+

WO2001007034A1 - Inhibiteurs de pompe a resistance multiple et utilisations - Google Patents

Inhibiteurs de pompe a resistance multiple et utilisations Download PDF

Info

Publication number
WO2001007034A1
WO2001007034A1 PCT/US2000/020001 US0020001W WO0107034A1 WO 2001007034 A1 WO2001007034 A1 WO 2001007034A1 US 0020001 W US0020001 W US 0020001W WO 0107034 A1 WO0107034 A1 WO 0107034A1
Authority
WO
WIPO (PCT)
Prior art keywords
mdr
multidrug resistance
composition
plant
compound
Prior art date
Application number
PCT/US2000/020001
Other languages
English (en)
Inventor
Kim Lewis
Frank R. Stermitz
Jeanne N. Tawara-Matsuda
Peter Lorenz
Lauren Zenewicz
Nathan R. Guz
Original Assignee
Colorado State University Research Foundation
Tufts University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Colorado State University Research Foundation, Tufts University filed Critical Colorado State University Research Foundation
Priority to AU63660/00A priority Critical patent/AU6366000A/en
Publication of WO2001007034A1 publication Critical patent/WO2001007034A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to a multidrug resistance pump inhibitor, method for detecting and isolating the same from a plant, and the use thereof.
  • Multidrug resistance (MDR) pumps i.e., active drug efflux systems
  • MDR pumps that extrude (i.e., excrete or remove from the cell interior) structurally unrelated toxins.
  • MDR pumps are responsible for many cases of clinically relevant multidrug resistance of microorganisms, including bacteria and fungi, such as yeast, and are believed to be the primary cause of multidrug resistant cancer.
  • the basic feature of a MDR pump in microorganisms is its ability to discriminate between a broad array of structurally unrelated antimicrobial compounds (i.e., toxins) and normal cellular compounds.
  • MDR pumps are thus very different from other enzymes and translocases that recognize a specific substrate or a group of substrates with a shared structure.
  • a "substrate” refers to a compound which is excreted by MDR pumps. Without being bound by any theory, it is believed that the mechanism of discrimination by MDR pumps is based largely on the polarity of a compound rather than its structure. It is believed that toxins with an intracellular target are generally amphiphilic in order to cross the cellular membrane, while cellular compounds are generally hydrophilic. See for example, Higgins and Fernman, Trends Biochem. Sci., 1992, 17, 8-21, and Lewis, Trends Biochem. Sci., 1994, 19, 119-123.
  • MDR pumps can be very effective in protecting microorganisms from toxins.
  • the EmrAB pump of E. coli confers approximately a 60-fold increase in resistance to the antibiotic thiolactomycine, Furukawa et al., J. Bacteriol, 1993, 175, 3723-3729; the AcrAB pump o ⁇ E. coli confers approximately a 60-100 fold resistance to novobiocin and erythromycin, Ma et al., Mol. Microbiol, 1995, 16, 45-55; and the MexAB pump of P. aeruginosa confers approximately a 50-100 fold resistance to some quinolones and hydrophobic antibiotics, Poole, Animicrob. Agents Chemother., 1994, 40, 2021-2028.
  • These levels of resistance rival specific resistance mechanisms such as tetracycline efflux by a dedicated pump; or inactivation of kanamycin by a specific acetyl transferase.
  • amphiphilic cations in particular antimicrobial isoquinoline alkaloids, including berberine and protoberberinium compounds, are one of the natural substrates for MDR pumps. See, for example, Lewis, Current Biol, 1999, 9, R403-R407.
  • a "natural” compound refers to a compound which is known to be produced in nature without the aid of man.
  • protoberberinium refers to a compound having a similar core ring structure as that of berberine.
  • an "inhibitor” refers to a compound which inhibits the efflux action of MDR pumps.
  • MDR pump(s) which effectively renders current antimicrobial compounds virtually useless.
  • using a MDR pump inhibitor in combination with an antimicrobial compound in animals or plants will overcome some of the ineffectiveness currently observed in treating drug resistant microorganism infections.
  • the present invention provides a method for determining the presence of a multidrug resistance (MDR) pump inhibitor in a natural antibiotic producing source including plants and microorganisms, such as fungi and bacteria.
  • MDR multidrug resistance
  • methods of the present invention generally involves extracting the plant, and culturing a cell having at least one multidrug resistance pump with a culture medium which comprises the plant extract and a multidrug resistance pump substrate at a concentration below minimum inhibitory concentration (MIC) level.
  • the plant is selected from plants which produce amphiphilic cations such as isoquinolines, in particular, berberine and protoberberinium alkaloids.
  • the present invention also provides flavolignans and porphyrin derivative compounds which have been identified by the method of the present invention as being MDR pump inhibitors. Specifically, the present invention provides a multidrug resistance pump inhibitor which is selected from a compound of the formula:
  • Ar 1 is aryl; each of R 1 and R 2 is independently H, C t -C alkyl or a hydroxy protecting group; each of R 3 , R J is independently H, C 1 -C 4 alkyl, a hydroxy protecting group or R 3 and R 4 together form a moiety of the formula:
  • each of R 9 and R 10 is independently C ⁇ -C 4 alkyl or C 6 -C ⁇ o aryl; each of Y and Z is H, hydroxy or C 1 -C4 alkoxy; each of R 1 ', R 12 , R 14 , R 16 , R 19 and R 20 is independently H or C r C 6 alkyl; each of R 13 and R 15 is independently H, C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl or C 2 - C 6 alkynyl; and each of R 17 and R 18 is a bond or C ⁇ -C 6 alkyl ene.
  • a MDR pump inhibitor is a compound of formula:
  • each of Q 1 and Q 2 is independently H or -OH.
  • Z is a hydroxy group.
  • each of R and R 10 is independently hydroxymethyl or 4- hydroxy-3-methoxyphenyl.
  • R 9 is hydroxymethyl and R 10 is 4-hydroxy-3-methoxyphenyl.
  • R 9 is 4-hydroxy-3- methoxyphenyl and R 10 is hydroxymethyl.
  • a MDR pump inhibitor is a compound of formula:
  • each of Q 1 and Q 2 is independently H or -OH.
  • Y is methoxy
  • each of R 9 and R 10 is independently hydroxymethyl or 4- hydroxy-3-methoxyphenyl.
  • R 9 is hydroxymethyl and R 10 is 4-hydroxy-3 -methoxyphenyl.
  • R 9 is 4-hydroxy-3- methoxyphenyl and R 10 is hydroxymethyl.
  • R 9 is hydroxymethyl and R 10 is 4-hydroxy-3 -methoxyphenyl.
  • a MDR pump inhibitor is a compound of the formula:
  • each of R 11 , R 12 , R 14 , R 16 and R 20 is independently d-C 6 alkyl. More preferably, R 1 ⁇ R 12 , R 14 , R 16 and R 20 are methyl.
  • R 19 is H or C ⁇ -C 6 alkyl. More preferably, R 19 is H.
  • each of R 13 and R 15 is independently C -C 6 alkenyl or C 2 -C 6 alkynyl. More preferably, each of R and R is independently C -C 6 alkenyl. And most preferably, R 13 and R 15 are ethenyl.
  • R 17 is a bond.
  • R 18 is C]-C 6 alkylene. More preferably, R 18 is ethylene.
  • a MDR pump inhibitor is pheophorbide a, which has the following structure:
  • a MDR pump inhibitor is a compound of the formula:
  • R 9 is hydroxymethyl and R 10 is 4-hydroxy-3 -methoxyphenyl; or R 9 is 4-hydroxy-3-methoxyphenyl and R 10 is hydroxymethyl.
  • Ar 1 is phenyl.
  • Z is H.
  • the present invention also provides a composition containing any of the above described MDR pump inhibitors and an antimicrobial compound, and a method for treating a microorganism infection using the same.
  • Figure 1 shows exemplary amphiphilic cations and other NorA (a multidrug resistance pump) substrates
  • Figure 2 is a schematic model of MDR pumps from different bacterial families
  • Figure 3a is a graph showing efflux of ethidium bromide from wild type and nor A strains of S. aureus
  • Figure 3b is a graph showing inhibition of ethidium bromide efflux in wild type cells by 5'-methoxyhydnocarpin-D (i.e., 5'-MHC-D, MHC-D or MHC);
  • Figure 4a is a flow diagram of a process for extracting MDR pump inhibitors from M. fremontii leaves
  • Figure 4b is a flow diagram of another process for extracting MDR pump inhibitors from M. fremontii leaves;
  • Figure 5 is a graph showing the MDR pump inhibitory activity of the silica gel separation fractions obtained by the process shown in Figure 4b;
