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Combinations of PARP, hedgehog and HDAC inhibitors with standard drugs
Available online at www.sciencedirect.com
Combinations of PARP, hedgehog and HDAC inhibitors with standard drugs Beverly A Teicher Objective methods for data analysis for the assessment of additivity, synergy, and antagonism that can be applied rigorously to cell-based and in vivo preclinical data have been defined. The Combination Index method has been most widely applied. This brief report discusses poly(ADP-ribose) polymerase (PARP) inhibitors, hedgehog inhibitors, and histone deacetylase inhibitors as examples of experimental agents used in combinations where additivity and synergy have been assessed. PARP, hedgehog, and HDAC inhibitors have demonstrated single agent activity in a limited spectrum of malignancies. Expanded application of these compounds may come from combination regimens. Several PARP inhibitors appear to be additive to synergistic with standard agents in cell culture; however, no determinations for additivity/synergy have been applied with these compounds in vivo. Hedgehog inhibitors have demonstrated interesting activity in some preclinical models and have been studied in vivo with gemcitabine. However, no analyses for additivity were conducted in these studies. Numerous studies have demonstrated additivity/synergy with several HDAC inhibitors and standard anticancer drugs in cell culture. Clinical trials with combination regimens are on-going with compounds from each of these classes based primarily upon limited cell culture data. Thus, there is still a distance to go in making optimal use of preclinical models to aid in the selection of combination regimens worthy of clinical trial. Address Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701-9322, United States Corresponding author: Teicher, Beverly A ([email protected])
Current Opinion in Pharmacology 2010, 10:397–404 This review comes from a themed issue on Cancer Edited by Raymond Winquist and Diane Boucher Available online 22nd May 2010 1471-4892/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2010.04.014
One of the main tenets of cancer therapeutics is that combinations of anticancer agents with different targets or different mechanisms of action and varied normal tissue toxicities will produce better therapeutic outcomes. Frequently proving that combination regimens result in www.sciencedirect.com
better treatment outcomes preclinically and clinically is not easy. The premise of controlled randomized trials, a paradigm used automatically in preclinical studies and often required in clinical studies, is that there is uncertainty whether any of the alterative treatments may have more benefit. Indeed, very large studies analyzing outcomes from multiple clinical trials most often find no substantive differences in the treatment arms lending support to the continuing need for randomized trials [1]. Sometimes the benefit of combination regimens can only be discerned through meta-analyses of many clinical trials. Gemcitabine as a single agent compared with gemcitabine in combination regimens for the treatment of pancreatic cancer is an example where the benefit of combination regimens could only be confirmed after tens of trials and thousands of patients were accrued [2– 6]. In some cases, gemcitabine combination preclinical data were described where synergy did not occur and was not claimed but were later interpreted by others as demonstrating synergy [7,8]. The combination of gemcitabine and navelbine was assessed for additivity in mice bearing the Lewis lung carcinoma. Gemcitabine was well tolerated over the range from 40 mg/kg 3 to 80 mg/ kg 3 [9]. Navelbine was well tolerated at total doses of 10, 15 and 22.5 mg/kg. Isobologram methodology [10] was used to determine whether the combinations of gemcitabine and navelbine achieved additive antitumor activity. At gemcitabine doses of 40 and 60 mg/kg, the combination achieved additivity. At the highest gemcitabine dose, the combination produced less than additive tumor growth delay [9,11]. The resulting phase 1 clinical trial concluded that this drug combination has potential as a front-line or second-line regimen for advanced lung cancer; however, hematologic toxicities would require intermittent dosing. The promise is that increased understanding of malignant disease and normal tissue effects and application of objective data analysis methods will allow better selection of combinations in the preclinical setting that will translate into an improved rate of success in clinical trial. Human tumor xenografts have been the primary models for the demonstration of in vivo efficacy for new compounds and other anticancer therapeutics for more than 30 years [12–18]. Appealing properties of human tumor xenograft models are that the malignant cells are human and that most grow slowly enough in immunodeficient mice to allow a reasonable course of an experimental treatment over one to three weeks to be administered to the mouse. For most of the more than 30 years during Current Opinion in Pharmacology 2010, 10:397–404
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which the cancer research and drug discovery community has relied on human tumor xenografts, little has been known about the tumor lines used except that many were originated in the late 1970s and early 1980s from patients with a known malignancy [12,13]. However, in recent years many lines have been characterized for gene expression, mutations, amplifications and other molecular alterations. Thus, the tumor model can be matched with the experimental therapeutic with great accuracy and confidence. The hope is that this knowledge and personalization of therapy will carry into clinical application [14,19,20]. The best models and methods for data analysis for defining drug combinations with the most clinical potential are topics of on-going investigation [14,21–23]. Objective methods for the assessment of additivity, synergy, and antagonism include combination index, median effect, isobolograms, continuous measures, Bliss methodology, and varied response surface techniques have been defined [24–37]. These methods can be applied rigorously to cellbased and in vivo preclinical data. This brief report will discuss poly(ADP-ribose) polymerase (PARP) inhibitors, hedgehog inhibitors, and histone deacetylase inhibitors as examples of experimental agents used in combinations, some of which have been translated to the clinic.
PARP inhibitors PARP is a family of nuclear enzymes involved in the detection and repair of DNA damage. Eighteen putative PARP family members have been identified; however, only PARP-1 and PARP-2 contain a DNA-binding domain that facilitates localization to the site of DNA damage. In addition to DNA repair, the PARP enzymes are involved in the modulation of chromatin structure, transcription regulation, DNA telomere maintenance, mitotic control, cellular transport, and cell death [38]. PARP-1 recognizes single-strand breaks and acts in base excision repair (BER). PARP-1 and PARP-2 catalyze the transfer of ADP-ribose units from intracellular nicotinamide adenine dinucleotide (NAD+) as a key step in DNA repair. Several small molecule inhibitors of the PARP-1 activity have been developed as potential agents to use mainly in combination with anticancer therapies that cause damage to DNA [39,40,41]. Temozolomide has frequently been used to cause DNA damage to allow the activity of PARP inhibitors to be manifest in preclinical models. HCT116 cells exposed to the combination of temozolomide with ABT-888 (500 nM), a benzimidazole PARP inhibitor, require 17–24 hours to achieve maximal cytotoxicity. The cell killing correlates with the level of double-stranded DNA breaks. In synchronized cells, exposure to temozolomide along with ABT-888 during the S phase resulted in high levels of DNA doublestranded breaks, most likely from single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides that were converted into double-stranded Current Opinion in Pharmacology 2010, 10:397–404
breaks during DNA replication. Thus, exposure of S phase cells to temozolomide and ABT-888 leads to high levels of cytotoxicity [38]. In mice bearing B16F10 melanoma ABT-888 (25 mg/kg/day, po) inhibited poly(ADPribose) formation and enhanced tumor growth delay by temozolomide (50 mg/kg/day, po) from 5 days for temozolomide alone to 10 days for the combination. In rats bearing intracranial 9L glioma, the combination of temozolomide (17.5 mg/kg) and ABT-888 (25 mg/kg) was more effective in slowing tumor growth than either drug alone. However, no analysis was performed to determine whether the increases in cytotoxicity and tumor growth delay were additive, subadditive, or synergistic. A breakthrough in understanding the potential of PARP inhibition as a therapeutic approach in cancer came with the realization that cell lines deficient in BRCA1 and BRCA2, proteins involved in DNA homologous recombination repair are very sensitive to PARP-1 inhibitors. Exposure to PARP inhibitors produces cell death in BRCA mutant cells [42,43]. BRCA1 and BRCA2 wildtype alleles are frequently lost in tumors of heterozygous carriers. Carriers of mutations in BRCA1 and BRCA2 are at elevated risk for breast, ovary, prostate, and pancreas cancers. The PARP inhibitor, AZD2281 has single agent cytotoxicity toward BRCA1-deficient breast cancer cell lines with IC50s of 200–800 nM. Several mouse tumor cell lines have been developed that are BRCA2 deficient [44]. AZD2281 was tested in combination with these clonal lines alone and along with a variety of anticancer agents and g-irradiation. A variant of the Combination Index method was applied to assess the additivity/synergy of the resulting cytotoxicity of the combinations. The combination of AZD2281 with cisplatin provided evidence of synergistic or greater-than-additive cytotoxicity. In a genetically engineered mouse model of BRCA1-associated breast cancer, treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity. However, long-term treatment of these mice with AZD2281 did not prevent tumor development [45]. Combination of AZD2281 with cisplatin or carboplatin was more effective than the single agents suggesting that AZD2281 adds to the effect of DNAdamaging agents. In a mouse with conditionally deleted BRCA2 and p53 in mammary epithelium daily AZD2281 produced some tumor growth delay; however, the combination of carboplatin with AZD2281 had no advantage over carboplatin monotherapy [46]. The combination of temozolomide (50 mg/kg/day) and AZD2281 (10 mg/kg/ day) was more effective than single agent temozolomide in the SW620 colon carcinoma xenograft [38]. Another PARP inhibitor AG014699 was combined in cell culture and in vivo with topotecan and temozolomide. In NB1691 and SHSY5Y neuroblastoma xenografts, temozolomide produced tumor growth delay, which was increased by co-administration of AG014699, and resulted in complete and sustained tumor regression in 6 of 10 mice [47]. www.sciencedirect.com
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Based upon these and other data PARP inhibitors have entered clinical trials. The early clinical trials group at NCI conducted a first phase 0 clinical trial of ABT-888 in patients with advanced malignancies [48]. ABT-888 was administered as a single oral dose of 10, 25, or 50 mg to determine the dose range and time course over which ABT-888 inhibits PARP activity in tumor samples and peripheral blood mononuclear cells, and to evaluate ABT888 pharmacokinetics. Blood samples and tumor biopsies were obtained pre-drug and post-drug administration for the evaluation of PARP activity and pharmacokinetics. Using data from the phase 0 trial, a phase 1 dose escalation study of the combination of ABT-888 and temozolomide was conducted in patients with metastatic melanoma and with BRCA-related breast and ovarian cancer. Patients were treated with escalating ABT-888 doses at a fixed temozolomide dose [49]. One patient with hepatocellular cancer had a partial response. Three patients had stable disease (1 neuroendocrine for 6 cycles and 2 patients with melanoma). The combination of ABT-888 and temozolomide was well tolerated. In a phase 1 trial of AZD2281 (olaparib) 60 patients (22 carriers of a BRCA1 or BRCA2 mutation and 1 with a strong family history of BRCAassociated cancer but declined to undergo mutational testing) were treated [50]. The olaparib dose was increased from 10 mg daily to 600 mg twice daily continuously. This led to enrollment of a cohort of BRCA1 or BRCA2 mutation carriers who received olaparib at a dose of 200 mg twice daily. Objective antitumor activity was reported only in mutation carriers, all of whom had ovarian, breast, or prostate cancer and had received multiple treatment regimens. Another PARP inhibitor, BSI-201 has completed phase 1 clinical trial and is undergoing a phase 2 randomized trial in triple negative breast cancer patients [51,52]. The early results indicated that patients treated with BSI-201 in combination with gemcitabine and carboplatin had an improved therapeutic outcome compared with patients treated with gemcitabine and carboplatin only.
