© 2004 by Oxford University Press
© 2004 Oxford University Press
ARTICLE |
Anticancer Chemosensitization and Radiosensitization by the Novel Poly(ADP-ribose) Polymerase-1 Inhibitor AG14361
Affiliations of authors: Northern Institute for Cancer Research, University of Newcastle upon Tyne, Medical School, Newcastle upon Tyne, U.K. (CRC, SB, MAB, AHC, BWD, SK, DRN, EN, HDT, LZW, NJC); Pfizer Global Research and Development/Agouron Pharmaceuticals, La Jolla, CA (RA, SCK, ZH, RAK, JL, KM, DS, SEW); School of Pharmacy, University of Manchester, Manchester, U.K. (IJS, KJW).
Correspondence to: Nicola J. Curtin, PhD, Northern Institute for Cancer Research, University of Newcastle upon Tyne, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K. (e-mail: n.j.curtin{at}newcastle.ac.uk)
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ABSTRACT |
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Background: Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates the repair of DNA strand breaks. Inhibiting PARP-1 increases the cytotoxicity of DNA-damaging chemotherapy and radiation therapy in vitro. Because classical PARP-1 inhibitors have limited clinical utility, we investigated whether AG14361, a novel potent PARP-1 inhibitor (inhibition constant <5 nM), enhances the effects of chemotherapy and radiation therapy in human cancer cell cultures and xenografts. Methods: The effect of AG14361 on the antitumor activity of the DNA alkylating agent temozolomide, topoisomerase I poisons topotecan or irinotecan, or x-irradiation or
-radiation was investigated in human cancer cell lines A549, LoVo, and SW620 by proliferation and survival assays and in xenografts in mice by tumor volume determination. The specificity of AG14361 for PARP-1 was investigated by microarray analysis and by antiproliferation and acute toxicity assays in PARP-1-/- and PARP-1+/+ cells and mice. After intraperitoneal administration, the concentration of AG14361 was determined in mouse plasma and tissues, and its effect on PARP-1 activity was determined in tumor homogenates. All statistical tests were two-sided. Results: AG14361 at 0.4 µM did not affect cancer cell gene expression or growth, but it did increase the antiproliferative activity of temozolomide (e.g., in LoVo cells by 5.5-fold, 95% confidence interval [CI] = 4.9-fold to 5.9-fold; P = .004) and topotecan (e.g., in LoVo cells by 1.6-fold, 95% CI = 1.3-fold to 1.9-fold; P = .002) and inhibited recovery from potentially lethal -radiation damage in LoVo cells by 73% (95% CI = 48% to 98%). In vivo, nontoxic doses of AG14361 increased the delay of LoVo xenograft growth induced by irinotecan, x-irradiation, or temozolomide by two- to threefold. The combination of AG14361 and temozolomide caused complete regression of SW620 xenograft tumors. AG14361 was retained in xenografts in which PARP-1 activity was inhibited by more than 75% for at least 4 hours. Conclusion: AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30) is an abundant 116-kd nuclear enzyme that is constitutively expressed. It is the first and most extensively characterized member of an expanding family of PARP enzymes (1). In response to DNA strand breaks, PARP-1 catalyzes the rapid synthesis of ADP-ribose polymers from the substrate NAD+, facilitating base excision repair of DNA and signaling to other critical cellular proteins, such as p53 (2). PARP-1 interacts with other components of the base excision repair complex, and cells lacking PARP-1 activity are deficient in base excision repair (3).
PARP-1 inhibitors have potential therapeutic applications (4), including the treatment of cancer (5). Enzyme-mediated DNA repair can cause resistance to DNA-damaging anticancer drugs and radiation, and so inhibition of DNA repair may be therapeutically beneficial (6). PARP-1 activation and subsequent poly-(ADP-ribosylation) are immediate cellular responses to drug- or radiation-induced DNA damage (2,7). Evidence from both inhibitor (7,8) and molecular genetic (9–11) studies indicates that inhibiting PARP-1-mediated DNA repair increases the cytotoxicity of DNA-damaging cancer therapeutics. However, the therapeutic potential of this approach has not been investigated because of the poor potency, solubility, and limited specificity of classical inhibitors, including 3-substituted benzamides [e.g., 3-aminobenzamide, inhibition constant (Ki) = 5 µM; Fig. 1, A, compound 1 (12)] and various fused-ring heterocyclic compounds [Fig. 1, A, compounds 2 and 3 (13,14)].
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We have developed several inhibitors of PARP-1 with potent chemo- and radiosensitizing activity in vitro, including NU1025 (Ki = 50 nM, Fig. 1, A, compound 4), that increase the cytotoxicity of the monofunctional DNA-alkylating agent temozolomide, the topoisomerase I inhibitor camptothecin, and
-irradiation in L1210 cells (15,16). Subsequent studies (17) demonstrated that NU1025 and NU1085 (Ki = 6 nM; Fig. 1, A, compound 5) increased the activity of temozolomide and the topoisomerase I inhibitor topotecan in 12 human tumor cell lines in a p53-independent manner. However, these compounds still lacked the potency and solubility for extensive preclinical evaluation, although a recent report (18) demonstrated that intracranial injection of NU1025 increased the temozolomide-induced survival of mice with brain lymphomas. A structural study of NU1085 co-crystallized with the catalytic domain of chicken PARP-1 (19) was used to direct the synthesis of additional compounds, with the resulting tricyclic indoles and benzimidazoles being very potent inhibitors of PARP-1 activity. Lead optimization studies (20) identified the tricyclic benzimidazole AG14361 (Fig. 1, A, compound 6) as an extremely potent inhibitor (Ki<5 nM) that is at least 1000-fold more potent than the benzamides.