  • Figure 6 is a graph showing bacterial growth inhibition by MHC at a variety of concentrations; and Figure 7 shows exemplary flavonolignans and possible intermediates in the synthesis of flavonolignans.
  • the present invention provides multidrug resistance pump inhibitors, a method for discovering and isolating naturally occurring multidrug resistance pump inhibitors from natural antibiotic producing sources, and the use thereof.
  • MDR pumps which remove antimicrobial compounds from their cytoplasm. It is believed that this excretion (i.e., efflux) of antimicrobial compounds by these MDR pumps is responsible for many drug resistances seen in microorganisms. In addition, the presence of MDR pumps in cancer cells is believed to be the primary cause of multidrug resistant cancers.
  • MDR pumps are thus very different from other enzymes and translocases that recognize a specific substrate or a group of substrates with a shared structure. Without being bound by any theory, it is believed that the mechanism of discrimination by MDR pumps is based largely on the polarity of a compound rather than its structure. Even though MDR pumps have the potential of protecting microorganisms from antimicrobial compounds, it is believed that this does not necessarily mean that drug (e.g., antimicrobial compound) resistance is the primary function of MDR pumps. For example, the Bit pump of B.
  • subtilis that protects the cells from a variety of amphiphilic cationic antimicrobials is part of an operon that codes for a putrescine acetyl transferase.
  • the Bit pump extrudes putrescine from the cell, suggesting that putrescine extrusion might be its natural, i.e., primary intended, function, and the drug resistance is a mere coincidental consequence.
  • other data indicate that drug resistance is the natural function of some MDR pumps.
  • the QacA MDR pump is found on broad host range plasmids that also carry specific gentamycin and trimethoprim resistance genes.
  • QacA pump is a dedicated drug resistance component of the plasmid. It has been recently reported that QacA expression is controlled by an upstream transcriptional repressor QacR belonging to the TetR repressor family. Grkovic et al, J. Biol Chem, 1998, 273, 18665-18673.
  • Resistance Nodulation Division pumps extrude natural antibiotics as well as a variety of artificial substrates.
  • bile acids that enter the cytoplasm of E. coli are extruded by the AcrAB pump which is believed to be responsible for protecting E. coli from bile acids.
  • Thanassi et al J. Bacteriol, 1997, 179, 2512- 2518.
  • Another example of a natural substrate for a resistance nodulation division pump has been described recently in a study of an IfeAB RND pump in Agrobacterium tumefaciens. Palumbo et al, J. Bacteriol, 1998, 180, 3107-3113.
  • amphiphilic compound is a compound containing
  • MDR pumps One of the simplest MDR pumps, shown in Figure 2, belongs to the Small Multidrug Resistance (SMR) pump family and are peptides of around 107 amino acids long that function as trimers. Paulsen et al, Mol. Microbiol, 1996, 19, 1167-1175. SMR pumps are unique in that there are no specific translocases in this family. It is believed that SMR pumps might have been the first dedicated MDR pumps to evolve. Lewis, Trends Biochem., 1994. 19, 119-123. Currently, amphiphilic cations are the only known substrates of SMR pumps. The Major Facilitator (MF) family of translocases has both uptake transporters like LacY and specific efflux pumps like TetA among its members.
  • SMR Small Multidrug Resistance
  • MDR pumps there are many MDR pumps in this family, and most of them exclusively extrude amphiphilic cations.
  • the QacA pump only extrudes amphiphilic cations
  • the NorA pump of S. aureus extrudes amphiphilic cations and to a lesser extent quinolones
  • the BMR pump of 5. subtilis extrudes primarily amphiphilic cations and neutral chloramphemcol. Paulsen et al, Microbiol. Rev., 1996, 60, 575-608; Lewis et al, ASM News, 1997, 63, 605-610.
  • EmrAB pump which appears to specialize in extruding hydrophobic substances such as uncouplers of oxidative phosphorylation and does not seem to protect the cell from amphiphilic cations. Lomovskaya and Lewis, Proc. Natl. Acad. Sci. USA, 1992, 89, 8938-8942.
  • RND pumps also extrude amphiphilic cations. Like EmrAB, the MDR pumps of the RND family are trans-envelope translocases of gram negative bacteria. Nikaido, J. Bacteriol, 1996, 178, 5853-5859. They export toxins across the outer membrane that is a permeability barrier for hydrophobic compounds as illustrated in Figure 2.
  • the substrate spectrum of RND pumps is broad and in the case of the E. coli AcrAB includes cationic acridine; anionic detergent sodium dodecylsulfate (SDS); and neutral ⁇ -lactam antibiotics, see for example, Nikaido, J. Bacteriol, 1996, 178, 5853- 5859.
  • the ATP Binding Cassette (ABC) family has both specific translocases and MDR pumps.
  • the human P-glycoprotein MDR pump which is largely responsible for multidrug resistance of tumors, belongs to this family.
  • the LmrA pump of L. lactis is a member of this MDR pump family and shares a substrate spectrum with P-glycoprotein. van Veen et al, Nature, 1998, 391, 291-295.
  • the preferred substrates for LmrA and P-glycoprotein pumps are amphiphilic cations, but some neutral compounds are extruded as well by these MDR pumps.
  • ABC MDR pumps there are many ABC MDR pumps in yeast, and at least 9 known ABC MDR pumps are present in S. cerevisiae alone.
  • the substrates of these pumps include amphiphilic cations, and neutral substances such as anti-yeast azoles.
  • MDR pumps like SMR pumps only export amphiphilic cations; MF MDR pumps export mainly cations; RND pumps and ABC pumps have broad spectra of specificity that include amphiphilic cations as preferred substrates. Generally, MDR pumps prefer amphiphilic cations to other substances, even though they belong to 4 unrelated protein families. Not only are MDR pumps unrelated, but even the general mechanisms of drug transport are different for different MDR pumps.
  • the P- glycoprotein pump that is homologous to a phospho lipid flippase acts by flipping a substrate from the inner to the outer leaflet of the membrane; the bacterial ABC transporter LmrA as well as MF transporters extrude drugs from the inner leaflet of the bilayer to the outside medium, while the RND translocases apparently extrude toxins from the outer leaflet of the inner membrane to the outer medium.
  • amphiphilic cations are believed to be among the most potent antimicrobials.
  • a positive charge leads to a considerable accumulation of a substance in the cell. According to the Nernst equation, there is a 10-fold accumulation of a cation
  • MDR pump substrates are generally overlooked. While using MDR mutants is a reasonable (if somewhat unpredictable) way to discover possible cationic antimicrobials, another approach is to search for possible MDR pump substrates among known compounds. Many natural substances have been identified as MDR pump substrates as a result of systematic chemical analysis of organisms. One can also use bioassay-driven purifications to identify MDR pump substrates by looking for substances that are amphiphilic cations of natural origin which have little or no antimicrobial activity. Using these criteria, the present inventors have identified a group of plant alkaloids whose members have weak or no antimicrobial activity against drug resistant microorganisms. These are the isoquinoline alkaloids (see Fig. 1) that are widely spread among the plant world and are found among many
  • Ranunculales species for example. These substances bear a resemblance to artificial MDR substrates such as ethidium bromide or benzalkonium chloride (see Fig. 1). They are amphiphilic and have a positive charge, a common feature of a good permeant cation. Microorganisms become sensitive to antimicrobial isoquinoline alkaloids in the absence of functional MDR pumps, and it is believed that a plant that makes antimicrobial isoquinoline alkaloids, such as berberine, would benefit from having a multidrug resistance pump inhibitor.
  • MDR substrates such as ethidium bromide or benzalkonium chloride (see Fig. 1). They are amphiphilic and have a positive charge, a common feature of a good permeant cation.
  • Microorganisms become sensitive to antimicrobial isoquinoline alkaloids in the absence of functional MDR pumps, and it is believed that a plant that makes antimicrobial isoquinoline alkaloids, such as
