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WO1990004177A1 - Procede permettant de deceler la presence de cellules tumorales a resistance multiple aux medicaments et sonde verapamil utile a cette fin - Google Patents

Procede permettant de deceler la presence de cellules tumorales a resistance multiple aux medicaments et sonde verapamil utile a cette fin Download PDF

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Publication number
WO1990004177A1
WO1990004177A1 PCT/US1989/004394 US8904394W WO9004177A1 WO 1990004177 A1 WO1990004177 A1 WO 1990004177A1 US 8904394 W US8904394 W US 8904394W WO 9004177 A1 WO9004177 A1 WO 9004177A1
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derivative
verapamil
polypeptide
sample
concentration
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Ahmad R. Safa
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Michael Reese Hospital and Medical Center
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Michael Reese Hospital and Medical Center
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9453—Cardioregulators, e.g. antihypotensives, antiarrhythmics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance

Definitions

  • the present invention relates to a method of detecting multiple drug resistance in tumor cells, a method of treatment of such tumors, and a method and kit for the detection of a verapamil binding polypeptide, P-glycoprotein, that is characteristically present as an integral membrane glycoprotein in multiple drug resistant (MDR) tumor cells. It also relates to derivatives of verapamil which are utilized to characterize this polypeptide and to a method of synthesis of such derivatives.
  • MDR multiple drug resistant
  • Affinity labeling of proteins with photoactive ligands is a powerful tool in probing biochemical targets.
  • photoaffinity labeling has been used for the identification, purification and characterization of mediators of biological, physiological and pharmacological activities.
  • the photoaffinity labeling technique allows for the investigation of drug-protein interactions with the general goal of identification of an acceptor molecule in a mixture of candidates.
  • a reversible complex presumably forms between the photoactive drug derivative and unique acceptor sites of specific polypeptides which preferentially recognize the characteristic structure of the drug derivative.
  • the drug derivative Upon irradiation with UV light, the drug derivative is converted into a highly reactive nitrene intermediate which covalently ' interacts with the acceptor sites.
  • a particular functional group at the acceptor site need not be present because the photogenerated species can react even with carbon-hydrogen bonds.
  • Multidrug resistance refers to patterns of cross-resistance that develop in tumor cells selected by using a single natural product drug. Exposure to natural product drugs such as vinblastine, vincristine, doxorubicin, or colchicine confers resistance to a wide range of compounds with no apparent structural or functional similarities to the selective agent.
  • MDR is frequently characterized by diminished drug accumulation in resistant cells compared to drug-sensitive cells. This reduced accumulation often correlates with the concomitant over-expression of a 150-180 kilodalton (kDa) molecular-weight integral membrane glycoprotein, P-glycoprotein (P-gp) or gp 150-180, which is produced in MDR cells in proportion to the cellular level of drug-resistance.
  • kDa kilodalton
  • P-gp P-glycoprotein
  • gp 150-180 150-180
  • verapamil which is known to be useful as a calcium channel blocker, has the property of binding to P-gp, and thereby the property of assaying P-gp when the verapamil is appropriately labelled.
  • the present invention contemplates labelled verapamil derivatives that correspond to the general structure
  • R' is a labeling group.
  • labeling groups are cross-linking agents, radio-labelled ligands and fluorescent radicals.
  • Cross-linking agents are contemplated to encompass photoaffinity ligands and chemoaffinity ligands.
  • Preferred labelled verapamil derivatives are
  • N-(p-azido salicyl)aminomethyl verapamil N-(p-azido-[3- 125 I]salicyl)aminomethyl verapamil, 5-[ (3,4-dimethoxyphenethyl)- methylaraino]-2-(3,4-dimethoxyphenethyl)-2-isopropyl- pentyl fluores ⁇ ein, and 5-[ (3,4-dimethoxyphenethyl)methylamino]-2-(3,4- dimethoxyphenyl)-2-isopropylpentyl bromo acetate.
