5 derivative and the other tetracycline-type composition together provides not only means for overcoming tetracycline resistance but also offers the capability to enhance the pharmacological activity of the known tetracycline-type composition to exert cidal activity and cidal effects upon the cell.
- the present method provides a synergistic combination of compositions which enhances the antibiotic activity of the tetracycline-type composition; and, for the first time, allows the enhanced tetracycline-type composition to exert
- bacteriocidal powers "cidal” capability, i.e., the ability to kill the cell rather than merely inhibit its growth, against a broad spectrum of bacteria.
- the general molar ratio of 13-S-derivative, the C5 derivative composition, or the 13,5 derivative to alternative tetracycline-type composition is expected generally to be from 0.01:100.0, and is preferably in the range from 0.05:2.0. It is most desirable, however, that in no instance should the dosage of the 13-S-derivative, the C5 derivative composition or the 13,5 derivative be employed in a concentration which is within the MIC or MLC values.
- the alternative, tetracycline-type composition should be administered in accordance with conventional practice for the efficacious therapeutic treatment of infection or disease in humans and/or animals.
- the daily dosage of 13-S-derivative, the C5 derivative composition or the 13,5 derivative for treatment of disease in living mammals is expected to lie in the range from 0.01-100 mg/kg (preferably from 15 to 30 mg/kg) of normal body weight while the dosage of the other tetracycline, analogue or derivative should continue to be given in the range from 500 milligrams to 2.0 grams per day depending upon the age, weight, and route of administration.
- the dosage employed is preferably that used in conventional tetracycline therapy.
- the methodology of the present invention is suitable for use with both tetracycline resistance attributable to an active efflux transport system utilizing one or more TET proteins which actively bind with tetracycline-type antibiotics and transport the tetracycline composition out of the cytoplasm of the cell; and also with tetracycline resistance which is a nonefflux system and typically involves a ribosome protection mechanism which causes a tetracycline antibiotic to fail to inhibit protein synthesis intracellularly.
- the present methodology is effective in overcoming tetracycline resistance and in rendering the cell tetracycline-sensitive.
- 13-S-derivative or the C5 derivative composition prepared in accordance with Formula I or Formula II or the 13,5 derivative and at least one other tetracycline antibiotic composition in accordance with Formula III causes an in situ conversion of the cell from a resistant state into a tetracycline-sensitive state.
- the resistance mechanism of the cell be it the active efflux system or the ribosome protection system - focuses upon and interacts with the 13-S-derivative, the C5 derivative composition or the 13,5 derivative primarily and predominantly; the concurrent or simultaneous presence of the other
- tetracycline-type antibiotic composition is relatively ignored and effectively overlooked by the tetracycline-resistance mechanism of the cell.
- the other tetracycline-type antibiotic composition is allowed to accumulate intracellularly in at least a minimum inhibitory concentration (and preferably in a minimum lethal concentration); and this other tetracycline-type antibiotic is able to bind to the ribosomes and to exert its recognized pharmacological activity intracellularly to prevent further protein synthesis within that cell.
- the administered 13-S-derivative of Formula I or C5 derivative composition of Formula II or 13,5 derivative is clearly the preferred composition for reaction with the tetracycline- resistance mechanism present; and by this preferred reactivity, acts as a blocking agent to engage and to divert the
- the present methodology is thus effective and useful by the cell's own preference for engagement and reaction with the 13-S-derivative, the C5 derivative composition or the 13,5 derivative, rather than with the concomitantly administered other tetracycline-type antibiotic. In this manner, the cumulative effect is to render the cell tetracycline-sensitive for therapeutic purposes.
- tetracycline for the 13-S-derivative or doxycycline for the C5 derivative was employed uniformly in combination with a variety of different C5 or 13 substituted mercapto-tetracyclines.
- doxycycline and tetracycline are employed merely as a representative of all the different compositions and embodiments of tetracyclines, tetracycline analogues, and tetracycline derivatives conforming to the definition of Formula III given previously; and that the present invention is not limited to the use of tetracycline alone as a specific chemical formulation and structure.