  • antimicrobial isoquinoline alkaloids represent natural substrates of some MDR pumps, and therefore some plants that make these antimicrobial isoquinoline alkaloids produce MDR pump inhibitors in order to counteract the effect of MDR pumps present in microorganisms.
  • One embodiment of the present invention provides a method for determining the presence of MDR pump inhibitors in a plant. The method involves obtaining a plant extract; culturing a cell (preferably a microorganism) with a culture
  • the plant extract can be derived from any part of the plant including roots, leaves, bark, stems, fruits (or berries) or mixtures thereof. Any plant can be tested to determine the presence of a multidrug resistance pump inhibitor; however, in order to increase the probability of finding a plant which produces a multidrug resistance pump inhibitor, it is preferred that a plant which produces an isoquinoline alkaloid is selected for testing.
  • Exemplary isoquinoline alkaloids include berberine, palmatine, jatrorhizine, berberubine, 13- methylberberine, coptisine, columbamine and other protoberberinium alkaloids. More preferably, a plant which produces an isoquinoline alkaloid selected from the group consisting of berberine, palmatine, jatrorhizine, berberubine, 13-methylberberine, coptisine, and columbamine is selected for testing. And most preferably, a plant which produces berberine is selected for testing.
  • the methods of the present invention can also include determining the minimum inhibitory concentrations (MICs) for these compounds for each microorganism strain. This allows the use of these compounds below MIC level, thus allowing more accurate determination of a multidrug resistance pump inhibition activity of a particular MDR pump inhibitor.
  • MICs minimum inhibitory concentrations
  • Some of the plants which are known to produce berberine are Mahonia fremontii (i.e., M. fremontii), Berberis fendleri, M. repens (Oregon grape), M. aquifolium (Oregon grape), Argemone sp., Chelidonium sp., Coptis sp., Hydrastis sp., Thalictrum sp., Corydalis sp., and others.
  • An added potential benefit to searching for antimicrobial compounds in berberine producing plants is the fact that some of these plants have edible berries known as "Oregon Grape.”
  • the MDR pump inhibitors are present in berries, they will most likely be relatively non-toxic to humans.
  • Mahonia and Berberis species of the Berberidaceae harbor the black rot fungus Puccinia graminis, which is periodically eradicated in order to control this pathogen that infects cereals and grasses.
  • the infection is also treated with propiconazole, an anti-fungal azole compound. See for example, Welty and Azevedo, Plant Dis., 1996, 80, 625-628. It is believed that the MDR pump inhibitors in combination with berberine are controlling the fungal infection in Mahonia plants.
  • an MDR pump inhibitor and berberine combination to suppress growth of Puccinia graminis in liquid culture can be readily tested using the procedures disclosed by Fasters et al., in Physiol Mol Plant Pathol, 1993, 42, 259-265.
  • a combination of a t multidrug resistance pump inhibitor and antimicrobial compound can be used in agricultural applications as well as in animal applications.
  • an array of bacteria in particular plant pathogenic bacteria, can be tested for susceptibility to a combination of MDR pump substrates (e.g., berberine) and MDR pump inhibitors.
  • Plant pathogenic bacteria fall into 23 different genera with numerous species. "Fundamentals of Bacterial Plant Pathology," M. Goto, Academic Press, 1992.
  • Exemplary plant pathogenic bacteria which are susceptible to a combination of a multidrug resistance pump inhibitor and an antimicrobial compound, such as berberine, which is present below its MIC level include the following genera/species:
  • Gram negative bacteria Agrobacterium: A. tumefaciens; Erwinia: E. amylovora; Xanthomonas: X. campestris; and Pseudomonas: P. syringae.
  • Gram positive bacteria Clavibacter: C. michiganensis; and Bacillus: B. megaterium pv. Cerealis.
  • the above bacteria can be cultured according to ATCC recommendations for each species, and susceptibility to antimicrobial isoquinoline alkaloids in the presence and absence of MDR inhibitors can be readily determined using the methods disclosed in the present invention.
  • the present inventors have discovered that determining the presence of MDR pump inhibitors and isolating the same from plants, or example, M. fremontii, can be achieved fairly rapidly. For example, using a microtiter plate assay, the present inventors have screened around 50 crude methanol and hexane/ethyl acetate extracts from an assorted plant collection and found MDR pump inhibitory activities ranging from about 35 ⁇ g/mL to about 1500 ⁇ g/mL in 15 of them (data not shown). In one particular embodiment, crude chloroform extracts of M. aquifolium and M. repens leaves were found to have MDR pump inhibitory activities of about 16 ⁇ g/mL and 69 ⁇ g/mL, respectively. Thus, plants that produce berberine are a good candidate for producing MDR pump inhibitors.
  • plants are separated into parts (roots, bark, stems, fruits (berries) and leaves) and dried in the shade at ambient temperature. It has been found that drying does not appear to decrease the yield and MDR pump inhibitory activity of extracts. However, the handling and extraction processes are facilitated when dry materials are used. During the collection process, plants are thoroughly examined in the shade at ambient temperature.
  • a general MDR pump inhibitor extraction process involves extracting a plant which produces an antimicrobial compound, such as an isoquinoline alkaloid, with an organic solvent, identifying the extract which contains an MDR pump inhibitor and isolating the MDR pump inhibitor.
  • the extraction process can involve a multiple extraction process using organic solvents with increasing polarity.
  • the plant material can be extracted initially with a solvent having dielectric constant, ⁇ , of less than about 3, preferably hexane, and followed by extracting the marc (i.e., the solid residue) with another organic solvent which has ⁇ of from about 3 to about 10, preferably ethyl acetate or chloroform.
  • the dielectric constant, ⁇ , of a solvent refers to the value at 20 °C.
  • the dielectric constant of a solvent can be found, for example, in Handbook of Chemistry and Physics, 63 rd Ed., CRC Press, 1983, pp. E-51 to E-54, which is incorporated herein by reference.
  • the residue from above can be further extracted with a relatively polar solvent (i.e., a solvent having ⁇ of from about 10 to about 40) such as methanol.
  • a relatively polar solvent i.e., a solvent having ⁇ of from about 10 to about 40
  • the relatively polar solvent extract is then acidified, extracted with an organic solvent having ⁇ of from about 3 to about 6, adjusting the pH of the aqueous layer to about pH 7 to about pH 12, and extracting the basic aqueous layer with a solvent having ⁇ of from about 3 to about 6 (preferably chloroform).
  • Microorganisms containing any of the known MDR pumps can be used in the methods of the present invention.
  • Exemplary microorganisms containing known MDR pumps can be used in the methods of the present invention.
  • MDR pumps are disclosed in, for example, Nikaido, Science, 1994, 264, 382-388, which is incorporated herein by reference in its entirety.
  • Other microorganisms lacking/expressing particular MDR pumps which can be used in the methods of the present invention are listed in Table 1.
  • NorA contributes to high levels of clinical resistance to quinolones in Staphylococcus aureus (S. aureus) infections and is thus an important target for therapy.
  • S. aureus Staphylococcus aureus
  • NorA inhibition by natural MDR inhibitors is of particular relevance to human health application.
  • cells e.g., microorganisms
  • a multidrug resistance pump is generally a better model for studying the kinetics of transport (i.e., efflux) and effects of inhibitors.
  • Studies with E. coli overexpressing the NorA pump have been reported. Ng et al., Antimicrob. Agents Chemother., 1994, 38, 1345-1355.
  • using a gram negative E. coli creates complications arising from transport of substrates and inhibitors across the outer membrane.
  • Preferred microorganisms of the present invention are those shown in Table 1 above and Staphylococcus aureus, Enter ococcus faecalis, Bacillus subtilis, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Saccharomyces cerevisiae and Candida albicans.