Hedgehog inhibitors Hedgehog is a secreted molecule that influences the differentiation of a variety of tissues during development and plays a pivotal role in tissue homeostasis in a variety of species. Hedgehog, its receptor patched, and many downstream members of the hedgehog signal transduction pathway were first elucidated as factors involved in fruit fly embryo development [53]. The hedgehog protein family includes sonic, Indian and desert hedgehogs. Patched-1 is the receptor for all of the hedgehog proteins. In the absence of a hedgehog, patched-1 inhibits smoothened, a G-protein-coupled receptor. When a hedgehog binds to patched-1, smoothened is activated and initiates a signaling cascade that results in the activation of a transcription factor, Gli-1. In humans, aberrant activation of the hedgehog pathway is associated with basal-cell carcinoma, chondrosarcoma, and medulloblastoma www.sciencedirect.com
[54,55]. Cyclopamine, a plant alkaloid, is a natural product antagonist of the hedgehog pathway that has been used to validate the role of the hedgehog pathway in disease. Several synthetic small molecule inhibitors of the hedgehog pathway have been developed [56,57]. IPI-926 is a seven-membered D-ring semisynthetic analog of cyclopamine that is a potent and orally active hedgehog pathway antagonist [57]. GDC-0449 is a novel fully synthetic functionalized 2-pyridyl amide inhibitor of the hedgehog pathway [56]. The COLO 357 cell line was developed in the late 1970s from a metastatic pancreatic adenocarcinoma [58]. In 1999, Bruns et al. selected sublines of COLO 357 by repeated injects into the spleen or pancreas of nude mice and selection of tumor nodules in the liver [59]. After repeating the cycle several times, two sublines designated L3.6sl (spleen to liver) and L3.6pl (pancreas to liver) were characterized. In a more recent effort, Embuscado et al. have developed new pancreatic adenocarcinoma cell lines from rapid autopsy specimens [60]. Using the L3.6pl line and a previously undescribed line designated E3LZ10.7 from a rapid autopsy isolate, a role for the hedgehog pathway in pancreatic cancer cell lines was reported [61,62]. Hedgehog inhibition with cyclopamine or IPI269609 resulted in inhibition of epithelial-to-mesenchymal transition and a reduction in invasion through Matrigel by cells in culture. In an orthotopic xenograft model, treatment with cyclopamine or IPI-260609 decreased metastasis. However, treatment with the combination of gemcitabine and cyclopamine or IPI-269609 did not alter the response of subcutaneous E3LZ10.7 tumors from that obtained with gemcitabine alone [61,62]. A genetically engineered mouse in which endogenous alleles of KrasG12D and Trp53R172H were conditionally activated in the pancreas of LSL-KrasG12D/+;LSL Trp53R172H/+;Pdx-1-Cre triple mutant mice was used to assess treatment with the combination of gemcitabine and IPI-926 [63]. These mice have a median survival time of five months [64]. In this model, treatment with IPI-926 for 10 days increased the microvessel density of the tumors allowing improved distribution of gemcitabine into the nodules. The combination of gemcitabine and IPI-926 resulted in eight to nine days increased survival compared with gemcitabine alone [63]. This study points out the importance of tumor stroma in therapy [65]. Clinical trials are on-going with GDC-0449 and IPI-926. In a phase 1 clinical trial, the safety and pharmacokinetics of GDC-0449 and responses of metastatic or locally advanced basal-cell carcinoma were determined. Thirty-three patients with metastatic or locally advanced basal-cell carcinoma received oral GDC-0449 at one of three doses: 17 patients received 150 mg/day, 15 patients received 270 mg/day, and 1 patient received 540 mg/day [66]. The median treatment duration was 9.8 months. Current Opinion in Pharmacology 2010, 10:397–404
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Of the 33 patients, 18 had an objective response to GDC0449, according to assessment on imaging (7 patients), physical examination (10 patients), or both (1 patient). Two of the responding patients had a complete response and 16 had a partial response. The other 15 patients had either stable disease (11 patients) or progressive disease (4 patients). In a case report, a 26-year-old man with metastatic medulloblastoma that was refractory to multiple therapies was treated with GDC-0449. The treatment resulted in rapid (although transient) tumor regression and reduction of symptoms [67]. Molecular analyses of tumor specimens obtained before treatment suggested hedgehog pathway activation, with loss of heterozygosity and somatic mutation of the gene encoding patched-1.
unknown whether targeting certain histone deacetylase isotypes selectively will reduce toxicity and achieve responses equivalent to broad spectrum inhibitors. In the clinic as single agents, histone deacetylase inhibitors have been effective against T-cell lymphomas. However, as single agents, histone deacetylase inhibitors have had little activity against solid tumors. A potential synergy between histone deacetylase inhibitors and chemotherapeutic or biologically active anticancer compounds can be demonstrated in cell culture experiments suggesting that combination strategies will be a major focus in future clinical studies although the molecular mechanisms that account for these greater-than-additive effects remain to be elucidated [73].
In another phase 1 clinical trial, GDC-0449 was administered orally as a single dose on day 1 with a one-week PK break, and then once daily continuously (cycle 1 = 35 days) to refractory solid tumor patients at 3 dose levels. Surrogate tissues were assessed for the expression of hedgehog downstream target gene Gli1. Of the 19 patients enrolled in the trial, 1 partial response (150 mg) and 2 stable disease patients (270 mg) in basal-cell carcinoma and adenocystic carcinoma were seen. Continuous oral dosing at 150 mg/day demonstrated safe, effective concentrations, pharmacodynamic activity, an objective response and clinical benefit, and was selected for phase 2 studies [68]. GDC-0449 is currently in multiple phase 2 clinical trials alone and in combination with varied chemotherapeutic agents.
The proteasome inhibitor bortezomib and the histone deacetylase inhibitors romidepsin or belinostat were studied in combination in cell culture in chronic lymphocytic leukemia (CLL) cells [74]. Primary and cultured CLL cells were exposed to the agents alone or in combination and cell death was estimated by 7-aminoactinomycin D flow cytometry. Exposure to romidepsin or belinostat in combination with bortezomib produced synergistic cell death in primary and cultured CLL cells as determined by combination index methodology. Cell death likely occurred through mechanisms involving NF-kB inactivation and perturbations in proapoptotic and antiapoptotic proteins. Human HL-60 and K562 leukemia cells in culture were exposed to cytosine arabinoside [1-b-Darabinofuranosylcytosine (ara-C)] and/or etoposide in combination with vorinostat and cell survival was estimated using a colorimetric or fluorescent dye endpoint [75]. Drug combination effects were analyzed by the combination index method and by a novel statistical method. Using the two independent data analysis methods, cytotoxic antagonism resulted when vorinostat was combined simultaneously with ara-C; however, when vorinostat exposure was first followed by a drug-free interval before ara-C exposure, the combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became greater when etoposide exposure occurred after vorinostat exposure. The cytotoxicity of the histone deacetylase inhibitor PCI-24781, a phenyl hydroxamic acid compound, alone and in combination with bortezomib was assessed in the L428 Hodgkin lymphoma cell line and the non-Hodgkin lymphoma cell lines Ramos (Burkitt lymphoma), HF1 (follicular lymphoma), and SUDHL4 (large B-cell lymphoma) in cell culture [76]. Cell viability was estimated morphologically after staining with trypan blue and by the analysis of apoptosis using flow cytometry after staining with Annexin V-FITC and propidium iodide. When the data were analyzed by the combination index method, combined PCI-24781 and bortezomib exposure resulted in synergistic apoptosis in all non-Hodgkin lymphoma lines and additivity in the Hodgkin lymphoma line.
Histone deacetylase inhibitors Histone deacetylases are a family of 4 classes of enzymes with 18 known members that catalyze the removal of acetyl groups, from histones leading to chromatin condensation and transcriptional repression [69]. Inhibition of histone deacetylases results in hyper-acetylated histones, cell cycle arrest at the G1/S boundary, and cell death, most frequently by apoptosis. Non-histone proteins (tumor suppressor p53) and several cytoplasmic proteins are also regulated by acetylation status [70,71]. Pan-histone deacetylase inhibitors in clinical trials include vorinostat, panobinostat, romidepsin, and belinostat. Isotype-selective histone deacetylase inhibitors in clinical trials include MGCD0103 and entinostat [72]. Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a hydroxamic acid derivative that inhibits both class I and II histone deacetylases. Vorinostat was approved by FDA for the treatment of relapsed and refractory cutaneous Tcell lymphoma. Panobinostat (LBH589) and belinostat (PXD101) are also hydroxamic acid derivatives. Romidepsin (FK228) is a prodrug that in cells is reduced to an active species that interacts with class I and II histone deacetylases. MGCD0103, an aminophenylbenzamide, inhibits class I and IV histone deacetylases selectively. Entinostat (MS-275) is a benzamide with predominant class I histone deacetylase inhibitory activity. It is Current Opinion in Pharmacology 2010, 10:397–404
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A combination of TRAIL and the histone deacetylase inhibitor LBH589 was studied in mesothelioma cell lines in culture [77,78]. Five mesothelioma cell lines and two normal cell types were tested for growth inhibition using a colorimetric assay and apoptosis in the presence of LBH589, TRAIL and the combination. Median effect and combination index methodology were used to determine whether the combinations resulted in additive or greater-than-additive growth inhibition. In mesothelioma cell lines, the combination of LBH589 and TRAIL produced synergistic cell growth inhibition and apoptosis and an additive effect in normal cells. Several investigations of histone deacetylase inhibitors in combination with other agents have been reported in cell lines and tumor models and have claimed synergy without appropriate data analysis [79,80]. Vorinastat is currently in phase 1, 2 and 3 trials in combination with bortezomib and in phase 1 and 2 clinical trials with azacitidine or decitabine in hematologic malignancies and solid tumors [53]. Entinostat is in phase 1 and 2 clinical trials with azacitidine in leukemias and lung cancer. Panbinostat is in a phase 1 clinical trial with bortezomib in multiple myeloma and with decitabine in leukemia. Romidepsin, belinostat, and MGCD0103 are in early combination clinical trials with bortzomib, azacitidine, decitabine, and azacitidine with all-transretinoic acid.
tivity in tumor response can be achieved in a relevant tumor in mice without decrease in therapeutic index, the combination regimen may be worthy of clinical trial.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of outstanding interest
References 1.
