- Email: [email protected]
Yet Another Nucleoside Analog for Pancreatic Cancer
Editorials continued
21. Gangula PR, Mukhopadhyay S, Pasricha PJ, et al. Sepiapterin reverses the changes in gastric nNOS dimerization and function in diabetic gastroparesis. Neurogastroenterol Motil 2010;22: 1325–13331. 22. Chandrasekharan B, Anitha M, Blatt R, et al. Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress. Neurogastroenterol Motil 2010 Oct 12 [Epub ahead of print]. 23. Vanderwinden JM, Mailleux P, Schiffmann SN, et al. Nitric oxide synthase activity in infantile pyloric stenosis. N Engl J Med 1992; 327:511–515. 24. Tomita R, Munakata K, Kurosu Y, et al. A role of nitric oxide in Hirschsprung’s disease. J Pediatr Surg 1995;30:437– 440. 25. Tomita R, Fujisaki S, Tanjoh K, et al. Role of nitric oxide in the internal anal sphincter of Hirschsprung’s disease. World J Surg 2002;26:1493–1498.
Reprint requests Address requests for reprints to: James J. Galligan, PhD, Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan 48824. e-mail: [email protected]; fax: 517-353-8915. Conflicts of interest The authors disclose no conflicts. Funding Supported by DK57039 (NIDDK). © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.12.016
Yet Another Nucleoside Analog for Pancreatic Cancer
See “Acycloguanosyl 5=-thymidyltriphosphate, a thymidine analogue prodrug activated by telomerase, reduces pancreatic tumor growth in mice” by Polvani S, Calamante M, Foresta V, et al, on page 709.
A
lthough personalized medicine and targeted therapy are attractive treatment options for patients with cancer, these approaches are not feasible when the pathophysiology of the disease is unknown, the biology of the malignancy is highly complex to target, or selective and specific agents for target(s) are not available. Adenocarcinoma of the pancreas (pancreatic cancer) is a paradigm of malignancies in which limited biology is known, etiology is intricate with influences from both genetic and environmental factors, the underlying molecular mechanisms of the disease and its interaction with the microenvironment remain elusive, and the pathophysiology is too complex to target by novel agents.1–3 Fortunately, genetically engineered mouse model systems that recapitulate the genesis, maintenance, and progression of the invasive pancreatic cancer are becoming available.4,5 These systems will provide unprecedented opportunities to understand the disease at molecular, genetic, and tumor environment levels as well as to test novel targeted chemotherapeutics. However, at present, physicians generally have to rely on the available conventional cytotoxic agents that have shown only limited efficacy. Since their introduction as cancer therapeutics ⬎50 years ago, nucleobases and nucleoside analogs have become more prevalent in the clinical treatment of cancer 400
and viral diseases than other mechanistically similar groups of drugs.6 The first nucleic acid antagonists to enter the clinic, the nucleobases 6-mercaptopurine and 5-fluorouracil, are still in use ⬎50 years after their introduction. Unlike nucleobases, nucleoside analogs have both an aglycone and a normal or modified sugar. This group of agents contains cytarabine, an established agent for pediatric and adult acute myelogenous leukemias and lymphomas; fludarabine which has become a gold standard for treatment of chronic lymphocytic leukemias and lymphomas; and cladribine, which is a curative single agent regimen for hairy cell leukemia. These erstwhile agents generated tremendous interest in this group of chemotherapeutic drugs, resulting in a second generation of analogs. In the last decade, 4 new nucleoside analogs— nelarabine, clofarabine, and hypomethylating drugs decitabine, and azacitidine—were approved by the US Food and Drug Administration (FDA). However, the revolutionary analog, which is a congener of cytarabine, was gemcitabine (2=,2=-difluorodeoxycytidine), which moved nucleoside analogs from liquid malignancies to solid tumors (Figure 1). The presence of dual fluorines, which has an atomic radius similar to hydrogen, in geminal configuration at the 2=-position in the sugar made this agent an efficient substrate for activation by deoxycytidine kinase7 as well as for DNA polymerases that incorporate this fraudulent nucleotide in to DNA. This highly favorable property resulted in a desired pharmacokinetic profile of the drug and may be the reason for its effectiveness in solid malignancies. Although the benefit of gemcitabine therapy was minor for patients with pancreatic cancer, a highly challenging malignancy to treat, the results were substantial enough to
Editorials continued
Figure 1. Chemical structures of antimetabolite or nucleoside analogs for pancreatic cancer.