  • microorganisms of the present invention are Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Saccharomyces cerevisiae and Candida albicans. And most preferred microorganism of the present invention is S. aureus. It is to be understood that a particular microorganism can be wild-type or mutant cells, and can be recombinant (i.e., transformed) or nonrecombinant.
  • the nor A locus can be amplified using polymerase chain reaction (PCR) from chromosomal DNA (as described in Hsieh et al, Proc. Natl. Acad. Sci. USA, 1998, 95, 6602-6606, which is incorporated herein by reference in its entirety) and cloned into the multicopy S. aureus/E. coli shuttle vector p52.1.
  • the recombinant plasmid can be transformed into the nor A lacking strain S. aureus KLE820.
  • the method of the present invention uses S. aureus KLE820, a nor A lacking mutant.
  • MDR pump substrates of the present invention can be any compound that is effectively excreted (i.e., removed) from the cytoplasm of microorganisms by MDR pump(s).
  • Exemplary MDR pump substrates include ethidium bromide, berberine, palmatine, tetraphenylphosphonium, pentamidine, benzalkonium chloride, chlorhexidine, norfloxacin, and other antibiotics.
  • Preferred MDR pump substrate is berberine and ethidium bromide.
  • MDR pump substrate of the present invention is a NorA substrate, in particular, the MDR pump substrate is ethidium bromide.
  • the presence of a MDR pump inhibitor in a plant extract can be determined by culturing microorganisms with a culture medium comprising the plant extract and a multidrug resistance pump substrate in a sub-inhibitory concentration, i.e., an amount less than the minimum inhibitory concentration (MIC).
  • a sub-inhibitory concentration i.e., an amount less than the minimum inhibitory concentration (MIC).
  • MIC minimum inhibitory concentration
  • the MDR pump substrate inhibits growth of microorganisms.
  • the presence of a MDR pump inhibitor in the extract can be readily determined. For example, if the combination of the extract and the MDR pump substrate inhibits the microorganism growth but the extract alone does not inhibit the microorganism growth, then the extract contains a MDR pump inhibitor.
  • MDR pump inhibitors can also be obtained from a traditional organic synthesis, thereby allowing a wide variety of other flavonolignan derivatives which may not be available from a natural source.
  • a method for preparing MDR pump inhibitors of flavonolignan derivatives includes coupling of coniferyl alcohol to an appropriate compound, e.g., to luteolin for synthesis of hydnocarpin.
  • Such coupling can be accomplished using, for example, horseradish peroxidase (i.e., HRP); thus, as shown in
  • regioisomer 5' -MHC-D (compound 5 of Figure 7) can be prepared by coupling coniferyl alcohol with 3,4-dihydroxy-4-methoxybenzaldehyde (compound 12) and further elaborating the flavonolignan via chalcone (compound 15) as shown in Scheme 2.
  • 5-MHC-D can be prepared by coupling selgin (compound 8 of Scheme 3) with coniferyl alcohol using a coupling agent, e.g., Ag 2 CO or HRP.
  • a coupling agent e.g., Ag 2 CO or HRP.
  • a single regioisomer i.e., 5-MHC-D
  • flavolignans also known as flavonolignans or flavanolignans
  • MDR pump inhibitors e.g., NorA pump inhibitors.
  • compounds 3, 4 and 5 of Figure 7 as well as other analogs are found to be inhibitors of the S. sureus NorA MDR pump.
  • porphyrin derivatives of formula Ul are also good MDR pump inhibitors.
  • pheophorbide a is also a good MDR pump inhibitor.
  • substituents on the compounds of the present invention can be present in the starting compounds, added to any one of the intermediates or added after formation of the final products by known methods of substitution or conversion reactions. If the substituents themselves are reactive, then the substituents can themselves be protected according to the techniques known in the art. A variety of protecting groups are known in the art, and can be employed. Examples of many of the possible groups can be found in Protective Groups in Organic Synthesis, 3rd edition, T.W. Greene and P.G.M. Wuts, John Wiley & Sons, New York, 1999, which is incorporated herein by reference in its entirety.
  • nitro groups can be added by nitration and the nitro group can be converted to other groups, such as amino by reduction, and halogen by diazotization of the amino group and replacement of the diazo group with halogen.
  • Acyl groups can be added by Friedel-Crafts acylation. The acyl groups can then be transformed to the corresponding alkyl groups by various methods, including the Wolff-Kishner reduction and Clemmensen reduction.
  • Amino groups can be alkylated to form mono- and di- alkylamino groups; and mercapto and hydroxy groups can be alkylated to form corresponding ethers.
  • Primary alcohols can be oxidized by oxidizing agents known in the art to form carboxylic acids or aldehydes, and secondary alcohols can be oxidized to form ketones.
  • substitution or alteration reactions can be employed to provide a variety of substituents throughout the molecule of the starting material, intermediates, or the final product, including isolated products.
  • Berberine has low toxicity and has been used in eye drops and at high concentrations orally as an emetic. It is present in many Native American, European and Oriental medicinal plants. MDR inhibitors of formula I, II and/or ILTI can be combined with berberine and other similar alkaloids to produce a natural "synergistic couple" as an effective disinfectant and antiseptic for use in human health applications as well as in agriculture.
  • the low toxicity of berberine and of MDR pump inhibitors allows for effective application of these agents, particularly in topical and systemic use.
  • present inventors have discovered that MDR pump inhibitors of the present invention can be used in conjunction with currently available antiseptics to provide a synergistic effect.
  • antiseptics which are currently available are generally listed in The Merck Index, 11 th Ed., Budavari ed., 1989, Merck & Co., Inc., Rahway, N.J., which is incorporated herein by reference in its entirety, and include benzalkonium chloride, and chlorhexidine.
  • the present inventors have found a synergistic effect against antibiotic resistant S. aureus using benzalkonium chloride and an MDR pump inhibitor of the present invention.
  • Such antiseptic composition can be used to reduce or eliminate the amount of bacteria present in various areas including hospital environments including surgical environments; household areas such as counters (e.g., kitchen or bathroom counters); and even topical applications on animals prior to surgery or other similar treatments.
  • MDR pump inhibitors are in potentiating the action of conventional antibiotics in those cases where MDR pumps are responsible for clinically significant drug resistance.
  • Exemplary clinical drug resistances include quinolone resistance of S. aureus and Streptococcus pneumoniae; broad-range resistance in P. aeruginosa; and azole resistance of Candida species. Lewis et al, ASM News, 1997, 63, 605-610.
  • the MDR pump inhibitors are also effective against P-glycoprotein MDR pump, and thus are useful as anti-cancer agents. Additionally, in some cases P- glycoprotein MDR pumps are believed to be responsible for preventing drugs from crossing the blood-brain barrier; therefore, MDR pumps are also effective in increasing the delivery of drugs across the blood-brain barrier.
  • MDR pump inhibitors are also useful in drug discovery.
  • MDR mutant microorganisms as sensitive tools for drug discovery. See for example, Hsieh et al, Proc. Natl. Acad. Sci. USA, 1998, 95, 6602-6606.
  • Using a broad-range MDR pump inhibitor that blocks many MDR pumps in drug discovery is a more effective tool in drug discovery than using MDR pump mutant microorganisms, especially in species like yeast and fungi that might have over a dozen different MDR pumps.
  • compositions including the compound of formula I, JJ and or III above and a pharmaceutical selected from the group consisting of antimicrobial compounds.
  • the antimicrobial compound is selected from the group consisting of antibiotics and antifungals.
