WO1991002084A1

WO1991002084A1 - Method for detection and treatment of multiple drug-resistant tumor cells and useful colchicine derivative probes

Info

Publication number: WO1991002084A1
Application number: PCT/US1990/004440
Authority: WO
Inventors: Ahmad R. Safa
Original assignee: Michael Reese Hospital And Medical Center
Priority date: 1989-08-09
Filing date: 1990-08-08
Publication date: 1991-02-21
Also published as: AU6179690A

Abstract

A method for the detection of multiple drug-resistant tumor cells is described which comprises the admixture of a sample of mammalian tumor cellular material with an effective binding amount of a labeled colchicine derivative to form a complex with any P-gp that is present, and then determining the amount of P-gp present by quantitating the amount of labeled colchicine derivative bound to P-gp. Photoaffinity and radiolabeled derivatives of colchicine and the syntheses of such derivatives are described, as are a method of treatment for multiple drug-resistant tumors and a diagnostic kit.

Description

METHOD FOR DETECTION AND TREATMENT OF MULTIPLE DRUG-RESISTANT TU¬ MOR CELLS AND USEFUL COLCHICINE DERIVATIVE PROBES

This application is a continuation-in-part of copending application Serial No. 252,746, filed October 3, 1988 whose disclosure is incorporated by reference. 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 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 colchicine 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 candidates.

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 UV light, the drug derivative is converted into a highly reactive nitrene intermediate which covaleivtly reacts with one or more atoms adjacent to the binding site. A particular functional group at the acceptor site need not be present because the photogenerated species can react even with carbon- hydrogen bonds.

Hultidrug 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 this 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. 8_3_, 3847-3850.

The binding of a radiolabeled, photoactive dihydropy idine calcium channel blocker, [³H]-azidopine, to P-gp was inhibited by the presence of verapamil, actino ycin D and adriamycin, but was not significantly inhibited by the presence of colchicine. Safa et al. (1987) J. Biol. Chem. 2 2.₁ 7884-7888. These results indicate that there is an identity or close relationship of the calcium channel blocker acceptor and the gp 150- 180 Vinca alkaloid acceptor (P-gp) .

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 [Cornwell et al., (1987) FASEB J. 2, 51-54], that the gene coding for this protein generates the MDR phenotype when transfected into drug-sensitive cells [Gros et al., (1986) Nature (London) 222., 728-731], and that the purified protein exhibits ATPase activity [Hamada et al., (1988) J. Biol. Chem. 261, 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 colchicine has the property of binding to P-gp to form a colchicine-polypeptide complex, and thereby the property of assaying P-gp when the colchicine is appropriately labeled.

The presence of a specific colchicine binding affinity for P-gp was a completely unexpected result because prior studies have shown that colchicine does not significantly inhibit the binding to P-gp of compounds such as verapamil and vinblastine.

The present invention contemplates labeled colchicine derivatives that correspond to Formula I

where R¹ is a labeling agent. The labeling agent R' contains either a labeling portion alone or a labeling portion attached to a spacing group where the spacing group is linked to the amine group of the deacetyl colchicine molecule.

If R' were an acetyl group, rather than a labeling agent, the compound of Formula I would be colchicine.

Examples of particular labeling agents are cross-linking agents, radiolabeled ligands and fluorescent radicals. Cross-linking agents are contemplated to encompass photoaffinity ligands and chemoaffinity ligands. As described above, a labeling portion of a colchicine derivative can be bonded or linked directly to the amine group of the colchicine portion of the molecule or can be linked thereto through a spacing group. Where a spacing group is utilized, as is preferred, the labeling portion and spacing group are together referred to as a labeling agent. Preferred labeled colchicine derivatives are: N-(p_-azidobenzoyl)aminohexanoyldeacetyl colchicine,

N-(β-azido-3,5-['ϋjbenzoyl)aminohexanoyl¬ deacetyl colchicine,

N-(p_-azido salicyl)aminohexanoyldeacetyl colchicine, and

N-(E~azido-3-[¹²⁵I]salicyl)aminohexanoyl¬ deacetyl colchicine.

It is particularly preferred that the colchicine derivative have a photoaffinity labeling portion coupled to it, and that the derivative is also radiolabeled. It is also contemplated that upon binding of the labeled colchicine derivative to a polypeptide to form a colchicine 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 and forms a polypeptide- bound colchicine derivative.

A diagnostic kit is also contemplated that contains a composition that contains an effective amount of a labeled colchicine derivative of the present invention to bind to P-gp together with an aqueous carrier. In a preferred embodiment, the diagnostic kit also contains instructions for use and an indicating means.

The present invention also contemplates a method for the detection of multiple drug-resistant tumor cells. This method assays for a colchicine binding polypeptide that is characteristically present in multiple drug-resistant (MDR) tumor cells as the P-glycoprotein (P-gp) . The present method utilizes a labeled colchicine derivative that binds to the P-glycoprotein, is subsequently detected, and preferably quantitate . Comparison of the concentration or amount of P-gp in normal (tumor-free tissue) and tumor tissue of the same type is carried out and provides a means for diagnosing multiple drug resistance in the tumor.