achieve a single agent approval by the FDA.8 Before gemcitabine and still today, the other commonly used clinical treatment option for patients with pancreatic cancer is a combination regimen of 5-fluorouracil and folinic acid (leucovorin). Overall, these systemic agents have become standard of care for adenocarcinoma of the pancreas. In fact, in a recent randomized study this couplet versus single-agent gemcitabine resulted in similar overall survival of patients with pancreatic cancer.9 Therapeutics that target epidermal growth factor receptors or vasculature of tumor tissue10 have been tested for this challenging tumor; although erlotinib was FDA approved, it still needed to be combined with gemcitabine.11,12 Other remedies that have been examined in the clinic with gemcitabine or fluorouracil include DNAdamaging agents (cisplatinum, oxaliplatinum, or radiation) and Ras-directed drugs (tipifarnib).13,14 Overall, these studies underscore the use and benefit of antimetabolites and nucleoside analogs for the treatment of 1 of the most insidious tumors, adenocarcinoma of the pancreas. A consequence of this albeit limited but only success in treating pancreatic cancer is that new nucleoside analogs are being synthesized and tested to find a better gemcitabine. Acyclovir (ACV), which is acycloguanosine, is a clinically used antiviral drug. Because ACV lacks a 3=-hydroxyl group, which is absolutely required for phosphodiester bond formation with the incoming deoxynucleotide, ACV incorporation leads to de facto chain termination, a
desired characteristic for any nucleoside analog. This analog is an extremely poor substrate for cellular thymidine kinase; the viral thymidine kinase has a 3000-fold greater affinity. Hence, tactics have been used to transfect viral thymidine kinase into tumor cells to facilitate phosphorylation of ACV.15,16 In tumor cells, such strategy has worked effectively in phosphorylating ACV and other analogs and inducing cytotoxicity.17 A primary requisite for the actions of any nucleoside analog, including gemcitabine and ACV, is transport into the cell followed by biotransformation to a triphosphorylated form using a 3-step process and kinases. In general, the first metabolic action in the cell, that is, conversion of analog to the corresponding 5=-monophosphate is the rate-limiting step. Analog triphosphate competes with normal natural congener (a deoxynucleoside triphosphate) for incorporation into DNA by DNA polymerases.7 The incorporation of the analog into DNA blocks or slows further extension of the nascent strand and causes stalling of replication forks. Once sensed at the molecular level, the stalled replication forks lead to activation of cell-cycle checkpoints, signaling for DNA repair responses, and either resistance to the genotoxic insult or cell death.18 Overall, the actions are directed toward DNA replication and hence these agents are Sphase specific, which leads to toxicity to tissues with a large proliferative fraction that include, but are not limited to, hematologic and gastrointestinal toxicities. 401
Editorials continued
In an attempt to design superior analogs, 3 approaches have been taken. Given that membrane penetration is a potentially physiologic as well as physical barrier, the first strategy was creation of analogs that are more cell membrane permeable. Nucleoside analogs as well as nucleobases are hydrophilic and require specific nucleoside transporters.19,20 Chemical modifications such as fatty acid ester derivatization have been synthesized for cytarabine and gemcitabine. These analogs with 5=-fatty acid chain were easily transported in the cells.21 Second, the design of analogs that do not require biotransformation to monophosphates by nucleoside kinases was employed as a strategy to circumvent limited nucleoside kinase expression and poor substrate characteristics of the analog. In fact, in most cases, these modified nucleoside analogs are phosphorylated analogs. Because phosphorylated nucleosides are subject to dephosphorylation by plasma enzymes, and significant quantities do not enter cells owing to negative charges, additional modifications are needed to permit entry into the cell. Examples of these include acyclic nucleoside phosphonate analogs that do not require the rate-limiting first phosphorylation step to produce the cytotoxic triphosphate.15 The guanine derivative of this modification, PMEG [9-(2phosphonylmethoxyethyl)guanine], has been tested in vitro in whole cells and in clinic.22 Similarly, bis[(pivaloyloxy)methyl]2=-deoxy-5-fluorouridine 5=-monophosphate was synthesized for passive diffusion entry in the cell followed by intracellular removal of 2 pivaloylomethoxy group and release of 2=-deoxy-5-fluorouridine 5=-monophosphate.23 Masking of negatively charged groups on the monophosphate has recently been used in the protide approach, which has created several molecules that have entered in the clinic as antiviral drugs.24 A third approach has been taken