The purpose of this study was to determine whether AG14361 increases the effect of anticancer chemotherapy and radiation therapy. We investigated the effects of AG14361 alone and in combination with ionizing radiation, temozolomide, and topoisomerase I poisons in cell culture and in tumor xenograft-bearing mice. These studies were supported by pharmacokinetic (analysis of the metabolic stability of AG14361 in vitro, and plasma and tissue concentrations of AG14361 after administration to tumor-bearing mice), pharmacodynamic (measurement of tumor PARP-1 inhibition by AG14361), and mechanistic investigations.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Temozolomide (a gift from the Cancer Research Campaign, London, U.K.), topotecan (SmithKline Beecham Pharmaceuticals, Philadelphia, PA), and AG14361 (20) were dissolved in dimethyl sulfoxide to allow addition to cell cultures to a final concentration of 1% dimethyl sulfoxide. For in vivo evaluation, drugs were dissolved immediately before administration as follows: temozolomide and irinotecan (Camptosar; Pharmacia Upjohn, Kalamazoo, MI) in normal saline, PD128763 (a gift from Warner-Lambert, Ann Arbor, MI) in 10% dimethyl acetamide in saline, and AG14361 (HCl salt) in saline. Other chemicals and reagents were obtained from Sigma (Poole, U.K.), unless otherwise stated.
Cell Lines and Culture
LoVo and SW620 colorectal cancer cells (American Type Culture Collection, Manassas, VA) and A549 non–small-cell lung carcinoma cells (National Cancer Institute, National Institutes of Health, Bethesda, MD) were maintained in RPMI-1640 medium containing 10% fetal calf serum (Life Technologies, Paisley, U.K.). Mouse embryonic fibroblasts, isolated from PARP-1–/– and PARP-1+/+ mice (11,21), were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum. Cells were mycoplasma-free (22).
Crystallographic Analysis of AG14361 Bound to the PARP-1 Catalytic Site
Crystals of the catalytic domain of chicken PARP-1 (6 mg/mL) and 200 µM AG14361, added in a 1 : 1 ratio with 100 mM Tris (pH 8.5), 8% 2-propanol, and 16%–30% polyethylene glycol (average molecular weight = 600), were grown at room temperature by the hanging-drop vapor diffusion method (23). X-ray diffraction data, collected at -4 °C, were measured with Cu K
radiation from a Rigaku rotating anode generator (Rigaku, The Woodlands, TX) and an MAR345 image plate (Mar Research, Evanston, IL). Crystals had a space group of P212121, with a = 59.09 Å, b = 64.35 Å, and c = 97.38 Å,and a resolution of 2.15 Å. The structure was solved from a previously determined structure by use of 2|Fobs| –|Fcalc| and |Fobs| –|Fcalc| maps and refinement (24), where Fobs is the amplitude of the experimentally measured structure factor, and Fcalc is the amplitude of the structure factor calculated from the model, in this case, a previously determined structure initially and the current model during refinement. The final R factor and R-free are 20.1% and 24.4%, respectively, with 0.006 Å and 1.23° root mean square deviations from ideal bond distances and angles, where the R factor is the sum of the absolute values of the differences between the observed and calculated structure factor amplitudes divided by the sum of the observed structure factor amplitudes and R-free is the R factor for a randomly selected subset of reflections that were excluded in the refinement. Molecular modeling of AG14361 with a chimeric human PARP-1 model, created from the co-crystal structure of AG14361 with the catalytic domain of chicken PARP-1 (humanized by changing residue 763 from glutamine to glutamate), used MacroModel version 5.5 (25). Fig. 1, B, was created with Insight II (2000) (Accelrys, San Diego, CA).
PARP-1 Activity Assays
The activity of full-length recombinant human PARP-1 was measured in a reaction mixture containing 20 nM PARP-1, 500 µM NAD+ plus [32P]NAD+ (0.1–0.3 µCi per reaction mixture; Amersham Pharmacia Biotech, Piscataway, NJ), and activated calf thymus DNA (10 µg/mL) at 25 °C; the reaction was terminated after 4 minutes by adding ice-cold 10% (wt/vol) trichloroacetic acid, as described previously (26). The reaction product [32P]ADP-ribose incorporated into acid-insoluble material was deposited onto Whatman GF/C glass fiber filters with a Bio-Dot microfiltration apparatus (Bio-Rad, Hercules, CA) and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Inhibition of PARP-1 activity by AG14361 at 0–600 nM was measured, and the Ki for AG14361 was calculated by nonlinear regression analysis.
We determined PARP-1 inhibition by AG14361 (0–1000 nM, see Fig. 2, A) in permeabilized cells, as described previously (27), and in intact cells. Briefly, SW620 cells (8 x 105 to 1 x 106 cells per reaction) were permeabilized with digitonin (0.15 mg/mL). PARP-1 activity was stimulated by the addition of 100 ng of 12-mer blunt-ended DNA double-stranded oligonucleotide (2.5 µg/mL) in the presence of 75 µM NAD+ and [32P]NAD+ (Amersham) for 6 minutes at 25 °C. Macromolecules were then precipitated with an ice-cold solution of 10% trichloroacetic acid and 10% sodium pyrophosphate, and incorporated radioactivity was measured to determine PARP-1 activity. PARP-1 inhibition was determined by addition of AG14361 (0–1000 nM) to the reaction mixture. Intact cells were incubated with AG14361 (1–1000 nM, see Fig. 2, A) for 10 minutes, rinsed, permeabilized, and assayed for PARP-1 activity as described above. The 50% PARP-1 inhibitory concentration (IC50) for AG14361 was calculated from computer-fitted curves (GraphPad Software, San Diego, CA). PARP-1 activity in tumor homogenates from untreated control and AG14361-treated (10 mg/kg) mice was determined by [32P]NAD+ incorporation into macromolecules as described for permeabilized cells.
