Patent Application
20100256136
The ATP-binding cassette protein ABCG2(breast cancer resistance protein) effluxes some of the photosensitizers used in photodynamic therapy (PDT) against hyperproliferative tissue such as tumors, and thus reduces efficacy of photodynamic therapy (PDT) using such photosensitizers.
Photodynamic therapy (PDT) is used for the treatment of many cancers. Photosensitizers are taken up by tumor cells and then activated by light (1), generating reactive oxygen species that cause cell death by necrosis or apoptosis (2). The outcome of PDT depends on accumulation of sufficient photosensitizer in tumor cells.
Expression of ATP-binding cassette (ABC) transport proteins renders tumor cells resistant to substrate chemotherapy drugs by virtue of drug efflux (3), and the effect of these transporters on intracellular photosensitizer accumulation has been examined as a potential cause of resistance to PDT. The ABC family transport protein that has been most thoroughly investigated is ABCB1, or P-glycoprotein (Pgp), but photosensitizers were found not to be substrates for ABCB1 (4-8), nor were they substrates for ABCC1, or multidrug resistance-associated protein-1 (MRP-1) (8). In contrast, another ABC family transport protein, ABCG2, or breast cancer resistance protein (BCRP), has been found to transport some photosensitizers and to decrease intracellular photosensitizer accumulation (8). Jonker et al. demonstrated that ABCG2 knock-out mice were photosensitive because of increased protoporphyrin IX (PpIX) levels (9). Robey et al. found that pheophorbide α (Pha) is a specific substrate for ABCG2 (10), and that ABCG2 also transports pyropheophorbide-a methyl ester, chlorin e6 and 5-aminolevulinic acid (ALA)-induced PpIX, but not hematoporphyrin IX, meso-tetra (3-hydroxyphenyl) porphyrin or meso-tetra (3-hydroxyphenyl) chlorin (8).
Tyrosine kinase inhibitors (TKIs), including imatinib mesylate (Gleevec) and gefitinib (Iressa) are novel agents in cancer treatment that have been found to reverse resistance to chemotherapy drugs by blocking their efflux by ABCG2 (9,11-13).
The invention is a method for treating hyperproliferative tissue in a mammal which tissue expresses ABCG2 including the steps of:
The photosensitizing compound is preferably a tetrapyrollic photosensitizer compound where the tetrapyrollic compound is a chlorin, bacteriochlorin, porphyrin, pheophorbide including pyropheophorbides, purpurinimide, or bacteriopurpurinimide and derivatives thereof; provided that, the photosensizing compound is not a meso-tetra (3-hydroxyphenyl) derivative, is not a saccharide derivative and is not a hematoporphyrin.
The photosensitizing compound is usually a protoporphyrin IX (PpIX), a pheophorbide α (Pha), a pyropheophorbide-a alkyl ester, a chlorin e6 or a 5-aminolevulinic acid (ALA)-induced PpIX.
ABCG2 protein is an ATP-binding cassette protein (known as a breast cancer resistance protein) that is a 655 amino acid peptide that effluxes some of the photosensitizers used in photodynamic therapy (PDT) against hyerproliferative tissue such as tumors, and thus reduces efficacy of photodynamic therapy (PDT) using such photosensitizers. This protein has been known for a number of years. Details concerning this protein can be found in Stand et al., International Journal of Biochemistry and Cell Biology 37 (2005) pp 720-725, incorporated herein by reference as background art.
As discussed above, tyrosine kinase inhibitors (TKI's) were investigated with respect to their effect upon improvement of PDT effect against tumor cell lines expressing ABCG2. While the primary TKI investigated was imatinab mesylate, it is understood that the invention includes the use of other tyrosine kinase inhibitors. Examples of such tyrosine kinase inhibitors include, but are not limited to: erlotinib, geitinib, imatinib and sunitinib. All of the foregoing are known to those skilled in the art. Erlotinib is chemically known as N-(3-ethynylphenyl)-6,7-bis(methoxyethoxy) quinazolin-4-amine. Gefitinib is chemically known as N-(3 -chloro-4-fluoro-phenyly)-7-methoxy-6(3 -morpholin-4-ylpropoxy) quinazolin-4-amine. Imatinib is chemically known as 4[(4-methyl-1-piperazininyl) methyl]-N-(4-methyl-3-[(4-(3-pyidinyl)-2-pyrimidinyl) amino)-phenyl] benzamide methane sulfonate. Sunitinib is chemically known as a 1:1 compound of hydroxybutanoic acid and N-(2-(diethylamine) ethyl-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-3h-indol-3-ylidine) methyl-carboxamide.