to synthesize analogs that are directed toward cancer cells while avoiding the normal, healthy cells. Gene therapy tactics that introduce unique metabolic enzymes such as viral thymidine kinase in the target cancer cell have demonstrated preclinical success when ACV was used as a substrate. This analog is phosphorylated to the monophosphate only by the herpes kinase and not by cellular thymidine kinase.15,16 To encompass all 3 desired characteristics, Polvani et al25 in this issue of GASTROENTEROLOGY have synthesized acycloguanosyl 5=-thymidyltriphosphate (ACV-TP-T). Their strategy was that the analog would enter pancreatic cell lines and would avoid the presence of specific transporters. It would then be metabolized to acycloguanosyl diphosphate in cells by the enzyme telomerase and then to triphosphate by cellular diphosphokinases, which are present in abundance. If effective, this would circumvent the need for phosphorylation by nucleoside kinases, in this case viral thymidine kinase, which is the rate-limiting step. Finally, because the expression of telomerase is 402
much higher in the target tumor tissue, the authors postulate that their analog will be more selective to tumor cells, thereby sparing normal cells. A parallel approach, albeit with the use of virus infected cells, has been demonstrated previously.17 ACV-TP-T–treated cells show growth inhibition measured by BrdU incorporation assay and clonogenicity procedures in variety of pancreatic cell lines. This seems to be associated with S-phase cell cycle arrest; BrdU incorporation inhibition was much more pronounced than caspase-3 activation. It is not clear whether the biological activity is comparable with that observed with ACV in viral thymidine kinase–infected cells. Additionally, no data are provided with gemcitabine for similar comparison. Hence, although these results provide some evidence that ACV-TP-T is effective in pancreatic cancer cell lines, they do not demonstrate that ACV-TP-T is equivalent or better than ACV (with viral TK) or gemcitabine. The authors present some data to elucidate their hypothesis that ACV-TP-T is converted to ACV-diphosphate by cellular telomerase. For example, there was a relationship between the level of endogenous telomerase in cell lines and cytotoxicity to ACV-TP-T. Additionally, knocking down this enzyme resulted in relative resistance to this analog. Conversely, over-expression of the enzyme made cells sensitive to this analog. Finally, ACV-TP-dCyd was not effective in cell lines because this was not the substrate of telomerase enzyme, which incorporates only T, A, and Gs. These data are correlative and suggestive of a role of telomerase, but do not provide a direct evidence to demonstrate intracellular conversion of ACV-TP-T to ACV-diphosphate by telomerases. At a cellular level, proof of concept could have been achieved by using dual-labeled [3H]ACV and [32P] alpha and beta phosphates of the ACV-TP-T. Metabolism of nucleosides could be defined by use of radioactive material. Double labeling with [32P] and [3H] has been cleverly used to identify and trace metabolites of nucleoside analogs.26 At a biochemical level, evidence could have been obtained by doing in vitro assays using telomerase with ACV-TP-T. At the structural level, molecular modeling could have been explored with the known crystal structure of telomerase27,28 and ACV-TP-T as substrate. The cytotoxic effect of the ACV-TP-T is presumed to be due to incorporation of ACV-TP in the nuclear DNA during DNA replication. This postulate could have been tested using radiolabeled ACV-TP-T and quantitating its incorporation into 3=-terminus of DNA as it is a de facto chain terminator. Again, this could have been tested by digesting the DNA with incorporated ACV moieties.29 Importantly, the authors extend their work in vivo in nude mice with xenografts from sensitive or resistant pancreatic cell lines. In both cases, there was reduction in
Editorials continued
tumor volume with minimal toxicity. Histochemical analyses show growth inhibition and apoptosis of the tumor tissue. Unfortunately, again comparison with established agents is not provided. In conclusion, the authors provide an intriguing and impressive new concept in analog synthesis that tackles transport into cells, bypassing the nucleoside kinase requirement, and selectivity to cancer cells. More work is required to provide a proof of principle for each of these 3 properties. If all 3 characteristics are achieved, this may provide yet another and tumor-targeted nucleoside analog for this insidious disease. Because pancreatic carcinoma is surrounded by desmoplastic stroma and sparse vasculature, pharmacologic manipulation of the microenvironment by interfering with Hedgehog signaling may be employed for maximal analog delivery to target tumor tissue.30 Because nucleoside analogs are used for varieties of malignancies, the unique mechanism-based approach used by the authors could benefit several diseases.
