WO1992013551A1

WO1992013551A1 - Inhibition of multidrug transport by transforming growth factor beta and uses thereof

Info

Publication number: WO1992013551A1
Application number: PCT/US1992/000974
Authority: WO
Inventors: Herman J. Schluesener; J. Gordon Foulkes
Original assignee: Oncogene Science Inc
Current assignee: OSI Pharmaceuticals LLC
Priority date: 1991-02-04
Filing date: 1992-02-04
Publication date: 1992-08-20
Anticipated expiration: 1993-08-04
Also published as: AU1426792A

Abstract

The present invention provides a method of inhibiting the proliferation of mammalian tumor cells, which are resistant to the anti-proliferative affects of a predetermined concentration of a chemotherapeutic agent, which concentration was capable of inhibiting the proliferation of such mammalian tumor cells when such cells were nonresistant to such concentration of such chemotherapeutic agent which comprises a) contacting the resistant mammalian tumor cells with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to such predetermined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells with the chemotherapeutic agent at the predetermined concentration so as thereby to inhibit proliferation of the mammalian tumor cells.

Description

INHIBITION OF MULTIDRUG TRANSPORT BY TRANSFORMING GROWTH FACTOR BETA AND U8ES THEREOF

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

BACKGROUND OF THE INVENTION

Many human cancers that are initially responsive to treatment by conventional chemotherapeutic drugs often become drug resistant following multiple rounds of chemotherapy. Resistance extends not only to the drug initially used for the treatment of the cancer but also to other cancer chemotherapeutic drugs. Such resistance is known as multiple drug resistance (MDR) and poses a major problem in chemotherapy (1, 2, 3). One major cause of MDR is the overexpression of a small gene family (the dr genes) which encode P-glycoproteins homologous to bacterial transport proteins (4, 5, 6). A heterogenous group of agents, which have been termed 'chemosensitizers* interfere with the function of the P-glycoprotein, e.g. by directly binding to the pl70 glycoprotein, and prevents extracellular transport across the plasma membrane (1 2 , 3, 7, 8). Such chemosensitizers are being developed to treat multidrug resistance.

The results of the experiments provided herein show the effect of transforming growth factor type Betas (TGF-βs) on multidrug transport using human glioblastoma cells and rat astrocytes as model examples. Glioblastoma cells are from rapidly growing tumors, usually confined to the cerebral hemispheres and composed of a mixture of spongioblasts, astroblasts, and astrocytes.

Transforming growth factor β (TGF-0) is part of a family of multifunctional proteins which appear to modulate, alone or in combination with other molecules, cell proliferation, differentiation and other effects on cells (9, 10, 11). Three homodimeric forms have been identified in humans, termed TGF-01 (12), TGF-02 (13) and TGF-33 (14).

On April 20, 1984, a patent application entitled "Tissue- derived Tumor Growth Inhibitors, Methods Of Preparation and Uses Thereof was filed with the United States Patent and Trademark Office under U.S. Serial No. 111,022, filed October 20, 1987, which is a continuation in part of U.S. Serial No. 922,121, filed October 20, 1986, which is a continuation in part of U.S. Serial No. 847,931, filed April 7, 1986, which is a continuation in part of U.S. Serial No. 725,003, filed April 19, 1985 (15). U.S. Serial No. 111,022 disclosed the nucleic acid sequence of mature, human TGF-B3.

TGF-J4 and TGF-05 have thus far only been identified in chickens and frogs, respectively (16, 17). Additionally, this tumor growth factor family further a heterodimer which contains a TGF-01 and a TGF-32 chain designated TGF-/81.2

Many problems have been encountered in the attempt to use chemotherapeutic drugs for the treatment of cancer (1-8) . Among the problems are (a) developed drug resistance following multiple rounds of chemotherapy, (b) de novo drug resistance either as observed in tumor types such as lung and colon tumors which fail to respond to current cytotoxic chemotherapy or in patients which present with a drug 1

3 resistant tumor of a type which normally respond t chemotherapy, and (c) gaining access to restricted location in the body, e.g. the brain. In order to overcome thes problems, the present invention provides methods fo overcoming drug resistance using TGF-βs . Additionally, thi invention also provides methods for enhancing access to th central nervous system and brain by pharmacologica molecules thereby facilitating transport through the blood brain barrier (BBB) . P-glycoproteins, the product of th mdr gene(s), have been shown previously to be expressed b the endothelial cells of the BBB (20) .