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Growth Inhibition and Cytotoxicity Assays In Vitro
Cell growth inhibition was estimated in exponentially growing LoVo, A549, and SW620 cells in 96-well plates. Cells were exposed to temozolomide (0–1000 µM, see Fig. 4, A) or topotecan (0–30 nM, see Fig. 4, B) in the presence or absence of 0.4 µM AG14361. Cells were also exposed to AG14361 (0–20 µM, see Fig. 4, C) alone or in the presence of 400 µM temozolomide. After 5 days of culture, these cells were fixed with 10% trichloroacetic acid and stained with sulforhodamine B (28). The concentration of temozolomide, topotecan, and AG14361 alone or in combination that inhibited growth by 50% (GI50) was calculated from computer-generated curves. Recovery from potentially lethal damage was measured in confluent LoVo cell cultures arrested in G1 phase (confirmed by flow cytometry, data not shown) to mimic the radiation-resistant quiescent cell population in tumors. Such cells were exposed to 8 Gy of -irradiation (Gammacell 1000 Elite; Nordion International, Kanata, Ontario, Canada) and then harvested and plated for colony formation assay immediately or maintained as growth-arrested confluent cultures for a 4-hour or 24-hour recovery period before harvesting and plating for the colony formation assay. Where indicated, 0.4 µM AG14361 was added 30 minutes before irradiation and was present in the recovery incubation.
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Microarray Analysis
Changes in gene expression after exposure to AG14361 (0.4 µM or 14 µM) were determined by use of RNA extracted from A549 cells with TRIzol (GIBCO/BRL Invitrogen, Carlsbad, CA) and the RNeasy mini-column (Qiagen, Valencia, CA), according to the manufacturers’ instructions. RNA was hybridized on human Hu6800 high-density oligonucleotide arrays (Affymetrix, Santa Clara, CA), as previously described (29,30). Analysis of the arrays by GeneChip 3.2 (Affymetrix) scaled to an average hybridization intensity of 200 is estimated to represent three to five copies per cell, and an expression ratio of twofold is the approximate limit of sensitivity (31).
AG14361 Metabolic Stability and Plasma and Tissue Distribution
The in vitro metabolic stability of AG14361 was determined in microsomal preparations (prepared by differential centrifugation of homogenized frozen liver tissue) from mouse, rat, dog, monkey, and human liver. AG14361 (5 µM) was incubated with microsomal preparations (0.5 mg of protein per mL) and an NADPH-generating system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and NADP) at 37 °C for 0.5 and 2 hours. The concentration of AG14361 remaining was determined by high-performance liquid chromatography–mass spectrometry/mass spectrometry (HPLC–MS/MS) (HP 1100DAD; Hewlett-Packard; mobile-phase and gradient 5%–61% acetonitrile in 0.1% formic acid with a Zorbax Eclipse C18 column, 2.1 x 50 mm) coupled to a Quattro II triple quadrupole mass spectrometer (Micromass). The initial and remaining concentrations of AG14361 were compared to determine its molecular stability.
The plasma concentration and tissue distribution of AG14361 was determined 30, 60, and 120 minutes (see Fig. 3, B) after intraperitoneal administration to CD-1 nude mice (Charles River, Margate, U.K.) bearing subcutaneous LoVo or SW620 xenografts (approximately 650 mm3). The concentration of AG14361 in acetonitrile-treated plasma and homogenized tissues was measured with reversed-phase HPLC (isocratic mobile phase = 40% acetonitrile in 0.1% ammonium formate; Hypersil BDS column of 4.6 x 250 mm and 5-µm particle size; Waters Alliance 2780 HPLC) by the method of addition (32). Animal care was in accordance with institutional guidelines.
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Acute Toxicity of AG14361 in PARP-1+/+ and PARP-1-/- Mice
Female PARP-1+/+ and PARP-1–/– mice, three mice per group [supplied by J. Menissier-de Murcia and G. de Murcia (11)], were treated with PD128763 or AG14361 (each at 50 mg/kg, intraperitoneally), and the effect on body temperature (an indicator of acute hypotension) was measured for up to 10 hours after treatment with a Testo 925 electronic rectal thermometer (Testo, Alton, U.K.).
Tumor Growth Inhibition
CD-1 nude mice bearing palpable, subcutaneous SW620 or LoVo xenografts were treated intraperitoneally with normal saline (control animals) or AG14361 (at 5 or 15 mg/kg) alone daily for 5 days (five mice per group). For drug combinations, AG14361 was administered intraperitoneally daily for 5 days immediately before administering the cytotoxic drug (temozolomide at 68 mg/kg orally or irinotecan at 2.5 mg/kg intraperitoneally) or 30 minutes before applying 2 Gy of x-irradiation locally to the tumor daily for 5 days. Tumor volumes, determined from two-dimensional caliper measurements and the equation a2 x b/2 (where a is the width and b is the length of the tumor), are presented as median relative tumor volume (RTV). That is, RTV1 is the tumor volume on the initial day of treatment (day 0), and RTV4 is the tumor volume 4 times that on the initial day of treatment. Tumor growth delay is defined as the time to RTV4 in drug-treated or irradiated mice compared with the time to RTV4 in control (vehicle alone) mice.
Median tumor volume is shown, rather than the mean, as this is generally accepted as the most statistically reliable representation of the average growth rate of tumors in a small group of mice, where a normal distribution of tumor volumes cannot be assumed. RTV4 was chosen as the most robust measurement that does not result in unacceptable suffering for the animals.