  • the verapamil derivative has a photoaffinity labeling group coupled to it, and that the derivative is also radio-labelled. It is also contemplated that upon binding of the labelled verapamil derivative to a polypeptide to form a derivative-polypeptide complex, covalent bonding is induced between the photoaffinity ligand and the polypeptide by irradiating the complex with ultraviolet or other actinic light. This irradiation supplies the activation energy necessary to enable the photoaffinity ligand to form a covalent linkage with the polypeptide.
  • a composition is also contemplated that contains a labelled verapamil derivative of the present invention together with an aqueous carrier.
  • the present invention also contemplates a method for the detection of multiple drug resistant tumor cells.
  • This method assays for a verapamil binding polypeptide that is characteristically present in multiple drug resistant (MDR) tumor cells as the P-glycoprotein (P-gp) .
  • MDR multiple drug resistant
  • P-gp P-glycoprotein
  • the present method utilizes labelled verapamil derivatives that bind to the P-glycoprotein, are subsequently detected and quantified. Comparison of the concentration of P-gp in normal and tumor tissue and provides a means for diagnosing multiple drug resistance in the tumors.
  • a sample of mammalian cellular material is admixed and maintained with an effective binding amount of a labelled verapamil derivative for a time period sufficient to permit a binding between the verapamil derivative and the polypeptide that may be present and to form a verapamil derivative- polypeptide complex.
  • a photoaffinity-labelled verapamil derivative is utilized and the admixture is thereafter irradiated with actinic light of an appropriate wavelength and in a sufficient amount to form a covalent bond between the reacted drug derivative and the polypeptide.
  • the concentration of the polypeptide present is quantified by measuring the concentration of bound verapamil derivatives.
  • a method of treatment is also contemplated in the present invention in which the concentration of verapamil binding polypeptide is determined in a sample tumor to be treated. If the concentration of the verapamil binding polypeptide in the tumor sample is about five fold greater than the concentration of verapamil binding polypeptide present in normal cells of the same cell type as the tumor, then the tumor is treated as a multiple drug resistant cell tumor.
  • Such a tumor is treated with a natural product-type chemotherapeutic agent together with a modulator compound, such as verapamil.
  • a method of determining the concentration of verapamil binding polypeptides in cellular material, as well as a diagnostic kit for carrying out the detection and quantifying of verapamil binding polypeptides in samples are also contemplated in the present invention.
  • the present invention has the benefit of enabling an early diagnosis and classification of a tumor as to whether it is of the multiple drug resistant phenotype. This rapid classification allows targeting of appropriate therapy against the tumor and helps to eliminate the initial utilization of chemotherapeutic drugs which would be ineffective toward the particular MDR tumor. In prior practice, MDR tumors were detected as a result of their resistance to the chemotherapeutic agents utilized. The present invention prevents the delay resultant from the prior trial and error approach. Description of Figures
  • Figure 1 illustrates the structural formulae of verapamil, and two radiolabelled photoaffinity derivatives of verapamil N-( ⁇ -azido-
  • Figure 2 is a two-part graph that illustrates the low resolution fast atom bombardment mass spectrograph of NABAV in which "m/z" is the mass to charge ratio.
  • Figure 3 is a two-part graph similar to that of Figure 2 that illustrates the low resolution fast atom bombardment mass spectrograph of NASAV.
  • Figure 4 illustrates the ultraviolet absorption spectra for NABAV and NASAV, respectively, in which the ordinates are in optical density units and the abscissas are in nanometers (nm) .
  • Figure 5 illustrates the infrared absorption spectra for verapamil, NABAV and NASAV, respectively, in which the abscissas are in units of centimeters -1 (cm -1 ) .
  • the present invention relates to (a) the synthesis of photoactive derivatives of verapamil, (b) the synthesis of the corresponding radioactive derivatives, (c) the synthesis of chemoaffinity derivatives of verapamil, (d) the use of these derivatives to detect multidrug resistant tumor cells and (e) the use of these compounds in the diagnosis and treatment of multiple drug resistant tumors.
  • Verapamil a phenylalkyla ine calcium channel blocker, ' has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells.