VARSHA GANDHI Department of Experimental Therapeutics and Department of Leukemia The University of Texas MD Anderson Cancer Center Houston, Texas References 1. Rustgi AK. The molecular pathogenesis of pancreatic cancer: clarifying a complex circuitry. Genes Dev 2006;20:3049 –3053. 2. Li D, Abbruzzese JL. New strategies in pancreatic cancer: emerging epidemiologic and therapeutic concepts. Clin Cancer Res 2010;16:4313– 4138. 3. Philip PA, Mooney M, Jaffe D, et al. Consensus report of the national cancer institute clinical trials planning meeting on pancreas cancer treatment. J Clin Oncol 2009;27:5660 –5669. 4. Hingorani SR, Petricoin EF, Maitra A, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 2003;4:437– 450. 5. Hingorani SR, Wang L, Multani AS, et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 2005;7:469 – 483. 6. Elion GB. The purine path to chemotherapy. Science 1989;244: 41– 47. 7. Plunkett W, Huang P, Searcy CE, et al. Gemcitabine: preclinical pharmacology and mechanisms of action. Semin Oncol 1996;23: 3–15. 8. Burris HA, 3rd, Moore MJ, Andersen J, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997;15:2403–2413. 9. Neoptolemos JP, Stocken DD, Bassi C, et al. Adjuvant chemotherapy with fluorouracil plus folinic acid vs gemcitabine following pancreatic cancer resection: a randomized controlled trial. JAMA 2010;304:1073–1081. 10. Van Cutsem E, Vervenne WL, Bennouna J, et al. Phase III trial of bevacizumab in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer. J Clin Oncol 2009; 27:2231–2237.
11. Moore MJ, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2007;25:1960 –1966. 12. Oettle H, Hilbig A. Does the addition of erlotinib to gemcitabine improve outcome in patients with advanced pancreatic cancer? Nat Clin Pract Oncol 2007;4:686 – 687. 13. Xiong HQ, Carr K, Abbruzzese JL. Cytotoxic chemotherapy for pancreatic cancer: Advances to date and future directions. Drugs 2006;66:1059 –1072. 14. Van Cutsem E, van de Velde H, Karasek P, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430 –1438. 15. De Clercq E. The design of drugs for HIV and HCV. Nat Rev Drug Discov 2007;6:1001–1018. 16. De Clercq E, Field HJ. Antiviral prodrugs—the development of successful prodrug strategies for antiviral chemotherapy. Br J Pharmacol 2006;147:1–11. 17. Shewach DS, Zerbe LK, Hughes TL, et al. Enhanced cytotoxicity of antiviral drugs mediated by adenovirus directed transfer of the herpes simplex virus thymidine kinase gene in rat glioma cells. Cancer Gene Ther 1994;1:107–112. 18. Ewald B, Sampath D, Plunkett W. Nucleoside analogs: molecular mechanisms signaling cell death. Oncogene 2008;27: 6522– 6537. 19. Ko AH, Tempero MA. Personalized medicine for pancreatic cancer: a step in the right direction. Gastroenterology 2009; 136:43– 45. 20. Zhang J, Visser F, King KM, et al. The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs. Cancer Metastasis Rev 2007;26:85–110. 21. Bergman AM, Kuiper CM, Voorn DA, et al. Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines. Biochem Pharmacol 2004;67:503–511. 