Tumor Perfusion Assay
AG14361 (10 mg/kg) or saline (control) was administered intraperitoneally to mice bearing SW620 xenografts 30 minutes before intravenous injection of Hoechst 33342 dye (15 mg/kg, dissolved in phosphate-buffered saline), followed 20 minutes later by the administration of carbocyanine (1 mg/kg, dissolved in 75% dimethyl sulfoxide); 5 minutes later, tumors were excised and frozen as described previously (33). To measure stained vessels, tumor sections (10 µm) were scanned under a Nikon Eclipse E800 microscope (excitation wavelengths = 340–380 nm and 450–490 nm; emission wavelengths = 480 nm and 510 nm, for Hoechst and carbocyanine, respectively) to determine the percentage of Hoechst- and carbocyanine-positive vessels.
Statistical Analysis
Cell growth inhibition and cytotoxicity assays were analyzed with paired or unpaired Student’s t tests when the variances were equal or with Wilcoxon rank tests or Mann–Whitney U tests, as appropriate. RTVs and tumor perfusion values were compared with the Mann–Whitney U test. All statistical tests were two-sided.
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RESULTS |
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Interaction of AG14361 With the Active Site of PARP-1
Crystallographic analysis of AG14361 bound to the catalytic domain of chicken PARP-1 (Fig. 1, B) showed that the tricyclic ring system of AG14361 was located in a pocket composed of amino acid residues Trp861, His862, Gly863, Tyr896, Phe897, Ala898, Lys903, Ser904, Tyr907, and Glu988. The 2-phenyl group extended outward and was exposed to bulk solvent on both faces; the 4'-dimethylaminomethyl moiety resided in a pocket composed of residues Gln759, Val762, Gln763, Asp766, Gly888, and Tyr889. AG14361 formed important hydrogen bonds with Ser904 and Gly863 and a water-mediated hydrogen bond with Glu988. The 4'-dimethylaminomethyl group formed an ion pair with Asp766 through a hydrogen bond between C-H and O and participated in a cation--
interaction with Tyr889. In human PARP-1, residue 763 is glutamate, which can form an additional ionic interaction with the 4'-dimethylaminomethyl group.
AG14361 and PARP-1 Inhibition In Vitro and in Whole Cells
AG14361 is a potent inhibitor of purified full-length human PARP-1 [Ki<5 nM; the limit of accurate kinetic determination for this homodimeric enzyme (34)]. The IC50 for AG14361 was 29 nM in permeabilized SW620 cells and 14 nM in intact SW620 cells (Fig. 2, A). To investigate whether AG14361 increased the effect of chemotherapeutic agents or radiation therapy, we used 0.4 µM AG14361, a concentration that inhibited PARP-1 activity by more than 85%.
Specificity of AG14361 for PARP-1 In Vitro and In Vivo
The specificity of AG14361 for PARP-1 was determined by investigating the effect of AG14361 on gene expression and cellular proliferation of human cancer cells and by investigating whether any growth-inhibitory effect or acute toxicity of AG14361 was related to PARP-1 in PARP-1–/– cells and mice compared with their wild-type counterparts. The GI50 of AG14361 alone (a 5-day continuous exposure) was 14 µM (95% confidence interval [CI] = 9 to 19 µM), 11.2 µM (95% CI = 8 to 14.4 µM), and more than 20 µM in cultured A549, LoVo, and SW620 cells, respectively. AG14361-induced growth inhibition was not attributed to PARP-1-related effects because maximal PARP-1 inhibition was observed at much lower concentrations (
1 µM; Fig. 2, A) than the GI50. We observed identical levels of AG14361-induced growth inhibition in cell lines derived from PARP-1–/– and PARP-1+/+ mice (mean GI50 = 66 and 65 µM, respectively; Fig. 2, B). In addition, 0.4 µM AG14361 did not substantially alter gene expression as shown by microarray analysis. A 17-hour exposure of A549 cells to 0.4 µM AG14361 did not change the expression of the 6800 genes studied by more than twofold. Changes of more than twofold were observed in the expression of 62 of these genes, including p21, at the GI50 of AG14361 (data not shown). Thus, although 0.4 µM AG14361 inhibited cellular PARP-1 activity by more than 85%, it essentially did not change gene expression and cell proliferation, indicating that the cellular effects of this low concentration of AG14361 are specific for PARP-1 inhibition. Higher, growth-inhibitory concentrations of AG14361 did affect gene expression, but these effects are not likely to be related to PARP-1 inhibition because cell proliferation was affected equally in PARP–/– and PARP-1+/+ cells.
Acute hypotension and hypothermia have been reported in mice treated with the PARP-1 inhibitor PD128763 (Fig. 1, A, compound 2) at doses that increased the antitumor activity of radiation therapy (35). To determine whether this result was caused by PARP-1 inhibition and whether AG14361, therefore, would have a similar side effect, we treated PARP-1–/– and PARP-1+/+ mice (three per group) with such doses (50 mg/kg) of PD128763 and AG14361 and evaluated their effect on mouse body temperature, an indicator of acute hypotension. Although PD128763 caused profound hypothermia in PARP-1+/+ mice (nadir = 28 °C, 95% CI = 27 to 29 °C) and PARP-1–/– mice (nadir = 27.1 °C, 95% CI = 26.7 to 27.3 °C) compared with control mice (temperature = 37.5 °C, 95% CI = 37.4 to 37.6 °C), AG14361 did not affect mouse body temperature in PARP-1+/+ (mean temperature = 37.4 °C, 95% CI = 37.2 to 37.6 °C) or PARP-1–/– mice (mean temperature = 37.4 °C, 95% CI = 37.3 to 37.5 °C) (Fig. 2, C). Because the more potent PARP-1 inhibitor AG14361 did not induce hypothermia and because PD128763-induced hypothermia was similar in PARP-1–/– and PARP-1+/+ mice, hypothermia apparently did not result from PARP-1 inhibition.
