CA2000082A1 - Method for the detection of multiple drug resistant tumor cells and verapamil probes useful therein - Google Patents
Method for the detection of multiple drug resistant tumor cells and verapamil probes useful thereinInfo
- Publication number
- CA2000082A1 CA2000082A1 CA 2000082 CA2000082A CA2000082A1 CA 2000082 A1 CA2000082 A1 CA 2000082A1 CA 2000082 CA2000082 CA 2000082 CA 2000082 A CA2000082 A CA 2000082A CA 2000082 A1 CA2000082 A1 CA 2000082A1
- Authority
- CA
- Canada
- Prior art keywords
- derivative
- verapamil
- polypeptide
- sample
- concentration
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
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- QCWXZFZXEGTLOV-UHFFFAOYSA-N n-propan-2-ylpentan-1-amine Chemical compound CCCCCNC(C)C QCWXZFZXEGTLOV-UHFFFAOYSA-N 0.000 description 1
- 229960001783 nicardipine Drugs 0.000 description 1
- HYIMSNHJOBLJNT-UHFFFAOYSA-N nifedipine Chemical compound COC(=O)C1=C(C)NC(C)=C(C(=O)OC)C1C1=CC=CC=C1[N+]([O-])=O HYIMSNHJOBLJNT-UHFFFAOYSA-N 0.000 description 1
- 229960001597 nifedipine Drugs 0.000 description 1
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- 125000003729 nucleotide group Chemical group 0.000 description 1
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- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940108519 trasylol Drugs 0.000 description 1
- ZEWQUBUPAILYHI-UHFFFAOYSA-N trifluoperazine Chemical compound C1CN(C)CCN1CCCN1C2=CC(C(F)(F)F)=CC=C2SC2=CC=CC=C21 ZEWQUBUPAILYHI-UHFFFAOYSA-N 0.000 description 1
- 229960002324 trifluoperazine Drugs 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
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Classifications
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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
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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
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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
- G01N33/68—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
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- 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
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- 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
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Abstract
Abstract of the Disclosure A method for the detection of multiple drug resistant tumor cells is described which comprises the incubation of a sample of mammalian tumor cellular material with an effective binding amount of a labelled verapamil derivative to form an complex with any P-gp that is present, and then determining the amount of P-gp present by quantifying the amount of labelled verapamil derivative bound in the complex. Photoaffinity, radiolabelled and chemoaffinity derivatives of verapamil and the syntheses of such derivatives are described, as are a method of treatment for multiple drug resistant tumors and a diagnostic kit.
Description
2~r.~
Method for the Detection of Multiple Drug Resistant Tumor Cells and VeraPamil Probes Useful Therein Technical Field 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 (~DR) tumor cellsO It also relates to derivatives of verapamil which are utilized to characterize this polypeptide and to a method of synthesis of such derivatives.
Background of the Invention Affinity labeling of proteins with photoactive ligands is a powerful tool in probing biochemical targets. In particular, 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 candidatesO ~-Under photoaffinity conditions, 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.
Upon irradiation with W light, the druq derivative is converted into a highly reactive nitrene intermediate which covalently interacts with the acceptor sites. A particular functional group at the ,, ...:~
.~'',`;: ~ .
, ~ ., 2~
acceptor site need not be present because the photogenerated species can react even with carbon-hydrogen bonds.
Multidrug resistance (MDR) 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.
Studies with vinblastine photoactive drug derivatives revealed the specific interaction of vinblastine with P-gp in plasma membranes from MDR
cells and suggested that thi~ protein may mediate cellular drug accumulation by binding to drugs and regulating their membrane transport. Safa, et al., ~1986) J. Biol. Chem. 261, 6137-6140; Cornwell, et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3847-3850.
Based on sequence data, it has been shown that significant homology exists between P-gp and bacterial transport proteins. It has also been shown that P-gp has two nucleotide binding domains that bind ATP ICornwell et al., (1987) FASEB J. 1, Sl-54], ~9 that the gene coding for this protein generates the 2~
MDR phenotype when transfected into drug-sensitive cells IGros et al., (1986) Nature ~London) 232, 728-731], and that the purified protein exhibits ATPase activity [Hamada et al., (1988) J. Biol. Chem.
263, 1454-1458]. These findings indicate that P-gp plays an important role in MDR and suggest that P-gp may function as an energy-dependent drug efflux pump.
Summary of the Invention In accordance with the present invention, it has been found that 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 2 0 CH,-NH-R T~3 /=<
~3U~2--U~2--U~2--~2--C~2~C)C~3 3S: C~3 ::
where R' is a labeling group. -Examples of particular 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-azidobenzoyl)aminomethyl verapamil, N-(p-azido-[3,5-3H]benzoyl)aminomethyl verapamil, - -N-~p-azido salicyl)aminomethyl verapamil, N-~p-azido-¦3-125I~salicyl)aminomethyl verapamil, 5-[(3,4-dimethoxyphenethyl)-methylamino]-2-(3,4-dimethoxyphenethyl)-2-isopropyl- `~
pentyl fluorescein, and z ~
....
~,..
z~
5-[(3,4-dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl bromo acetate.
It is particularly preferred that 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. ~his method assays for a verapamil binding p~lypeptide that is characteristically present in multiple drug resistant (MDR) tumor cells as the P-glycoprotein (P-gp). 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.
In one method of the present invention, a sample of mammalian cellular material is admixed and maintained with an effective bindinq 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.
~, . .~ . ..,; ,, , ~ - ;, , , . ,, . - : ,. - , . -2~
In a preferred embodiment, 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 guantified by measuring the concentration of bound verapamil derivatives. By comparing the concentration of the derivative-polypeptide complex present in the sample of tumor cells with the concentration of such a complex found with normal cells of the same tissue type as the tumor cells, multiple drug resistance is indicated if the concentration of the complex is elevated five fold relative to its concentration with normal cells.
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 concen~ration 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 30 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.
Method for the Detection of Multiple Drug Resistant Tumor Cells and VeraPamil Probes Useful Therein Technical Field 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 (~DR) tumor cellsO It also relates to derivatives of verapamil which are utilized to characterize this polypeptide and to a method of synthesis of such derivatives.