  • Some of the useful antibiotics and antifungals of the present invention are disclosed in The Merck Index, 11 th Ed., Budavari ed., 1989, Merck & Co., Inc., Rahway, N.J., pp. THER-9 to THER-11 and THER-13, pages of which are incorporated herein by reference.
  • Other useful antibiotics include berberine.
  • other useful antifungals include azole compounds such as fluconazole.
  • compositions of the present invention can be administered to a patient to achieve a desired physiological effect.
  • the patient is a plant or an animal, more preferably a crop or a mammal, and most preferably a crop or a human.
  • the compound can be administered in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally.
  • Parenteral administration in this respect includes administration by the following routes: intravenous; intramuscular; subcutaneous; intraocular; intrasynovial; transepithelially including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation and aerosol; intraperitoneal; and rectal systemic.
  • the composition can be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it can be enclosed in hard or soft shell gelatin capsules, or it can be compressed into tablets, or it can be incorporated directly with the food of the diet.
  • the active composition may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparation can contain at least 0.1% of active composition.
  • the percentage of the active compositions and preparation can, of course, be varied and can conveniently be between about 1 to about 10% of the weight of the unit.
  • the amount of active composition in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared such that an oral dosage unit form contains from about 1 to about 1000 mg of an antimicrobial compound and from about 1 to about 100 mg of a MDR pump inhibitor.
  • the tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin can be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, com starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin can be added or a flavoring agent such as
  • tablets, pills, or capsules can be coated with shellac, sugar or both.
  • a syrup or elixir can contain the active compound, sucrose as a sweetening agent, methyl and propylparabens a preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active composition can be incorporated into sustained-release preparations and formulation.
  • the active composition can also be administered parenterally. Solutions of the active composition as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent of dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants it will be preferable to include isotonic agents, e.g., sugars or sodium chloride.
  • Sterile injectable solutions are prepared by incorporating the active composition in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the therapeutic compositions of the present invention can be administered to a mammal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the composition, chosen route of administration and standard pharmaceutical practice.
  • the physician will determine the dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment and it will vary with the form of administration and the particular composition chosen, and also, it will vary with the particular patient under treatment.
  • the physician will generally wish to initiate treatment with small dosages by small increments until the optimum effect under the circumstances is reached.
  • the therapeutic dosage can generally be from about 0.1 to about 1000 mg/day (each of an antimicrobial compound and a MDR pump inhibitor), and preferably from about 10 to about 100 mg/day, or from about 0.1 to about 50 mg/Kg of body weight per day and preferably from about 0.1 to about 20 mg/Kg of body weight per day and can be administered in several different dosage units.
  • composition of the present invention can also be used for prophylaxis or treatment of plants.
  • the composition of the present invention can be diluted in a solvent, e.g., water and or suitable organic solvent(s), and sprayed on to the plant. Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting.
  • This example illustrates a method for determimng a MDR pump inhibition activity of a compound (or an extract) using ethidium bromide as the MDR pump substrate.
  • a compound (or an extract) to be tested is added to the suspension of cells preloaded with EtBr, and the rate of EtBr efflux is measured to determine the MDR pump inhibition activity of the compound.
  • a kinetic analysis is performed to provide information on saturating concentration of an inhibitor; apparent Ki; and the mode of action, i.e., competitive or non-competitive.
  • This example illustrates a method for determining a MDR pump inhibition activity of a compound (or an extract) using berberine as the MDR pump substrate.
  • Procedure of Example 1 is carried out using berberine as a MDR pump substrate. Similar to EtBr, berberine is a DNA intercalating agent and has been used as a
  • DNA fluorochrome Its fluorescence intensity increases sharply upon binding to DNA.
  • Microorganisms are loaded with berberine using the same procedure of Example 1 and efflux is followed fluorometrically in the presence of a compound (or an extract) which is to be tested for its
  • MDR pump inhibitory activity Inhibition of berberine efflux by the compound (or the extract) is determined.
  • A3 This example illustrates a method for determining the MDR pump inhibition activity of MDR pump inhibitor(s) isolated from M.fermontii using NorA as a model MDR pump.
  • Example 4 Using the procedure of Example 2, inhibition of berberine efflux by MDR pump inhibitor(s) isolated from M. fremontii is measured directly. A kinetic analysis is used to determine the Ki and the nature of inhibition (competitive or non-competitive). Example 4
  • This example illustrates a method for determining the MDR pump inhibition activity of a multidrug resistance pump inhibitor using a variety of isoquinoline alkaloids as the MDR pump substrate.
  • MDR pump representatives from all known families are tested for their ability to protect cells from isoquinoline alkaloids using a procedure similar to Example 1. Strains overexpressing and lacking the pumps are tested for this purpose. This survey shows whether isoquinoline alkaloids are natural MDR substrates. Potentiation of alkaloid action is studied in the same experiments in order to determine the spectrum of activity of the MDR pump inhibitors against different MDR pumps.
  • Example 5
  • This example illustrates a synergistic effect of a mixture of berberine and a MDR pump inhibitor against plant pathogens.
  • This example illustrates a method for determining the presence of berberine and or MDR pump inhibitior(s) in various parts of M. fremontii.
  • berberine and/or MDR inhibitor(s) is measured in different parts of M. fremontii (e.g., leaves, twigs, roots and berries) using a procedure similar to Example 2, in order to determine whether berberine and/or MDR pump inhibitor(s) are present in various parts of fremontii.
  • M. fremontii e.g., leaves, twigs, roots and berries
  • This example illustrates a method for determining whether a particular isoquinoline alkaloid is a MDR pump substrate.
  • This example also demonstrates the ability of MHC to inhibit a MDR pump.
  • S. aureus is chosen as a model organism to test the possibility of isoquinoline alkaloids acting as natural substrates of MDR pumps.
  • S. aureus has a well- characterizd NorA pump of the 12 transmembrane segment (TMS) subfamily of Major Facilitators that is chromosomally encoded. It is involved in resistance to cationic antiseptics and disinfectants and confers resistance to hydrophilic quinolones. See for example, Neyfakh et al, Antimicrob.
  • the MICs were determined in micro titer plates by the broth microdilution method in Mueller-Hinton (i.e., MH) medium.
  • Figure 3 a shows efflux of ethidium bromide from wild type and nor A strains of S. aureus in the absence of MHC.
  • the procedure generally involves deenergizing the cells with uncoupler and loading them with EtBr.
  • the graph in Figure 3 a depicts the loss of EtBr from cells resuspended in buffer without EtBr and uncoupler.
  • Figure 3b is a graph showing inhibition of EtBr efflux in wild type cells by MHC. The two experiments were performed on different days, which explains the variation in the absolute rates of active efflux. Fluorescence in both Figures 3a and 3b are in arbitrary units. At 10 ⁇ g mL, MHC appears to completely inhibit efflux (a small residual loss of EtBr in the presence of MHC is due to unavoidable passive diffusion).