Metabolic Stability of AG14361, Tissue Distribution, and Tumor PARP-1 Inhibition
The metabolic stability of AG14361 (5 µM) was tested in vitro in liver microsomal preparations from mice, rats, dogs, monkeys, and humans (Fig. 3, A). After a 2-hour incubation with such microsomal preparations, at least 50% of the initial concentration of AG14361 remained with the human preparations, but only 25% remained with the murine preparations.
Plasma and tissue distribution studies in mice bearing LoVo (Fig. 3, B) and SW620 (data not shown) xenografts after intraperitoneal administration of AG14361 at 50 mg/kg demonstrated that AG14361 was rapidly absorbed into the bloodstream and distributed to the tumor and liver with lower concentrations detected in the brain. Tissue-to-plasma concentration ratios indicated that AG14361 was retained in tumor tissue over time in both xenograft models, with tumor concentrations (
15 µM for 2 hours) in excess of that required to inhibit PARP-1 activity in vitro.
PARP-1 activity, detected by pharmacodynamic assay, in SW620 xenografts was inhibited by more than 75% for at least 4 hours after intraperitoneal administration of AG14361 (10 mg/kg), consistent with the concentration of AG14361 persisting in the tumor (Fig. 3, C). Inhibition of PARP-1 activity was confirmed at doses that increase the effect of chemotherapeutic agents and radiation therapy. At 30 minutes after intraperitoneal administration, AG14361 at 5 mg/kg inhibited PARP-1 activity by 75% (95% CI = 67% to 83%) and AG14361 at 15 mg/kg inhibited PARP-1 activity by 90% (95% CI = 89% to 91%).
Combination of Chemotherapy or Radiation Therapy With AG14361 in Cultured Human Cancer Cells
The effect of 0.4 µM AG14361 (>100 times the Ki and <5% of the GI50 values) on temozolomide- and topotecan-induced growth inhibition was measured in A549, LoVo, and SW620 cell cultures (Fig. 4, A and B). In combination with temozolomide, AG14361 statistically significantly reduced the temozolomide GI50 value 5.5-fold (95% CI = 4.9-fold to 5.9-fold; P = .004) in LoVo cells and reduced the GI50 value 2.1-fold or 2.3-fold (in two independent experiments) in A549 cells, but AG14361 failed to reduce the temozolomide GI50 value in SW620 cells. In combination with topotecan, AG14361 statistically significantly reduced the topotecan GI50 value 1.7-fold (95% CI = 1.3-fold to 2.1-fold; P = .020) in SW620 cells, 1.6-fold (95% CI = 1.3-fold to 1.9-fold; P = .002) in LoVo cells, and 1.9-fold or 2.1-fold (two independent experiments) in A549 cells. Growth inhibition of LoVo cells by AG14361 alone or in combination with 400 µM temozolomide (approximately half its GI50 concentration; temozolomide GI50 = 789 µM, 95% CI = 713 to 865 µM) demonstrated that concentrations in excess of 1.0 µM AG14361 alone were required to retard cell growth, whereas only 0.1 µM AG14361 was required for the maximum potentiation of temozolomide (Fig. 4, C).
A contributory factor to radiation resistance in vivo is the ability of quiescent cells to repair potentially lethal damage (36). In in vitro models of recovery from potentially lethal irradiation damage, the increased survival of growth-arrested cells is assessed by colony formation assay after a recovery period and compared with that of cells without the recovery period. Confluent LoVo cells, arrested in G1 phase, were exposed to 8 Gy of irradiation and then replated to assess their colony-forming ability immediately after irradiation or after a 4- or 24-hour recovery incubation in control medium or medium containing 0.4 µM AG14361 (Fig. 4, D). Survival increased approximately sevenfold for cells given a 24-hour recovery period in control medium before replating for the colony-forming assay compared with that of cells replated for colony formation immediately after irradiation. The increase in survival of cells exposed to AG14361 during the recovery period was statistically significantly reduced (P = .002) compared with that of cells not treated with AG14361, such that AG14361 inhibited the recovery of cells by 73% (95% CI = 48% to 98%) at 24 hours.