  • the pharmacologically active, radioactive, photoactive verapamil derivatives of the present invention are capable of covalently binding to unique cellular polypeptides which have high affinity for the parent compound. Tritiated and radioiodinated photoaffinity derivatives of verapamil were used to directly identify P-gp in MDR cells as a specific acceptor for verapamil.
  • photoactive radioactive drugs are important probes, and development of photoaffinity labeling technology with the photoactive derivatives of verapamil facilitates detection of P-glycoprotein in a large number of human tumor samples, expedites the evaluation of large numbers of potential modulating agents, and leads to greater understanding of the function of P-glycoprotein.
  • Such studies enable physicians and researchers to identify therapeutic compounds which circumvent MDR and thus aid in the design of agents whose major and perhaps only pharmacological activity is the reversal of MDR.
  • the compounds of the present invention are valuable in identifying cellular receptors which may be involved in novel, as well as in known, mechanisms of verapamil action.
  • Two compounds of the present invention are radiolabelled photoactive derivatives of verapamil: 1) N-(£-azido[3,5 H]benzoyl)aminomethyl verapamil or N-(p_-azido-[3,5 H]benzoyl-5-[ (3,4-dimethoxyphenethyl)methy1 amino]-2(3,4-dimethoxyphenyl)-2-isopropylpentyl-
  • Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive derivatives as well as with verapamil.
  • exemplary photoaffinity labeling agents are: 4-azidobenzoi ⁇ a ⁇ id-N-hydroxysuccinimide ester.
  • P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 micromolar (uM) compared to that by verapamil at 8 uM.
  • Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubi ⁇ in and actinomycin D, at 100 uM, but not by colchicine.
  • a method for assaying tumor cells to ascertain whether they are multiple drug resistant is provided by the present invention. Since multiple drug resistant cell tumors are refractory to conventional chemotherapeutic approaches, the foreknowledge of whether the tumor is MDR can minimize the chances that ineffective therapy will be pursued. This allows a method of treatment to be utilized in the present invention whereby tumors are assayed for multiple drug resistance by the detection of elevated concentrations of verapamil binding protein (P-gp) in the tumors.
  • P-gp verapamil binding protein
  • a sample of tumor tissue is admixed with a composition containing a labelled verapamil derivative.
  • concentration of the verapamil derivative is about 4 to about 100 nanomolar (nM) in an aqueous buffer such as 40 mM potassium phosphate (pH 7.0).
  • the admixture is maintained at about 25°C. for about 30 minutes.
  • the tissue is separated from the admixture, such as by centrifugation, and resuspended in buffer. If a photoaffinity labelled verapamil derivative was used, the suspension is then irradiated for 15 minutes with ultraviolet light, as described hereinafter.
  • the concentration of P-gp is then determined by an appropriate means such as scintillation counting, autoradiography or spectrofluorimetry. A determination that the concentration of P-gp in the tumor tissue is significantly elevated (by over five fold) over the concentration present in cells derived from the same tissue type but not multiple drug resistant is an indication that the tumor is multiple drug resistant.
  • a natural product type chemotherapeutic agent e.g., vinblastine, vincristine and adriamycin.
  • exemplary modulator compounds are verapamil and trifluoperazine.
  • the present invention also encompasses a diagnostic kit for the determination of the concentration of verapamil binding polypeptide (P-gp) present in a sample.
  • This kit includes at least one package that contains a binding composition comprising a labelled verapamil derivative and a carrier.
  • the labelled verapamil derivative is present in an amount sufficient to carry out at least one assay.
  • Instructions for use of the kit, and an indicating means to enable detection and quantification of the formation of a complex formed between the labelled verapamil derivative and the polypeptide are typically also included in a kit.
  • the binding composition contains a verapamil derivative as described in the present invention.
  • Such verapamil derivative may have attached to it a photoaffinity or chemoaffinity ligand.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperatures, buffer conditions and the like.
  • the "indicating means”, as used herein, in its various grammatical forms refers to single atoms or molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of the derivative-polypeptide complex. Particularly preferred indicating means are radiolabels or fluorescent molecules attached to the verapamil derivative.