Soares HP, Kumar A, Daniels S, Swann S, Cantor A, Hozo I, Clark M, Serdarevic F, Gwede C, Trotti A, Djulbegovic B: Evaluation of new treatments in radiation oncology: Are they better than standard treatments? J Am Med Assoc 2005, 293:970-978.
2.
Di Maio M, Chiodini P, Georgoulias V, Hatzidaki D, Takeda K, Wachters FM, Gebbia V, Smit EF, Morabito A, Gallo C et al.: Metaanalysis of single-agent chemotherapy compared with combination chemotherapy as second-line treatment of advanced non-small cell lung cancer. J Clin Oncol 2009, 27:1836-1843.
3.
Sultana A, Ghaneh P, Cunningham D, Starling N, Neoptolemos JP, Smith CT: Gemcitabine based combination chemotherapy in advanced pancreatic cancer-indirect comparison. BMC Cancer 2008, 8:192-196.
4.
Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, Natale RB, Schiller JH, von Pawel J, Pluzanska A et al.: Gefitinib in combination with gemcitabine and cisplatin in advanced non-small cell lung cancer: a phase III trial-INTACT 1. J Clin Oncol 2004, 22:777-784.
5.
Heinemann V, Boeck S, Hinke A, Labianca R, Louvet C: Metaanalysis of randomized trials: evaluation of benefit from gemcitabine-based combination chemotherapy applied in advanced pancreatic cancer. BMC Cancer 2008, 8:82-92.
6.
Li J, Podoltsev N, Saif MW: Management of advanced pancreatic cancer. Expert Rev Clin Pharmacol 2009, 2:527-541.
7.
Ng SSW, Tsao MS, Nicklee T, Hedley DW: Effects of the epidermal growth factor receptor inhibitor OSI-774, tarceva, on downstream signaling pathways and apoptosis in human pancreatic adenocarcinoma. Mol Cancer Ther 2002, 1:777-783.
8.
Bayraktar S, Bayraktar UD, Rocha-Lima CM: Recent developments in palliative chemotherapy for locally advanced and metastatic pancreas cancer. World J Gastroenterol 2010, 16:673-682.
9.
Herbst RS, Lynch C, Vasconcelles M, Teicher BA, Strauss G, Elias A, Anderson I, Zacarola P, Dang NH, Leong T et al.: Gemcitabine and vinorelbine in patients with advanced lung cancer: preclinical studies and report of a phase I trial. Cancer Chemother Pharmacol 2001, 48:151-159.
Conclusion These recent examples of the translation of preclinical combination studies to clinical trial indicate that there is still a distance to go in making optimal use of preclinical models to aid in the selection of combinations to apply in clinical trial. The few cell culture studies cited herein have made use of the combination index method to assess the additivity/synergy of the combinations; however, the limitations of the well-controlled cell culture assay to mimic the dynamics of the in vivo situation were not acknowledged in most studies [81]. The analysis of the additivity/synergy of same combinations in bone marrow CFU-GM or other stem cell culture assays is under utilized as a method to estimate whether the combination will have an improved therapeutic index or not. In fact, there were no examples of in vivo studies demonstrating additivity or synergy with the drug combinations currently in clinical trial. Mice are not patients, however, experiments in which tumor volumes and body weight measurements are made can often allow determination of increased efficacy, tumor response without increased toxicity, or determination of increased tumor response and increased toxicity (body weight loss and hematology). Such preclinical in vivo experiments with combinations of kinase inhibitors and other combinations showed increased tumor response, the cost being increased body weight loss [82,83]. In vivo synergy is a very rare result and subadditivity of combinations is quite frequent. If addiwww.sciencedirect.com
10. Teicher BA, Herman TS, Holden SA, Eder JP: Chemotherapeutic potentiation through interaction at the level of DNA. In Synergism and Antagonism in Chemotherapy. Edited by Chou TC, Rideout DC. Orlando, FL: Academic Press; 1991:541-583. 11. Teicher BA, Frei E III: Laboratory models to evaluate new agents for the systemic treatment of lung cancer. In Multimodality Treatment of Lung Cancer. Edited by Skarin AT. New York, NY: Marcel Dekker Inc; 2000:301-336. 12. Teicher BA: Tumor models for efficacy determination. Mol Cancer Ther 2006, 5:2435-2443. 13. Teicher BA: Human tumor xenografts and mouse models of human tumors: re-discovering the models. Expert Opin Drug Discov 2009, 4:1-11. 14. Sausville EA, Burger AM: Contributions of human tumor xenografts to anticancer drug development. Cancer Res 2006, 66:3351-3354. Current Opinion in Pharmacology 2010, 10:397–404
402 Cancer
15. Garber K: Realistic rodents? Debate grows over new mouse models of cancer. J Natl Cancer Inst 2006, 98:1176-1178.
diamminedichloroplatinum and 1-b-Darabinofuransylcytosine. Cancer Res 1992, 52:4558-4565.
16. Suggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clin Cancer Res 2005, 11:971-981.
35. Chou TC: Theoretical basis, experimental design and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006, 58:621-681.
17. Fiebig HH, Maier A, Burger AM: Clonogenic assay with established human tumor xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery. Eur J Cancer 2004, 40:802-820.
36. Steel GG, Peckham MJ: Exploitable mechanisms in combined radiotherapy chemotherapy: the concept of additivity. Int J Radiat Oncol Biol Phys 1979, 5:85-91.
18. Kerbel RS: Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans. Cancer Biol Ther 2003, 2(Suppl 1):S134-S139. 19. Korrat A, Greiner T, Maurer M, Metz T, Fiebig HH: Gene signaturebased prediction of tumor response to cyclophosphamide. Cancer Genom Proteom 2007, 4:187-196. 20. Fiebig HH, Schuler J, Bausch N, Hofmann M, Metz T, Korrat A: Gene signature developed from patient tumor explants grown in nude mice to predict tumor response to 11 cytotoxic drugs. Cancer Genom Proteom 2007, 4:197-210. 21. Lee EM, Bachmann PS, Lock RB: Xenograft models for the preclinical evaluation of new therapies in acute leukemia. Leukemia Lymphoma 2007, 48:659-668. 22. Sausville EA, Elsayed Y, Monga M, Kim G: Signal transductiondirected cancer treatments. Annu Rev Pharmacol Toxicol 2003, 43:199-231. 23. Gitler MS, Monks A, Sausville EA: Preclinical models for defining efficacy of drug combinations: mapping the road to the clinic. Mol Cancer Ther 2003, 2:929-932. 24. Buck E, Eyzaguirre A, Brown E, Petti F, McCormack S, Haley JD, Iwata KK, Gibson NW, Griffin G: Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in nonsmall-cell lung, pancreatic, colon and breast tumors. Mol Cancer Ther 2006, 5:2676-2684. 25. Duska LR, Hamblin MR, Miller JL, Hasan T: Combination photoimmuno-therapy and cisplatin: effects on human ovarian cancer ex vivo. J Natl Cancer Inst 1999, 91:1557-1563. 26. Borisy AA, Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER, Serbedzija G, Zimmermann GR, Foley MA, Stockwell BR, Keith CT: Systematic discovery of multicomponent therapeutics. Proc Natl Acad Sci U S A 2003, 100:7977-7982. 27. Tan M, Fang HB, Tian GL, Houghton PJ: Experimental design and sample size determination for testing synergism in drug combination studies based on uniform measures. Stats Med 2003, 22:2091-2100. 28. Grabovsky Y, Tallarida RJ: Isobolographic analysis for combinations of a full and partial agonist: curved isoboles. J Pharmacol Exp Ther 2004, 310:981-986. 29. Zhao L, Wientjes MG, Au JLS: Evaluation of combinations chemotherapy: integration of nonlinear regression, curve shift, isobologram and combination index analyses. Clin Cancer Res 2004, 10:7994-8004. 30. Kong M, Lee JJ: A generalized response surface model with varying relative potency for assessing drug interaction. Biometrics 2006, 62:986-995. 31. Zhao L, Au JLS, Wientjes MG: Comparison of methods for evaluating drug-drug interaction. Front Biosci 2010, 2:241-249. 32. Levasseur LM, Delon A, Greco WR, Faury P, Bouquet S, Couet W: Development of a new quantitative approach for the isobolographic assessment of the convulsant interaction between perfloxacin and theophylline in rats. Pharm Res 1998, 15:1069-1076. 33. Greco WR, Faessel H, Levasseur L: The search for cytotoxic synergy between anticancer agents: a case of Dorothy and the ruby slippers? J Natl Cancer Inst 1996, 88:699-700. 34. Berenbaum MC: Correspondence re: WR Greco et al., application of a new approach for the quantitation of drug synergism to the combination of cisCurrent Opinion in Pharmacology 2010, 10:397–404
37. Berenbaum MC: Synergy, additivism and antagonism in immunosuppression. Clin Exp Immunol 1977, 28:1-18. 38. Liu X, Shi Y, Guan R, Donawho C, Luo Y, Palma J, Zhu G, Johnson EF, Rodriguez LE, Ghoreishi-Haack N et al.: Potentiation of temozolomide cytotoxicity by poly(ADP)ribose polymerase inhibitor ABT-888 requires a conversion of single-stranded DNA damage to double-stranded DNA breaks. Mol Cancer Res 2008, 6:1621-1629. 39. Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, Cranston A, Dillon KJ, Drzewiecki J, Garman S, Gomez S et al.: 4[3-(4-Cyclopropoanecarbonylpiperazine-1-carbonyl)-4fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem 2008, 51:6581-6591. This study describes the discovery of the PARP inhibitor ABT-888 along with testing in enzyme activity assays, cell culture and in vivo models as a single agent and in combination with temozolomide is described. 40. Penning TD, Zhu GD, Gandhi VB, Gong JC, Liu XS, Shi Y, Klinghofer V, Johnson EF, Bouska JJ, Osterling DJ et al.: Discovery of the poly(ADP-ribose) polymerase (PARP) inhibitor 2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4carboxamide (ABT-888) for the treatment of cancer. J Med Chem 2009, 52:514-523. 41. Tong Y, Bouska JJ, Ellis PA, Johnson EF, Leverson J, Liu X, Marcotte PA, Olson AM, Osterling DJ, Przytulinska M et al.: Synthesis and evaluation of a new generation of orally efficacious benzimidazole-based poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors as anticancer agents. J Med Chem 2009, 52:6803-6813. 42. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T: Specific killing of BRCA2-deficient tumors with inhibitors of poly(ADP-ribose) polymerase. Nature 2005, 434:913-917. 43. Farmer H, McCabe N, Lord CJ, Tutt ANJ, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C et al.: Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005, 434:917-921. 44. Evers B, Drost R, Schut E, de Bruin M, van der Burg E, Derksen PWB, Holstege H, Liu X, van Drunen E, Beverloo HB et al.: Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin. Clin Cancer Res 2008, 14:3916-3925. 45. Rottenberg S, Jaspers JE, Kersbergenh A, van der Burg E, Nygren AOH, Zander SAL, Derksen PWB, de Bruin M, Zevenhoven J, Lau A et al.: High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl Acad Sci U S A 2008, 105:17079-17084. This study describes a genetically engineered mouse model for BRCA1associated breast cancer and demonstrates that the PARP inhibitor AZD2281 has single agent activity in this disease as well as in combination with cisplatin or carboplatin. 46. Hay T, Matthews JR, Pietzka L, Lau A, Cranston A, Nygren AOH, Douglas-Jones A, Smith GCM, Martin NMB, O’Connor M, Clarke AR: Poly(ADP-ribose) polymerase-1 inhibitor treatment regresses autochthonous Brca2/p553-mutant mammary tumors in vivo and delays tumor relapse in combination with carboplatin. Cancer Res 2009, 69:3850-3855. 47. Daniel RA, Rozanska AL, Thomas HD, Mulligan EA, Drew Y, Caselbuono DJ, Hostomsky Z, Plummer ER, Boddy AV, Tweddle DA et al.: Inhibition of poly(ADP-ribose) polymerase-1 enhances temozolomide and topotecan activity against childhood neuroblastoma. Clin Cancer Res 2009, 15:1241-1249. 48. Kummar S, Kinders R, Gutierrez ME, Rubinstein L, Parchment RE, Phillips LR, Ji J, Monks A, Low JA, Chen A et al.: Phase 0 clinical www.sciencedirect.com