The tyrosine kinase inhibiting compound may be systemically introduced by ingestion or injection.
Broadly, the photosensitizing compounds for use in accordance with the invention are those photosensitizing compounds whose cell retention is adversely affected by a tyrosine kinase, especially ABCG2, or breast cancer resistance protein (BCRP). Such photosensitizing compounds generally include tetrapyrollic photosensitizer compounds where the tetrapyrollic compound is a chlorin, bacteriochlorin, porphyrin, pheophorbides including pyropheophorbides, purpurinimide, or bacteriopurpurinimide excluding meso-tetra (3-hydroxyphenyl), and saccharide derivatives and excluding hematoporphyrins. The photosensitizing compound is usually a protoporphyrin IX (PpIX), a pheophorbide α (Pha), a pyropheophorbide-a alkyl ester, a chlorin e6 or a 5-aminolevulinic acid (ALA)-induced PpIX. The photosensitizing compound is preferably a pyropheophorbide such as HPPH.
The photosensitizing compound is commonly a tetrapyrollic pharmaceutically acceptable compound that acts as a substrate for ABC family transport protein ABCG2 and that has a preferential light absorbance frequency and that has the chemical formula:
where R1 and R2 are each independently substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, —C(O)Ra or —COORa or —CH(CH3)(ORa) or —CH(CH3)(O(CH2)nXRa) where Ra is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted cycloalkyl where R2 may be
CH═CH2, CH(OR20)CH3, C(O)Me, C(═NR20)CH3 or CH(NHR20)CH3;
where X is an aryl or heteroaryl group;
n is an integer of 0 to 6;
where R20 is methyl, ethyl, butyl, heptyl, docecyl or 3,5-bis(trifluoromethyl)-benzyl; and
R1a and R2a are each independently hydrogen or substituted or unsubstituted alkyl, or together form a covalent bond;
R3 and R4 are each independently hydrogen or substituted or unsubstituted alkyl;
R3a and R4a are each independently hydrogen or substituted or unsubstituted alkyl, or together form a covalent bond;
R5 is hydrogen or substituted or unsubstituted alkyl;
R6 and R6a are each independently hydrogen or substituted or unsubstituted alkyl, or together form ═O;
R7 is a covalent bond, alkylene, azaalkyl, or azaaraalkyl or ═NR21 where R21 is —CH2X-R1 or —YR1 where Y is an aryl or heteroaryl group and R1 is —H or lower alkyl;
R8 and R8a are each independently hydrogen or substituted or unsubstituted alkyl or together form =O;
R9 and R10 are each independently hydrogen, or substituted or unsubstituted alkyl and R9 may be —CH2CH2COORa where Ra is an alkyl group;
each of Ra-R10, when substituted, is substituted with one or more substituents each independently selected from Q, where Q is alkyl, haloalkyl, halo, pseudohalo, or —COORb where Rb is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, or ORc where Rc is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or CONRdRe where Rd and Re are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NRfRg where Rf and Rg are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or ═NRh where Rh is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue;
each Q is independently unsubstituted or is substituted with one or more substituents each independently selected from Q1, where Q1 is alkyl, haloalkyl, halo, pseudohalo, or —COORb where Rb is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, or ORc where Rc is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or CONRdRe where Rd and Re are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NRfRg where Rf and Rg are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or =NRh where Rh is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue;
provided that, the photosensizing compound is not a meso-tetra (3-hydroxyphenyl) derivative, is not a saccharide derivative and is not a hematoporphyrin. In a preferred embodiment, R7 is a covalent bond and the compound is a pyropheophorbide.
Usually in the method of the invention two through four doses of tyrosine kinase inhibiting compound at about 100 to about 300 mg/kg body weight is orally administered at intervals separated by from about 4 to about 12 hours in step a) and about 0.1 to about 0.3 μmol/kg of body weight of a pyropheophorbide photosensitizer is administered in step b) by injection at from about one to about three hours after completion of administration of the tyrosine kinase inhibiting compound.
Where the pyropheophorbide photosensitizer is HPPH and 24 hours after administration of the HPPH, the tumors were treated with 665 nm light from an argon ion laser-pumped dye laser with a fluence of about 50 to about 100 J/cm2 delivered at a rate of about 10 to about 25 mW/cm2.