- Tc s tetracycline sensitive
- Tc r tetracycline resistant strains of E. coli. S. aureus. and E. faecalis.
- the general protocol for performing these experiments is as follows: Cultures were grown up fresh in L broth in the morning from an overnight culture. After 4-6 hours of growth, each bacterial culture was diluted to an A 530 of 0.2-0.5 depending on the strain (E. coli, 0.5; S. aureus, 0.4; E.
- 13-S-derivatives were effective alone within therapeutic ranges. They were about as active as tetracycline and minocycline (except perhaps the cyclohexyl derivative). All 13-S-derivatives showed bacteriocidal activity better than tetracycline or minocycline of which 4 showed, bacteriocidal activity at a level of about 5 ⁇ g/ml.
- cyclohexyl, and cyclopentyl derivatives also had MIC values below minocycline and within therapeutic levels (Table E3, Column 3). Bacteriocidal activity was observed, but above therapeutic levels.
- Charts 1-5 in which: Chart 1 represents the concurrent administration of 13-cyclopentyl sulfide derivative of methacycline in varying proportional ratios to tetracycline; Chart 2 represents
- Chart 3 represents varying proportional ratios with tetracycline
- Chart 4 represents varying proportional ratios of 13-cyclohexyl-sulfide derivatives of methacycline and tetracycline administered concurrently;
- Chart 4 represents varying proportional ratios of 13-benzyl-sulfide derivatives of methacycline delivered
- Chart 5 illustrates the concurrent administration of varying proportions of
- E. coli strain D1-299
- synergy was observed.
- the most effective analogues were cyclopentyl, cyclohexyl, and ethyl. These all inhibited growth at concentrations of 5 ⁇ g/ml or less of analogue and tetracycline.
- Synergy was also demonstrated in bacteriocidal activity, although the amounts of the 13-S-derivatives needed were higher than 5 ⁇ g/ml in order to kill 99.9% of the cells with 4-5 ⁇ g/ml of tetracycline.
- 13-S-derivatives show synergy, both in growth inhibition and in bacteriocidal activity for gram-positive as well as gram-negative susceptible and resistant strains.
- aureus and E. faecalis chiefly the benzyl, cyclohexyl, and cyclopentyl derivatives.
- Everted vesicles to which the TET proteins responsible for tetracycline efflux are attached, provide a reliable method for measuring efflux from tetracycline-resistant bacteria. By exposing the vesicles to different concentrations of
- the affinity of tetracycline for the efflux system may be determined.
- exposure of the vesicles to solutions having both tetracycline and a potential efflux protein blocking agent produces a competition between
- 3H-tetracycline in a volume of 300 ⁇ l.
- Different potential blocking agents with substitutions at the C5 position were separately tested at concentrations of 0.2, 0.5 and 2 ⁇ g/ml.
- a control experiment, wherein no blocking agent was used, was also performed. After incubation for 2.5 minutes, the vesicles were collected on membrane filters and the effect of the blocking agent on uptake of 3 H-tetracycline was assessed by liquid scintillation counting of the radioactivity on the filters.
- the assay showed the relative inhibition of tetracycline by the different drugs vis a vis drug amounts (Table E4 below).
- the IC 50 of the analogs was determined.
- the IC 50 of different C5 esters ranged from 0.2 ⁇ M (5 proprionate methacycline) to 9.4 ⁇ M (5 cyclopropanoate methacycline).
- MLC ⁇ tiniinal lethal concentration
- Chart 7 MIC/MLC ( ⁇ g/ml) dosages for tetracycline resistant strains using 5 prqprionate doxycycline with and without doxycycline.
- Chart 8 MIC/MLC ( ⁇ g/ml) dosages for tetracycline resistant strains using 13-cycl ⁇ pentyl-thio-5-proprionate tetracycline with and without doxycycline.
- Chart 9 MIC/MLC ( ⁇ g/ml) dosages for tetracycline resistant strains using 13-propyl-thio-5-proprionate tetracycline with and without doxycycline.
- the analog concentration is given in columns A and H. It is this concentration which is within the squares 2-10.