In one method of the present invention, a sample of mammalian cellular material is admixed and maintained with an effective binding amount of a colchicine derivative for a time period sufficient to permit binding between the colchicine derivative and the P-gp polypeptide that may be present, and to form a colchicine derivative-polypeptide complex.

In a preferred embodiment, a radiolabeled photoaffinity-labeled colchicine derivative is utilized and the admixture is thereafter irradiated with actinic light of an appropriate wavelength and in a sufficient amount to form a reaction product having a covalent bond between the reacted colchicine derivative and the polypeptide*, producing a polypeptide-bound colchicine derivative. Any unreacted colchicine derivative is separated from the cellular material-containing portion, and the presence of covalently-bound colchicine derivative (reaction product) is assayed for to thereby provide a qualitative assay for the presence (detection) of the P-gp polypeptide. More preferably, the concentration or amount of the polypeptide present is quantitated by measuring the concentration or amount of polypeptide-bound colchicine derivative. By comparing the concentration or amount of the colchicine derivative-polypeptide reaction product present in the sample of tumor cells with the concentration or amount of polypeptide-bound colchicine derivative found with normal cells of the same tissue type as the tumor cells, multiple drug resistance is indicated if the reaction product is present^" and its concentration or amount is elevated about five-fold or more relative to its concentration or amount in normal cells.

A method of treatment is also contemplated in the present invention as a further embodiment of the above method. Here, the concentration of P-gp is determined in a sample tumor to be treated using the quantitative aspect of the above method. If the concentration of the P-gp in the tumor sample is about five-fold or more greater than the concentration of P-gp present in normal cells of the same cell type as the tumor, then the tumor is treated as a multiple drug- resistant cell tumor. A patient with such a tumor is treated with a natural product-type chemotherapeutic agent together with a modulator compound.

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 permits 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, which resistance typically is found after weeks or months of chemotherapy. The present invention helps to prevent the delay resultant from the trial and error approach previously practiced. Description of Fiσures

In the figures forming a portion of this disclosure:

Figure 1 contains three portions that illustrate the structural formulae of colchicine (1A) , and two radiolabeled photoaffinity derivatives of colchicine: N-(p-azido-3,5-[³H]benzoyl)- aminohexanoyldeacetyl colchicine (IB, sometimes referred to as [^- BC) and N-(]≥-azido-3-

[¹²⁵I]salicyl)aminohexanoyldeacetyl colchicine (1C, sometimes referred to as [¹²⁵I]-NASC) , respectively.

Figure 2 is a photograph of a SDS-PAGE (5-15 percent gel containing 4.5 M urea) fluorograph of [~E] ~ NABC photoaffinity labeled membrane vesicles (25 ug of protein) of drug-sensitive Chinese hamster lung DC-3F cells (lanes 1 and 2), drug-resistant DC-3F/VCRd-5L cells (lanes 3 and 4) and of material immunoprecipitated with P-gp monoclonal antibody C 219 (lane 5) . Photoaffinity labeling was carried out with 0.5 uM [³H]- NABC (47.7 Ci/mmol) in the absence (lanes 1, 3) and presence (lanes ϊ'and 4) of 1000 uM colchicine (COL). For immunoprecipitation, 200 ug of ['HJ-NABC photolabeled deoxycholate-solubilized membrane vesicles were incubated with 10 ug of the P-gp monoclonal antibody C 219 and after subsequent incubation with Protein A-Sβpharose, samples were processed for SDS-PAGE and autoradiography (lane 5) . The positions of molecular weight standards in kDa are present at the left.

Figure 3 is a photograph of a SDS-PAGE (5-15 percent gel cbntaining 4.5 M urea) fluorograph of [¹²⁵I]- NASC photolibeϊed membrane vesicles (25 ug of protein) of drug-sensitive Chinese hamster lung DC-3F cells (lanes 1 and 2) and drug-resistant DC-3F/VCRd-5L cells

(lanes 3-11) in the absence ( , lanes 1 and 3) and presence of 100 uM colchicine (COL, lanes 2 and 4) , 1000 uM colchicine (lane 5) , or 100 uM vincristine (VCR, lane 6) , vinblastine (VBL, lane 7) , doxorubicin (DOX, lane 8) , actinomycin D (ACT, lane 9) , and methotrexate (MTX, lane 10) . For immunoprecipitation (MCA, lane 11) , 200 ug of [¹²⁵I]-NASC photolabeled deoxycholate-solubilized membrane vesicles and monoclonal antibody C 219 were used. Figure 4 is a representation of the high resolution fast atom bombardment mass εpectrograph for NABC.

Figure 5 is a representation of the high resolution fast atom bombardment εpectrograph for NASC.

Figure 6 is a representation of the ultraviolet-visible absorbance spectra for NABC (Panel A) and NASC (Panel B) . Detailed Description of the Invention

The present invention relates to (a) the synthesis of radiolabeled derivatives of colchicine, (b) the use of these derivatives to detect multidrug- resistant tumor cells and (c) the use of these compounds in the diagnosis and treatment of multiple drug- resistant tumors.