Background of the Invention Affinity labeling of proteins with photoactive ligands is a powerful tool in probing biochemical targets. In particular, 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 candidatesO ~-Under photoaffinity conditions, 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.
Upon irradiation with W light, the druq derivative is converted into a highly reactive nitrene intermediate which covalently interacts with the acceptor sites. A particular functional group at the ,, ...:~
.~'',`;: ~ .
, ~ ., 2~
acceptor site need not be present because the photogenerated species can react even with carbon-hydrogen bonds.
Multidrug resistance (MDR) 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.
Studies with vinblastine photoactive drug derivatives revealed the specific interaction of vinblastine with P-gp in plasma membranes from MDR
cells and suggested that thi~ protein may mediate cellular drug accumulation by binding to drugs and regulating their membrane transport. Safa, et al., ~1986) J. Biol. Chem. 261, 6137-6140; Cornwell, et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3847-3850.
Based on sequence data, it has been shown that significant homology exists between P-gp and bacterial transport proteins. It has also been shown that P-gp has two nucleotide binding domains that bind ATP ICornwell et al., (1987) FASEB J. 1, Sl-54], ~9 that the gene coding for this protein generates the 2~
MDR phenotype when transfected into drug-sensitive cells IGros et al., (1986) Nature ~London) 232, 728-731], and that the purified protein exhibits ATPase activity [Hamada et al., (1988) J. Biol. Chem.
263, 1454-1458]. These findings indicate that P-gp plays an important role in MDR and suggest that P-gp may function as an energy-dependent drug efflux pump.
Summary of the Invention In accordance with the present invention, it has been found that 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 2 0 CH,-NH-R T~3 /=<
~3U~2--U~2--U~2--~2--C~2~C)C~3 3S: C~3 ::
where R' is a labeling group. -Examples of particular 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-azidobenzoyl)aminomethyl verapamil, N-(p-azido-[3,5-3H]benzoyl)aminomethyl verapamil, - -N-~p-azido salicyl)aminomethyl verapamil, N-~p-azido-¦3-125I~salicyl)aminomethyl verapamil, 5-[(3,4-dimethoxyphenethyl)-methylamino]-2-(3,4-dimethoxyphenethyl)-2-isopropyl- `~
pentyl fluorescein, and z ~
....
~,..
z~
5-[(3,4-dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl bromo acetate.
It is particularly preferred that 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. ~his method assays for a verapamil binding p~lypeptide that is characteristically present in multiple drug resistant (MDR) tumor cells as the P-glycoprotein (P-gp). 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.
In one method of the present invention, a sample of mammalian cellular material is admixed and maintained with an effective bindinq 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.
~, . .~ . ..,; ,, , ~ - ;, , , . ,, . - : ,. - , . -2~
In a preferred embodiment, 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 guantified by measuring the concentration of bound verapamil derivatives. By comparing the concentration of the derivative-polypeptide complex present in the sample of tumor cells with the concentration of such a complex found with normal cells of the same tissue type as the tumor cells, multiple drug resistance is indicated if the concentration of the complex is elevated five fold relative to its concentration with normal cells.
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 concen~ration 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 30 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.
3~ `~
.,~; ~ .
,,., . ~ . .
2 ~
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 ~hemotherapeutic drugs which would be ineffective toward the particular MDR tumor. In prior practice, MDR tum~rs 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 Fi~ures In the figures forming a portion of this disclosure:
Figure 1 iilustrates the structural formulae of verapamil, and two radiolabelled photoaffinity derivatives of verapamil N-~p-azido-[3,5-3~]benzoyl)-aminomethyl verapamil ~[3H]
NAB~V) and N-(p-azido-13-125I]salicyl)aminomethyl verapamil ([12SI~ NASAV), respectively.
Figure 2 is a two-part graph that illustrate 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, 35 in which the abscissas are in units of -~
centimeters -1 (cm~
~; , .. , ., .. . . . -.. -~ . . . .. .
Z~
Detailed Description of the Invention 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 phenylalkylamine 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 1~ 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.
These 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 ~DR.
_....~.....
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. ~nowing the properties of the parent drug, plus the ability to photoactivate the derivative, permits one to easily probe specific receptors for the parent drugs in the cells. Without such derivatives which are photoactive and radioactive, it is impossible to probe the interactions of verapamil with its receptors in cells and in tissue homogenates.
Two compounds of the present invention are radiolabelled photoactive derivatives of verapamil: 1) N~ azido[3,53H]benzoyl)aminomethyl verapamil or N-(~-azido-[3,53H]benzoyl-5-[(3,4-dimethoxyphenethyl)methyl-amino]-2(3,4-dimethoxyphenyl)-2-isopropylpentyl-amine, also referred to as [3H]NABAV; and 2) N-~-azido[3-125I]salicyl)-aminomethyl verapamil or N-(p-azido-[3-125I]salicyl-5-[(3,4-dimethoxyphenethyl)-methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine, also referred to as [125I NASAV]. Specifics for the preparation of these two compounds are provided hereinafter.
The compounds can be used to photoaffinity-label a 150-180kD membrane protein which is present in MDR tumor cells. The resulting specifically labelled 150-180 kD
membrane protein in drug resistant cells has been immunoprecipitated with a monoclonal antibody, C 219, specific for P-glycoprotein. Xartner et al (1985) Nature (London) 316:820-823.
Phenylalkylamine binding specificity was established by competitive blocking of spPcific photolabeling with the nonradioactive photoactive ~`
derivatives as well as with verapamil. Exemplary photoaffinity labeling agents are~
.,~; ~ .
,,., . ~ . .
2 ~
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 ~hemotherapeutic drugs which would be ineffective toward the particular MDR tumor. In prior practice, MDR tum~rs 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 Fi~ures In the figures forming a portion of this disclosure:
Figure 1 iilustrates the structural formulae of verapamil, and two radiolabelled photoaffinity derivatives of verapamil N-~p-azido-[3,5-3~]benzoyl)-aminomethyl verapamil ~[3H]
NAB~V) and N-(p-azido-13-125I]salicyl)aminomethyl verapamil ([12SI~ NASAV), respectively.