22. Rose WC, Crosswell AR, Bronson JJ, et al. In vivo antitumor activity of 9-[(2-phosphonylmethoxy)ethyl]-guanine and related phosphonate nucleotide analogues. J Natl Cancer Inst 1990;82: 510 –512. 23. Farquhar D, Khan S, Srivastva DN, et al. Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2’-deoxy-5-fluorouridine 5’monophosphate (FdUMP): a strategy to introduce nucleotides into cells. J Med Chem 1994;37:3902–3909. 24. Mehellou Y, Balzarini J, McGuigan C. Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. ChemMedChem 2009;4: 1779 –1791. 25. Polvani S, Calamante M, Foresta V, et al. Acycloguanosyl 5=thymidyltriphosphate, a thymidine analogue pro-drug for pancreatic cancer therapy. Gastroenterology 2011;140:709 –720. 26. Plunkett W, Lapi L, Ortiz PJ, et al. Penetration of mouse fibroblasts by the 5’-phosphate of 9-beta-D-arabinofuranosyladenine and incorporation of the nucleotide into DNA. Proc Natl Acad Sci U S A 1974;71:73–77. 27. Jacobs SA, Podell ER, Cech TR. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase. Nat Struct Mol Biol 2006;13:218 –225. 28. Mitchell M, Gillis A, Futahashi M, et al. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat Struct Mol Biol 2010;17:513–518. 29. Rodriguez CO, Jr., Stellrecht CM, Gandhi V. Mechanisms for T-cell selective cytotoxicity of arabinosylguanine. Blood 2003;102: 1842–1848. 403
Editorials continued
30. Olive KP, Jacobetz MA, Davidson CJ, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 2009;324:1457–1461.
Reprint requests Address requests for reprints to: Varsha Gandhi, PhD, Department of Experimental Therapeutics and Department of Leukemia, The
404
University of Texas MD Anderson Cancer Center, Houston, Texas 77030. e-mail: [email protected]; fax: 713-794-4316. Conflicts of interest The author discloses no conflicts. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.12.018
21. Gangula PR, Mukhopadhyay S, Pasricha PJ, et al. Sepiapterin reverses the changes in gastric nNOS dimerization and function in diabetic gastroparesis. Neurogastroenterol Motil 2010;22: 1325–13331. 22. Chandrasekharan B, Anitha M, Blatt R, et al. Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress. Neurogastroenterol Motil 2010 Oct 12 [Epub ahead of print]. 23. Vanderwinden JM, Mailleux P, Schiffmann SN, et al. Nitric oxide synthase activity in infantile pyloric stenosis. N Engl J Med 1992; 327:511–515. 24. Tomita R, Munakata K, Kurosu Y, et al. A role of nitric oxide in Hirschsprung’s disease. J Pediatr Surg 1995;30:437– 440. 25. Tomita R, Fujisaki S, Tanjoh K, et al. Role of nitric oxide in the internal anal sphincter of Hirschsprung’s disease. World J Surg 2002;26:1493–1498.
Reprint requests Address requests for reprints to: James J. Galligan, PhD, Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan 48824. e-mail: [email protected]; fax: 517-353-8915. Conflicts of interest The authors disclose no conflicts. Funding Supported by DK57039 (NIDDK). © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.12.016
Yet Another Nucleoside Analog for Pancreatic Cancer
See “Acycloguanosyl 5=-thymidyltriphosphate, a thymidine analogue prodrug activated by telomerase, reduces pancreatic tumor growth in mice” by Polvani S, Calamante M, Foresta V, et al, on page 709.