A. The Compounds

Colchicine (N-(5,6,7,9-tetrahydro-l,2,3,10- tetramethoxy-9-oxobenzo[a]heptalen-7-yl)-acetamide) is an alkaloid which was originally derived from the plant Colchicum autumnale. Colchicine is principally utilized in the treatment of acute gouty arthritis and is a useful an i i otic agent as a result of its inhibition of spindle formation during mitosis. The antimitotic properties of colchicine are similar to those of Vinca alkaloids such as vincristine and vinblastine. As used herein, a colchicine derivative of the present invention is a colchicine molecule in which the acetyl group has been removed and replaced by a labeling agent that is attached at one end to the deacetyl colchicine by an amide linkage. Such a compound is referred to herein as a "colchicine derivative" or a "deacetyl colchicine," which terms are used interchangeably. A preferred compound of the present invention is a pharmacologically active, radiolabeled, photoactive colchicine derivative which, after photoactivation, is capable of covalently reacting with the unique cellular polypeptide (P-gp) that has a binding affinity for the parent colchicine compound.

A colchicine derivative of the present invention Corresponds to Formula I

where R¹ is a labeling agent. The labeling agent R¹ is attached to the amino group of deacetyl- colchicine and contains either a labeling portion alone or a labeling portion attached to a spacing group where the spacing group Is linked to the amino group of deacetyl cβlchicine. Exemplary labeling agents are cross-linking agents, radiolabeled ligands and fluorescent radicals.

As used herein, a "cross-linking agent" is a compound or functional group that is capable of producing a bridge-like attachment between a P-gp molecule^"and a colchicine derivative by the formation of a chemical bond between the molecules. Illustrative of such cross-linking agents are chemoaffinity ligands, such as the amino acid cysteine which forms a disulfide cystine bond with proteins and polypeptides, and photoaffinity ligands such as azido groups which when irradiated by light form a covalent bond with adjacent proteins and polypeptides.

A labeling portion of a colchicine derivative of the present invention can be bonded or linked directly to the amino group of deacetyl colchicine or can be linked thereto through a spacing group. Where a spacing group is utilized, as is preferred, the labeling portion and the spacing group are together referred to as a labeling agent.

Specific tritiated and radioiodinated photoaffinity derivatives of colchicine are used herein illustratively to directly identify P-gp in MDR cells as a specific acceptor for colchicine.

These photoactive radiolabeled drugs are important probes, and development of photoaffinity labeling technology with the photoactive derivatives of colchicine 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.

A compound of the present invention is valuable in identifying cellular receptors which may be involved in novel, as well as in known, mechanisms of colchicine action. Knowing the properties of colchicine, plus the ability to label the derivative. permits one to easily probe specific receptors in the cells for colchicine. Without such labeled derivativeε, preferably those which are both photoactive and radioactive, it is difficult or impossible to probe the interactions of colchicine with its receptors in cells and in tissue homogenates.

Two specific compounds of the present invention are radiolabeled photoactive derivatives of colchicine: 1) N-(p.-azido-3,5-['HJbenzoyl)amino¬ hexanoyldeacetyl colchicine also referred to as [ i] - NABC; and

2) N-(p_-azido-3-[¹²⁵I]salicyl)aminohexanoyldeacetyl colchicine also referred to as [¹²⁵I]-NASC. Specifics for the preparation of these two compounds are provided hereinafter_*

The UV-visible absorption spectra of NABC and NASC were composites of the spectra of their component chromophores, i.e.,. the ->-azidobenzoyl group (lambda max 270 nm) or the azidosalicyl group (lambda max 280 and 310 mn) and colchicine (lambda max 350 nm) . The IR spectra showed a strong resonance at 2130 cm^"1 indicative of the presence of azide. The elemental composition was found by high resolution mass spectroscopy. The structures of colchicine, [hi]-NABC and [¹²⁵I]-NASC are shown in FIG. 1.

The compounds can be used to photoaffinity- label a 150-180 kDa membrane protein which is present in MDR tumor cells. The resulting specifically labeled 150-180 kDa membrane protein in drug-resistant cells has been immunoprecipitated with a monoclonal antibody, C219, specific for P-glycoprotein. Kartner et al. (1985) Nature (London) HI, 820-823.

Binding specificity was established by competitive blocking of specific photolabeling with the nonradioac€ive photoactive derivatives as well as with colchicine. Specifics of these studies are provided hereinafter.

The colchicine portion of a useful colchicine molecule is linked to a photoaffinity labeling portion (group) of the derivative either directly, or more preferably by means of a spacing group that contains a chain of 3 to about 8 atoms. The spacing group provides added rotational or other flexional freedom to the photo-induced nitrene so that that entity can react with P-gp, and together with the photoaffinity labeling portion (group) forms a photoaffinity labeling agent.

Typical spacing groups contain two reactive functional groups; a first of which reacts with the deacetylated amino group of the colchicine portion and a second of which reacts with a functional group of the photoaffinity labeling portion. Exemplary of such spacing groups are dicarboxylic acids, dialdehydes, diisocyanates and amino acids.