  • This example illustrates action of berberine on yeast S. cerevisiae.
  • S. aureus is a human pathogen, and may encounter plant alkaloids consumed by the host, or when it is between hosts in the external environment. Organisms that come into direct contact with plants are expected to have an even stronger incentive to harbor resistance mechanisms against amphiphilic cations. We thus tested the possible action of berberine on yeast S. cerevisiae.
  • Yeast have a large complement of MF MDR pumps and ABC MDR pumps. At least 9 of them contribute to antimicrobial resistance. See for example, Kolaczkowski and Goffeau, Pharm. Therap., 1997, 76, 219-242 and Goffeau, Society for Industrial Microbiology Annual Meeting 1998, Abstract S14. S. cerevisiae appeared fairly resistant to berberine (MIC 120 ⁇ g/mL), but in the presence of a MDR pump inhibitor the MIC dropped significantly.
  • This example illustrates a method for isolating MDR inhibitors from M. fremontii, a producer of berberine.
  • a simple procedure for screening MDR inhibitors is developed using any of the known MDR inhibitor.
  • S aureus cells are inoculated in Mueller-Hinton broth in microtiter plates, as for standard MIC determination by microdilution.
  • Berberine is added at 30 ⁇ g/mL (1/4 MIC) in the presence and absence of the known MDR pump inhibitor. After an overnight incubation, there is comparable growth in control wells (no additions; the known MDR pump inhibitor alone; berberine alone) and no growth in berberine + the known MDR pump inhibitor.
  • plant extracts are added at a series of dilutions into control wells; and into wells containing 30 ⁇ g/mL berberine. Lack of growth in a well with berberine + extract, and normal growth with extract alone signifies a possible presence of an MDR pump inhibitor.
  • M. fremontii, M. repens and M. aquifolium are plants native to the western United States.
  • a preliminary screening of M. fremontii for alkaloids in 1987 was positive for the presence of protoberberinium compounds, including berberine.
  • Identification of alkaloids, including berberine from M. repens, was previously reported by Suess and Stermitz, J. Nat. Prod., 1981, 44, 680-687.
  • the literature contains many references to alkaloid composition of M. aquifolium since it has been cultivated throughout the world as an ornamental. It also contains protoberberinium alkaloids, including berberine.
  • the first treatment of M. fremontii leaves is designed to provide both alkaloid and alkaloid-free extracts.
  • 23 g of dry ground leaves are successively extracted with hexanes and then ethyl acetate to remove lipid and the more nonpolar materials (See flow diagram Fig 4a).
  • the residue from a subsequent methanol extract is triturated with 0.1 M aq. HC1 and the acidic solution is then extracted with chloroform. Evaporation of the chloroform layer yields about 159 mg of an "alkaloid-free" residue.
  • the pH of the aqueous layer is adjusted to about pH 10 with hydroxide and then reextracted with chloroform.
  • the chloroform layer upon evaporation, yields about 16 mg of an "alkaloid-containing" residue.
  • the 159 mg of alkaloid-free residue shows MDR inhibitory activity at 75 ⁇ g/mL when tested against S. aureus in the presence of a sub- MIC concentration of berberine.
  • the 16 mg of alkaloid-containing residue shows MDR inhibitory activity a 163 ⁇ g/mL in a similar test. These two fractions show no antimicrobial activity when added at a concentration of 500 ⁇ g/mL in the absence of berberine.
  • This example illustrates another method for isolating MDR inhibitors from M. fremontii, a producer of berberine.
  • Tetracycline is not an MDR pump substrate, and the fraction has no effect on tetracycline's ability to inhibit the growth of microorganisms (See Table in in Example 12) indicating that the extract appears to contain a specific MDR pump inhibitor.
  • This example illustrates the ability of the MDR inhibitor containing fraction from M. fremontii to potentiate the action of a variety of antimicrobial compounds.
  • MDR inhibitor(s) to potentiate the action of berberine, norfloxacin, pentamidine and tetracycline is tested and the results are shown in Table IV.
  • Example 13 This example illustrates characterization of an MDR pump inhibitor isolated from M. fremontii.
  • Fractions 4-14 of the chromatography separation (Fig. 5) containing the chloroform-soluble MDR inhibitor are used singly or in combination for purification of the active species. Further separations are conducted on reverse phase (C ⁇ 8 ) silica gel columns, using an eluting mixture of chloroform/ethyl acetate/acetone 7:1:2 containing a trace of acetic acid. This yields two active materials, 5'-methoxyhydnocarpin-D and a mixture dominated by the porphyrin derivative compound, pheophorbide a. MHC-D is identified by NMR, mass spectroscopy and UV spectroscopy. The spectral data correlate well with literature data.
  • a bioassay-driven purification of the second MDR inhibitor from the main alkaloid-containing fraction is also undertaken.
  • the initial method for separation of an extract into alkaloid-containing vs. alkaloid-free fractions via differential pH extraction is a preferred method.
  • the basified solution is extracted with chloroform for the isolation of the inhibitor.
  • high pressure liquid chromatography is a preferred method.
  • Separation procedures and choice of detection methods depend on the chemical nature of the active component(s) as determined by nuclear magnetic resonance (NMR) and mass spectrometric (MS) analysis of fractions obtained in the preliminary column chromatographic separations. See “Detection and isolation of bioactive natural products. In Bioactive natural products. Detection, isolation and structural determination," by Ghisalberti, CRC Press, Boca Raton 1993, Clegate and Molyneux eds.
  • This example illustrates synthesis of 7,8,4'-flavonolignan.
  • This synthetic method is applicable for preparing other flavonolignan derivatives.
  • the reaction was placed in a 55-60 °C oil bath and stirred for 20 min.
  • 0.625 g (0.228 mmol) of Ag 2 CO 3 was added to the reaction flask and the reaction stirred 24 h.
  • Example 19 This example illustrates synthesis of 3-[2-hydroxymethyl-8-methoxy-3-(3- methoxy-4-methoxymethoxy-phenyl)-2,3-dihydro-benzo[l,4]dioxin-6-yl]-l-(2,4,6-tris- methoxymethoxy-phenyl)-propenone (compound 14 of Scheme 2).
  • This example illustrates synthesis of 3-[2-hydroxymethyl-8-methoxy-3-(3- methoxy-4-methoxymethoxy-phenyl)-2,3-dihydro-benzo[l,4]dioxin-6-yl]-l -(2,4,6- trihydroxy-phenyl)-propenone (compound 15 of Scheme 2).
  • compound 14 To a 25 mL three-neck round-bottomed flask was added 0.042 g of compound 14, 5 mL of methanol, and 2 drops of concentrated HC1. This solution was stirred at rt for 17 h, poured into a saturated sodium bicarbonate solution, and extracted with EtOAc.
  • This example illustrates synthesis of 5,7-dihydroxy-2-[3-(4-hydroxy-3- methoxy-phenyl)-2-hydroxymethyl-8-methoxy-2,3,-dihydro-benzo[l,4]dioxin-6-yl]- chroman-4-one (compound 16 of Scheme 2).
  • compound 16 of Scheme 2 To a 50 mL three-neck round-bottomed flask was added 0.021 g (0.043 mmol) of compound 15, 15 mL of methanol, and 0.035 g (0.43 mmol) of NaOAc. This solution was heated at reflux for 3 h, poured into a saturated sodium bicarbonate solution, and extracted with EtOAc.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé permettant de déterminer la présence d'un inhibiteur de pompe à résistance multiple (MDR) dans une source produisant des antibiotiques naturels, dont les plantes et les micro-organismes, tels que les champignons et les bactéries. La plante est sélectionnée parmi celles produisant des cations amphiphiles, tels que des isoquinolines, p. ex. la berbérine et les alcaloïdes protoberberinium. La présente invention concerne également des flavolignans et des composés dérivés de la porphyrine ayant été identifiés selon le procédé de la présente invention comme étant des inhibiteurs de pompe MDR.
PCT/US2000/020001 1999-07-23 2000-07-21 Inhibiteurs de pompe a resistance multiple et utilisations WO2001007034A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63660/00A AU6366000A (en) 1999-07-23 2000-07-21 Multidrug resistance pump inhibitors and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14547499P 1999-07-23 1999-07-23
US60/145,474 1999-07-23