The BBB in a normal brain effectively restricts transpor between blood and the central nervous system of certai molecules. Small foreign molecules introduced into th circulation rapidly distribute themselves throughout th body's extracellular fluids; however, they may be unable t penetrate the tissues of the brain (19) .

The BBB is a functional barrier between the brai capillaries and the brain tissue which allows som substances from the blood to enter the brain rapidly whil other substances appear to either enter slowly or not a all. Further, the BBB effectively restricts transpor between blood and the central nervous system of certai molecules; especially those which are water soluble charged, and larger than about 200 daltons. The BBB ha been found to function over all anatomical regions of th central nervous system. The basis for this barrier appear to be embodied in the endothelial cells of the bloo capillaries in the brain (19) .

The BBB is not a fixed barrier. It can be influenced by th metabolic requirements of the brain, in addition to insult such as mechanical trauma, cerebral embolism, hypercapnia 1

4 hypoxia, extensive stress, radiation, electroconvulsive shock, explosive decompression, and various toxic substances (19) . All of these conditions may alter the permeability of the barriers and, subsequently, the composition of the extracellular fluid.

Various substances are transported across the BBB either by passive diffusion or, more often, by a carrier-mediated or active form of transport. Accordingly, this invention is important because it facilitates transport across the BBB.

SUMMARY OF THE INVENTION;

This invention provides a method of inhibiting the proliferation of mammalian tumor cells, which are resistant to the anti-proliferative affects of a predetermined concentration of a chemotherapeutic agent, which concentration was capable of inhibiting the proliferation of such mammalian tumor cells when such cells were nonresistant to such concentration of such chemotherapeutic agent. The method comprises a) contacting the resistant mammalian tumor cells with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to such predetermined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells with the chemotherapeutic agent at the predetermined concentration so as to inhibit proliferation of the mammalian tumor cells.

Additionally, the invention provides a method of inhibiting the proliferation of mammalian tumor cells of a certain type which are resistant to the anti-proliferative affects of a defined concentration of a chemotherapeutic agent normally capable of inhibiting the proliferation of mammalian tumor cells of such type. This method comprises a) contacting the resistant mammalian tumor cells of such type with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to the defined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells of such type with the chemotherapeutic agent at the defined concentration so as to inhibit proliferation of the mammalian tumor cells.

Further, this invention also provides a method of inhibiting the proliferation of mammalian tumor cells of a certain type which type of cells are resistant to the anti-proliferative effects of a defined concentration of a chemotherapeutic agent normally capable of inhibiting the proliferation of other types of mammalian tumor cells. This method comprises a) contacting the resistant mammalian tumor cells of such type with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to the defined concentration of the chemotherapeutic agent; and b) treating the resulting human tumor cells of such type with the chemotherapeutic agent at the defined concentration so as to inhibit proliferation of the mammalian tumor cells.

This invention further provides a method of enhancing the intracellular accumulation of a molecule within a cell, which molecule does not naturally occur in such cell, but is capable of entering such cell. This method comprises a) contacting the cell with a sufficient concentration of a TGF-Beta to inhibit extracellular transport of the molecule from the cell; and b) contacting the resulting cell with the molecule so as to effect intracellular accumulation of the molecule within the cell.