Combination of Chemotherapy or Radiation Therapy With AG14361 in Human Tumor Xenografts
The effect of AG14361 on the antitumor activity of temozolomide, irinotecan, and x-irradiation was determined in mice bearing established subcutaneous human LoVo or SW620 tumor xenografts (five mice per treatment group). At the doses used (5 and 15 mg/kg), AG14361 alone did not induce toxicity or affect tumor growth. Temozolomide alone at 68 mg/kg given daily for 5 days [approximately equivalent to the recommended schedule in patients of 200 mg/m2 per day (37)] induced a tumor growth delay (time to RTV4) of 3 days (95% CI = 0 to 6 days) for LoVo xenografts and 24 days (95% CI = 16 to 32 days) for SW620 xenografts (Fig. 5, A and B
). Increasing the temozolomide dose to 136 mg/kg (Fig. 5, A and B) or increasing the duration of administration to 68 mg/kg given daily for 10 days (data not shown) did not markedly augment its activity. In contrast, coadministration of AG14361 with temozolomide statistically significantly increased temozolomide activity against LoVo xenografts, with the tumor growth delay being increased from 3 days to 9 days (95% CI = 7 to 11 days; P = .032) by AG14361 at 5 mg/kg and to 10 days (95% CI = 8 to 12 days; P = .032) by AG14361 at 15 mg/kg (Fig. 5, A). This effect on temozolomide activity was even more pronounced in mice bearing SW620 xenografts, with a 100% complete remission rate observed with temozolomide and AG14361, an effect that persisted for 60 days in mice receiving temozolomide at 68 mg/kg plus AG14361 at 5 mg/kg or until 100 days in mice receiving temozolomide at 68 mg/kg plus AG14361 at 15 mg/kg, when mice were killed (Fig. 5, B). Limited toxicity, reflected by loss of body weight, was observed in mice treated with temozolomide alone at 68 mg/kg (nadir body weight = 92% of initial weight, 95% CI = 85% to 99%), which was not statistically significant (P = .310); this value was not statistically significantly increased by AG14361 at 5 mg/kg (nadir body weight = 93% of initial weight, 95% CI = 88% to 98%). However, statistically significant loss of body weight was observed in mice treated with temozolomide at 138 mg/kg (nadir = 88% of initial weight, 95% CI = 84% to 92%; P = .032) and the combination of AG14361 at 15 mg/kg and temozolomide at 68 mg/kg (nadir = 80% of initial weight, 95% CI = 71% to 88%; P = .016), including one toxic death.
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In mice bearing LoVo xenografts, irinotecan (2.5 mg/kg given daily for 5 days intraperitoneally) delayed xenograft tumor growth by 4 days (95% CI = 2 to 7 days). When AG14361 at 5 or 15 mg/kg was administered with irinotecan, the growth delay was statistically significantly increased to 9 days (95% CI = 7 to 11 days; P = .016) or to 11 days (95% CI = 9 to 13 days; P = .008), respectively (Fig. 5, C). Similarly, in mice bearing SW620 xenografts, irinotecan alone delayed xenograft tumor growth 8 days (95% CI = 6 to 10 days). When AG14361 at 5 or 15 mg/kg was administered with irinotecan, the delay was statistically significantly increased to 14 days (95% CI = 12 to 16 days; P = .001) or 16 days (95% CI = 15 to 17 days; P = .002), respectively (Fig. 5, D). No statistically significant loss of body weight was observed in the single-agent irinotecan groups, and only limited loss of body weight was observed in the irinotecan combination groups (nadir body weight in the high-dose combination group = 91% of initial weight, 95% CI = 86% to 96%; P = .69). Diarrhea, the normal indicator of gastrointestinal tract toxicity associated with irinotecan, was not observed in these animals.
AG14361 treatment before irradiation statistically significantly increased the sensitivity to radiation therapy of mice bearing LoVo xenografts (P = .008). Local tumor irradiation (2 Gy daily for 5 days) alone caused a 19-day tumor growth delay (95% CI = 16 to 22 days) that was extended to a 37-day delay (95% CI = 35 to 39 days) in mice treated with AG14361 at 15 mg/kg before irradiation (Fig. 5, E). Local toxicity in normal tissues was not observed in either group.
The increased antitumor activity observed with the combination of temozolomide and AG14361 in SW620 xenografts (Fig. 5, B) was not predicted from the in vitro data (Fig. 4, A). Consequently, we investigated whether AG14361 has vasoactive properties, as observed with nicotinamide (a weak PARP-1 inhibitor) and other benzamide analogs (38). Fluorescent markers of vascular perfusion detected a "mismatch" (lack of overlap) of vessels stained with both dyes (resulting from intermittent blood flow) in control SW620 tumors that was statistically significantly reduced by pretreatment with AG14361 (P = .022) (Fig. 6). Blood flow was maintained in 88% (95% CI = 78% to 98%) of the Hoechst-stained vessels in the AG14361-treated group but was maintained in 58% (95% CI = 31% to 84%) of those in the control tumors (seven tumors per group). AG14361 therefore statistically significantly increased blood flow in xenografts and thus potentially increased drug delivery to tumor xenografts.
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The implications of DNA repair inhibitors for anticancer therapy are well recognized (39), and the role of PARP-1 in DNA damage repair has been extensively characterized. First-generation PARP-1 inhibitors, such as 3-aminobenzamide, lacked the potency, specificity, and pharmacologic properties required for detailed preclinical evaluation of their ability to increase the sensitivity of tumors to anticancer chemotherapy and radiation therapy (5,6). Although researchers in a limited number of in vivo anticancer studies of second-generation PARP-1 inhibitors in combination with cytotoxic agents have observed increased survival compared with chemotherapy alone (4,18,40), most in vivo investigations of second-generation PARP-1 inhibitors have focused on protecting normal tissue from oxidative damage by using a variety of models of human pathophysiologic states [for review, see (41)].
AG14361 is more than 1000 times more potent than 3-aminobenzamide (20), and x-ray crystallographic analysis shows that AG14361 makes several critical interactions within the active site of PARP-1. AG14361 at 0.4 µM inhibited PARP-1 activity by more than 85% without affecting gene expression or cell proliferation (Fig. 2). At this low concentration, AG14361 increased the activity of DNA-damaging chemotherapeutic agents, temozolomide and topotecan, and the activity of radiation therapy in vitro (Fig. 4). AG14361 at this concentration also inhibited the recovery of growth-arrested cells from potentially lethal irradiation damage by approximately 75% (Fig. 4, D). This sensitization of growth-arrested cells is important for tumor radiation therapy because experimental and clinical evidence indicate that the growth-arrested cell fraction within a tumor is radiation-resistant and capable of re-entering the cell proliferation cycle and thereby re-populating the tumor after radiation therapy (36,42).