Figure 2 is a two-part graph that illustrate 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, 35 in which the abscissas are in units of -~
centimeters -1 (cm~
~; , .. , ., .. . . . -.. -~ . . . .. .
Z~
Detailed Description of the Invention 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 phenylalkylamine 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 1~ 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.
These 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 ~DR.
_....~.....
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. ~nowing the properties of the parent drug, plus the ability to photoactivate the derivative, permits one to easily probe specific receptors for the parent drugs in the cells. Without such derivatives which are photoactive and radioactive, it is impossible to probe the interactions of verapamil with its receptors in cells and in tissue homogenates.
Two compounds of the present invention are radiolabelled photoactive derivatives of verapamil: 1) N~ azido[3,53H]benzoyl)aminomethyl verapamil or N-(~-azido-[3,53H]benzoyl-5-[(3,4-dimethoxyphenethyl)methyl-amino]-2(3,4-dimethoxyphenyl)-2-isopropylpentyl-amine, also referred to as [3H]NABAV; and 2) N-~-azido[3-125I]salicyl)-aminomethyl verapamil or N-(p-azido-[3-125I]salicyl-5-[(3,4-dimethoxyphenethyl)-methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine, also referred to as [125I NASAV]. Specifics for the preparation of these two compounds are provided hereinafter.
The compounds can be used to photoaffinity-label a 150-180kD membrane protein which is present in MDR tumor cells. The resulting specifically labelled 150-180 kD
membrane protein in drug resistant cells has been immunoprecipitated with a monoclonal antibody, C 219, specific for P-glycoprotein. Xartner et al (1985) Nature (London) 316:820-823.
Phenylalkylamine binding specificity was established by competitive blocking of spPcific photolabeling with the nonradioactive photoactive ~`
derivatives as well as with verapamil. Exemplary photoaffinity labeling agents are~
4-azidobenzoic acid-N-hydroxysuccinimide ester, .. ... : .~.
-... . . .
... . . :~ -2~r3~f~2 N-(5-azido-2-nitrobenzyloxy)succinimide, 6-(4-azido-2-nitrophenylamino)hexanoic-acid-N-hydroxysuccinimide ester, p-azidophenacyl bromide - 3-(4-azidophenyldithio)propionic acid-N-hydroxysuccinimide S ester and 2-Diazo-333-trifluoropropionic-acid-p-nitrophenylester. Photoaffinity labeling was also inhibited by 50 uM concentrations of the calcium channel blockers nimodipine, nifedipine nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine (Sigma Chem. Co., St~ Louis, MO). Bay K86-44, (Miles Pharmaceuticals, New Haven, CT) a calcium channel agonist, also inhibited P-glycoprotein photolabeling.
Moreover, 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, doxorubicin and acti~omycin D, at 10~ uM, but not by colchicine.
These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.
A method for assaying tumor cells to ascertain whether they are multiple drug resistant is provided by the present invention7 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 ;; ~ .
~ ,:
~ ~ ,, - .
2 ~
of elevated concentrations of verapamil binding protein (P-gp) in the tumors.
In this method a sample of tumor tissue is admixed with a composition containing a labelled verapamil derivative. The concentration of the verapamil derivative is about 4 to about 100 nanomolar ~nN) in an aqueous buffer such as 40 mM
potassium phosphate (pH 7.0). The admixture is maintained at about 25C. 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 i5 significantly elevated (by over five 20 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.
If such elevated levels of P-gp are found then appropriate therapy utilizing either a natural product type chemotherapeutic agent together with a modulator. Exemplary natural product type chemotherapeutic agents are vinblastine, vincristine and adriamycin. ~xemplary modulator compounds are 30 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 i .. - . ~ .
. . - . ~ - . ~ . . . -.. . .
n ~
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 guantification 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.
nInstructions 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-polypep~ide complex. Particularly preferred indicating means are radiolabels or fluorescent molecules attached to the verapamil derivative.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
,~, ~, .
~ - .
. "
-.`. ~, '' '' ' .
Example 1: Synthesis of (~)-5-1(3,4-Dimethoxy-phenethyl)methylamino]-2-(3,4-dimethoxy-phenyl)-2-isopropylpentylamine (DMDIL.
- This compound is prepared according to the following procedure. Verapamil hydrochloride (1.5 gm, 3.05 mmol) in dry tetrahydrofuran (T~F) (50 ml) was admixed with lithiu~ aluminum hydride (500 mg (13.2 mmol) 0.5 M solution in diglyme). The mixture was stirred for 4 hours with refluxing at 70-75C , 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 wa6 washed with ether (50 ml). The organic phases were comb;ned, dried over MgS04, filtered, and concentrated to produce a colorless oil. The product produced a single W
absorbing spot on silica gel TLC with fluorescent indicator. Solvent I utilized consisted of chloroform:methanol:H2O, 80:20:3 and had a Rf=0.23; solvent II consisted of chloroform:methanol: 40% methylamine, 80:20:4 and had a Rf=0.8.
Example 2: Synthesis of N-(p-azidobenzoyl)-aminomethyl verapamil ~ -The photoactive phenylalkylamine derivative N-(p-azidobenzoyl-5-[(3,4-dimethoxyphenethyl~methyl-amino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine or N-(~-azidobenzoylaminomethyl verapamil (NABAV3 was synthesized by azidobenzoylation of (+)-5-[(3,4 - dimethoxyphenethyl)methylamino]-2-(3~4-dirnethoxyphenyl) -2 isopropyl-pentylamine with N-hydroxysuccinimidyl-4-azidobenzoate. The synthesis was carried out by dissolving DMDI (5 uM) in chloroform (1 ml). To this 2~3~f~ Z
solution was added N-hydroxysuccinimidyl-4-azidobenzoate tNAB) ~10 umol) (Pierce Chemical Co., Rockford, IL). The reaction mixture was maintained at 40C 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. The product gave a single UV-absorbing spot on a silica gel TLC plate with fluorescence indicator run in solvent I
chloroform/methanol/H2O, 80:20:3 (vol/vol), =0.76]. Reversed phase high performance liquid chromatography (HPLC) analysis was conducted by the method described in ~orn et al., (1987) Arzeim.
Forsch. Drug Res. 37, 956-959 and indicated a single product peak that accounted for 99% of the eluted absorbance measured at 254 nm, and had a retention time of 19.94 minutes.