Illustrative dicarboxylic acids include oxalic, malonic, succinic, fumaric, glutaric, adipic and terephthalic acids, their anhydrides, acid halides and activated esters such as N-hydroxysuccinimido ethers. Where a dicarboxylic acid spacing group is employed, the photoaffinity labeling portion contains a suitable reactive functional group such as an alcohol, amine or mercaptan to form an ester, amide or thioester linkage with the dicarboxylic acid.

Illustrative dialdehydes include the aldehydes of the above dicarboxylic acids. Where dialdehydes are used, the photoaffinity labeling portion contains an amine functional group and the colchicine and photoaffinity labeling agent portions are joined by reactive alkylation as with sodium borohydride. It is usually preferred that one of the aldehyde groups be blocked with a removable group such as an acetal group so that the two portions of the molecule can be joined separately without destroying both aldehyde groups when either colcliicine^*or a photoaffinity labeling agent is first reacted with the dialdehyde.

Diisocyanates form a urea linkage with the amine group of the colchicine portion of the molecule and can form a urethane, urea or thiourethane with the photoaffinity labeling agent. Illustrative diisocyanates include tolylene 2,4- and 2,6- diisocyanates, methylene dicyclohexane diisocyanate, isophorone diisocyanate, and diphenyl ethylene diisocyanate and the like.

The spacing group illustrated herein is an amino acid. Exemplary amino acid spacing groups include beta-alanine. ^"σamma-aminobutyric acid, epsilon- aminocaproic acid (specifically utilized herein) and oroega-aminocaprylic acid. Here, the carboxylic acid functional group forms an amide with the amine of the colchicine-portion and the amine functional group forms an amide with a carboxylic acid group of the photoaffinity labeling portion. The primary amine can also form a secondary amino group when reacted with a photoaffinity labeling portion such as p-azidophenaryl bromide. ^> '

As noted earlier, the spacing group contains a chain of 3 to about 8 atoms. The "chain" is intended to include the atoms provided by the spacing group such as an amϊne nitrogen, carboxyl carbon and carbamyl groups, and to exc.ft_.de~atoms provided by colchicine (the amine) and photoaffinity labeling agent. Where the spacing group contains a ring structure such as a substituted benzene ring, "chain" length is deemed to be the lowest number of atoms linking the two functional groups. Thus, the "chain" in terephthalic acid contains six atoms, four for the ring and one each for the carboxyl carbon atoms, whereas the "chain" for 2,4-tolylene diisocyanate contains a total of seven atoms, three for the ring and two each for the carbamyl groups. Exemplary photoaffinity labeling groups and agents are commercially available and include 4-azidobenzoic acid- N-hydroxysuccinimide ester, N-(5-azido-2- nitrobenzyloxy)succinimide, 6-(4-azido-2- nitrophenylamino)hexanoic-acid-N-hydroxysuccinimide ester, β-azidophenacyl bromide, 3-(4-azidophenyldi- thio)propionic acid-N-hydroxysuccinimide ester and 2- diazo-3,3,3-trifluoropropionic-acid-->-nitrophenylester.

Fluorescent colchicine derivatives are prepared by coupling a fluorescent molecule (such as fluorescein isothiocyanate) to a deacetyl colchicine molecule directly or through a spacing group or through a cross-linking agent.

B. Methods

A method for assaying tumor cells to ascertain whether they are multiple drug-resistant is provided by the present invention. Since multiple drug-resistant tumor cells 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 permits a method of treatment to be utilized in accordance with another method of the present invention whereby tumors are assayed for multiple drug resistance by the detection of elevated concentrations of P-gp in the tumors.

In the first method, a sample of tumor tissue is admixed with a composition containing a labeled colchicine derivative. For example, the concentration of the colchicine derivative is about 0.5 micromolar (uM) NABC or about 40 nanomolar (nM) NASC in an aqueous buffer such as 10 millimolar (mM) Tris-HCl (pH 7.4) containing about 4 percent dimethylεulfoxide (DMSO) . The admixture is maintained at about 25 degrees C for about 30 minutes. The tissue is separated from the admixture, such as by centrifugation, and resuspended in buffer. If a photoaffinity labeled colchicine derivative is used, the suspension is then irradiated (e.g. 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 εpectrofluorimetry. A determination that the concentration of P-gp in the tumor tissue is significantly elevated (by five-fold or more) over the concentration present in non-MDR cells derived from the same tissue type is an indication that the tumor is multiple drug-resistant.

If such elevated levels of P-gp are found, then a further embodiment of the above method is to treat the human or other animal having the tumor with a natural product type chemotherapeutic agent together with a modulator. Exemplary natural product type chemotherapeutic agents are vinblastine, vincristine and adriamycin. Exemplary modulator compounds are verapamil and trifluoperazine. This type of therapy is itself well known to skilled workers as are the doses of the specific chemotherapeutic agents and modulators, and the entire treatment regimens. The important and unexpected aspect of this therapy is that MDR can be assayed for early in the treatment period so that important treatment time is not wasted in treating a MDR tumor by standard therapy to which the tumor is refractory.