Pharmacologic studies demonstrated limited metabolic degradation of AG14361 by human liver microsomes in vitro, the retention of AG14361 in the tumor, and the inhibition of tumor PARP-1 activity by 75% or more for at least 4 hours after administration of AG14361 (Fig. 3). The acutely toxic effects, found with other PARP-1 inhibitors, were not observed in AG14361-treated mice. AG14361 at well-tolerated doses increased the antitumor activity of temozolomide threefold or more in LoVo xenograft tumors, and the combination resulted in complete remission of SW620 xenograft tumors. The addition of AG14361 increased the antitumor activity of both irinotecan and fractionated x-irradiation approximately twofold in both LoVo and SW620 xenografts (Fig. 5). Alternative strategies to increase the antitumor activity of temozolomide by inhibiting the DNA repair protein O6-alkylguanine DNA alkyltransferase (ATase) have been reported (43). Unlike ATase inhibitors, which can only sensitize cells proficient in mismatch repair (44), AG14361 increased the activity of temozolomide in mismatch repair-deficient LoVo cells in vitro and in vivo. Because AG14361 doubled the radiation-induced tumor-growth delay, PARP-1 inhibition may be an effective alternative to other radiosensitization approaches, such as the use of bioreductively activated drugs (45,46) or the inactivation or inhibition of epidermal growth factor receptor tyrosine kinase (47,48). Moreover, at radiosensitizing doses, AG14361 was nontoxic alone or in combination with radiation therapy.
AG14361 did not increase the antiproliferative effect of temozolomide in SW620 cells in vitro (Fig. 4, A), but the combination of AG14361 and temozolomide resulted in the complete regression of SW620 xenografts (Fig. 5, B). SW620 cells are hypersensitive to temozolomide by virtue of low ATase levels and mismatch repair proficiency (49,50), because resistance to DNA alkylating agents is associated with both high ATase and loss of mismatch repair [for respective reviews, see (51) and (52)]. Similar studies have shown that, whereas 3-aminobenzamide and another base excision repair inhibitor (methoxyamine) can sensitize mismatch repair-deficient cells to temozolomide (53,54), 3-aminobenzamide is ineffective in cells that are hypersensitive to temozolomide because of low ATase activity and mismatch repair proficiency (53,55). The marked increase in the antitumor activity of temozolomide against SW620 xenografts by coadministration of AG14361 may arise from an effect of AG14361 on the tumor microenvironment. AG14361 increased transient perfusion (Fig. 6), potentially altering the microdistribution and delivery of chemotherapeutic agents (56). PARP-1 inhibitors may therefore have greater chemosensitizing potential in vivo than indicated by the in vitro data. Nicotinamide (a weak PARP-1 inhibitor) similarly inhibits contraction of vascular smooth muscle in tumors by a mechanism that does not involve nitric oxide release or Ca2+ signaling (57,58); nicotinamide is in advanced clinical trials to evaluate its ability to increase sensitivity to radiation therapy (59).
In conclusion, AG14361 is, to our knowledge, the first PARP-1 inhibitor with the pharmacologic properties of high potency, specificity, stability, and in vivo activity that are necessary for its use in anticancer chemotherapy and radiation therapy in humans. We describe an improved therapeutic index with AG14361 in combination with temozolomide, irinotecan, and radiation in a human colon tumor xenograft model. Measurement of PARP-1 activity in cells and tissue homogenates provides a reliable and quantitative biomarker to support the performance and evaluation of clinical trials with PARP-1 inhibitors. Such trials should allow a mechanism-based evaluation of PARP-1 inhibition as a therapeutic treatment for cancer and could be a major step forward in the development of chemotherapy directed at the DNA-damage response of cancer cells.
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NOTES |
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Editor’s note: Agouron Pfizer approved this manuscript. R. Almassy, Z. Hostomsky, J. Li, K. Maegley, and D. Skalitsky hold stock in Pfizer, owner of AG14361. A. H. Calvert, N. J. Curtin, and D. R. Newell are eligible for an employee reimbursement of a fraction of any royalty paid to the University of Newcastle upon Tyne should a PARP inhibitor from this program generate such royalty. I. J. Stratford and K. J. Williams are conducting research sponsored by Agouron Pfizer.
Supported by Cancer Research UK (formerly Cancer Research Campaign).
We are grateful to M. North (Pfizer) for the gene array analysis data, to Brian Telfer (Manchester) for excellent technical assistance, and to Josiane Menissier-de Murcia and Gilbert de Murcia for supplying PARP-1-/- and PARP-1+/+ mice.