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trial of the poly (ADP-ribose) polymerase inhibitor ABT-888 in patients with advanced malignancies. J Clin Oncol 2009, 27:2705-2711. 49. Molina J, Erlichman C, Northfelt DW, Lensing J, Giranda V: Ongoing Phase 1 study of a novel PARP inhibitor, ABT-888 in combination with temozolomide: pharmacokinetics, safety and antitumor activity. Proc Am Assoc Cancer Res 2009, 50: Abstr 3602. 50. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O’Connor MJ et al.: Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009, 316:123-134. This phase 1 clinical trial in a study population enriched in carrier of a BRCA1 or BRCA2 mutation demonstrates the objective single agent activity of AZD2281 in these patients. 51. Kopetz S, Mita MM, Mok I, Sankhala KK, Moseley J, Sherman BM, Bradley CR, Tolcher AW: First in human phase I study of BSI201, a small molecule inhibitor of poly ADP-ribose polymerase (PARP) in subjects with advanced solid tumors. J Clin Oncol 2008, 26: abstr 3577. 52. O’Shaughnessy J, Osborne C, Pippen J, Yoffe M, Patt D, Monaghan G, Rocha C, Ossovskaya V, Sherman B, Bradley C: Efficacy of BSI-201, a poly (ADP-ribose) polymerase-1 (PARP1) inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): results of a randomized phase II trial. J Clin Oncol 2009, 27: abstr 3. 53. Bale AE: Hedgehog signaling and human disease. Annu Rev Genom Hum Genet 2002, 3:47-65. 54. Tiet TD, Hopyan S, Nadesan P, Gokgoz N, Poon R, Lin AC, Yan T, Andrulis IL, Alman BA, Wunder JS: Constitutive hedgehog signaling in chondrosarcoma up-regulates tumor cell proliferation. Am J Pathol 2006, 168:321-330. 55. Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter JA, Rubin LL, Wang FY: Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. Proc Natl Acad Sci U S A 2003, 100:4616-4621. 56. Robarge KD, Brunton SA, Castanedo GM, Cui Y, Dina MS, Goldsmith R, Gould SE, Guichert O, Gunzner JL, Halladay J et al.: GDC-0449 — a potent inhibitor of the hedgehog pathway. Biorg Med Chem Lett 2009, 19:5576-5581. 57. Tremblay MR, Lescarbeau A, Grogan MJ, Tan E, Lin G, Austad BC, Yu LC, Behnke ML, Nair SJ, Hagel M et al.: Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926). J Med Chem 2009, 52:4400-4418. 58. Morgan RT, Woods LK, Moore GE, Quinn LA, McGravan L, Gordon SG: Human cell line (COLO 357) of metastatic pancreatic adenocarcinoma. Int J Cancer 1980, 25:591-598. 59. Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ: In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia (NY) 1999, 1:50-62. 60. Embuscado EE, Laheru D, Ricci F, Yun KJ, de Boom Witzel S, Seigel A, Flicklinger K, Hidalgo M, Bova GS, IacobuzioDonahue CA: Immortalizing the complexity of cancer metastasis. Cancer Biol Ther 2005, 4:548-554. 61. Feldmann G, Dhara S, Fendrich V, Bedja D, Beaty R, Mullendore M, Karikari C, Alvarez H, Iacobuzio-Donahue C, Jimeno A et al.: Blockade of hedgehog signaling inhibits pancreatic cancer invasion and metastases: a new paradigm for combination therapy in solid cancers. Cancer Res 2007, 67:2187-2196. 62. Feldmann G, Fendrich V, McGovern K, Bedja D, Bisht S, Alvarez H, Koorstra JBM, Habbe N, Karikari C, Mullendore M et al.: An orally bioavailable small-molecule inhibitor of hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer. Mol Cancer Ther 2008, 7:2725-2735. 63. Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D et al.: Inhibition of hedgehog signaling enhances delivery of www.sciencedirect.com
chemotherapy in a mouse model of pancreatic cancer. Science 2009, 324:1457-1461. This study demonstrates the antiangiogenic effect of the hedgehog inhibitor IPI-926 in a genetically engineered mouse model of pancreatic ductal adenocarcinoma and shows that IPI-926 depletes tumor-associated stromal tissue thus allowing higher intratumoral concentration of gemcitabine. 64. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, Hruban RH, Rustgi AK, Chang S, Tuveson DA: Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 2005, 7:469-482. 65. Von Hoff DD, Korn R, Mousses S: Pancreatic cancer — could it be that simple? A different context of vulnerability. Cancer Cell 2009, 16:7-8. 66. Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR et al.: Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Eng J Med 2009, 36:1164-1172. This phase 1 trial of the hedgehog inhibitor GDC-0449 in patients with metastatic or locally advanced basal-cell carcinomas shows that 18/33 patients had an objective response to treatment with GDC-0449 as a single agent. Two patients had complete responses and 16 patients had partial responses. 67. Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, Holcomb T, Stinson J, Gould SE, Coleman B et al.: Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 2009, 316:1173-1178. 68. LoRusso PM, Rudin CM, Borad MJ, Vernillet L, Darbonne WC, Mackey H, DiMartino JF, de Sauvage F, Low JA, Von Hoff DD: A first-in human, first-in-class, phase I study of systemic hedgehog pathway antagonist, GD-0449, in patients with advanced solid tumors. J Clin Oncol 2008, 26: abstr 3516. 69. Bolden JE, Peart MJ, Johnstone RW: Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006, 5:769-784. 70. Minucci S, Pelicci PG: Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006, 6:38-51. 71. Schrump DS: Cytotoxicity mediated by histone deacetylase inhibitors in cancer cells: mechanisms and potential clinical implications. Clin Cancer Res 2009, 15:3947-3957. 72. Prince HM, Bishton MJ, Harrison SJ: Clinical studies of histone deacetylase inhibitors. Clin Cancer Res 2009, 15:3958-3969. 73. Bots M, Johnstone RW: Rational combinations using HDAC inhibitors. Clin Cancer Res 2009, 15:3970-3977. 74. Dai Y, Chen S, Kramer LB, Funk VL, Dent P, Grant S: Interactions between bortezomib and romidepsin and belinostat in chronic lymphocytic leukemia cells. Clin Cancer Res 2008, 14:549-558. 75. Shiozawa K, Nakanishi T, Tan M, Fang HB, Wang WC, Edelman MJ, Carlton D, Gojo I, Sausville EA, Ross DD: Preclinical studies of vorinostat (suberoylanilide hydroxamic acid) combined with cytosine arabinoside and etoposide for treatment of acute leukemias. Clin Cancer Res 2009, 15:16981707. This report elegantly demonstrates the application of analyses for additivity/synergy to the study of the HDAC inhibitor vorinostat in combination with standard drugs in cell culture. Two methods of data analysis are applied, sequencing of combinations is examined and mechanistic questions are addressed. The implications of the findings for clinical trial design are discussed. 76. Bhalla S, Balasubramanian S, David K, Sirisawad M, Buggy J, Mauro L, Prachand S, Miller R, Gordon LI, Evens AM: PCI-24781 induces caspase and reactive oxygen species-dependent apoptosis through NF-kB mechanisms and is synergistic with bortezomib in lymphoma cells. Clin Cancer Res 2009, 15:3354-3365. 77. Symanowski JT, Giraud Y, Dino P, Atadja P, Vogelzang N, Sharma S: Design and analysis methods for assessing drug synergy: illustration using histone deacetylase (HDAC) inhibitor LBH589 combined with CDDP in mesothelioma (meso) cell lines. J Clin Oncol 2007, 25(Suppl) abstr# 18108. Current Opinion in Pharmacology 2010, 10:397–404
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78. Symanowski J, Vogelzang N, Zawel L, Atadja P, Pass H, Sharma S: A histone deacetylase inhibitor LBH589 downregulates XIAP in mesothelioma cell lines which is likely responsible for increased apoptosis with TRAIL. J Thorac Oncol 2009, 4:149-160. This report describes experiments with the HDAC inhibitor LBH589 in combination with TRAIL in five mesothelioma cell lines and two normal cell types. The combination index method was used for data analysis of additivity/synergy. 79. Whitecross KF, Alsop AE, Cluse LA, Wiegmans A, Banks K-M, Coomans C, Peart MJ, Newbold A, Lindemann RK, Johnstone RW: Defining the target specificity of ABT-737 and synergistic antitumor activities in combination with histone deacetylase inhibitors. Blood 2009, 113:1982-1991. 80. Frew AJ, Lindemann RK, Martin BP, Clarke CJP, Sharkey J, Anthony DA, Banks K-M, Haynes NM, Gangatirkar P, Stanley K
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et al.: Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist. Proc Natl Acad Sci U S A 2008, 105:11317-11322. 81. Reynolds CP, Maurer BJ: Evaluating response to antineoplastic drug combinations in tissue culture models.Methods in Molecular Medicine: Chemosensitivity2005:. 110:173–183. 82. Carter CA, Chen C, Brink C, Vincent P, Maxuitenko YY, Gilbert KS, Waud WR, Zhang X: Sorafenib is efficacious and tolerated in combination with cytotoxic or cytostatic agents in preclinical models of human non-small cell lung carcinoma. Cancer Chemother Pharmacol 2007, 59:183-195. 83. Teicher BA, Chen V, Shih C, Menon K, Forler PA, Phares VG, Amsrud T: Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts. Clin Cancer Res 2000, 6:1016-1023.