Example 3: Synthesis of N-(p-azidosalicyl) aminomethyl verapamil The photoactive derivative N-(p-a~idosalicyl)-aminomethyl verapamil (NASAV) was synthesized from (~)-5-[(3,4-dimethoxyphenethyl) methylamino]-2(3,4-dimethoxyphenyl)-2-isopropylpentyl-amine and N-hydroxysuccinimidyl-4-a~idosalicylate 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.
The chemical structures of NABAV and NASAV
~Fig. 1) were analyzed by low and high-resolution fast atom bombardment (FAB) mass spectroscopy, 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 ,:: , .
,.:
. . ,~, . .
- 2~ 8~
~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). Fragment ions corresponding to (MH-CO) and (MH-CgH12O2) are si~nificant under FAB conditions. The accurate mass measured for the (MH)+ of NABAV was 604.3504 which corresponds to an elemental composition of (C34H45N505+H). Similarly, the accurate mass of (MH)+ ion from NASAV was measured as 620.3456 (Fi~. 3) which correspond to an elemental composition of (C34H45N505+H). The High Resolution/FAB/Mass Spectrometry data confirmed the molecular structures for NABAV and NASAV.
The photoactive derivatives exhibited a -UV-visible absorption spectrum consisting of a composite of thè absorption spectra of verapamil and -~
20 either the azidobenzoate or azidosalicylate -~
chromophore (Fig. 4). Upon W irradiation, the absorption peak between 2~0 and 400 nm was lost, yielding a spectrum similar to verapamil. The IR
spectrum showed a strong resonance at 2130 cm 1 -~
indicating the presence of azide (Fig. 5).
Example 4: Synthesis of N-(p-azido-3,5-[3H]-benzoyl)aminomethyl verapamil The photoactive, radioactive N-(~-azido-3,5]3H]-benzoyl-5-[(3,4-dimethoxy-phenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine or N-(p-azido-3,5-[3H]-benzoyl)-aminomethyl verapamil ([3H]NABAV) was synthesized as follows. N-Hydroxysuccinimidyl- -4-azido-[3,5-3H]-benzoate (47.7 Ci/mmol) in 0.05 ml - ~::: ' ' . :
2~
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 4C 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 with NABAV by TLC run in solvent I.
Example 5: Synthesis of N-(p-azido-[3-125I]-salicyl)aminometh~l vera~amil The photoactive, radioactive N~ -azido-[3,125I]salicyl-5-13,4-dimethoxyphenethyl) methylamino~-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl-amine or N-(~-azido-[3,125I]salicyl)aminomethyl verapamil ~1125]NASAV) was synthesized by two step reactions. -First Reaction:
N-hydroxysuccinimidyl-4-azidosalicylate (NAS) (1.67 n~ole) (Pierce Chemical Co., Rockford, IL) was dissolved in acetonitrile (15 ul~ . Five microliters of 0.5 M sodium phosphate (pH 7) and 5 mCi of Nal2~I in 10 ul of 0.1 N NaOH (Amersham Corporation, Arlington Heights, IL) was added.
Chloramine T 2.5 nmoles in 10 ul of a mixture of acetonitrile and dimethylformamide (1:1) was added and the mixture was maintained for 2 minutes at room temperature. Three hundred microliters of 10% NaCl was added and the reaction mixture was extracted with 300 ul of ethyl acetate. The extract was evaporated under nitrogen and the reaction mixture was chromatographed on a silica gel G thin layer using a running solvent of benzene:chloroform:ethylacetate:
acetic acid (1:1:1:0.1, v/v). The product (N-hydroxy-''.~ ~' ~.' '' . ''.
.
2~
succinimidyl-~3-125I]-4-azidosalicylate), gave a radioactive spot by autoradiography that accounted for 90% of the total radioactivity (Rf=0.4).
Second Reaction:
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 4C. The progress of the reaction was monitored by TLC (run in Solvent II, Rf=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 15 column with 5% methanol in chloroform. The product ~ -gave a single radioactive spot on silica gel TLC with a Rf=0.67 when run in solvent II as determined by autoradiography. -Example 6: Synthesis of Chemoaffinity Ligands of Verapamil Chemoaffinity ligands of verapamil are synthesized by coupling the intermediate DMDI to appropriate group-specific reagents (e.g., bromoacetyl electrophile using N-succimidylbromoacetate). Verapamil has cyano group that forms a primary alkylamine that can be readily linked to various functional groups. Initially, 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-succinimidyl bromoacetate in chloroform provides ~-(3,4-dimethoxyphenethyl)-.
.:
. :, '~ '. O
Z~
methylamino]-2-~3,4-dimethoxyphenyl)-2-isopropylpentyl-bromol2- H] acetate which is reactive with cysteine and methionine.
Corresponding radioactive derivatives are prepared using N-succinimidylbromo [2-3~]acetate.
Verapamil derivatives containing maleimide reactive groups are prepared by coupling aminomethyl verapamil to commercially available N-alkylmaleimide carboxylic acids by carbodiimide catalyzed condensations.
Example 7: Synthesis of Fluorescent Verapamil Derivatives A fluorescent verapamil derivative, 5-[3~4-Dimethoxyphenethyl)methylamirlo]-2-(3,4-dimethoxy phenyl)-2-isopropylpentyl 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.
Example 8: Photoaffinity Labeling of Cells Sensitive DC3F Chinese hamster lung cells and vincristine-resistant variant DC-3F/VCD-5L cells that were selected for primary resistance to ~incristine (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-Xettering Cancer Center, New York) and were :. --~ :, , : -. . -: . .: . .
~: .
2~
maintained as described in Peterson et al., (1983) Cancer Res. 43, 222-228; and Meyers et al., (1985) J. Cell Biology, 100, 588-597 Drug resistant cells -were treated weekly with a maintenance concentration S of vincristine (40 ug/ml). Vincristine was removed from cultures 8-10 days before experiments.
Exponentially growing cells were harvested by ~craping culture plates with a rubber blade.