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S. Miknyoczki, H. Chang, J. Grobelny, S. Pritchard, C. Worrell, N. McGann, M. Ator, J. Husten, J. Deibold, R. Hudkins, et al. The selective poly(ADP-ribose) polymerase-1(2) inhibitor, CEP-8983, increases the sensitivity of chemoresistant tumor cells to temozolomide and irinotecan but does not potentiate myelotoxicity Mol. Cancer Ther., August 1, 2007; 6(8): 2290 - 2302. [Abstract] [Full Text] [PDF]
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J. M. Albert, C. Cao, K. W. Kim, C. D. Willey, L. Geng, D. Xiao, H. Wang, A. Sandler, D. H. Johnson, A. D. Colevas, et al. Inhibition of Poly(ADP-Ribose) Polymerase Enhances Cell Death and Improves Tumor Growth Delay in Irradiated Lung Cancer Models Clin. Cancer Res., May 15, 2007; 13(10): 3033 - 3042. [Abstract] [Full Text] [PDF]
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C. K. Donawho, Y. Luo, Y. Luo, T. D. Penning, J. L. Bauch, J. J. Bouska, V. D. Bontcheva-Diaz, B. F. Cox, T. L. DeWeese, L. E. Dillehay, et al. ABT-888, an Orally Active Poly(ADP-Ribose) Polymerase Inhibitor that Potentiates DNA-Damaging Agents in Preclinical Tumor Models Clin. Cancer Res., May 1, 2007; 13(9): 2728 - 2737. [Abstract] [Full Text] [PDF]
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H. D. Thomas, C. R. Calabrese, M. A. Batey, S. Canan, Z. Hostomsky, S. Kyle, K. A. Maegley, D. R. Newell, D. Skalitzky, L.-Z. Wang, et al. Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial Mol. Cancer Ther., March 1, 2007; 6(3): 945 - 956. [Abstract] [Full Text] [PDF]
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K. Ratnam and J. A. Low Current Development of Clinical Inhibitors of Poly(ADP-Ribose) Polymerase in Oncology Clin. Cancer Res., March 1, 2007; 13(5): 1383 - 1388. [Abstract] [Full Text] [PDF]
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M. S. Bentle, K. E. Reinicke, E. A. Bey, D. R. Spitz, and D. A. Boothman Calcium-dependent Modulation of Poly(ADP-ribose) Polymerase-1 Alters Cellular Metabolism and DNA Repair J. Biol. Chem., November 3, 2006; 281(44): 33684 - 33696. [Abstract] [Full Text] [PDF]
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 D. Martin-Oliva, R. Aguilar-Quesada, F. O'Valle, J. A. Munoz-Gamez, R. Martinez-Romero, R. Garcia del Moral, J. M. Ruiz de Almodovar, R. Villuendas, M. A. Piris, and F. J. Oliver Inhibition of Poly(ADP-Ribose) Polymerase Modulates Tumor-Related Gene Expression, Including Hypoxia-Inducible Factor-1 Activation, during Skin Carcinogenesis Cancer Res., June 1, 2006; 66(11): 5744 - 5756. [Abstract] [Full Text] [PDF]
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Y. Zhao, H. D. Thomas, M. A. Batey, I. G. Cowell, C. J. Richardson, R. J. Griffin, A. H. Calvert, D. R. Newell, G. C.M. Smith, and N. J. Curtin Preclinical Evaluation of a Potent Novel DNA-Dependent Protein Kinase Inhibitor NU7441. Cancer Res., May 15, 2006; 66(10): 5354 - 5362. [Abstract] [Full Text] [PDF]
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G. Noel, C. Godon, M. Fernet, N. Giocanti, F. Megnin-Chanet, and V. Favaudon Radiosensitization by the poly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide is specific of the S phase of the cell cycle and involves arrest of DNA synthesis. Mol. Cancer Ther., March 1, 2006; 5(3): 564 - 574. [Abstract] [Full Text] [PDF]
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A. Poonepalli, L. Balakrishnan, A. K. Khaw, G. K. M. Low, M. Jayapal, R. N. Bhattacharjee, S. Akira, A. S. Balajee, and M. P. Hande Lack of Poly(ADP-Ribose) Polymerase-1 Gene Product Enhances Cellular Sensitivity to Arsenite Cancer Res., December 1, 2005; 65(23): 10977 - 10983. [Abstract] [Full Text] [PDF]
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L. M. Smith, E. Willmore, C. A. Austin, and N. J. Curtin The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks Clin. Cancer Res., December 1, 2005; 11(23): 8449 - 8457. [Abstract] [Full Text] [PDF]
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T. Hay, H. Jenkins, O. J. Sansom, N. M.B. Martin, G. C.M. Smith, and A. R. Clarke Efficient Deletion of Normal Brca2-Deficient Intestinal Epithelium by Poly(ADP-Ribose) Polymerase Inhibition Models Potential Prophylactic Therapy Cancer Res., November 15, 2005; 65(22): 10145 - 10148. [Abstract] [Full Text] [PDF]
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E. R. Plummer, M. R. Middleton, C. Jones, A. Olsen, I. Hickson, P. McHugh, G. P. Margison, G. McGown, M. Thorncroft, A. J. Watson, et al. Temozolomide Pharmacodynamics in Patients with Metastatic Melanoma: DNA Damage and Activity of Repair Enzymes O6-Alkylguanine Alkyltransferase and Poly(ADP-Ribose) Polymerase-1 Clin. Cancer Res., May 1, 2005; 11(9): 3402 - 3409. [Abstract] [Full Text] [PDF]
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L. Catley, Y.-T. Tai, R. Shringarpure, R. Burger, M. T. Son, K. Podar, P. Tassone, D. Chauhan, T. Hideshima, L. Denis, et al. Proteasomal Degradation of Topoisomerase I Is Preceded by c-Jun NH2-Terminal Kinase Activation, Fas Up-Regulation, and Poly(ADP-Ribose) Polymerase Cleavage in SN38-Mediated Cytotoxicity against Multiple Myeloma Cancer Res., December 1, 2004; 64(23): 8746 - 8753. [Abstract] [Full Text] [PDF]
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N. J. Curtin, L.-Z. Wang, A. Yiakouvaki, S. Kyle, C. A. Arris, S. Canan-Koch, S. E. Webber, B. W. Durkacz, H. A. Calvert, Z. Hostomsky, et al. Novel Poly(ADP-ribose) Polymerase-1 Inhibitor, AG14361, Restores Sensitivity to Temozolomide in Mismatch Repair-Deficient Cells Clin. Cancer Res., February 1, 2004; 10(3): 881 - 889. [Abstract] [Full Text] [PDF]
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