When the concentration or amount of P-gp in a tumor iε not elevated by five-fold or more over the concentration in normal tissue, the tumor is classified as not exhibiting multiple drug resistance and is treated by standard chemotherapy. Standard chemotherapy consists of the administration to a patient of one or more drugs selected from five principal groups. The five groups are alkylating agents, antimetabolites, plant alkaloids, antitumor antibiotics and other agents.

The alkylating agents include compounds such as nitrogen mustard, cyclophosphamide, melphalan and chlorambucil. These compounds function by attacking electron-rich regions of molecules in the tumor, adding alkyl groups to oxygen, nitrogen and sulfur atoms present in proteins and nucleic acids.

The antimetabolites include compounds that function as purine, pyrimidine and folate antagonists. These compounds interfere with the progression of the tumor cells through the cell cycle. Exemplary purine antagonists are 6-mercaptopurine and 6-thioguanine which inhibit de novo purine synthesis and purine interconversions. Exemplary pyrimidine antagonists include cytarabine, 5-fluorouracil and 5-floxuridine. Cytarabine inhibits DNA synthesis by inhibiting the enzymes deoxycytidylate kinase and DNA polymerase. 5-Fluorouracil and 5-floxuridine inhibit DNA synthesis by inhibiting thymidylate synthetase. An exemplary folate antagonist is methotrexate which binds tightly to the enzyme dihydrofolate acid reductase and inhibits the formation of tetrahydrofolic acid, which is the reduced and active form of folic acid necessary for pyrimidine biosynthesis.

The plant alkaloids include the Vinca alkaloids, podophyllotoxins and colchicine. The Vinca alkaloids (such as vincristine and vinblastine) and colchicine inhibit mitosis in cells by interaction with microtubular proteins. The podophyllotoxinε (εuch as etoposide and teniposide) inhibit both DNA εynthesis and RNA-dependent protein synthesis. The plant alkaloids are administered to patients by intravenous infusion and have plasma half lives of from 15 minutes (vincristine) to 11.5 hours (etoposide).

The antitumor antibiotics include compoundε which intercalate between base pairε in DNA (εuch aε dactinomycin, doxorubicin, and adriamycin) , produce εcission of the DNA (such as bleomycin) and covalently bind to DNA by acting as a bifunctional alkylating agent (such as mitomycin) .

Other compounds which have useful chemotherapeutic properties are cisplatin (which is an inorganic salt that functions as a bifunctional alkylating agent and a DNA intercalator) , biologic response modulators (such as the interferons) , and various enzymes and endocrine hormones for specific types of tumors.

.The above, standard, chemotherapeutic drugε and treatment regimens are also well known to skilled workers and need not be dealt with further herein.

The present invention also encompasses a diagnostic kit for the determination of the concentration of P-gp present in a sample. This kit includes at least one package that contains a binding compoεition comprising a labeled colchicine derivative as described before and a carrier. The labeled colchicine 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 quantitat on of the formation of a complex formed between the labeled colchicine derivative and the polypeptide are typically also included in a kit.

The binding composition contains a colchicine derivative as described in the present invention. Such colchicine derivative can have attached to it a photoaffinity or chemoaffinity ligand.

"Instructionε for uεe" typically include a tangible expression describing the reagent concentration or at least one asεay 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 polypeptide-bound colchicine derivative. Particularly preferred indicating meanε are radiolabels or fluorescent molecules attached to the bound colchicine 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.

All reagents used in the following examples were commercially available. Actinomycin D, doxorubicin, colchicine and methotrexate were purchased from Sigma Chemical Co. (St. Louis, MO) . ϋ-hydroxysuccinimidyl-4-azidobenzoate and ϋ-hydroxysuccinimidyl-4-azidosalicylate were obtained from Pierce Chemical Co. (Rockford, IL) . Vinblastine was obtained from Eli Lilly and Co. (Indianapolis, IN), and monoclonal antibody C219 was purchaεed from Centacor (Malvern, PA) . All other chemicals, unless otherwise noted, were obtained commercially and were of reagent grade. EXAMPLE 1: Synthesis of

benzoyl)aminohexanoyldeacetyl colchicine

The photoactive, radioactive N-(]≥-azido-3,5- [³H]-benzoyl)z_minohexanoyldeacetyl colchicine ([³H]NABC) was εynthesized as follows. N-Hydroxysuccinimidyl-4- azido-3,5-["H]-benzoate (47.7 Ci/mmol) in 0.05 ml isopropanol (New England Nuclear, Boston, MA) was added to 0.45 ml of a solution of aminohexanoyldeacetyl colchicine trifluoro acetate (Molecular Probes Inc., Eugene, OR) (0.5-0.7 uM) in chloroform in the presence of triethyla ine and the mixture waε maintained at 4 degrees C for 24 hours. The product was purified by silica gel column chromatography, with 2 percent (vol/vol) methanol in chloroform as eluate. The product gave a single UV-absorbing spot on a silica gel thin- layer chromatography (TLC) plate with fluorescence indicator (Anal tek, Newark, DE) , run in chlorofom methanol:water (80:20:3 v/v) , R_f «- 0.8; and benzene: methanol (3:1 v/v), R_f « 0.5. Identity and purity of the compound was confirmed by co- chromatography with NABC by TLC run in the same solvent.