Trypan blue viable (greater than 90%) cell suspensions (2-5 x 105 cells per assay) in Ca2l/Mg2~-free Dulbecco's phosphate-~uffered saline containing 4~ (vol/vol) dimethylsulfoxide and 50-100 nM 13H]NABAV or 4 nM 1125I]NASAV in a - ~-final volume of 0.050 ml were photolabelled after lS preincubation for 30 minutes at 25C 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, Ultravio;et Products, San Gabriel, CA). The samples labelled with the photoactive derivatives were then solubilized for sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodS04/PAGE ) .
Example 9: Immunoprecipitation Cell membrane vesicles were prepared by nitrogen cavitation and differential centrifugation as described by Lever, (1977) J. 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
[125I] NASAV in a final volume of 0.05 ml were photolabelled after preincubation for 30 minutes at x - ~
, ., ~, .. .
2~
25C 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, C~).
Immunoprecipitations of 1125I]NASAV
photolabelled membrane vesicles (20 ug of protein) solubilized in a deoxycholate buffer [100 ml. of 50 mM tris-HCl buffer (pH 7.0) containing 150 mM NaCl, 1% Triton X-100, 13 sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 10 mg/ml trasylol] were performed as described in Safa et al., (1986) J. Biol. Chem. 261, 6137-6140, with monoclonal antibody C 219, specific for P-gp, or 30 ug of normal m~use serum as a control.
The photolabelled samples were electrophoresed on NaDodSO4/5-15~ polyacrylamide gels containing 4.5 M urea and processed for fluorography or autoradiography.
.
, " ~, . ,. :
'.~.:' '
-... . . .
... . . :~ -2~r3~f~2 N-(5-azido-2-nitrobenzyloxy)succinimide, 6-(4-azido-2-nitrophenylamino)hexanoic-acid-N-hydroxysuccinimide ester, p-azidophenacyl bromide - 3-(4-azidophenyldithio)propionic acid-N-hydroxysuccinimide S ester and 2-Diazo-333-trifluoropropionic-acid-p-nitrophenylester. Photoaffinity labeling was also inhibited by 50 uM concentrations of the calcium channel blockers nimodipine, nifedipine nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine (Sigma Chem. Co., St~ Louis, MO). Bay K86-44, (Miles Pharmaceuticals, New Haven, CT) a calcium channel agonist, also inhibited P-glycoprotein photolabeling.
Moreover, 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, doxorubicin and acti~omycin D, at 10~ uM, but not by colchicine.
These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.
A method for assaying tumor cells to ascertain whether they are multiple drug resistant is provided by the present invention7 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 ;; ~ .
~ ,:
~ ~ ,, - .
2 ~
of elevated concentrations of verapamil binding protein (P-gp) in the tumors.
In this method a sample of tumor tissue is admixed with a composition containing a labelled verapamil derivative. The concentration of the verapamil derivative is about 4 to about 100 nanomolar ~nN) in an aqueous buffer such as 40 mM
potassium phosphate (pH 7.0). The admixture is maintained at about 25C. 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 i5 significantly elevated (by over five 20 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.
If such elevated levels of P-gp are found then appropriate therapy utilizing either a natural product type chemotherapeutic agent together with a modulator. Exemplary natural product type chemotherapeutic agents are vinblastine, vincristine and adriamycin. ~xemplary modulator compounds are 30 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 i .. - . ~ .
. . - . ~ - . ~ . . . -.. . .
n ~
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 guantification 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.
nInstructions 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-polypep~ide complex. Particularly preferred indicating means are radiolabels or fluorescent molecules attached to the verapamil derivative.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
,~, ~, .
~ - .
. "
-.`. ~, '' '' ' .
Example 1: Synthesis of (~)-5-1(3,4-Dimethoxy-phenethyl)methylamino]-2-(3,4-dimethoxy-phenyl)-2-isopropylpentylamine (DMDIL.
- This compound is prepared according to the following procedure. Verapamil hydrochloride (1.5 gm, 3.05 mmol) in dry tetrahydrofuran (T~F) (50 ml) was admixed with lithiu~ aluminum hydride (500 mg (13.2 mmol) 0.5 M solution in diglyme). The mixture was stirred for 4 hours with refluxing at 70-75C , 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 wa6 washed with ether (50 ml). The organic phases were comb;ned, dried over MgS04, filtered, and concentrated to produce a colorless oil. The product produced a single W
absorbing spot on silica gel TLC with fluorescent indicator. Solvent I utilized consisted of chloroform:methanol:H2O, 80:20:3 and had a Rf=0.23; solvent II consisted of chloroform:methanol: 40% methylamine, 80:20:4 and had a Rf=0.8.
Example 2: Synthesis of N-(p-azidobenzoyl)-aminomethyl verapamil ~ -The photoactive phenylalkylamine derivative N-(p-azidobenzoyl-5-[(3,4-dimethoxyphenethyl~methyl-amino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine or N-(~-azidobenzoylaminomethyl verapamil (NABAV3 was synthesized by azidobenzoylation of (+)-5-[(3,4 - dimethoxyphenethyl)methylamino]-2-(3~4-dirnethoxyphenyl) -2 isopropyl-pentylamine with N-hydroxysuccinimidyl-4-azidobenzoate. The synthesis was carried out by dissolving DMDI (5 uM) in chloroform (1 ml). To this 2~3~f~ Z
solution was added N-hydroxysuccinimidyl-4-azidobenzoate tNAB) ~10 umol) (Pierce Chemical Co., Rockford, IL). The reaction mixture was maintained at 40C 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. The product gave a single UV-absorbing spot on a silica gel TLC plate with fluorescence indicator run in solvent I
chloroform/methanol/H2O, 80:20:3 (vol/vol), =0.76]. Reversed phase high performance liquid chromatography (HPLC) analysis was conducted by the method described in ~orn et al., (1987) Arzeim.
Forsch. Drug Res. 37, 956-959 and indicated a single product peak that accounted for 99% of the eluted absorbance measured at 254 nm, and had a retention time of 19.94 minutes.
Example 3: Synthesis of N-(p-azidosalicyl) aminomethyl verapamil The photoactive derivative N-(p-a~idosalicyl)-aminomethyl verapamil (NASAV) was synthesized from (~)-5-[(3,4-dimethoxyphenethyl) methylamino]-2(3,4-dimethoxyphenyl)-2-isopropylpentyl-amine and N-hydroxysuccinimidyl-4-a~idosalicylate 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.