- the deoxycycline concentration in the control is in Column 11 and its concentration in each of the boxes is given as small numbers within each of the squares. All four tetracycline derivatives effectively inhibit growth of sensitive Staph aureus and E. faecalis when used alone. None was effective alone against susceptible E. coli (Table E5).
- the C5 prqprionate esters were also effective alone against resistant S. aureus and E. faecalis. and showed further efficacy in additiveness and synergy when used with doxycycline in MIC and MIC.
- Chart 6B the combination of 1.55 ⁇ g/ml doxycycline and 1.56 ⁇ g/ml 5-proprionate methacycline or 6.23 ⁇ g/ml doxycycline and 0.78 ⁇ g/ml analog achieved MIC.
- the growth inhibition included cells bearing different tetracycline efflux systems (Class A, B, K & L) and a ribosomal protection system (Class M). These unexpected results confirm that the substitution at the C5 position produces an effective efflux blocking agent which demonstrates synergistic
- Methacycline hydrochloride (5.0 g, 10.4 mmol) was placed in a round-bottom flask and suspended in 100 mL of EtOH. Twenty mL of propanethiol (16.8 g, 0.270 mol) and AIBN 250 mg, were added and the reaction inixture refluxed with stirring for 12 h while under N 2 . The mixture was reduced to 1/5 volume by
- This compcund was prepared substantially as described in Example 1. Purification was either by column chr ⁇ matography on EDTA silica, extraction pH 4.5 into CH 2 Cl 2 , or by HPLC
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Abstract
L'invention se rapporte à une méthodologie améliorée au moyen de laquelle il est possible thérapeutiquement de supprimer la résistance à la tétracycline dans des cellules vivantes, y compris des bactéries, des parasites, des champignons et des rickettsiae. Ladite méthodologie utilise un agent bloquant, tel que des dérivés d'esters C5 ou des dérivés 6-déoxy 13-mercapto substitué de la tétracycline combiné à d'autres antibiotiques du type tétracycline en tant que combinaison synergique de compositions à administrer simultanément, séquentiellement ou parallèlement. Dans un autre mode de réalisation de l'invention, certaines nouvelles compositions peuvent être administrées seules contre, par exemple, une souche sensible ou résistante de bactéries à gram positif, tel que S. aureus et E. faecalis. Les compositions administrées de façon concomitante supprime efficacement les mécanismes de résistance à la tétracycline, de telle façon que la cellule est effectivement convertie depuis un état de résistance à la tétracycline enun état de sensibilité à la tétracycline.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/232,247 US5589470A (en) | 1990-02-26 | 1992-10-16 | Reducing tetracycline resistance in living cells |
US09/504,697 US6756365B2 (en) | 1991-11-06 | 2000-02-16 | Reducing tetracycline resistance in living cells |
US10/770,883 US7414041B2 (en) | 1991-11-06 | 2004-02-02 | Reducing tetracycline resistance in living cells |
US12/194,362 US7732429B2 (en) | 1991-11-06 | 2008-08-19 | Reducing tetracycline resistance in living cells |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US78869391A | 1991-11-06 | 1991-11-06 | |
US788,693 | 1991-11-06 |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US78869391A Continuation | 1982-11-18 | 1991-11-06 | |
US78869391A Continuation-In-Part | 1982-11-18 | 1991-11-06 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/232,247 Continuation US5589470A (en) | 1982-11-18 | 1992-10-16 | Reducing tetracycline resistance in living cells |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993008806A1 true WO1993008806A1 (fr) | 1993-05-13 |
Family
ID=25145279
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1992/008965 WO1993008806A1 (fr) | 1990-02-26 | 1992-10-16 | Procede de diminution de la resistance a la tetracycline dans des cellules vivantes |
Country Status (2)