The unlabeled colchicine derivative, NABC, was prepared by the εame procedure. The abεorbance εpectrum of NABC (Figure 6, Panel A) shows absorbance peaks at about 245 nm and 350 nm. The elemental composition of NABC was found by high resolution fast atom bombardment mass εpectroεcopy to exhibit a protonated molecular ion at M/Z 616, with a (MH)+ maεs of 616.2754, corresponding to an elemental composition of C^D^S^_γ+H. The resultε are illustrated in Figure 4.

EXAMPLE 2: Syntheεiε of N-(p-azido-3-[¹²⁵I]- εalicyl)aminohexanoyldeacetyl colchicine The photoactive, radioactive N-(p_-azido-3- [¹²⁵I]εalicyl)aminohexanoyldeacetyl colchicine ([¹⁵I]- NASC) waε εynthesized by two step reactions.

First Reaction:

N-hydroxysuccinimidyl-4-azidosalicylate (NAS) (1.67 nmole) (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 Na¹²⁵I in 10 microliters (ul) of 0.1 N NaOH (2200 Ci/mmole, Amersham Corporation, Arlington Heights, IL) were added. Chlbramine 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 percent aqueous NaCl were added and the reaction mixture was extracted with 300 ul of ethyl acetate. The extract was evaporated under nitrogen and the reaction mixture was chro atographed on a silica gel G thin layer using a running solvent of benzene:chloroform:ethyl acetate: acetic acid (1:1:1:0.1, v/v). The product (N-hydroxy- succι •nι•mι•dyl-3-[125I]-4-azι•doεalι•cylate) , gave a radioactive εpot by autoradiography that accounted for 90 percent of the total radioactivity (R_f = 0.4).

Second Reaction:

The product of the first reaction, above, was dissolved in chloroform (0.45 ml), aminohexanoyldeacetyl colchicine trifluoro acetate (Molecular Probes, Eugene, OR) (0.5 umole) was added and the mixture was maintained at 4 degrees C. The progresε of the reaction waε monitored by TLC [run in benzene:methanol (3:1), R_f = 0.57]. The reaction mixture waε then applied to a column (0.5 x 7 cm) of silica gel equilibrated in chloroform. After washings with chloroform (5 ml) and of 1 percent methanol in chloroform (5 ml) , the product was eluted from the column with 5 percent methanol in chloroform. The product gave a single radioactive spot on silica gel TLC with a R_f ** 0.57 when run in a solvent of benzene:methanol (3:1) as determined by autoradiography.

The unlabeled compound, NASC, was prepared by reaction of unlabeled N-hydroxysuccinimidyl-4- azidosalicylate with aminohexamoyldeacetyl colchicine trifluoro acetate as described above. The absorbance spectrum of NASC (Figure 6, Panel B) shows a shoulder at about 245 nm, a broad absorbance peaks at 310-320 nm and a second absorbance peak at 350 nm. The elemental composition of NASC was found by high resolution fast atom bombardment maεs spectroscopy to be: C₃₃0₈N₅H₃₇+H, with a molecular ion mass of 632.276, exhibited at M/Z 632. The resultε are illustrated in Figure 5.

EXAMPLE 3: Photoaffinity Labeling of Cells

Drug-senεitive DC-3F Chineεe hamεter lung cellε and vincriεtine-reεistant variant DC-3F/VCD-5L cells (MDR cells) that were selected for primary resistance to vincristine (2750-fold resistant) and crosε-reεistance 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 deεcribed in Peterεon et al., (1983) Cancer Reε. 3, 222-228; and Meyerε et al., (1985) J. Cell Biology, 10_), 588-597. Drug-resistant cells were treated weekly with a maintenance concentration of vincristine (40 ug/ml) . Vincristine waε removed from cultureε 8-10 dayε before εtudies were carried out using them. Exponentially growing cellε were harvested by scraping culture plates with a rubber blade. Membrane vesicles of cell suspensions were prepared by nitrogen cavitation and differential centrifugation as described by Lever, (1977) J. Biol. Chem. 202, 1990-1997. Protein concentrations were determined by the procedure of Lowry et al., (1951) J. Biol. Chem. 193. 265-275. Membrane vesicles (about 50 ug of protein) in 10 mM Tris-HCl buffer (pH 7.4) containing 4 percent (vol/vol) dimethylεulfoxide, 250 mM sucrose, 10 mM NaCl, 1.5 mM MgCl₂, and 1 mM ATP, and about 500 nM [³H]-NABC (47.7 Ci/mmol) or 40 nM [¹²⁵I]-NASC (220 Ci/mmol) in a final volume of 0.05 ml were photolabeled after preincubation (admixture and maintenance) for 30 minutes at 25 degrees C in the absence or presence of a nonradioactive competing ligand. Samples were then irradiated for 20 minutes at 25 degrees C with two 15 watt self-filtering 302 nanometer (nm) or 366 nm lampε (model XX-15, Ultraviolet Productε, San Gabriel, CA) .