Publications (1)

Publication Number Publication Date
WO2001007034A1 true WO2001007034A1 (fr) 2001-02-01

Family

ID=22513282

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/020001 WO2001007034A1 (fr) 1999-07-23 2000-07-21 Inhibiteurs de pompe a resistance multiple et utilisations

Country Status (2)

Country Link
AU (1) AU6366000A (fr)
WO (1) WO2001007034A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079257A3 (fr) * 2000-04-14 2002-03-21 Phytera Inc Pompes d'ecoulement mdr
US7011957B2 (en) 2001-09-26 2006-03-14 Northeastern University Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon
EP1591112A4 (fr) * 2003-02-04 2006-06-14 Yakult Honsha Kk Inhibiteur de la proteine resistant au cancer du sein
EP2014651A1 (fr) * 2007-07-12 2009-01-14 Exonhit Therapeutics SA Composants et procédés de modulation de Rho GTPases
EP2687097A1 (fr) * 2012-07-16 2014-01-22 Universite D'angers Agents de potentialisation pour protéger des plantes contre des infections fongiques
WO2014077224A1 (fr) * 2012-11-13 2014-05-22 学校法人順天堂 Agent antimicrobien
WO2015184296A1 (fr) * 2014-05-30 2015-12-03 Friedhoff Lawrence T Compositions pharmaceutiques antimicrobiennes possédant des propriétés inhibitrices de la multirésistance aux médicaments
CN109364071A (zh) * 2019-01-02 2019-02-22 李萍 一种促唾液分泌治疗口腔干燥综合征的药物及用途
CN110639017A (zh) * 2019-09-19 2020-01-03 安徽中医药大学 一种抑制白色念珠菌的药物组合物
US20210246126A1 (en) * 2018-06-09 2021-08-12 Jiangsu Atom Bioscience Pharmaceutical Co., Ltd. Compound for treatment or prevention of liver diseases
CN114105963A (zh) * 2021-12-16 2022-03-01 贵州大学 一种抗结核杆菌化合物及其制备方法与应用
US11566260B2 (en) 2012-07-16 2023-01-31 Universite D'angers Potentiating agents for protecting plants from fungal infections
CN116270489A (zh) * 2023-03-22 2023-06-23 遵义市中医院 一种姜功胃安颗粒及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648365A (en) * 1993-01-21 1997-07-15 Merrell Pharmaceuticals Inc. Diarylalkyl piperidines useful as multi-drug resistant tumor agents
US5665780A (en) * 1992-02-06 1997-09-09 Merrell Pharmaceuticals Inc. Reversal of multi-drug resistance by tetraarylethylenes
EP0813872A1 (fr) * 1996-06-18 1997-12-29 Kureha Chemical Industry Co., Ltd. Dérivés de la berbérine pour l'inhibition de la production de hsp 27
US5763443A (en) * 1994-04-05 1998-06-09 Universiteit Van Pretoria MDR resistance treatment and novel pharmaceutically active riminophenazines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5665780A (en) * 1992-02-06 1997-09-09 Merrell Pharmaceuticals Inc. Reversal of multi-drug resistance by tetraarylethylenes
US5648365A (en) * 1993-01-21 1997-07-15 Merrell Pharmaceuticals Inc. Diarylalkyl piperidines useful as multi-drug resistant tumor agents
US5763443A (en) * 1994-04-05 1998-06-09 Universiteit Van Pretoria MDR resistance treatment and novel pharmaceutically active riminophenazines
EP0813872A1 (fr) * 1996-06-18 1997-12-29 Kureha Chemical Industry Co., Ltd. Dérivés de la berbérine pour l'inhibition de la production de hsp 27