Also, this invention further provides a method of enabling a molecule to cross the blood brain barrier and accumulate in the central nervous system, which molecule is not normally capable of crossing such barrier and accumulating in the central nervous system. This method comprises a) contacting the blood brain barrier with a sufficient concentration of a TGF-Beta to enable the molecule to cross the barrier; and b) contacting the resulting barrier with the molecule so as to enable the molecule to cross the barrier. BRIEF DESCRIPTION OF THE FIGURES

Figure l is a flow cytometry profile which shows inhibitio of MDR in human glioblastoma cells (designated pat-1) b TGF-03 (time course) .

(A) control without fluorescent dye rhodamine 123 (R123) ;

(B) R123, 1 hour (1 h) 37^βC; (C) R123, 1 h 37^βC + verapamil (lOμg/ml) ;

(D) 1 day after addition of TGF-_/93;

(E) 2 days after addition of TGF-,93;

(F) 4 days after addition of TGF-,93;

(G) 5 days after addition of TGF-,93; and (H) 6 days after addition of TGF-,93.

TGF-,93 was used at a concentration of lng/ml R123 transpor was measured as follows: glioblastoma cells were stained fo 15 min with R123, washed in dye-free medium and afte incubation at 37°C 1 h, centrifuged, washed and processe for cytofluorography using a FACScan cytofluorometer. Horizontal axis: Fluorescence intensity (log); Vertica axis: cell number (relative) .

Figure 2 is a flow cytometry profile which shows th inhibition of MDR in human glioblastoma cells (designated pat 1-mdr) by TGF-,93 (dose response) .

(A) control (without R123) ;

(B) R123, 0-4^βC;

(C) R123, 1 h 37^βC; (D) R123, 1 h 37^βC + verapamil (10/_g/ml) ;

(E) 5 days with 30 ng/ml TGF-,93;

(F) 5 days with 0.1 ng/ml TGF-,93;

(G) 5 days with 0.03 ng/ml TGF-,93; (H) 5 days with 0.003 ng/ml TGF-,93; (I) 5 days with 0.003 μg/ml TGF-B3. 8

The cells were processed for cytofluorography as described in Figure 1. Horizontal axis: Fluorescence intensity (log) ; Vertical axis: cell number (relative) .

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of inhibiting the proliferation of mammalian tumor cells, which are resistant to the anti-proliferative affects of a predetermined concentration of a chemotherapeutic agent, which concentration was capable of inhibiting the proliferation of such mammalian tumor cells when such cells were nonresistant to such concentration of such chemotherapeutic agent. The method comprises a) contacting the resistant mammalian tumor cells with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to such predetermined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells with the chemotherapeutic agent at the predetermined concentration so as to inhibit proliferation of the mammalian tumor cells. Suitable examples of mammalian tumor types include myelomas, lymphomas, and neuroblastomas.

Further, the term "resistant" in the context of the invention means withstanding the growth inhibitory effects of a drug thereby continuing proliferation of tumor cells.

Additionally, the present also provides a method of inhibiting the proliferation of mammalian tumor cells of a certain type which are resistant to the anti-proliferative affects of a defined concentration of a chemotherapeutic agent normally capable of inhibiting the proliferation of mammalian tumor cells of such type. The method comprises a) contacting the resistant mammalian tumor cells of such type with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to the defined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells of such type with the chemotherapeutic agent at the defined concentration so as to 10 inhibit proliferation of the mammalian tumor cells. Tumor cells typical of this type include breast tumor cells.

Also, this invention provides a method of inhibiting the proliferation of mammalian tumor cells of a certain type (e.g. colon, hepatic, lung or renal tumor type) which type of cells are resistant to the anti-proliferative effects of a defined concentration of a chemotherapeutic agent normally capable of inhibiting the proliferation of other types of mammalian tumor cells. This method comprises a) contacting the resistant mammalian tumor cells of such type with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to the defined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells of such type with the chemotherapeutic agent at the defined concentration so as to inhibit proliferation of the mammalian tumor cells.