The chemical structures of NABAV and NASAV
~Fig. 1) were analyzed by low and high-resolution fast atom bombardment (FAB) mass spectroscopy, 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 ,:: , .
,.:
. . ,~, . .
- 2~ 8~
~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). Fragment ions corresponding to (MH-CO) and (MH-CgH12O2) are si~nificant under FAB conditions. The accurate mass measured for the (MH)+ of NABAV was 604.3504 which corresponds to an elemental composition of (C34H45N505+H). Similarly, the accurate mass of (MH)+ ion from NASAV was measured as 620.3456 (Fi~. 3) which correspond to an elemental composition of (C34H45N505+H). The High Resolution/FAB/Mass Spectrometry data confirmed the molecular structures for NABAV and NASAV.
The photoactive derivatives exhibited a -UV-visible absorption spectrum consisting of a composite of thè absorption spectra of verapamil and -~
20 either the azidobenzoate or azidosalicylate -~
chromophore (Fig. 4). Upon W irradiation, the absorption peak between 2~0 and 400 nm was lost, yielding a spectrum similar to verapamil. The IR
spectrum showed a strong resonance at 2130 cm 1 -~
indicating the presence of azide (Fig. 5).
Example 4: Synthesis of N-(p-azido-3,5-[3H]-benzoyl)aminomethyl verapamil The photoactive, radioactive N-(~-azido-3,5]3H]-benzoyl-5-[(3,4-dimethoxy-phenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentylamine or N-(p-azido-3,5-[3H]-benzoyl)-aminomethyl verapamil ([3H]NABAV) was synthesized as follows. N-Hydroxysuccinimidyl- -4-azido-[3,5-3H]-benzoate (47.7 Ci/mmol) in 0.05 ml - ~::: ' ' . :
2~
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 4C 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 with NABAV by TLC run in solvent I.
Example 5: Synthesis of N-(p-azido-[3-125I]-salicyl)aminometh~l vera~amil The photoactive, radioactive N~ -azido-[3,125I]salicyl-5-13,4-dimethoxyphenethyl) methylamino~-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl-amine or N-(~-azido-[3,125I]salicyl)aminomethyl verapamil ~1125]NASAV) was synthesized by two step reactions. -First Reaction:
N-hydroxysuccinimidyl-4-azidosalicylate (NAS) (1.67 n~ole) (Pierce Chemical Co., Rockford, IL) was dissolved in acetonitrile (15 ul~ . Five microliters of 0.5 M sodium phosphate (pH 7) and 5 mCi of Nal2~I in 10 ul of 0.1 N NaOH (Amersham Corporation, Arlington Heights, IL) was added.
Chloramine T 2.5 nmoles in 10 ul of a mixture of acetonitrile and dimethylformamide (1:1) was added and the mixture was maintained for 2 minutes at room temperature. Three hundred microliters of 10% NaCl was added and the reaction mixture was extracted with 300 ul of ethyl acetate. The extract was evaporated under nitrogen and the reaction mixture was chromatographed on a silica gel G thin layer using a running solvent of benzene:chloroform:ethylacetate:
acetic acid (1:1:1:0.1, v/v). The product (N-hydroxy-''.~ ~' ~.' '' . ''.
.
2~
succinimidyl-~3-125I]-4-azidosalicylate), gave a radioactive spot by autoradiography that accounted for 90% of the total radioactivity (Rf=0.4).
Second Reaction:
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 4C. The progress of the reaction was monitored by TLC (run in Solvent II, Rf=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 15 column with 5% methanol in chloroform. The product ~ -gave a single radioactive spot on silica gel TLC with a Rf=0.67 when run in solvent II as determined by autoradiography. -Example 6: Synthesis of Chemoaffinity Ligands of Verapamil Chemoaffinity ligands of verapamil are synthesized by coupling the intermediate DMDI to appropriate group-specific reagents (e.g., bromoacetyl electrophile using N-succimidylbromoacetate). Verapamil has cyano group that forms a primary alkylamine that can be readily linked to various functional groups. Initially, 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-succinimidyl bromoacetate in chloroform provides ~-(3,4-dimethoxyphenethyl)-.
.:
. :, '~ '. O
Z~
methylamino]-2-~3,4-dimethoxyphenyl)-2-isopropylpentyl-bromol2- H] acetate which is reactive with cysteine and methionine.
Corresponding radioactive derivatives are prepared using N-succinimidylbromo [2-3~]acetate.
Verapamil derivatives containing maleimide reactive groups are prepared by coupling aminomethyl verapamil to commercially available N-alkylmaleimide carboxylic acids by carbodiimide catalyzed condensations.
Example 7: Synthesis of Fluorescent Verapamil Derivatives A fluorescent verapamil derivative, 5-[3~4-Dimethoxyphenethyl)methylamirlo]-2-(3,4-dimethoxy phenyl)-2-isopropylpentyl 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.
Example 8: Photoaffinity Labeling of Cells Sensitive DC3F Chinese hamster lung cells and vincristine-resistant variant DC-3F/VCD-5L cells that were selected for primary resistance to ~incristine (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-Xettering Cancer Center, New York) and were :. --~ :, , : -. . -: . .: . .
~: .
2~
maintained as described in Peterson et al., (1983) Cancer Res. 43, 222-228; and Meyers et al., (1985) J. Cell Biology, 100, 588-597 Drug resistant cells -were treated weekly with a maintenance concentration S of vincristine (40 ug/ml). Vincristine was removed from cultures 8-10 days before experiments.
Exponentially growing cells were harvested by ~craping culture plates with a rubber blade.
Trypan blue viable (greater than 90%) cell suspensions (2-5 x 105 cells per assay) in Ca2l/Mg2~-free Dulbecco's phosphate-~uffered saline containing 4~ (vol/vol) dimethylsulfoxide and 50-100 nM 13H]NABAV or 4 nM 1125I]NASAV in a - ~-final volume of 0.050 ml were photolabelled after lS preincubation for 30 minutes at 25C 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, Ultravio;et Products, San Gabriel, CA). The samples labelled with the photoactive derivatives were then solubilized for sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodS04/PAGE ) .