Country | Link |
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AU (1) | AU2907192A (fr) |
WO (1) | WO1993008806A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0618190A1 (fr) * | 1993-04-02 | 1994-10-05 | American Cyanamid Company | 9-(Glycyle substitué)amido-6-(substitué)-5-hydroxy-6-déoxytétracyclines |
WO1999037307A1 (fr) * | 1998-01-23 | 1999-07-29 | Trustees Of Tufts College | Composes actifs du point de vue pharmaceutique et leurs methodes d'utilisation |
WO2001052858A1 (fr) * | 2000-01-24 | 2001-07-26 | Trustees Of Tufts College | Composes de tetracycline pour le traitement de troubles afferents au cryptosporidium parvum |
Citations (3)
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US3454697A (en) * | 1965-06-08 | 1969-07-08 | American Cyanamid Co | Tetracycline antibiotic compositions for oral use |
US4806529A (en) * | 1982-11-18 | 1989-02-21 | Trustees Of Tufts College, Tufts University | Tetracycline activity enhancement |
US5064821A (en) * | 1982-11-18 | 1991-11-12 | Trustees Of Tufts College | Method and compositions for overcoming tetracycline resistance within living cells |
-
1992
- 1992-10-16 AU AU29071/92A patent/AU2907192A/en not_active Abandoned
- 1992-10-16 WO PCT/US1992/008965 patent/WO1993008806A1/fr active Application Filing
Patent Citations (4)
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US3454697A (en) * | 1965-06-08 | 1969-07-08 | American Cyanamid Co | Tetracycline antibiotic compositions for oral use |
US4806529A (en) * | 1982-11-18 | 1989-02-21 | Trustees Of Tufts College, Tufts University | Tetracycline activity enhancement |
US5021407A (en) * | 1982-11-18 | 1991-06-04 | Trustees Of Tufts College | Tetracycline activity enhancement |
US5064821A (en) * | 1982-11-18 | 1991-11-12 | Trustees Of Tufts College | Method and compositions for overcoming tetracycline resistance within living cells |
Non-Patent Citations (3)
Title |
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CHEM. ABS., 1971, (KUCK et al.), 74:74941g. * |
CHEM. ABS., 1974, (LEIGH et al.), 115200e. * |
RESEARCH AND THERAPY, 1981, (GRASSI et al.), New Trends in Antibiotics, pp. 3-54. * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0618190A1 (fr) * | 1993-04-02 | 1994-10-05 | American Cyanamid Company | 9-(Glycyle substitué)amido-6-(substitué)-5-hydroxy-6-déoxytétracyclines |
EP1435352A2 (fr) * | 1993-04-02 | 2004-07-07 | Wyeth Holdings Corporation | 9-[(Glycyle substitué)amido]-6-(substitué)-5-hydroxy-6-déoxytétracyclines |
EP1435352A3 (fr) * | 1993-04-02 | 2004-11-03 | Wyeth Holdings Corporation | 9-[(Glycyle substitué)amido]-6-(substitué)-5-hydroxy-6-déoxytétracyclines |
WO1999037307A1 (fr) * | 1998-01-23 | 1999-07-29 | Trustees Of Tufts College | Composes actifs du point de vue pharmaceutique et leurs methodes d'utilisation |
EP1047431A1 (fr) * | 1998-01-23 | 2000-11-02 | Trustees Of Tufts College | Composes actifs du point de vue pharmaceutique et leurs methodes d'utilisation |
EP1047431A4 (fr) * | 1998-01-23 | 2004-12-08 | Tufts College | Composes actifs du point de vue pharmaceutique et leurs methodes d'utilisation |
WO2001052858A1 (fr) * | 2000-01-24 | 2001-07-26 | Trustees Of Tufts College | Composes de tetracycline pour le traitement de troubles afferents au cryptosporidium parvum |
US6833365B2 (en) | 2000-01-24 | 2004-12-21 | Trustees Of Tufts College | Tetracycline compounds for treatment of Cryptosporidium parvum related disorders |
US7202235B2 (en) | 2000-01-24 | 2007-04-10 | Trustees Of Tufts College | Tetracycline compounds for treatment of cryptosporidium parvum related disorders |
US7807660B2 (en) | 2000-01-24 | 2010-10-05 | Trustees Of Tufts College | Tetracycline compounds for treatment of Cryptosporidium parvum related disorders |
Also Published As
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AU2907192A (en) | 1993-06-07 |
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