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Combinations of PARP, hedgehog and HDAC inhibitors with standard drugs Beverly A Teicher Objective methods for data analysis for the assessment of additivity, synergy, and antagonism that can be applied rigorously to cell-based and in vivo preclinical data have been defined. The Combination Index method has been most widely applied. This brief report discusses poly(ADP-ribose) polymerase (PARP) inhibitors, hedgehog inhibitors, and histone deacetylase inhibitors as examples of experimental agents used in combinations where additivity and synergy have been assessed. PARP, hedgehog, and HDAC inhibitors have demonstrated single agent activity in a limited spectrum of malignancies. Expanded application of these compounds may come from combination regimens. Several PARP inhibitors appear to be additive to synergistic with standard agents in cell culture; however, no determinations for additivity/synergy have been applied with these compounds in vivo. Hedgehog inhibitors have demonstrated interesting activity in some preclinical models and have been studied in vivo with gemcitabine. However, no analyses for additivity were conducted in these studies. Numerous studies have demonstrated additivity/synergy with several HDAC inhibitors and standard anticancer drugs in cell culture. Clinical trials with combination regimens are on-going with compounds from each of these classes based primarily upon limited cell culture data. Thus, there is still a distance to go in making optimal use of preclinical models to aid in the selection of combination regimens worthy of clinical trial. Address Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701-9322, United States Corresponding author: Teicher, Beverly A ([email protected])
Current Opinion in Pharmacology 2010, 10:397–404 This review comes from a themed issue on Cancer Edited by Raymond Winquist and Diane Boucher Available online 22nd May 2010 1471-4892/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2010.04.014
One of the main tenets of cancer therapeutics is that combinations of anticancer agents with different targets or different mechanisms of action and varied normal tissue toxicities will produce better therapeutic outcomes. Frequently proving that combination regimens result in www.sciencedirect.com
better treatment outcomes preclinically and clinically is not easy. The premise of controlled randomized trials, a paradigm used automatically in preclinical studies and often required in clinical studies, is that there is uncertainty whether any of the alterative treatments may have more benefit. Indeed, very large studies analyzing outcomes from multiple clinical trials most often find no substantive differences in the treatment arms lending support to the continuing need for randomized trials [1]. Sometimes the benefit of combination regimens can only be discerned through meta-analyses of many clinical trials. Gemcitabine as a single agent compared with gemcitabine in combination regimens for the treatment of pancreatic cancer is an example where the benefit of combination regimens could only be confirmed after tens of trials and thousands of patients were accrued [2– 6]. In some cases, gemcitabine combination preclinical data were described where synergy did not occur and was not claimed but were later interpreted by others as demonstrating synergy [7,8]. The combination of gemcitabine and navelbine was assessed for additivity in mice bearing the Lewis lung carcinoma. Gemcitabine was well tolerated over the range from 40 mg/kg 3 to 80 mg/ kg 3 [9]. Navelbine was well tolerated at total doses of 10, 15 and 22.5 mg/kg. Isobologram methodology [10] was used to determine whether the combinations of gemcitabine and navelbine achieved additive antitumor activity. At gemcitabine doses of 40 and 60 mg/kg, the combination achieved additivity. At the highest gemcitabine dose, the combination produced less than additive tumor growth delay [9,11]. The resulting phase 1 clinical trial concluded that this drug combination has potential as a front-line or second-line regimen for advanced lung cancer; however, hematologic toxicities would require intermittent dosing. The promise is that increased understanding of malignant disease and normal tissue effects and application of objective data analysis methods will allow better selection of combinations in the preclinical setting that will translate into an improved rate of success in clinical trial. Human tumor xenografts have been the primary models for the demonstration of in vivo efficacy for new compounds and other anticancer therapeutics for more than 30 years [12–18]. Appealing properties of human tumor xenograft models are that the malignant cells are human and that most grow slowly enough in immunodeficient mice to allow a reasonable course of an experimental treatment over one to three weeks to be administered to the mouse. For most of the more than 30 years during Current Opinion in Pharmacology 2010, 10:397–404
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which the cancer research and drug discovery community has relied on human tumor xenografts, little has been known about the tumor lines used except that many were originated in the late 1970s and early 1980s from patients with a known malignancy [12,13]. However, in recent years many lines have been characterized for gene expression, mutations, amplifications and other molecular alterations. Thus, the tumor model can be matched with the experimental therapeutic with great accuracy and confidence. The hope is that this knowledge and personalization of therapy will carry into clinical application [14,19,20]. The best models and methods for data analysis for defining drug combinations with the most clinical potential are topics of on-going investigation [14,21–23]. Objective methods for the assessment of additivity, synergy, and antagonism include combination index, median effect, isobolograms, continuous measures, Bliss methodology, and varied response surface techniques have been defined [24–37]. These methods can be applied rigorously to cellbased and in vivo preclinical data. This brief report will discuss poly(ADP-ribose) polymerase (PARP) inhibitors, hedgehog inhibitors, and histone deacetylase inhibitors as examples of experimental agents used in combinations, some of which have been translated to the clinic.
PARP inhibitors PARP is a family of nuclear enzymes involved in the detection and repair of DNA damage. Eighteen putative PARP family members have been identified; however, only PARP-1 and PARP-2 contain a DNA-binding domain that facilitates localization to the site of DNA damage. In addition to DNA repair, the PARP enzymes are involved in the modulation of chromatin structure, transcription regulation, DNA telomere maintenance, mitotic control, cellular transport, and cell death [38]. PARP-1 recognizes single-strand breaks and acts in base excision repair (BER). PARP-1 and PARP-2 catalyze the transfer of ADP-ribose units from intracellular nicotinamide adenine dinucleotide (NAD+) as a key step in DNA repair. Several small molecule inhibitors of the PARP-1 activity have been developed as potential agents to use mainly in combination with anticancer therapies that cause damage to DNA [39,40,41]. Temozolomide has frequently been used to cause DNA damage to allow the activity of PARP inhibitors to be manifest in preclinical models. HCT116 cells exposed to the combination of temozolomide with ABT-888 (500 nM), a benzimidazole PARP inhibitor, require 17–24 hours to achieve maximal cytotoxicity. The cell killing correlates with the level of double-stranded DNA breaks. In synchronized cells, exposure to temozolomide along with ABT-888 during the S phase resulted in high levels of DNA doublestranded breaks, most likely from single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides that were converted into double-stranded Current Opinion in Pharmacology 2010, 10:397–404
breaks during DNA replication. Thus, exposure of S phase cells to temozolomide and ABT-888 leads to high levels of cytotoxicity [38]. In mice bearing B16F10 melanoma ABT-888 (25 mg/kg/day, po) inhibited poly(ADPribose) formation and enhanced tumor growth delay by temozolomide (50 mg/kg/day, po) from 5 days for temozolomide alone to 10 days for the combination. In rats bearing intracranial 9L glioma, the combination of temozolomide (17.5 mg/kg) and ABT-888 (25 mg/kg) was more effective in slowing tumor growth than either drug alone. However, no analysis was performed to determine whether the increases in cytotoxicity and tumor growth delay were additive, subadditive, or synergistic. A breakthrough in understanding the potential of PARP inhibition as a therapeutic approach in cancer came with the realization that cell lines deficient in BRCA1 and BRCA2, proteins involved in DNA homologous recombination repair are very sensitive to PARP-1 inhibitors. Exposure to PARP inhibitors produces cell death in BRCA mutant cells [42,43]. BRCA1 and BRCA2 wildtype alleles are frequently lost in tumors of heterozygous carriers. Carriers of mutations in BRCA1 and BRCA2 are at elevated risk for breast, ovary, prostate, and pancreas cancers. The PARP inhibitor, AZD2281 has single agent cytotoxicity toward BRCA1-deficient breast cancer cell lines with IC50s of 200–800 nM. Several mouse tumor cell lines have been developed that are BRCA2 deficient [44]. AZD2281 was tested in combination with these clonal lines alone and along with a variety of anticancer agents and g-irradiation. A variant of the Combination Index method was applied to assess the additivity/synergy of the resulting cytotoxicity of the combinations. The combination of AZD2281 with cisplatin provided evidence of synergistic or greater-than-additive cytotoxicity. In a genetically engineered mouse model of BRCA1-associated breast cancer, treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity. However, long-term treatment of these mice with AZD2281 did not prevent tumor development [45]. Combination of AZD2281 with cisplatin or carboplatin was more effective than the single agents suggesting that AZD2281 adds to the effect of DNAdamaging agents. In a mouse with conditionally deleted BRCA2 and p53 in mammary epithelium daily AZD2281 produced some tumor growth delay; however, the combination of carboplatin with AZD2281 had no advantage over carboplatin monotherapy [46]. The combination of temozolomide (50 mg/kg/day) and AZD2281 (10 mg/kg/ day) was more effective than single agent temozolomide in the SW620 colon carcinoma xenograft [38]. Another PARP inhibitor AG014699 was combined in cell culture and in vivo with topotecan and temozolomide. In NB1691 and SHSY5Y neuroblastoma xenografts, temozolomide produced tumor growth delay, which was increased by co-administration of AG014699, and resulted in complete and sustained tumor regression in 6 of 10 mice [47]. www.sciencedirect.com
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Based upon these and other data PARP inhibitors have entered clinical trials. The early clinical trials group at NCI conducted a first phase 0 clinical trial of ABT-888 in patients with advanced malignancies [48]. ABT-888 was administered as a single oral dose of 10, 25, or 50 mg to determine the dose range and time course over which ABT-888 inhibits PARP activity in tumor samples and peripheral blood mononuclear cells, and to evaluate ABT888 pharmacokinetics. Blood samples and tumor biopsies were obtained pre-drug and post-drug administration for the evaluation of PARP activity and pharmacokinetics. Using data from the phase 0 trial, a phase 1 dose escalation study of the combination of ABT-888 and temozolomide was conducted in patients with metastatic melanoma and with BRCA-related breast and ovarian cancer. Patients were treated with escalating ABT-888 doses at a fixed temozolomide dose [49]. One patient with hepatocellular cancer had a partial response. Three patients had stable disease (1 neuroendocrine for 6 cycles and 2 patients with melanoma). The combination of ABT-888 and temozolomide was well tolerated. In a phase 1 trial of AZD2281 (olaparib) 60 patients (22 carriers of a BRCA1 or BRCA2 mutation and 1 with a strong family history of BRCAassociated cancer but declined to undergo mutational testing) were treated [50]. The olaparib dose was increased from 10 mg daily to 600 mg twice daily continuously. This led to enrollment of a cohort of BRCA1 or BRCA2 mutation carriers who received olaparib at a dose of 200 mg twice daily. Objective antitumor activity was reported only in mutation carriers, all of whom had ovarian, breast, or prostate cancer and had received multiple treatment regimens. Another PARP inhibitor, BSI-201 has completed phase 1 clinical trial and is undergoing a phase 2 randomized trial in triple negative breast cancer patients [51,52]. The early results indicated that patients treated with BSI-201 in combination with gemcitabine and carboplatin had an improved therapeutic outcome compared with patients treated with gemcitabine and carboplatin only.