  • Example 1 Synthesis of (+)-5-[ (3,4-Dimethoxy- phenethyl)methylamino]-2-(3,4-dimethoxy- phenyl)-2-isopro ⁇ ylpentylamine (DMDI) .
  • DMDI isopro ⁇ ylpentylamine
  • Verapamil hydrochloride 1.5 gm, 3.05 mmol in dry tetrahydrofuran (THF) (50 ml) was admixed with lithium aluminum hydride (500 mg (13.2 mmol) 0.5 M solution in diglyme) .
  • the mixture was stirred for 4 hours with refluxing at 70-75°C , cooled, and quenched with 5% NaOH (30 ml).
  • the mixture was then stirred for 30 minutes at room temperature, placed in a glass syringe and filtered through a solvent resistant Milex-SR 0.5 mM filter unit (Milipore) . The residue was washed with ether (50 ml).
  • the photoactive phenylalkylamine derivative N-(£-azidobenzoyl-5-[ (3,4-dimethoxyphenethyl)methyl- amino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine or N-(£-azidobenzoylaminomethyl verapamil (NABAV) was synthesized by azidobenzoylation of (+)-5-[(3,4 dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl) -2-isopropyl-pentylamine with N-hydroxysu ⁇ cinimidyl-4- azidobenzoate.
  • the synthesis was carried out by dissolving DMDI (5 uM) in chloroform (1 ml). To this solution was added N-hydroxysuccinimidyl-4- azidobenzoate (NAB) (10 umol) (Pierce Chemical Co. , Rockford, IL) . The reaction mixture was maintained at 40°C overnight. Progress of the reaction was checked by silica gel thin layer chromatography (TLC) with a fluorescent indicator. The product was purified by silica gel column chromatography with 2% (vol/vol) methanol in chloroform.
  • NAB N-hydroxysuccinimidyl-4- azidobenzoate
  • N- (p-azidosali ⁇ yl)-aminomethyl verapamil was synthesized from (+)-5-[ (3,4-dimethoxyphenethyl) methylamino]-2(3,4-dimethoxyphenyl)-2-isopropylpentyl ⁇ amine and N-hydroxysuccinimidyl-4-azidosalicylate and was purified according to the method described for NABAV in Example 2, with the exception that 3% methanol in chloroform was used during purification.
  • Fig. 1 were analyzed by low and high-resolution fast atom bombardment (FAB) mass spe ⁇ troscopy, as described by (Safa et al., (1987) J. Biol. Chem. 262, 1261-1267) , using m-nitro benzyl alcohol (NBA) as a matrix.
  • the accurate mass measurements for the (M+H) ions from these samples were obtained using a cluster ion of NBA at m/z 613 (613.178197) as reference under high resolution conditions.
  • the low resolution FAB spectra of NABAV and NASAV exhibited the protonated molecular ions at m/z 604 and 620, respectively (Fig. 2) .
  • the photoactive derivatives exhibited a UV-visible absorption spectrum consisting of a composite of the absorption spectra of verapamil and either the azidobenzoate or azidosalicylate chromophore (Fig. 4). Upon UV irradiation, the absorption peak between 250 and 400 nm was lost, yielding a spectrum similar to verapamil. The IR spectrum showed a strong resonance at 2130 cm indicating the presence of azide (F.ig. 5) .
  • N-Hydroxysuccinimidyl- 4-azido-[3,5- 3 H]-benzoate (47.7 Ci/mmol) in 0.05 ml isopropanol (New England Nuclear, Boston, MA) was added to 0.45 ml solution of DMDI (0.5-0.7 umol) in chloroform and the mixture was kept at 4°C for 24 hours.
  • the product was purified by silica gel column chromatography, according to the method described for the non-radioactive compound in Example 2. Identity and purity of the compound were confirmed by co-chromatography. ith NABAV by TLC run in solvent I.