In accordance with the practice of this invention the resistant mammalian tumor cells may exhibit multiple drug resistance. Further, the mammalian tumor cells may be human tumor cells.

Also, in accordance with the practice of this invention, the TGF-Beta may be TGF-Beta 1. Alternatively, the TGF-Beta may be TGF-Beta 2. Further alternatively, the TGF-Beta may be TGF-Beta 3. As used herein, the TGF-Beta may be naturally- occurring or recombinantly made by genetic engineering methods or otherwise.

Additionally, in accordance with the practice of this invention, the chemotherapeutic agent includes a vinca alkaloid, an anthracycline, an epipodophyllotoxin and dactinomycin. This invention further provides a method of enhancing the intracellular accumulation of a molecule within a cell, which molecule does not naturally occur in such cell, but is capable of entering such cell. This method comprises a) contacting the cell with a sufficient concentration of a TGF-Beta to inhibit extracellular transport of the molecule from the cell; and b) contacting the resulting cell with the molecule so as to effect intracellular accumulation of the molecule within the cell.

In accordance with the practice of this invention, such molecules include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mito ycin, etoposide, tenoposide, vincristine, vinblastine, coltricin, doxirubicin, daunorubicin,^" dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propraolol, and puromycin.

As used herein the term "enhancing the intracellular accumulation of a molecule" means increasing the intracellular molecule level in the cell by means of preventing the exodus of the drug from the cell.

In accordance with this invention the cell may be a tumor cell. In one example, the tumor cell is a neural cell. Further, in accordance with the practice of this method the TGF-Beta may be TGF-Beta 1, TGF-Beta 2, or TGF-Beta 3.

Also, this invention further provides a method of enabling a molecule to cross the blood brain barrier and accumulate in the central nervous system, which molecule is not normally capable of crossing such barrier and accumulating in the central nervous system. This method comprises a) contacting the blood brain barrier with a sufficient 12 concentration of a TGF-Beta to enable the molecule to cross the barrier; and b) contacting the resulting barrier with the molecule so as to enable the molecule to cross the barrier.

In accordance with the practice of the invention the TGF- Beta may be TGF-Beta 1, TGF-Beta 2, or TGF-Beta 3. Moreover, in one example of the invention, the TGF-Beta contacts the endothelial cells of the barrier.

Further, in accordance with the practice of this invention, examples of suitable chemotherapeutic agents includes vinca alkaloids, anthracyclines, epipodophyllotoxins, or dactinomycin.

This invention is illustrated in the Experimental Details section which follows. This section is set forth to aid an understand of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow.

13

EXPERIMENTAL DETAILS

Abbreviation and technical terms.

BMP 2 (bone morphogenetic protein 2)

DMEM (Dulbecco's modified Eagle's medium)

FACS (fluorescence activated cell sorter)

FCS (fetal calf, serum)

FGF (fibroblast growth factor)

IFN (interferon)

IL (interleukin) mdr (the multiple drug resistance, typically mdr-l and mdr-2)

MDR (multi drug resistance) mRNA (messenger ribonucleic acid)

PBS (phosphate buffered saline)

PCR (polymerase chain reaction)

R123 (rhodamine 123)

TGF-β (transforming growth factor-beta)

TNF (tumor necrosis factor)

EXAMPLE 1

Culture of human glioblastoma cells. Cell lines were established from stereotactic biopsies of glial tumors. Brain tissue were minced and cultured in DMEM plus 10% fetal calf serum (FCS) in flat-bottomed microtiter plates.

After 4-6 weeks, tumor cells were subcultured in DMEM/FCS or in DMEM/FCS supplemented with vincristine sulfate. One cell line, pat 1, was spontaneously resistant to mitomycin C, vincristine, vinblastine and other cytostatic agents. Furthermore, these cells rapidly aαapted to increasing doses of vincristine (subline pat 1-mdr) . mdr mRNA transcripts (mdr 1 and mdr 3) were identified in pat-1 and pat-1 mdr cells by PCR with mdr oligonucleotide primers and by Northern blotting. Additionally, P-glycoprotein was detected in pat-1 cells and pat-1 mdr cells, but not in the other glioma cell lines by immunocytochemistry, using an antibody specific for P-glycoprotein.