Hedgehog inhibitors Hedgehog is a secreted molecule that influences the differentiation of a variety of tissues during development and plays a pivotal role in tissue homeostasis in a variety of species. Hedgehog, its receptor patched, and many downstream members of the hedgehog signal transduction pathway were first elucidated as factors involved in fruit fly embryo development [53]. The hedgehog protein family includes sonic, Indian and desert hedgehogs. Patched-1 is the receptor for all of the hedgehog proteins. In the absence of a hedgehog, patched-1 inhibits smoothened, a G-protein-coupled receptor. When a hedgehog binds to patched-1, smoothened is activated and initiates a signaling cascade that results in the activation of a transcription factor, Gli-1. In humans, aberrant activation of the hedgehog pathway is associated with basal-cell carcinoma, chondrosarcoma, and medulloblastoma www.sciencedirect.com
[54,55]. Cyclopamine, a plant alkaloid, is a natural product antagonist of the hedgehog pathway that has been used to validate the role of the hedgehog pathway in disease. Several synthetic small molecule inhibitors of the hedgehog pathway have been developed [56,57]. IPI-926 is a seven-membered D-ring semisynthetic analog of cyclopamine that is a potent and orally active hedgehog pathway antagonist [57]. GDC-0449 is a novel fully synthetic functionalized 2-pyridyl amide inhibitor of the hedgehog pathway [56]. The COLO 357 cell line was developed in the late 1970s from a metastatic pancreatic adenocarcinoma [58]. In 1999, Bruns et al. selected sublines of COLO 357 by repeated injects into the spleen or pancreas of nude mice and selection of tumor nodules in the liver [59]. After repeating the cycle several times, two sublines designated L3.6sl (spleen to liver) and L3.6pl (pancreas to liver) were characterized. In a more recent effort, Embuscado et al. have developed new pancreatic adenocarcinoma cell lines from rapid autopsy specimens [60]. Using the L3.6pl line and a previously undescribed line designated E3LZ10.7 from a rapid autopsy isolate, a role for the hedgehog pathway in pancreatic cancer cell lines was reported [61,62]. Hedgehog inhibition with cyclopamine or IPI269609 resulted in inhibition of epithelial-to-mesenchymal transition and a reduction in invasion through Matrigel by cells in culture. In an orthotopic xenograft model, treatment with cyclopamine or IPI-260609 decreased metastasis. However, treatment with the combination of gemcitabine and cyclopamine or IPI-269609 did not alter the response of subcutaneous E3LZ10.7 tumors from that obtained with gemcitabine alone [61,62]. A genetically engineered mouse in which endogenous alleles of KrasG12D and Trp53R172H were conditionally activated in the pancreas of LSL-KrasG12D/+;LSL Trp53R172H/+;Pdx-1-Cre triple mutant mice was used to assess treatment with the combination of gemcitabine and IPI-926 [63]. These mice have a median survival time of five months [64]. In this model, treatment with IPI-926 for 10 days increased the microvessel density of the tumors allowing improved distribution of gemcitabine into the nodules. The combination of gemcitabine and IPI-926 resulted in eight to nine days increased survival compared with gemcitabine alone [63]. This study points out the importance of tumor stroma in therapy [65]. Clinical trials are on-going with GDC-0449 and IPI-926. In a phase 1 clinical trial, the safety and pharmacokinetics of GDC-0449 and responses of metastatic or locally advanced basal-cell carcinoma were determined. Thirty-three patients with metastatic or locally advanced basal-cell carcinoma received oral GDC-0449 at one of three doses: 17 patients received 150 mg/day, 15 patients received 270 mg/day, and 1 patient received 540 mg/day [66]. The median treatment duration was 9.8 months. Current Opinion in Pharmacology 2010, 10:397–404
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Of the 33 patients, 18 had an objective response to GDC0449, according to assessment on imaging (7 patients), physical examination (10 patients), or both (1 patient). Two of the responding patients had a complete response and 16 had a partial response. The other 15 patients had either stable disease (11 patients) or progressive disease (4 patients). In a case report, a 26-year-old man with metastatic medulloblastoma that was refractory to multiple therapies was treated with GDC-0449. The treatment resulted in rapid (although transient) tumor regression and reduction of symptoms [67]. Molecular analyses of tumor specimens obtained before treatment suggested hedgehog pathway activation, with loss of heterozygosity and somatic mutation of the gene encoding patched-1.
unknown whether targeting certain histone deacetylase isotypes selectively will reduce toxicity and achieve responses equivalent to broad spectrum inhibitors. In the clinic as single agents, histone deacetylase inhibitors have been effective against T-cell lymphomas. However, as single agents, histone deacetylase inhibitors have had little activity against solid tumors. A potential synergy between histone deacetylase inhibitors and chemotherapeutic or biologically active anticancer compounds can be demonstrated in cell culture experiments suggesting that combination strategies will be a major focus in future clinical studies although the molecular mechanisms that account for these greater-than-additive effects remain to be elucidated [73].
In another phase 1 clinical trial, GDC-0449 was administered orally as a single dose on day 1 with a one-week PK break, and then once daily continuously (cycle 1 = 35 days) to refractory solid tumor patients at 3 dose levels. Surrogate tissues were assessed for the expression of hedgehog downstream target gene Gli1. Of the 19 patients enrolled in the trial, 1 partial response (150 mg) and 2 stable disease patients (270 mg) in basal-cell carcinoma and adenocystic carcinoma were seen. Continuous oral dosing at 150 mg/day demonstrated safe, effective concentrations, pharmacodynamic activity, an objective response and clinical benefit, and was selected for phase 2 studies [68]. GDC-0449 is currently in multiple phase 2 clinical trials alone and in combination with varied chemotherapeutic agents.
The proteasome inhibitor bortezomib and the histone deacetylase inhibitors romidepsin or belinostat were studied in combination in cell culture in chronic lymphocytic leukemia (CLL) cells [74]. Primary and cultured CLL cells were exposed to the agents alone or in combination and cell death was estimated by 7-aminoactinomycin D flow cytometry. Exposure to romidepsin or belinostat in combination with bortezomib produced synergistic cell death in primary and cultured CLL cells as determined by combination index methodology. Cell death likely occurred through mechanisms involving NF-kB inactivation and perturbations in proapoptotic and antiapoptotic proteins. Human HL-60 and K562 leukemia cells in culture were exposed to cytosine arabinoside [1-b-Darabinofuranosylcytosine (ara-C)] and/or etoposide in combination with vorinostat and cell survival was estimated using a colorimetric or fluorescent dye endpoint [75]. Drug combination effects were analyzed by the combination index method and by a novel statistical method. Using the two independent data analysis methods, cytotoxic antagonism resulted when vorinostat was combined simultaneously with ara-C; however, when vorinostat exposure was first followed by a drug-free interval before ara-C exposure, the combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became greater when etoposide exposure occurred after vorinostat exposure. The cytotoxicity of the histone deacetylase inhibitor PCI-24781, a phenyl hydroxamic acid compound, alone and in combination with bortezomib was assessed in the L428 Hodgkin lymphoma cell line and the non-Hodgkin lymphoma cell lines Ramos (Burkitt lymphoma), HF1 (follicular lymphoma), and SUDHL4 (large B-cell lymphoma) in cell culture [76]. Cell viability was estimated morphologically after staining with trypan blue and by the analysis of apoptosis using flow cytometry after staining with Annexin V-FITC and propidium iodide. When the data were analyzed by the combination index method, combined PCI-24781 and bortezomib exposure resulted in synergistic apoptosis in all non-Hodgkin lymphoma lines and additivity in the Hodgkin lymphoma line.
Histone deacetylase inhibitors Histone deacetylases are a family of 4 classes of enzymes with 18 known members that catalyze the removal of acetyl groups, from histones leading to chromatin condensation and transcriptional repression [69]. Inhibition of histone deacetylases results in hyper-acetylated histones, cell cycle arrest at the G1/S boundary, and cell death, most frequently by apoptosis. Non-histone proteins (tumor suppressor p53) and several cytoplasmic proteins are also regulated by acetylation status [70,71]. Pan-histone deacetylase inhibitors in clinical trials include vorinostat, panobinostat, romidepsin, and belinostat. Isotype-selective histone deacetylase inhibitors in clinical trials include MGCD0103 and entinostat [72]. Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a hydroxamic acid derivative that inhibits both class I and II histone deacetylases. Vorinostat was approved by FDA for the treatment of relapsed and refractory cutaneous Tcell lymphoma. Panobinostat (LBH589) and belinostat (PXD101) are also hydroxamic acid derivatives. Romidepsin (FK228) is a prodrug that in cells is reduced to an active species that interacts with class I and II histone deacetylases. MGCD0103, an aminophenylbenzamide, inhibits class I and IV histone deacetylases selectively. Entinostat (MS-275) is a benzamide with predominant class I histone deacetylase inhibitory activity. It is Current Opinion in Pharmacology 2010, 10:397–404
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A combination of TRAIL and the histone deacetylase inhibitor LBH589 was studied in mesothelioma cell lines in culture [77,78]. Five mesothelioma cell lines and two normal cell types were tested for growth inhibition using a colorimetric assay and apoptosis in the presence of LBH589, TRAIL and the combination. Median effect and combination index methodology were used to determine whether the combinations resulted in additive or greater-than-additive growth inhibition. In mesothelioma cell lines, the combination of LBH589 and TRAIL produced synergistic cell growth inhibition and apoptosis and an additive effect in normal cells. Several investigations of histone deacetylase inhibitors in combination with other agents have been reported in cell lines and tumor models and have claimed synergy without appropriate data analysis [79,80]. Vorinastat is currently in phase 1, 2 and 3 trials in combination with bortezomib and in phase 1 and 2 clinical trials with azacitidine or decitabine in hematologic malignancies and solid tumors [53]. Entinostat is in phase 1 and 2 clinical trials with azacitidine in leukemias and lung cancer. Panbinostat is in a phase 1 clinical trial with bortezomib in multiple myeloma and with decitabine in leukemia. Romidepsin, belinostat, and MGCD0103 are in early combination clinical trials with bortzomib, azacitidine, decitabine, and azacitidine with all-transretinoic acid.
tivity in tumor response can be achieved in a relevant tumor in mice without decrease in therapeutic index, the combination regimen may be worthy of clinical trial.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of outstanding interest
References 1.
Soares HP, Kumar A, Daniels S, Swann S, Cantor A, Hozo I, Clark M, Serdarevic F, Gwede C, Trotti A, Djulbegovic B: Evaluation of new treatments in radiation oncology: Are they better than standard treatments? J Am Med Assoc 2005, 293:970-978.
2.