The photosensizer is usually systemically administered by injection.
The invention may be illustrated by the following specific examples showing preparation of reagents for use in accordance with the invention and use thereof in determining improvement in PDT efficacy.
5-Aminolevulinic acid hydrochloride (ALA), PpIX and cyclosporin A (CsA) are known compounds and were purchased from Sigma-Aldrich (St. Louis, MO.). 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH; Photochlor®), HPPH-lactose conjugate and benzoporphyrin derivative monoacid ring A (BPD-MA) were synthesized at Roswell Park Cancer Institute. Porfimer sodium (Photofrin®), a known commercially available compound, was obtained from Axcan Scandipharm, Inc. (Birmingham, AL). Imatinib mesylate (Gleevec®), a known commercially available compound, was provided by Novartis Pharmaceuticals (Basel, Switzerland) and fumitremorgin C (FTC) was provided by Dr. Susan Bates (NIH, Bethesda, MD). Gefitinib (Iressa) is a known material and was manufactured by AstraZeneca (Bristol, England).
In general known cell lines were used. FaDu human hypopharyngeal squamous cell carcinoma, RIF-1 murine radiation-induced fibrosarcoma and Colo 26 murine colon carcinoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). BCC-1/KMC, a human basal cell carcinoma cell line (14), was provided by Dr. Tak-Wah Wong, National Cheng Kung University Hospital, Tainan, Taiwan. HEK-293 cells transfected with either an empty pcDNA3 vector or a pcDNA3 vector containing full-length ABCG2 (HEK-293 pcDNA or HEK-293 482R) were provided by Dr. Susan Bates at the U.S. National Institute of Health, Bethesda, MD..
FaDu cells were grown in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 1% penicillin-streptomycin, 100 mM non-essential amino acids and 1 mM MEM sodium pyruvate. RIF-1 cells were grown in MEM-α medium and BCC-1 cells and Colo 26 cells in RPMI 1640; both media were supplemented with 10% FBS, 200 mM L-glutamine and 1% penicillin-streptomycin. HEK-293 pcDNA and HEK-293 R482 cells were grown in EMEM supplemented with 10% FBS, 200 mM L-glutamine, 1% penicillin-streptomycin and 2 mg/ml G-418.
Aliquots of cell extracts were separated on 8% SDS-polyacrylamide gels by Western Blot Analysis. Protein was prepared in 30 μg quantities from all cell lines, except for HEK-293 482R cells, from which 2 μg protein were used. Proteins were transferred to Protran® membranes (Schleicher & Schuell, Riviera Beach, FL), and the membranes were reacted with antibodies to ABCB1, ABCC1 and ABCG2 (BXP-53) (Alexis Biochemicals, San Diego, CA) and β-actin (Sigma-Aldrich, St. Louis, MO). Reaction with horseradish peroxidase (HRP)-labeled secondary antibodies (ICN Biomedicals, Inc., Aurora, OH) was performed in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 5% milk. Immune complexes were visualized by an enhanced chemiluminescence (ECL) reaction (Amersham Biosciences, Piscataway, NJ). The ECL images were recorded on X-ray films with various exposure lengths.
Cells were plated in 6-well plates at a density of 3×105 cells per well and incubated overnight. To study photosensitizer accumulation, cells were exposed to ABCG2 modulators including 10 μM imatinib mesylate, FTC (15) and CsA (16) for 1 hour prior to the addition of photosensitizers, which included HPPH (0.4−0.8 μM), HPPH-lactose (0.8 μM), Photofrin (2 μg/ml) and ALA (0.4-0.8 mM in 1% FCS medium). Cells were cultured for an additional 4 hours, then washed with cold culture medium and with PBS. Photosensitizer levels were determined using Solvable® (Perkin Elmer, Boston, MA) extraction (17). Briefly, the cells were solubilized in 0.5 ml Solvable® at 37° C. overnight. The Solvable® extract then was diluted 1:1 with PBS, the photosensitizer levels were determined by fluorometry, and concentrations were extrapolated from standard curves. Intracellular photosensitizer levels were normalized to intracellular protein content. To study photosensitizer efflux, cells were incubated with photosensitizer for 4 hours, then washed once with cold medium, resuspended in drug-free medium, placed at 37° C. or 4° C. for 1 hour and washed once with cold PBS. Photosensitizer levels were then determined using Solvable® extraction, as above.