Cytofluorometry of multidrug transport. For measurement of multidrug transport the fluorescent probe, rhodamine 123 (R123) was used. This fluorescent dye can be removed from the cell via P-glycoprotein and probe export can be competitively inhibited by various agents binding to P- glycoprotein (i.e. verapamil) (21). For analysis of multidrug transport, cells were removed from tissue culture flasks by mild trypsinization and stained for 15 min with rhodamine 123 (R123; lxlO⁶ cells/ml, 5μg/ml R123 in DMEM, 5% FCS and 25mm HEPES) . Cells were then washed with dye-free medium, centrifuged and resuspended in ice-cold PBS (phosphate buffered saline) , 1% serum albumin. One control sample was kept on ice in the presence of 0.2% sodium azide to block multidrug transport which is ATP dependent (Figure 2B) . Another sample was incubated for 1 h at 37°C as a control for export of fluorescent probe (Figures IB, 2C) . Another control sample was incubated at 37°C in the presence of verapamil, an agent that competes with R123 for cellular export using P-glycoprotein (Figures 1C, 2D) . After 1 hour cells were centrifuged, washed in PBS and processed for cytofluorography with a FACS cytofluorometer (Becton and Dickinson, Mountainview, CA, USA) .

In pat-1 cells the dye efflux could be blocked by lOμg/ml verapamil (Figure 1C) . However, in the pat-1 mdr cells, which overexpress the type 1 and type 3 mdr genes, verapamil at this concentration is inefficient in blocking R123 efflux. Overexpression of mdr gene 1 and 3 may result in a considerable increase in. multidrug transport and thereby 15 confer resistance to inhibition by such concentrations of chemosensitizers. Alternatively, the vincristine selection of pat 1 mdr cells may have induced another non-P- glycoprotein mediated mechanism for drug transport.

Growth factors and cytokines (TGF-øs, acidic and basic FGF, BMP-2, IL-1 alpha and beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, TNF-alpha and beta, interferon gamma) were tested for effects on multidrug transport using the pat-1 and pat-l mdr glioma cells in combination with R123 cytofluorometry as an assay system. The cells treated with factors were handled exactly as described above; incubated for 1 h at 37^βC after R123 staining for 15 min and processed for cytofluorometry.

Treatment of pat-1 cells with TGF-03 (lμg/ l) for different time periods (1-6 days) showed that after a latency period of about 1 day (Figure ID) the multidrug transport of pat-1 cells decreased with a complete inhibition 5 days after addition of TGF-03 (Figure 1G) . Treatment of pat-1 mdr cells with a range of doses of TGF-93 (0.003 - 30ng/ml) for 5 days showed that the concentration necessary to achieve maximal inhibition of multidrug transport is as low as 0.03 ng/ml (Figure 2) . Interestingly, verapamil at 10 μg/ml was unable to block the R123 transport in these cells.

Identical effects were observed with the other members of TGF-9 family, including TGF-,91, TGF-02 and the heterodimeric TGF-01.2. Additionally, the multidrug transport in rat brain astrocyte cell lines was also inhibited by TGF-.S.

These effects of TGF-β on multidrug transport were unique. The related factor BMP-2, interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8) and other cytokines (TNF-α and β , interferon gamma, basic and acidic FGF, TGF-α) did not inhibit multidrug transport, using this assay system. 51

16

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Claims

18What is claimed is:

1. A method of inhibiting the proliferation of mammalian tumor cells, which are resistant to the anti- proliferative affects of a predetermined concentration of a chemotherapeutic agent, which concentration was capable of inhibiting the proliferation of such mammalian tumor cells when such cells were nonresistant to such concentration of such chemotherapeutic agent which comprises: a) contacting the resistant mammalian tumor cells with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to such predetermined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells with the chemotherapeutic agent at the predetermined concentration so as to inhibit proliferation of the mammalian tumor cells.