Di Maio M, Chiodini P, Georgoulias V, Hatzidaki D, Takeda K, Wachters FM, Gebbia V, Smit EF, Morabito A, Gallo C et al.: Metaanalysis of single-agent chemotherapy compared with combination chemotherapy as second-line treatment of advanced non-small cell lung cancer. J Clin Oncol 2009, 27:1836-1843.
3.
Sultana A, Ghaneh P, Cunningham D, Starling N, Neoptolemos JP, Smith CT: Gemcitabine based combination chemotherapy in advanced pancreatic cancer-indirect comparison. BMC Cancer 2008, 8:192-196.
4.
Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, Natale RB, Schiller JH, von Pawel J, Pluzanska A et al.: Gefitinib in combination with gemcitabine and cisplatin in advanced non-small cell lung cancer: a phase III trial-INTACT 1. J Clin Oncol 2004, 22:777-784.
5.
Heinemann V, Boeck S, Hinke A, Labianca R, Louvet C: Metaanalysis of randomized trials: evaluation of benefit from gemcitabine-based combination chemotherapy applied in advanced pancreatic cancer. BMC Cancer 2008, 8:82-92.
6.
Li J, Podoltsev N, Saif MW: Management of advanced pancreatic cancer. Expert Rev Clin Pharmacol 2009, 2:527-541.
7.
Ng SSW, Tsao MS, Nicklee T, Hedley DW: Effects of the epidermal growth factor receptor inhibitor OSI-774, tarceva, on downstream signaling pathways and apoptosis in human pancreatic adenocarcinoma. Mol Cancer Ther 2002, 1:777-783.
8.
Bayraktar S, Bayraktar UD, Rocha-Lima CM: Recent developments in palliative chemotherapy for locally advanced and metastatic pancreas cancer. World J Gastroenterol 2010, 16:673-682.
9.
Herbst RS, Lynch C, Vasconcelles M, Teicher BA, Strauss G, Elias A, Anderson I, Zacarola P, Dang NH, Leong T et al.: Gemcitabine and vinorelbine in patients with advanced lung cancer: preclinical studies and report of a phase I trial. Cancer Chemother Pharmacol 2001, 48:151-159.
Conclusion These recent examples of the translation of preclinical combination studies to clinical trial indicate that there is still a distance to go in making optimal use of preclinical models to aid in the selection of combinations to apply in clinical trial. The few cell culture studies cited herein have made use of the combination index method to assess the additivity/synergy of the combinations; however, the limitations of the well-controlled cell culture assay to mimic the dynamics of the in vivo situation were not acknowledged in most studies [81]. The analysis of the additivity/synergy of same combinations in bone marrow CFU-GM or other stem cell culture assays is under utilized as a method to estimate whether the combination will have an improved therapeutic index or not. In fact, there were no examples of in vivo studies demonstrating additivity or synergy with the drug combinations currently in clinical trial. Mice are not patients, however, experiments in which tumor volumes and body weight measurements are made can often allow determination of increased efficacy, tumor response without increased toxicity, or determination of increased tumor response and increased toxicity (body weight loss and hematology). Such preclinical in vivo experiments with combinations of kinase inhibitors and other combinations showed increased tumor response, the cost being increased body weight loss [82,83]. In vivo synergy is a very rare result and subadditivity of combinations is quite frequent. If addiwww.sciencedirect.com
10. Teicher BA, Herman TS, Holden SA, Eder JP: Chemotherapeutic potentiation through interaction at the level of DNA. In Synergism and Antagonism in Chemotherapy. Edited by Chou TC, Rideout DC. Orlando, FL: Academic Press; 1991:541-583. 11. Teicher BA, Frei E III: Laboratory models to evaluate new agents for the systemic treatment of lung cancer. In Multimodality Treatment of Lung Cancer. Edited by Skarin AT. New York, NY: Marcel Dekker Inc; 2000:301-336. 12. Teicher BA: Tumor models for efficacy determination. Mol Cancer Ther 2006, 5:2435-2443. 13. Teicher BA: Human tumor xenografts and mouse models of human tumors: re-discovering the models. Expert Opin Drug Discov 2009, 4:1-11. 14. Sausville EA, Burger AM: Contributions of human tumor xenografts to anticancer drug development. Cancer Res 2006, 66:3351-3354. Current Opinion in Pharmacology 2010, 10:397–404
402 Cancer
15. Garber K: Realistic rodents? Debate grows over new mouse models of cancer. J Natl Cancer Inst 2006, 98:1176-1178.
diamminedichloroplatinum and 1-b-Darabinofuransylcytosine. Cancer Res 1992, 52:4558-4565.
16. Suggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clin Cancer Res 2005, 11:971-981.
35. Chou TC: Theoretical basis, experimental design and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006, 58:621-681.
17. Fiebig HH, Maier A, Burger AM: Clonogenic assay with established human tumor xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery. Eur J Cancer 2004, 40:802-820.
36. Steel GG, Peckham MJ: Exploitable mechanisms in combined radiotherapy chemotherapy: the concept of additivity. Int J Radiat Oncol Biol Phys 1979, 5:85-91.
18. Kerbel RS: Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans. Cancer Biol Ther 2003, 2(Suppl 1):S134-S139. 19. Korrat A, Greiner T, Maurer M, Metz T, Fiebig HH: Gene signaturebased prediction of tumor response to cyclophosphamide. Cancer Genom Proteom 2007, 4:187-196. 20. Fiebig HH, Schuler J, Bausch N, Hofmann M, Metz T, Korrat A: Gene signature developed from patient tumor explants grown in nude mice to predict tumor response to 11 cytotoxic drugs. Cancer Genom Proteom 2007, 4:197-210. 21. Lee EM, Bachmann PS, Lock RB: Xenograft models for the preclinical evaluation of new therapies in acute leukemia. Leukemia Lymphoma 2007, 48:659-668. 22. Sausville EA, Elsayed Y, Monga M, Kim G: Signal transductiondirected cancer treatments. Annu Rev Pharmacol Toxicol 2003, 43:199-231. 23. Gitler MS, Monks A, Sausville EA: Preclinical models for defining efficacy of drug combinations: mapping the road to the clinic. Mol Cancer Ther 2003, 2:929-932. 24. Buck E, Eyzaguirre A, Brown E, Petti F, McCormack S, Haley JD, Iwata KK, Gibson NW, Griffin G: Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in nonsmall-cell lung, pancreatic, colon and breast tumors. Mol Cancer Ther 2006, 5:2676-2684. 25. Duska LR, Hamblin MR, Miller JL, Hasan T: Combination photoimmuno-therapy and cisplatin: effects on human ovarian cancer ex vivo. J Natl Cancer Inst 1999, 91:1557-1563. 26. Borisy AA, Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER, Serbedzija G, Zimmermann GR, Foley MA, Stockwell BR, Keith CT: Systematic discovery of multicomponent therapeutics. Proc Natl Acad Sci U S A 2003, 100:7977-7982. 27. Tan M, Fang HB, Tian GL, Houghton PJ: Experimental design and sample size determination for testing synergism in drug combination studies based on uniform measures. Stats Med 2003, 22:2091-2100. 28. Grabovsky Y, Tallarida RJ: Isobolographic analysis for combinations of a full and partial agonist: curved isoboles. J Pharmacol Exp Ther 2004, 310:981-986. 29. Zhao L, Wientjes MG, Au JLS: Evaluation of combinations chemotherapy: integration of nonlinear regression, curve shift, isobologram and combination index analyses. Clin Cancer Res 2004, 10:7994-8004. 30. Kong M, Lee JJ: A generalized response surface model with varying relative potency for assessing drug interaction. Biometrics 2006, 62:986-995. 31. Zhao L, Au JLS, Wientjes MG: Comparison of methods for evaluating drug-drug interaction. Front Biosci 2010, 2:241-249. 32. Levasseur LM, Delon A, Greco WR, Faury P, Bouquet S, Couet W: Development of a new quantitative approach for the isobolographic assessment of the convulsant interaction between perfloxacin and theophylline in rats. Pharm Res 1998, 15:1069-1076. 33. Greco WR, Faessel H, Levasseur L: The search for cytotoxic synergy between anticancer agents: a case of Dorothy and the ruby slippers? J Natl Cancer Inst 1996, 88:699-700. 34. Berenbaum MC: Correspondence re: WR Greco et al., application of a new approach for the quantitation of drug synergism to the combination of cisCurrent Opinion in Pharmacology 2010, 10:397–404
37. Berenbaum MC: Synergy, additivism and antagonism in immunosuppression. Clin Exp Immunol 1977, 28:1-18. 38. Liu X, Shi Y, Guan R, Donawho C, Luo Y, Palma J, Zhu G, Johnson EF, Rodriguez LE, Ghoreishi-Haack N et al.: Potentiation of temozolomide cytotoxicity by poly(ADP)ribose polymerase inhibitor ABT-888 requires a conversion of single-stranded DNA damage to double-stranded DNA breaks. Mol Cancer Res 2008, 6:1621-1629. 39. Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, Cranston A, Dillon KJ, Drzewiecki J, Garman S, Gomez S et al.: 4[3-(4-Cyclopropoanecarbonylpiperazine-1-carbonyl)-4fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem 2008, 51:6581-6591. This study describes the discovery of the PARP inhibitor ABT-888 along with testing in enzyme activity assays, cell culture and in vivo models as a single agent and in combination with temozolomide is described. 40. Penning TD, Zhu GD, Gandhi VB, Gong JC, Liu XS, Shi Y, Klinghofer V, Johnson EF, Bouska JJ, Osterling DJ et al.: Discovery of the poly(ADP-ribose) polymerase (PARP) inhibitor 2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4carboxamide (ABT-888) for the treatment of cancer. J Med Chem 2009, 52:514-523. 41. Tong Y, Bouska JJ, Ellis PA, Johnson EF, Leverson J, Liu X, Marcotte PA, Olson AM, Osterling DJ, Przytulinska M et al.: Synthesis and evaluation of a new generation of orally efficacious benzimidazole-based poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors as anticancer agents. J Med Chem 2009, 52:6803-6813. 42. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T: Specific killing of BRCA2-deficient tumors with inhibitors of poly(ADP-ribose) polymerase. Nature 2005, 434:913-917. 43. Farmer H, McCabe N, Lord CJ, Tutt ANJ, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C et al.: Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005, 434:917-921. 44. Evers B, Drost R, Schut E, de Bruin M, van der Burg E, Derksen PWB, Holstege H, Liu X, van Drunen E, Beverloo HB et al.: Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin. Clin Cancer Res 2008, 14:3916-3925. 45. Rottenberg S, Jaspers JE, Kersbergenh A, van der Burg E, Nygren AOH, Zander SAL, Derksen PWB, de Bruin M, Zevenhoven J, Lau A et al.: High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl Acad Sci U S A 2008, 105:17079-17084. This study describes a genetically engineered mouse model for BRCA1associated breast cancer and demonstrates that the PARP inhibitor AZD2281 has single agent activity in this disease as well as in combination with cisplatin or carboplatin. 46. Hay T, Matthews JR, Pietzka L, Lau A, Cranston A, Nygren AOH, Douglas-Jones A, Smith GCM, Martin NMB, O’Connor M, Clarke AR: Poly(ADP-ribose) polymerase-1 inhibitor treatment regresses autochthonous Brca2/p553-mutant mammary tumors in vivo and delays tumor relapse in combination with carboplatin. Cancer Res 2009, 69:3850-3855. 47. Daniel RA, Rozanska AL, Thomas HD, Mulligan EA, Drew Y, Caselbuono DJ, Hostomsky Z, Plummer ER, Boddy AV, Tweddle DA et al.: Inhibition of poly(ADP-ribose) polymerase-1 enhances temozolomide and topotecan activity against childhood neuroblastoma. Clin Cancer Res 2009, 15:1241-1249. 48. Kummar S, Kinders R, Gutierrez ME, Rubinstein L, Parchment RE, Phillips LR, Ji J, Monks A, Low JA, Chen A et al.: Phase 0 clinical www.sciencedirect.com