The samples labeled with the photoactive derivatives were then solubilized for sodium dodecyl sulfate/polyacrylamide gel electrophoreεiε (SDS/PAGE) . Fluorography εhowed a photolabeled

150-180 kDa polypeptide in membrane veεicleε from MDR cellε (Figure 2, laneε 3 and 4, Figure 3, laneε 3, 4 and 5) that waε not detectable in the drug-εensitive parent DC-3F cells. (FIGURE 2, lanes 1 and 2, Figure 3, laneε 1 and 2).

[³H]-NABC photoaffinity labeling in the preεence of 1000 uM non-radioactive colchicine produced an 82 percent inhibition of photolabeling of the 150-180 kDa polypeptide in the MDR cells. (FIGURE 2, laneε 2 and 4)

Autoradiographε of [¹²⁵I]-NASC photolabeled membrane veεicleε from these cells indicated that P-gp waε faintly .labeled in the parental drug-sensitive DC-3F cells, whereas radiolabeling of the MDR variant was increased εubεtantially. (Figure 3)

[¹²⁵I]-NASC radiolabeling of P-gp in the presence of 100 and 1000 uM colchicine was reduced by 45 and 85.5 percent, reεpectively, indicating the εpecificity of [¹²⁵I]-NASC photolabeling for P-gp. Furthermore, vijicriεtine, the drug uεed to εelect the resistant variant, reduced photolabeling of P-gp by 88.8 percent. Vinblastine, doxorubicin and actinomycin D, drugs to which these cellε display crosε-resistance, inhibited the [¹²⁵I]-NASC photolabeling by 91.1, 61.6 and 51 percent, reεpectively. (FIGURE 3) Methotrexate, an antitumor agent to which the reεiεtant cell line demonstrates no crosε-reεiεtance, had no εignificant effect on the [¹²⁵I]-NASC photolabeling of P-gp. Theεe results inicate th^t several classes of cytotoxic drugs interact with the colchicine binding site on P-gp and that this membrane drug acceptor has an important role in maintaining the MDR. phenotype.

Although colchicine does not inhibit the binding of erapamil or vinblastine to P-gp in MDR cells, this study shows that colchicine photolabeling is inhibited by vinblastine. Furthermore, verapamil can also reduce colchicine photolabeling of P-gp suggeεting that colchicine may bind to P-gp at a εite which iε diεtincjt from the vinblaεtine and/or verapamil binding site(s), or that these agents bind to overlapping εiteε. It iε alεo poεεible that P-gp may have a common drug-acceptor εite diεplaying high affinity for Vinca alkaloidε and lower affinity for colchicine. Alternatively, the inhibitory effect cauεed by vinblaεtine may alεo be the reεult of a negative alloεteric effect of thiε drug on [hi]-NABC or [¹²⁵I]-NASC labeling by binding to a separate site on P-gp thereby increasing the binding and coupling of these photoactive analogs to P-gp.

EXAMPLE 4: Immunoprecipitation

Cell membrane vesicleε were prepared by nitrogen cavitation and differential centrifugation and were photoaffinity labeled in the preεence or abεence of competing drugs as described in EXAMPLE 3.

Immunoprecipitations of [^J-NABC- or [¹²⁵I]-NASC-photolabeled membrane veεicleε (20 ug of protein) εolubilized in a deoxycholate buffer [100 ml of 50 mM tris-HCl buffer (pH 7.0) containing 150 mM NaCl, 1 percent Triton X-100, 1 percent εodium deoxycholate, 0.1 percent SDS, 1 mM EDTA, 1 mM phenylmethylεulfonyl fluoride and 10 mg/ml Trasylol] were performed as described in Safa et al., (1986) J. Biol. Chem. 261. 6137-6140, with monoclonal antibody C219, specific for P-gp, or 30 ug of normal non-immune mouεe εerum as a control. The radiolabeled 150-180 kDa polypeptide was immunoprecipitated by the C219 antibody indicating its identity with P-gp. No radioactivity was detected in the precipitate when non-immune mouse εerum waε used.

The photolabeled samples were electrophoresed on SDS/5-15 percent polyacrylamide gradient gelε containing 4.5 M urea and processed for fluorography or autoradiography. (FIGURE 2, lane 5 and FIGURE 3, lane 11).

The foregoing description and the EXAMPLES are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily preεent themselves to those skilled in this art.

Claims

I Claim:

1. A colchicine derivative correεponding to the general εtructural formula:

where _.' is a labeling group.