Example 9: Immunoprecipitation Cell membrane vesicles were prepared by nitrogen cavitation and differential centrifugation as described by Lever, (1977) J. 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
[125I] NASAV in a final volume of 0.05 ml were photolabelled after preincubation for 30 minutes at x - ~
, ., ~, .. .
2~
25C 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, C~).
Immunoprecipitations of 1125I]NASAV
photolabelled membrane vesicles (20 ug of protein) solubilized in a deoxycholate buffer [100 ml. of 50 mM tris-HCl buffer (pH 7.0) containing 150 mM NaCl, 1% Triton X-100, 13 sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 10 mg/ml trasylol] were performed as described in Safa et al., (1986) J. Biol. Chem. 261, 6137-6140, with monoclonal antibody C 219, specific for P-gp, or 30 ug of normal m~use serum as a control.
The photolabelled samples were electrophoresed on NaDodSO4/5-15~ polyacrylamide gels containing 4.5 M urea and processed for fluorography or autoradiography.
.
, " ~, . ,. :
'.~.:' '
Claims (53)
1. A method for assaying for multiple drug resistance of mammalian tumor cells comprising a) admixing a sample of mammalian tumor cellular material with an effective binding amount of a verapamil derivative;
b) maintaining said admixture for a time period sufficient to permit the formation of a derivative-polypeptide complex between said verapamil derivative and a verapamil binding polypeptide that may be present;
c) separating said sample of cellular material containing said derivative-polypeptide complex from said verapamil derivative that remains unbound in said admixture;
d) determining the concentration of said polypeptide present in said sample of cellular material; and e) comparing the concentration of said derivative-polypeptide complex found in step (d) to the concentration of said derivative-polypeptide complex found with normal cells of the same tissue type as said tumor cells, an increased concentration of said derivative-polypeptide complex in said tumor cells relative to the concentration in said normal cells indicating multiple drug resistance.
b) maintaining said admixture for a time period sufficient to permit the formation of a derivative-polypeptide complex between said verapamil derivative and a verapamil binding polypeptide that may be present;
c) separating said sample of cellular material containing said derivative-polypeptide complex from said verapamil derivative that remains unbound in said admixture;
d) determining the concentration of said polypeptide present in said sample of cellular material; and e) comparing the concentration of said derivative-polypeptide complex found in step (d) to the concentration of said derivative-polypeptide complex found with normal cells of the same tissue type as said tumor cells, an increased concentration of said derivative-polypeptide complex in said tumor cells relative to the concentration in said normal cells indicating multiple drug resistance.
2. The method according to claim 1 wherein said verapamil derivative is a photoaffinity labelled derivative.
3. The method according to claim 1 wherein said labelled derivative is radiolabelled.
4. The method according to claim 2, wherein said photoaffinity labelled derivative is N-(p-azidobenzoylaminomethyl verapamil or N-(p-azidosalicyl)aminomethyl verpamil.
5. The method according to claim 3, wherein said derivative is radio iodinated.
6. The method according to claim 3, wherein said derivative is tritiated.
7. The method according to claim 5, wherein said derivative is N-(p-azido-[3-125I]-salicyl)aminomethyl verapamil.
8. The method according to claim 6, wherein said derivative is N-(p-azido-[3,5-3H]benzoyl)aminomethyl verapamil.
9. The method according to claim 3, further comprising irradiating said sample with an effective amount of ultraviolet light to induce covalent bonding in said derivative-polypeptide complex.
10. The method according to claim 9 wherein said determination of the amount of said polypeptide present in said sample is quantified by autoradiography.
11. The method according to claim 1 wherein said sample of mammalian tumor cellular material is human tumor tissue.
12. The method according to claim 1 wherein said sample of mammalian tumor cellular material comprises cellular membranes.
13. The method according to claim 1, wherein said verapamil binding polypeptide is a polypeptide that has a molecular weight of about 150 to about 180 kilodaltons, and is identical to the P-glycoprotein that is present in multidrug resistant cells.
14. The method according to claim 1 wherein said verapamil derivative is 5-[(3,4-dimethoxy-phenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-iso-propylpentyl-bromo acetate.
15. The method according to claim 14 wherein said derivative is tritiated.
16. The method according to claim 1 wherein said derivative is a fluorescent verapamil derivative.
17. The method according to claim 16 wherein said derivative is 5-[(3,4-dimethoxyphenethyl) methylamino]-2-(3,4-dimethoxyphenethyl)-2-isopropyl-pentyl fluorescein.
18. The method according to claim 1, wherein said separation of said sample of cellular material is by centrifugation of said admixture.
19. A method of treatment for a mammalian tumor comprising:
a) admixing a sample of said tumor with an effective binding amount of a verapamil derivative;
b) maintaining said admixture for a time period sufficient to permit the formation of a derivative-polypeptide complex between said verapamil derivative and a verapamil-binding polypeptide that may be present;
c) separating said tumor sample containing said derivative-polypeptide complex from said admixture;
d) determining the concentration of said polypeptide present in said sample;
e) comparing the concentration of said derivative-polypeptide complex found in step (d) to the concentration of said derivative-polypeptide complex found with normal cells of the same tissue type as said tumor; and f) when said concentration found in (d) is about five fold greater than said concentration present with normal cells, treating said tumor as a multiple drug resistant cell tumor.
a) admixing a sample of said tumor with an effective binding amount of a verapamil derivative;
b) maintaining said admixture for a time period sufficient to permit the formation of a derivative-polypeptide complex between said verapamil derivative and a verapamil-binding polypeptide that may be present;
c) separating said tumor sample containing said derivative-polypeptide complex from said admixture;
d) determining the concentration of said polypeptide present in said sample;
e) comparing the concentration of said derivative-polypeptide complex found in step (d) to the concentration of said derivative-polypeptide complex found with normal cells of the same tissue type as said tumor; and f) when said concentration found in (d) is about five fold greater than said concentration present with normal cells, treating said tumor as a multiple drug resistant cell tumor.
20. The method of treatment according to claim 19 wherein said multiple drug resistant cell tumor is treated with a natural product-type chemotherapeutic agent together with a modulator compound.