PARP, hedgehog and HDAC inhibitors with standard drugs Teicher 403
trial of the poly (ADP-ribose) polymerase inhibitor ABT-888 in patients with advanced malignancies. J Clin Oncol 2009, 27:2705-2711. 49. Molina J, Erlichman C, Northfelt DW, Lensing J, Giranda V: Ongoing Phase 1 study of a novel PARP inhibitor, ABT-888 in combination with temozolomide: pharmacokinetics, safety and antitumor activity. Proc Am Assoc Cancer Res 2009, 50: Abstr 3602. 50. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O’Connor MJ et al.: Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009, 316:123-134. This phase 1 clinical trial in a study population enriched in carrier of a BRCA1 or BRCA2 mutation demonstrates the objective single agent activity of AZD2281 in these patients. 51. Kopetz S, Mita MM, Mok I, Sankhala KK, Moseley J, Sherman BM, Bradley CR, Tolcher AW: First in human phase I study of BSI201, a small molecule inhibitor of poly ADP-ribose polymerase (PARP) in subjects with advanced solid tumors. J Clin Oncol 2008, 26: abstr 3577. 52. O’Shaughnessy J, Osborne C, Pippen J, Yoffe M, Patt D, Monaghan G, Rocha C, Ossovskaya V, Sherman B, Bradley C: Efficacy of BSI-201, a poly (ADP-ribose) polymerase-1 (PARP1) inhibitor, in combination with gemcitabine/carboplatin (G/C) in patients with metastatic triple-negative breast cancer (TNBC): results of a randomized phase II trial. J Clin Oncol 2009, 27: abstr 3. 53. Bale AE: Hedgehog signaling and human disease. Annu Rev Genom Hum Genet 2002, 3:47-65. 54. Tiet TD, Hopyan S, Nadesan P, Gokgoz N, Poon R, Lin AC, Yan T, Andrulis IL, Alman BA, Wunder JS: Constitutive hedgehog signaling in chondrosarcoma up-regulates tumor cell proliferation. Am J Pathol 2006, 168:321-330. 55. Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter JA, Rubin LL, Wang FY: Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. Proc Natl Acad Sci U S A 2003, 100:4616-4621. 56. Robarge KD, Brunton SA, Castanedo GM, Cui Y, Dina MS, Goldsmith R, Gould SE, Guichert O, Gunzner JL, Halladay J et al.: GDC-0449 — a potent inhibitor of the hedgehog pathway. Biorg Med Chem Lett 2009, 19:5576-5581. 57. Tremblay MR, Lescarbeau A, Grogan MJ, Tan E, Lin G, Austad BC, Yu LC, Behnke ML, Nair SJ, Hagel M et al.: Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926). J Med Chem 2009, 52:4400-4418. 58. Morgan RT, Woods LK, Moore GE, Quinn LA, McGravan L, Gordon SG: Human cell line (COLO 357) of metastatic pancreatic adenocarcinoma. Int J Cancer 1980, 25:591-598. 59. Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ: In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia (NY) 1999, 1:50-62. 60. Embuscado EE, Laheru D, Ricci F, Yun KJ, de Boom Witzel S, Seigel A, Flicklinger K, Hidalgo M, Bova GS, IacobuzioDonahue CA: Immortalizing the complexity of cancer metastasis. Cancer Biol Ther 2005, 4:548-554. 61. Feldmann G, Dhara S, Fendrich V, Bedja D, Beaty R, Mullendore M, Karikari C, Alvarez H, Iacobuzio-Donahue C, Jimeno A et al.: Blockade of hedgehog signaling inhibits pancreatic cancer invasion and metastases: a new paradigm for combination therapy in solid cancers. Cancer Res 2007, 67:2187-2196. 62. Feldmann G, Fendrich V, McGovern K, Bedja D, Bisht S, Alvarez H, Koorstra JBM, Habbe N, Karikari C, Mullendore M et al.: An orally bioavailable small-molecule inhibitor of hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer. Mol Cancer Ther 2008, 7:2725-2735. 63. Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D et al.: Inhibition of hedgehog signaling enhances delivery of www.sciencedirect.com
chemotherapy in a mouse model of pancreatic cancer. Science 2009, 324:1457-1461. This study demonstrates the antiangiogenic effect of the hedgehog inhibitor IPI-926 in a genetically engineered mouse model of pancreatic ductal adenocarcinoma and shows that IPI-926 depletes tumor-associated stromal tissue thus allowing higher intratumoral concentration of gemcitabine. 64. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, Hruban RH, Rustgi AK, Chang S, Tuveson DA: Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 2005, 7:469-482. 65. Von Hoff DD, Korn R, Mousses S: Pancreatic cancer — could it be that simple? A different context of vulnerability. Cancer Cell 2009, 16:7-8. 66. Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR et al.: Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Eng J Med 2009, 36:1164-1172. This phase 1 trial of the hedgehog inhibitor GDC-0449 in patients with metastatic or locally advanced basal-cell carcinomas shows that 18/33 patients had an objective response to treatment with GDC-0449 as a single agent. Two patients had complete responses and 16 patients had partial responses. 67. Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, Holcomb T, Stinson J, Gould SE, Coleman B et al.: Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 2009, 316:1173-1178. 68. LoRusso PM, Rudin CM, Borad MJ, Vernillet L, Darbonne WC, Mackey H, DiMartino JF, de Sauvage F, Low JA, Von Hoff DD: A first-in human, first-in-class, phase I study of systemic hedgehog pathway antagonist, GD-0449, in patients with advanced solid tumors. J Clin Oncol 2008, 26: abstr 3516. 69. Bolden JE, Peart MJ, Johnstone RW: Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006, 5:769-784. 70. Minucci S, Pelicci PG: Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006, 6:38-51. 71. Schrump DS: Cytotoxicity mediated by histone deacetylase inhibitors in cancer cells: mechanisms and potential clinical implications. Clin Cancer Res 2009, 15:3947-3957. 72. Prince HM, Bishton MJ, Harrison SJ: Clinical studies of histone deacetylase inhibitors. Clin Cancer Res 2009, 15:3958-3969. 73. Bots M, Johnstone RW: Rational combinations using HDAC inhibitors. Clin Cancer Res 2009, 15:3970-3977. 74. Dai Y, Chen S, Kramer LB, Funk VL, Dent P, Grant S: Interactions between bortezomib and romidepsin and belinostat in chronic lymphocytic leukemia cells. Clin Cancer Res 2008, 14:549-558. 75. Shiozawa K, Nakanishi T, Tan M, Fang HB, Wang WC, Edelman MJ, Carlton D, Gojo I, Sausville EA, Ross DD: Preclinical studies of vorinostat (suberoylanilide hydroxamic acid) combined with cytosine arabinoside and etoposide for treatment of acute leukemias. Clin Cancer Res 2009, 15:16981707. This report elegantly demonstrates the application of analyses for additivity/synergy to the study of the HDAC inhibitor vorinostat in combination with standard drugs in cell culture. Two methods of data analysis are applied, sequencing of combinations is examined and mechanistic questions are addressed. The implications of the findings for clinical trial design are discussed. 76. Bhalla S, Balasubramanian S, David K, Sirisawad M, Buggy J, Mauro L, Prachand S, Miller R, Gordon LI, Evens AM: PCI-24781 induces caspase and reactive oxygen species-dependent apoptosis through NF-kB mechanisms and is synergistic with bortezomib in lymphoma cells. Clin Cancer Res 2009, 15:3354-3365. 77. Symanowski JT, Giraud Y, Dino P, Atadja P, Vogelzang N, Sharma S: Design and analysis methods for assessing drug synergy: illustration using histone deacetylase (HDAC) inhibitor LBH589 combined with CDDP in mesothelioma (meso) cell lines. J Clin Oncol 2007, 25(Suppl) abstr# 18108. Current Opinion in Pharmacology 2010, 10:397–404
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78. Symanowski J, Vogelzang N, Zawel L, Atadja P, Pass H, Sharma S: A histone deacetylase inhibitor LBH589 downregulates XIAP in mesothelioma cell lines which is likely responsible for increased apoptosis with TRAIL. J Thorac Oncol 2009, 4:149-160. This report describes experiments with the HDAC inhibitor LBH589 in combination with TRAIL in five mesothelioma cell lines and two normal cell types. The combination index method was used for data analysis of additivity/synergy. 79. Whitecross KF, Alsop AE, Cluse LA, Wiegmans A, Banks K-M, Coomans C, Peart MJ, Newbold A, Lindemann RK, Johnstone RW: Defining the target specificity of ABT-737 and synergistic antitumor activities in combination with histone deacetylase inhibitors. Blood 2009, 113:1982-1991. 80. Frew AJ, Lindemann RK, Martin BP, Clarke CJP, Sharkey J, Anthony DA, Banks K-M, Haynes NM, Gangatirkar P, Stanley K
Current Opinion in Pharmacology 2010, 10:397–404
et al.: Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist. Proc Natl Acad Sci U S A 2008, 105:11317-11322. 81. Reynolds CP, Maurer BJ: Evaluating response to antineoplastic drug combinations in tissue culture models.Methods in Molecular Medicine: Chemosensitivity2005:. 110:173–183. 82. Carter CA, Chen C, Brink C, Vincent P, Maxuitenko YY, Gilbert KS, Waud WR, Zhang X: Sorafenib is efficacious and tolerated in combination with cytotoxic or cytostatic agents in preclinical models of human non-small cell lung carcinoma. Cancer Chemother Pharmacol 2007, 59:183-195. 83. Teicher BA, Chen V, Shih C, Menon K, Forler PA, Phares VG, Amsrud T: Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts. Clin Cancer Res 2000, 6:1016-1023.
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