Cells were plated in 96-well plates at a density of 1×104 cells per well. After overnight incubation, they were exposed to ABCG2 modulators including imatinib mesylate, FTC and CsA at 10 μM and gefitinib at 5 μM, for one hour prior to the addition of photosensitizers, which included HPPH (0.4 or 0.8 μM), ALA (0.4 or 0.8 mM), BPD-MA (0.14 μM) or Photofrin (2 μg/ml), for an additional 4 hours. Cells were then irradiated with a filtered xenon arc lamp (600-700 nm) at a fluence rate of 14 mW/cm2 for HPPH and BPD-MA, or with a red light (570-700 nm) at a fluence rate of 6.3 mW/cm2 for ALA and Photofrin. Cell viability was evaluated by the 1,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 48 hours after irradiation.
Eight-week-old female C3H/HeJCr mice were injected intradermally with 4×105 RIF-1 tumor cells. When the tumors reached a diameter of 4 mm, groups of 5 mice received tail vein injections of 0.2 μmol/kg body weight HPPH alone or HPPH preceded by four doses of imatinib mesylate, 200 mg/kg body weight, administered by oral gavage 26, 14, 8 and 2 hours before the HPPH. To determine photosensitizer levels, samples of tumor, skin and muscle tissue were harvested 24 hours after the HPPH administration and dissolved in Solvable at 63° C. overnight. HPPH levels were measured by fluorometry as described above. In other experiments mice were administered 14C-labelled HPPH and photosensitizer levels in the harvested tissues were determined by scintillation counting. For PDT, groups of 5 tumor-bearing mice received HPPH or HPPH preceded by imatinib mesylate, as above. After 24 hours, the tumors were treated with 665 nm light from an argon ion laser-pumped dye laser (Spectra Physics, Mountain View CA) with a fluence of 72 J/cm2 delivered at a rate of 14 mW/cm2. Additional control mice received no treatment or imatinib mesylate alone, without HPPH. Tumors were measured every 1 to 3 days, and mice were sacrificed when tumor volumes exceeded 400 mm3.
HPPH efflux was studied in cell lines with a range of levels of ABCG2 expression. Expression of ABCG2 was highest in HEK-293 R482 and BCC-1 cells, and also was high in Colo 26 and RIF-1 cells, but not in FaDu cells or HEK-293 pcDNA controls (
Effects of imatinib mesylate on intracellular levels of different photosensitizers were studied. Imatinib mesylate had no effect on HPPH accumulation in HEK-293 pcDNA cells, but increased intracellular HPPH levels in HEK-293 R482 cells (p<0.05) (
Consistent with the higher photosensitizer levels, increases in phototoxicity were observed in the presence of ABCG2 modulators in cells that expressed ABCG2. HEK-293 pcDNA cells were more sensitive to HPPH-PDT than HEK-293 R482 cells, and pretreatment with 10 μM imatinib mesylate increased phototoxicity 2- to 8-fold in HEK-293 R482 cells, depending on the light doses used, but had no effect on the sensitivity of HEK-293 pcDNA3 cells to HPPH-PDT (
In mice bearing subcutaneous RIF-1 tumors, imatinib mesylate increased median HPPH levels in the tumors 1.8 fold (p<0.001), but had less effect on skin and muscle (
Two photosensitizers (
Structure-specific active transport of three clinically used second-generation photosensitizers by ABCG2 and inhibition of ABCG2-mediated photosensitizer transport and enhancement of both in vitro and in vivo PDT through administration of the TKI imatinib mesylate have been demonstrated. TKIs increase intracellular photosensitizer accumulation and enhance phototoxicity in cells that express ABCG2. TKIs have previously been found to inhibit ABCG2-mediated transport of chemotherapy drugs and sensitize cells to chemotherapy (11-13), but the present invention provides the first demonstration that a clinically applicable TKI, imatinib mesylate, selectively increases accumulation of photosensitizer and enhances both in vitro and in vivo PDT in ABCG2+ tumor cells.