21. The method of treatment according to claim 19 wherein said multiple drug resistant cell tumor is treated with vincristine and verapamil.
22. The method of treatment according to claim 20 wherein said natural product-type chemotherapeutic agent is a compound of the class consisting of vinblastine, vincristine and adriamycin .
23. A method for the determination of the concentration of a verapamil binding polypeptide in cellular material comprising a) admixing a sample of said cellular material with an effective binding amount of a verapamil derivative;
b) maintaining said admixture for a time period sufficient to permit the formation of a derivative-polypeptide complex between said verapamil derivative and said verapamil binding polypeptide that may be present; and c) determining the concentration of said polypeptide present in said sample.
b) maintaining said admixture for a time period sufficient to permit the formation of a derivative-polypeptide complex between said verapamil derivative and said verapamil binding polypeptide that may be present; and c) determining the concentration of said polypeptide present in said sample.
24. The method according to claim 23 wherein said verapamil derivative is a photoaffinity labelled derivative.
25. The method according to claim 24 wherein said photoaffinity labelled derivative is radiolabelled.
26. The method according to claim 24, wherein said photoaffinity labelled derivative is N-(p-azidobenzoylaminomethyl verapamil or N-(p-azidosalicyl)aminomethyl verpamil.
27. The method according to claim 25, wherein said derivative is radio iodinated.
28. The method according to claim 25, wherein said derivative is tritiated.
29. The method according to claim 27, wherein said derivative is N-(p-azido-[3-125I]-salicyl)aminomethyl verapamil.
30. The method according to claim 28, wherein said derivative is N-(p-azido-[3,5-3H]benzoyl)aminomethyl verapamil.
31. The method according to claim 25, further comprising irradiating said sample with an effective amount of ultraviolet light to induce covalent bonding in said derivative-polypeptide complex.
32. The method according to claim 31 wherein said determination of the amount of said polypeptide present in said sample is quantified by autoradiography.
33. The method according to claim 23 wherein said sample of mammalian tumor cellular material is human tumor tissue.
34. The method according to claim 23 wherein said sample of mammalian tumor cellular material comprises cellular membranes.
35. The method according to claim 23, wherein said verapamil binding polypeptide is a polypeptide that has a molecular weight of about 150 to about 180 kilodaltons, and is identical to the P-glycoprotein that is present in multidrug resistant cells.
36. The method according to claim 23 wherein said verapamil derivative is 5-[(3,4-dimethoxy-phenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylpentyl-bromo acetate.
37. The method according to claim 36 wherein said derivative is tritiated.
38. The method according to claim 23 wherein said derivative is a fluorescent verapamil derivative.
39. The method according to claim 38 wherein said derivative is 5-[(3,4-dimethoxyphenethyl) methylamino]-2-(3,4-dimethoxyphenethyl)-2-isopropyl-pentyl fluorescein.
40. N-(p-azido-[3,5-3H]benzoyl)aminomethyl verapamil
41. N-(p-azidobenzoyl)aminomethyl verapamil.
42. N-(p-azido-13-125I]salicyl)aminomethyl verapamil.
43. N-(p-azido salicyl) aminomethyl verapamil.
44. A diagnostic kit for the determination of the concentration of a verapamil binding polypeptide present in a sample comprising at least one package comprising a binding composition comprising a labelled verapamil derivative and a carrier.
45. The kit according to claim 44, further comprising:
instructions for use; and an indicating means, such that when said sample is admixed and maintained in contact with said binding composition for a time period sufficient to permit the formation of a derivative-polypeptide complex, said complex may be detected and the concentration of said polypeptide determined by use of said indicating means.
instructions for use; and an indicating means, such that when said sample is admixed and maintained in contact with said binding composition for a time period sufficient to permit the formation of a derivative-polypeptide complex, said complex may be detected and the concentration of said polypeptide determined by use of said indicating means.
46. The kit according to claim 44 wherein said verapamil derivative has a photoaffinity ligand attached to it.
47. The kit according to claim 44 wherein said indicating means is a fluoresent radical such that the concentration of said radical present can be spectrofluorometrically determined.
48. The kit according to claim 46 wherein said indicating means is a radiolabel attached to said photoaffinity ligand.
49. The kit according to claim 46, wherein sid derivative-polypeptide complex is irradiated with an effective amount of ultraviolet light to induce covalent bonding in said complex.
50. A verapamil derivative corresponding to the general structural formula:
where R' is a labeling group.
where R' is a labeling group.
51. The verapamil derivative according to claim 50 where R' is a photoaffinity ligand.
52. The verapamil derivative according to claim 51 wherein said photoaffinity ligand is radiolabelled.
53. The verapamil derivative according to claim 50 wherein R' is a fluorescent ligand.
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US25274688A | 1988-10-03 | 1988-10-03 | |
US252,746 | 1988-10-03 |
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CA 2000082 Abandoned CA2000082A1 (en) | 1988-10-03 | 1989-10-03 | Method for the detection of multiple drug resistant tumor cells and verapamil probes useful therein |
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AU (1) | AU4518889A (en) |
CA (1) | CA2000082A1 (en) |
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IT1258273B (en) * | 1992-04-06 | 1996-02-22 | Anna Maria Villa | METHOD FOR DETERMINING MULTI-DRUG RESISTANCE IN LIVING CELLS. |
US5436243A (en) * | 1993-11-17 | 1995-07-25 | Research Triangle Institute Duke University | Aminoanthraquinone derivatives to combat multidrug resistance |
FR2729569B1 (en) * | 1995-01-19 | 1997-07-25 | Consultants Internationaux Sur | PHARMACEUTICAL COMPOSITIONS USEFUL FOR THE REVERSION OF MULTIPLE DRUG RESISTANCE |
WO1998004250A1 (en) * | 1996-07-25 | 1998-02-05 | Consultants Internationaux Sur Le Medicament (S.A.R.L.) | Pharmaceutical compositions for the reversion of multiple resistance to drugs |
KR20020045445A (en) * | 2000-12-11 | 2002-06-19 | 최철희 | Screening methods for the detection of multidrug resistance protein inhibitors and reactive oxygen species-producing cytotoxic substances using the doxorubicin-resistant acute myelocytic leukemia subline AML-2/DX100 |
-
1989
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