  • the product of the first reaction, above, was dissolved in chloroform (0.45 ml), DMDI (0.5 umole) was added and the mixture was maintained at 4°C. The progress of the reaction was monitored by TLC (run in Solvent II, R f 0.67). The reaction mixture was then applied to a column (0.5 x 7 cm) of silica gel equilibrated in chloroform. After washing with chloroform (5 ml) and of 1% methanol in chloroform (5 ml) , the product was eluted from the column with 5% methanol in chloroform. The product gave a single radioactive spot on silica gel TLC with a when run in solvent II as determined by autoradiography.
  • Chemoaffinity ligands of verapamil are synthesized by coupling the intermediate DMDI to appropriate group-specific reagents (e.g., bromoacetyl electrophile using
  • Verapamil has cyano group that forms a primary alkylamine that can be readily linked to various functional groups.
  • nonradioactive compounds are synthesized and tested for their ability to block verapamil photolabeling.
  • Compounds exhibiting apparent specific effects are then prepared with radioactive label for the identification of verapamil binding sites.
  • the reaction of DMDI with N-su ⁇ inimidyl bromoacetate in chloroform provides 5-(3,4-dimethoxyphenethyl)- methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl-
  • Radioactive derivatives are prepared using N-succinimidylbromo [2- H]acetate.
  • Verapamil derivatives containing maleimide reactive groups are prepared by coupling aminomethyl verapamil to commercially available N-alkylraaleimide ⁇ arboxyli ⁇ acids by carbodiimide catalyzed condensations.
  • Example 7 Synthesis of Fluorescent Verapamil Derivatives
  • a fluorescent verapamil derivative, 5-[3,4-Dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxy ⁇ henyl)-2-isopro ⁇ yl ⁇ entyl fluorescein (fluorescein verapamil) is synthesized and used 1) to detect P-gp in MDR cells in human tumors and 2) to investigate the subcellular distribution and/or the intracellular processing of verapamil specific P-gp.
  • the fluorescent verapamil is synthesized by the addition of 1 ml of 1.0 mM DMDI to a chloroform solution of 7 mM fluorescein isothiocyanate. The mixture is incubated overnight at room temperature, and the reaction mixture is separated by TLC. The product is purified by reversed phase HPLC.
  • Sensitive DC3F Chinese hamster lung cells and vincristine-resistant variant DC-3F/VCD-5L cells that were selected for primary resistance to vin ⁇ ristine (2750-fold resistant) and cross-resistance to doxorubicin (220-fold) , actinomycin D (1000-fold) , and colchicine (1000-fold) were supplied by Dr. June L. Biedler (Memorial Sloan-Kettering Cancer Center, New York) and were maintained as described in Peterson et al. , (1983)
  • Exponentially growing cells were harvested by scraping culture plates with a rubber blade.
  • Cell membrane vesicles were prepared by nitrogen cavitation and differential centrifugation as described by Lever, (1977) _. Biol. Chem. 252, 1990-1997. Protein concentrations were determined by the procedure of Lowry et al., (1951) J. Biol. Chem. 193, 265-275. Membrane vesicles (5-10 mg of protein) in 40 mM potassium phosphate buffer (pH 7.0) containing 4% (vol/vol) dimethylsulfoxide and 4 nM [ 1 5 I] NASAV in a final volume of 0.05 ml were photolabelled after preincubation for 30 minutes at 25°C in the absence or presence of nonradioactive competing ligand. Samples were then irradiated for 15 minutes with two 15 watt self-filtering 302 nm or 366 nm lamps (model XX-15, Ultraviolet Products, San Gabriel, CA) .

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Abstract

On décrit un procédé permettant de déceler la présence de cellules tumorales à résistance multiple aux médicaments qui consiste à mélanger un échantillon de tissu cellulaire tumoral de mammifère avec une quantité suffisante de dérivé liant de vérapamil étiqueté pour former un complexe des P-gp (glycoprotéines) présentes dans l'échantillon, et à déterminer ensuite la quantité de P-gp présente en quantifiant la quantité de dérivé de vérapamil étiqueté lié dans le complexe. On décrit des dérivés de vérapamil étiquetés par photoaffinité, irradiation et affinité chimique ainsi que les procédés de synthèse de tels dérivés, de même qu'un procédé de traitement pour des tumeurs résistant aux médicaments multiples et un nécessaire pour les diagnostiquer.