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079257A3 (fr) * 2000-04-14 2002-03-21 Phytera Inc Pompes d'ecoulement mdr
US7011957B2 (en) 2001-09-26 2006-03-14 Northeastern University Isolation and cultivation of microorganisms from natural environments and drug discovery based thereon
EP1591112A4 (fr) * 2003-02-04 2006-06-14 Yakult Honsha Kk Inhibiteur de la proteine resistant au cancer du sein
EP2014651A1 (fr) * 2007-07-12 2009-01-14 Exonhit Therapeutics SA Composants et procédés de modulation de Rho GTPases
US11566260B2 (en) 2012-07-16 2023-01-31 Universite D'angers Potentiating agents for protecting plants from fungal infections
EP2687097A1 (fr) * 2012-07-16 2014-01-22 Universite D'angers Agents de potentialisation pour protéger des plantes contre des infections fongiques
WO2014012766A1 (fr) * 2012-07-16 2014-01-23 Université d'Angers Agents potentialisateurs pour protéger des plantes d'infections fongiques
US10405550B2 (en) 2012-07-16 2019-09-10 Universite D'angers Potentiating agents for protecting plants from fungal infections
WO2014077224A1 (fr) * 2012-11-13 2014-05-22 学校法人順天堂 Agent antimicrobien
WO2015184296A1 (fr) * 2014-05-30 2015-12-03 Friedhoff Lawrence T Compositions pharmaceutiques antimicrobiennes possédant des propriétés inhibitrices de la multirésistance aux médicaments
US11643405B2 (en) * 2018-06-09 2023-05-09 Jiangsu Atom Bioscience And Pharmaceutical Co., Ltd. Compound for treatment or prevention of liver diseases
US20210246126A1 (en) * 2018-06-09 2021-08-12 Jiangsu Atom Bioscience Pharmaceutical Co., Ltd. Compound for treatment or prevention of liver diseases
CN109364071A (zh) * 2019-01-02 2019-02-22 李萍 一种促唾液分泌治疗口腔干燥综合征的药物及用途
CN110639017A (zh) * 2019-09-19 2020-01-03 安徽中医药大学 一种抑制白色念珠菌的药物组合物
CN114105963A (zh) * 2021-12-16 2022-03-01 贵州大学 一种抗结核杆菌化合物及其制备方法与应用
CN114105963B (zh) * 2021-12-16 2023-08-18 贵州大学 一种抗结核杆菌化合物及其制备方法与应用
CN116270489A (zh) * 2023-03-22 2023-06-23 遵义市中医院 一种姜功胃安颗粒及其制备方法
CN116270489B (zh) * 2023-03-22 2023-09-08 遵义市中医院 一种姜功胃安颗粒及其制备方法

Also Published As

Publication number Publication date
AU6366000A (en) 2001-02-13

Similar Documents

Publication Publication Date Title
Urbina et al. Inhibitors of the fungal cell wall. Synthesis of 4-aryl-4-N-arylamine-1-butenes and related compounds with inhibitory activities on β (1–3) glucan and chitin synthases
Clark et al. Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance
WO2001007034A1 (fr) Inhibiteurs de pompe a resistance multiple et utilisations
Svetaz et al. Antifungal activity of Zuccagnia punctata Cav.: evidence for the mechanism of action
Lee et al. 5-Hydroxyindole-type alkaloids, as Candida albicans isocitrate lyase inhibitors, from the tropical sponge Hyrtios sp.
EP0282507B1 (fr) Compositions a base de discorhabdine et procedes d'utilisation
Cho et al. Spatholobus suberectus Dunn. constituents inhibit sortase A and Staphylococcus aureus cell clumping to fibrinogen
Li et al. Discovery of novel simplified isoxazole derivatives of sampangine as potent anti-cryptococcal agents
JP5956439B2 (ja) 抗菌剤組成物
KR20000075672A (ko) 항균제
WO2002036203A2 (fr) Composes antifongiques et utilisations associees
Rząd et al. A new 1-nitro-9-aminoacridine derivative targeting yeast topoisomerase II able to overcome fluconazole-resistance
Alaqeel et al. Antimicrobial activities of novel class of dispirooxindolopyrrolidine grafted indanedione hybrid heterocycles against carbapenemase producing Klebsiella pneumoniae (CKP)
Prats et al. Antifungal activity of a new phenolic compound from capitulum of a head rot-resistant sunflower genotype
Choi et al. Isolation of (-)-olivil-9′-O-β-d-glucopyranoside from Sambucus williamsii and its antifungal effects with membrane-disruptive action
Souza et al. A New Prenylisoflavone from the Antifungal Extract of Leaves of Vatairea guianensis Aubl.
WO1999017760A2 (fr) Inhibiteurs de pompes d'efflux fongiques
KR20120079281A (ko) 펩티드 디포밀라제 저해 및 항균 활성을 갖는 신규한 프라비마이신 화합물
US5128344A (en) Sampangine and derivatives useful as an antifungal agent
Thissera et al. Potent antiplasmodial alkaloids from the rhizobacterium Pantoea agglomerans as hemozoin modulators
US4965272A (en) Antimicrobial compound and compositions particularly effective against candida albicans
KR101772680B1 (ko) 마그노로사이드 에이 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항진균용 조성물
Insuasty et al. Fungicide Activity of 5‐(4‐Chlorobenzylidene)‐(Z)‐2‐dimethylamino‐1, 3‐thiazol‐4‐one against Cryptococcus Neoformans
Dischler Investigations of targeted natural sources in search of bioactive metabolites
Abdulrazik et al. Structure elucidation and anti-Klebsiella activity with in silico ADME prediction and molecular docking of the bioactive metabolite from Aspergillus sp. 3MAG

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载