2. A method of inhibiting the proliferation of mammalian tumor cells of a certain type which are resistant to the anti-proliferative affects of a defined concentration of a chemotherapeutic agent normally capable of inhibiting the proliferation of mammalian tumor cells of such type which comprises: a) contacting the resistant mammalian tumor cells of such type with a sufficient concentration of a TGF-Beta so as to overcome the resistance of the cells to the defined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells of such type with the chemotherapeutic agent at the defined concentration so as to inhibit proliferation of the mammalian tumor cells.

3. A method of inhibiting the proliferation of mammalian tumor cells of a certain type which type of cells are resistant to the anti-proliferative effects of a defined concentration of a chemotherapeutic agent normally capable of inhibiting the proliferation of other types of mammalian tumor cells which comprises: a) contacting the resistant mammalian tumor cells of such type with a sufficient concentration of a

TGF-Beta so as to overcome the resistance of the cells to the defined concentration of the chemotherapeutic agent; and b) treating the resulting mammalian tumor cells of such type with the chemotherapeutic agent at the defined concentration so as to inhibit proliferation of the mammalian tumor cells.

4. The method of claim 1, 2, or 3, wherein the resistant human tumor cells exhibit multiple drug resistance.

5. The method of claim 1, 2, or 3, wherein the mammalian tumor cells are human tumor cells.

6. The method of claim 1, 2, or 3, wherein the TGF- Beta is Beta 1.

7. The method of claim 1, 2, or 3, wherein the TGF- Beta is Beta 2.

8. The method of claim 1, 2, or 3, wherein the TGF- Beta is Beta 3.

9. The method of claim 1, 2, or 3, wherein the _{2 Q}

chemotherapeutic agent is a vinca alkaloid, an anthracycline, an epipodophyllotoxin or dactino ycin.

10. The method of claim 3, wherein the tumor type is a lung, colon, hepatic or renal tumor.

11. The method of claim 1, wherein the mammalian tumor type is a lympho a, myeloma, or neural tumor.

12. A method of enhancing the intracellular accumulation of a molecule within a cell, which molecule does not naturally occur in such cell, but is capable of entering such cell, which comprises: a) contacting the cell with a sufficient concentration of a TGF-Beta to inhibit extracellular transport of the molecule from the cell; and b) contacting the resulting cell with the molecule so as to effect intracellular accumulation of the molecule within the cell.

13. The method of claim 12, wherein the cell is a tumor cell.

14. The method of claim 13, wherein the tumor cell is a neural cell.

15. The method of claim 12, wherein the TGF-Beta is TGF-Beta 1. 21

16. The method of claim 12, wherein the TGF-Beta is TGF-Beta 2.

17. The method of claim 12, wherein the TGF-Beta is TGF-Beta 3.

18. A method of enabling a molecule to cross the blood brain barrier and accumulate in the central nervous system, which molecule is not normally capable of crossing such barrier and accumulating in the central nervous system, which comprises: a) contacting the blood brain barrier with a sufficient concentration of a TGF- Beta to enable the molecule to cross the barrier; and b) contacting the resulting barrier with the molecule so as to enable the molecule to cross the barrier.

19. The method of claim 18, wherein the TGF-Beta is TGF-Beta 1.

20. The method of claim 18, wherein the TGF-Beta is TGF-Beta 2.

21. The method of claim 18, wherein the TGF-Beta is TGF-Beta 3.

22. The method of claim 18, wherein the TGF-Beta contacts the endothelial cells of the barrier.

23. The method of claim 1, 2, or 3, wherein the chemotherapeutic agent is selected from a group consisting of vinca alkaloids, anthracyclines, epipodophyllotoxins, and dactinomycin.