PCT/US1989/004394 1988-10-03 1989-10-03 Procede permettant de deceler la presence de cellules tumorales a resistance multiple aux medicaments et sonde verapamil utile a cette fin Ceased WO1990004177A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0568126A1 (fr) * 1992-04-06 1993-11-03 Silvia Maria Doglia Procédé pour la détermination de la résistance multidrogue dans des cellules vivantes
US5436243A (en) * 1993-11-17 1995-07-25 Research Triangle Institute Duke University Aminoanthraquinone derivatives to combat multidrug resistance
FR2729569A1 (fr) * 1995-01-19 1996-07-26 Consultants Internationaux Sur Compositions pharmaceutiques utiles pour la reversion de la resistance multiple aux drogues
WO1998004250A1 (fr) * 1996-07-25 1998-02-05 Consultants Internationaux Sur Le Medicament (S.A.R.L.) Compositions pharmaceutiques utiles pour la reversion de la resistance multiple aux drogues
KR20020045445A (ko) * 2000-12-11 2002-06-19 최철희 독소루비신 내성 급성 골수성 백혈병 아세포주를 이용한내성억제물질과 반응성 산소종 생성 세포독성물질의선별방법

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 43, issued 1983, TSURUO et al.: "Potentiation of Vincristine and Adriamycan Effects in Human Hemopooetic Tumor Cell Lines by Calcium Antagonists and Calmodulin Inhibitors", pages 2267-2272. *
CANCER RESEARCH, Volume 46, issued 1986, KLOHS et al.: "Resistance to Anthrapyrazoles and Anthracyclines in Multidrug-resistant P388 Murine Leukemia Cells: Reversal by Calcium Blockers and Calmodulin Antogonists", pages 4352-4356. *
CANCER TREAT. REP., Volume 67, issued 1983, TSURUO, T.: "Reversal of acquired resistance to vinca alkaloids and anthracycline antibiotics", pages 889-894, Abstract. *
CELL, Volume 47, issued 1985, GROSS et al.: "Mammalian Multidrug Resistance Gene: Complete cDNA Sequence Indicates Strong Homology to Bacterial Transport Proteins", pages 371-380, see p. 371 and 374, column 1. *
J. NEUROSURG., Volume 63, issued 1985, KABA et al.: "Potentiation of vincristine effect in human and murine gliomas by calcium channel blockers or calmodulin inhibitors", pages 905-911, Abstract. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 261, issued 1986, SAFA et al.: "Vinblastine Photoaffinity Labeling of a High Molecular Weight Surface Membrane Glycoprotein Specific for Multidrug-resistant Cells", page 6137-6140. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 262, issued 1987, SAFA et al.: "Specific Vinca Alkaloid-binding Polypeptides Identified in Calf Brain by Photoaffinity Labeling", page 1261-1267. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, Volume 83, issued 1986, CORNWELL et al.: "Membrane vesicles from multidrug -resistant human cancer cells contain a specific 150-to 170-KDa protein detected by photoaffinity labeling", pages 3847-3850. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0568126A1 (fr) * 1992-04-06 1993-11-03 Silvia Maria Doglia Procédé pour la détermination de la résistance multidrogue dans des cellules vivantes
US5436243A (en) * 1993-11-17 1995-07-25 Research Triangle Institute Duke University Aminoanthraquinone derivatives to combat multidrug resistance
FR2729569A1 (fr) * 1995-01-19 1996-07-26 Consultants Internationaux Sur Compositions pharmaceutiques utiles pour la reversion de la resistance multiple aux drogues
WO1998004250A1 (fr) * 1996-07-25 1998-02-05 Consultants Internationaux Sur Le Medicament (S.A.R.L.) Compositions pharmaceutiques utiles pour la reversion de la resistance multiple aux drogues
KR20020045445A (ko) * 2000-12-11 2002-06-19 최철희 독소루비신 내성 급성 골수성 백혈병 아세포주를 이용한내성억제물질과 반응성 산소종 생성 세포독성물질의선별방법

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