2. The colchicine derivative according to claim 1 where R' is a photoaffinity ligand.

3. The colchicine derivative according to claim 3 wherein said photoaffinity ligand is radiolabeled.

4. N-(p-azido-3,5-[^benzoyl)aminohexanoyl¬ deacetyl colchicbie.

5. N-(p-azido-3-[¹²⁵I]salicyl)aminohexanoyl¬ deacetyl colchicine.

6. A method for assaying for multiple drug resistance (MDR) of mammalian tumor cellε compriεing: a) admixing a εample of mammalian tumor cellular material with an effective binding amount of a labeled colchicine derivative; b) maintaining said admixture for a time period sufficient to permit the formation of a colchicine derivative-polypeptide complex between said colchicine derivative and a MDR-specific binding polypeptide that may be present to produce a polypeptide-bound colchicine derivative; c) separating said sample of cellular material containing said polypeptide-bound colchicine derivative from said colchicine derivative that remains unbound in said admixture; d) determining the concentration of said polypeptide-bound colchicine derivative present in said sample of cellular material; and e) comparing the concentration of said polypeptide-bound colchicine derivative found in step (d) to the concentration of said polypeptide-bound colchicine derivative found with non-MDR cells of the εame tissue type as said tumor cells, an increased concentration of said polypeptide-bound colchicine derivative in said tumor cells relative to the concentration in εaid normal cellε indicating multiple drug resistance.

7. The method according to claim 6 wherein said colchicine derivative is a photoaffinity labeled derivative.

8. The method according to claim 7 wherein said labeled colchicine derivative is radiolabeled.

9. The method according to claim 7, wherein said photoaffinity labeled colchicine derivative is N- (E-azidobenzoyl)aminohexanoyldeacetyl colchicine or N- (E-azidoεalicyl)aminohexanoyldeacetyl colchicine.

10. The method according to claim 8, wherein said colchicine derivative is radio-iodinated.

11. The method according to claim 8, wherein εaid colchicine derivative iε tritiated.

12. The method according to claim 10, wherein εaid colchicine derivative iε N-(p.-azido-3-[ ⁵I]- εalicyl)aminohexanoyldeacetyl colchicine.

13. The method according to claim 11, wherein said colchicine derivative is N-(E-azido-3,5- [^Jbenzoyl)aminohexanoyldeacetyl colchicine.

14. The method according to claim 8, further compriεing irradiating εaid sample with an effective amount of ultraviolet light to induce covalent bonding in said colchicine derivative-polypeptide complex.

15. The method according to claim 14 wherein εaid determination of the amount of εaid polypeptide present in εaid sample is quantitated by autoradiography.

16. The method according to claim 6 wherein said sample of mammalian tumor cellular material is human tumor tissue.

17.. The method according to claim 6 wherein said sample of mammalian tumor cellular material comprises cellular membrane vesicles.

18. The method according to claim 6, wherein εaid MDR-εpecific binding polypeptide iε 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 cellε.

19. The method according to claim 6, wherein εaid separation of said sample of cellular material is by centrifugation of said admixture.

20. A method of treatment for a mammalian tumor comprising: a) admixing a sample of said tumor with an effective binding amount of a labeled colchicine derivative; b) maintaining said admixture for a time period sufficient to permit the formation of a colchicine derivative-polypeptide complex between said colchicine^.derivative and a MDR-specific binding polypeptide that may be present to produce a polypeptide-bound colchicine derivative; c) separating said tumor sample containing said polypeptide-bound colchicine derivative from εaid admixture; d) determining the concentration of εaid polypeptide-bound colchicine derivative present in εaid sample; e) comparing the concentration of said polypeptide-bound colchicine derivative found in step (d) to the concentration of εaid polypeptide-bound colchicine derivative found with normal cellε of the εame tissue type as said tumor; and f) when said concentration found in (d) iε about five-fold greater than said concentration present with normal cells, treating εaid tumor as a multiple drug-reεiεtant cell tumor.

21. The method of treatment according to claim 20 wherein εaid multiple drug-reεiεtant cell tumor iε treated with a natural product-type chemotherapeutic agent together with a modulator compound.

22. The method of treatment according to claim 21 wherein εaid natural product-type chemotherapeutic agent iε a compound εelected from the group conεiεting of vinblastine, vincristine and adriamycin.

23. The method of treatment according to claim 20 wherein εaid modulator compound iε verapamil or trifluoperazine.

24. The method of treatment according to claim 21 wherein εaid multiple drug-resistant cell tumor is treated with vincristine and verapamil.

25. A diagnoεtic kit for the determination of the concentration of a MDR-εpecific binding polypeptide present in a ^'sample comprising at least one package comprising: a binding compoεition compriεing a labeled colchicine derivative and a carrier.

26. The kit according to claim 25, further compriεing*: inεtructionε for uεe; and an indicating means, such that when said sample is admixed and maintained in contact with εaid binding compoεition for a time period εufficient to permit the formation of a colchicine derivative-polypeptide complex, εaid complex may be detected and the concentration of εaid polypep^'tidfe determined by uεe of εaid indicating meanε.

21. The kit according to claim 25 wherein εaid colchicine derivative haε a photoaffinity ligand attached to it.

28, The kit according to claim 27 wherein εai^'d ±nciicating*meanε iε a radiolabel attached to εaid photόafunity ligand.

29. The kit according to claim 27, wherein εaid colchicine derivative-polypeptide complex iε irradiated with an effective amount of ultraviolet light to induce covalent bonding in εaid complex.