ABCG2+ cells including Colo 26, RIF-1, BCC-1 and ABCG2-transfected HEK-293 cells, exhibited decreased intracellular levels of HPPH, BPD-MA and ALA/PpIX, and resistance to PDT with these agents. In contrast, transport of these photosensitizers was not found in FaDu cells, which do not express ABCG2, or in plasmid-transfected HEK-293 cells. Note that Colo 26 cells reproducibly become ABCG2+ after about 20 passages; early passage cells are ABCG2−. Since HPPH is a derivative of pyropheophorbide-a, the results for this agent, which is in promising Phase II trials (19,20), are consistent with Robey et al.'s recent report that pyropheophorbide-a is a substrate of ABCG2 (8). The amount of HPPH transport was not directly proportional to the expression of ABCG2 measured by Western blot analysis, as exemplified by BCC-1 cells, which had higher levels of ABCG2 expression but exhibited a lesser degree of HPPH transport than the other cell lines with ABCG2 expression. Discordance between expression and function of ABCG2 has been previously demonstrated in cancer cells (21).
The mechanism(s) by which imatinib mesylate and other TKIs inhibit transport of ABCG2 substrates are being studied. Houghton et al. (12) and Jordanides et al. (22) found that imatinib mesylate inhibits ABCG2 function but is not an ABCG2 substrate (12), while Burger et al. found imatinib mesylate to be an ABCG2 substrate that inhibits pump activity by competitive inhibition (23). Ozvegy-Laczka et al. demonstrated that imatinib mesylate inhibits ABCG2 ATPase activity, possibly consistent with it not being a substrate (11). Finally, Nakanishi et al. found that imatinib decreases expression of ABCG2 protein, but not mRNA, in bcr-abl+ cells through inhibition of the PI3K-Akt pathway (24); this mechanism also might apply in malignant cells with other aberrant signaling mechanisms.
PDT acts by directly killing tumor cells, and, in many cases, by shutting down the microvasculature feeding the tumor (2). Treatment selectivity is based on higher photosensitizer levels within the target than in surrounding normal tissues, and ABCG2 expression in tumors (25,26) and on capillaries (27) can decrease both efficacy and selectivity. In addition to baseline ABCG2 expression, hypoxia, which is very common in tumors, has been found to upregulate expression of ABCG2 and to increase cell survival by decreasing intracellular accumulation of heme and other porphyrins (28). Therefore hypoxia may inhibit PDT not only because the photodynamic process requires oxygen (2), but also through ABCG2-mediated decreases in intracellular photosensitizer levels. Importantly, ABCG2+ cancer stem cells (e.g. 29, 30, 31) are expected to be relatively resistant to PDT with photosensitizers that are substrates for the ABCG2 transporter, and they may be responsible for late tumor recurrences (29,30). While ABCG2-mediated transport might be overcome by administering higher photosensitizer doses, this approach may cause unacceptable normal tissue damage. Thus, with photosensitizers that are ABCG2 substrates, inhibiting transport is likely to be a more successful approach to enhancing clinical PDT.
Administration of imatinib mesylate or other ABCG2 inhibitors in conjunction with PDT has significant potential for enhancing the efficacy of this therapeutic modality in the treatment of tumors that express ABCG2, including gastrointestinal, genitourinary, lung and head and neck cancers (25,26). Because transporter inhibition is only necessary during the interval between photosensitizer dosing and photoillumination (0.5 to 48 hours), toxicities should be minimal in relation to those associated with chronic administration of the TKI. Pump inhibition may allow lower photosensitizer doses and may improve selectivity and decrease normal tissue damage. Imatinib mesylate also may increase the levels of endogenous porphyrins in ABCG2-expressing tumors, potentially enhancing diagnosis with devices that measure endogenous fluorescence, such as Laser-Induced Fluorescence Endoscopy (LIFE) (32). Finally, it is evident that ABCG2 transport is an important, previously unconsidered factor for the design of new photosensitizers. It is not surprising that multimeric Photofrin® is not a substrate. With newer, monomeric agents, carbohydrate conjugation to a pyropheophorbide molecule blocks transport, as do the modifications in meso-tetra(3-hydroxyphenyl) porphyrin and meso-tetra(3-hydroxyphenyl) chlorin (8).
The above results show that certain second-generation photosensitizers in clinical use, especially derivatives of pyropheophorbide-a and its derivatives, are transported out of cells by ABCG2, and this effect can be abrogated by co-administration of imatinib mesylate. By increasing intracellular photosensitizer levels in ABCG2+ tumors, imatinib mesylate or other agents inhibiting ABCG2 transport may enhance efficacy and selectivity of clinical PDT.
The following references are incorporated herein by reference as background art.
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/819,773, filed Jul. 10, 2006.
This work was supported by the NIH (USA) Grant CA55791. The United States Government may have certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/15263 | 6/29/2007 | WO | 00 | 4/19/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60819773 | Jul 2006 | US |