A
lthough personalized medicine and targeted therapy are attractive treatment options for patients with cancer, these approaches are not feasible when the pathophysiology of the disease is unknown, the biology of the malignancy is highly complex to target, or selective and specific agents for target(s) are not available. Adenocarcinoma of the pancreas (pancreatic cancer) is a paradigm of malignancies in which limited biology is known, etiology is intricate with influences from both genetic and environmental factors, the underlying molecular mechanisms of the disease and its interaction with the microenvironment remain elusive, and the pathophysiology is too complex to target by novel agents.1–3 Fortunately, genetically engineered mouse model systems that recapitulate the genesis, maintenance, and progression of the invasive pancreatic cancer are becoming available.4,5 These systems will provide unprecedented opportunities to understand the disease at molecular, genetic, and tumor environment levels as well as to test novel targeted chemotherapeutics. However, at present, physicians generally have to rely on the available conventional cytotoxic agents that have shown only limited efficacy. Since their introduction as cancer therapeutics ⬎50 years ago, nucleobases and nucleoside analogs have become more prevalent in the clinical treatment of cancer 400
and viral diseases than other mechanistically similar groups of drugs.6 The first nucleic acid antagonists to enter the clinic, the nucleobases 6-mercaptopurine and 5-fluorouracil, are still in use ⬎50 years after their introduction. Unlike nucleobases, nucleoside analogs have both an aglycone and a normal or modified sugar. This group of agents contains cytarabine, an established agent for pediatric and adult acute myelogenous leukemias and lymphomas; fludarabine which has become a gold standard for treatment of chronic lymphocytic leukemias and lymphomas; and cladribine, which is a curative single agent regimen for hairy cell leukemia. These erstwhile agents generated tremendous interest in this group of chemotherapeutic drugs, resulting in a second generation of analogs. In the last decade, 4 new nucleoside analogs— nelarabine, clofarabine, and hypomethylating drugs decitabine, and azacitidine—were approved by the US Food and Drug Administration (FDA). However, the revolutionary analog, which is a congener of cytarabine, was gemcitabine (2=,2=-difluorodeoxycytidine), which moved nucleoside analogs from liquid malignancies to solid tumors (Figure 1). The presence of dual fluorines, which has an atomic radius similar to hydrogen, in geminal configuration at the 2=-position in the sugar made this agent an efficient substrate for activation by deoxycytidine kinase7 as well as for DNA polymerases that incorporate this fraudulent nucleotide in to DNA. This highly favorable property resulted in a desired pharmacokinetic profile of the drug and may be the reason for its effectiveness in solid malignancies. Although the benefit of gemcitabine therapy was minor for patients with pancreatic cancer, a highly challenging malignancy to treat, the results were substantial enough to
Editorials continued
Figure 1. Chemical structures of antimetabolite or nucleoside analogs for pancreatic cancer.
achieve a single agent approval by the FDA.8 Before gemcitabine and still today, the other commonly used clinical treatment option for patients with pancreatic cancer is a combination regimen of 5-fluorouracil and folinic acid (leucovorin). Overall, these systemic agents have become standard of care for adenocarcinoma of the pancreas. In fact, in a recent randomized study this couplet versus single-agent gemcitabine resulted in similar overall survival of patients with pancreatic cancer.9 Therapeutics that target epidermal growth factor receptors or vasculature of tumor tissue10 have been tested for this challenging tumor; although erlotinib was FDA approved, it still needed to be combined with gemcitabine.11,12 Other remedies that have been examined in the clinic with gemcitabine or fluorouracil include DNAdamaging agents (cisplatinum, oxaliplatinum, or radiation) and Ras-directed drugs (tipifarnib).13,14 Overall, these studies underscore the use and benefit of antimetabolites and nucleoside analogs for the treatment of 1 of the most insidious tumors, adenocarcinoma of the pancreas. A consequence of this albeit limited but only success in treating pancreatic cancer is that new nucleoside analogs are being synthesized and tested to find a better gemcitabine. Acyclovir (ACV), which is acycloguanosine, is a clinically used antiviral drug. Because ACV lacks a 3=-hydroxyl group, which is absolutely required for phosphodiester bond formation with the incoming deoxynucleotide, ACV incorporation leads to de facto chain termination, a
desired characteristic for any nucleoside analog. This analog is an extremely poor substrate for cellular thymidine kinase; the viral thymidine kinase has a 3000-fold greater affinity. Hence, tactics have been used to transfect viral thymidine kinase into tumor cells to facilitate phosphorylation of ACV.15,16 In tumor cells, such strategy has worked effectively in phosphorylating ACV and other analogs and inducing cytotoxicity.17 A primary requisite for the actions of any nucleoside analog, including gemcitabine and ACV, is transport into the cell followed by biotransformation to a triphosphorylated form using a 3-step process and kinases. In general, the first metabolic action in the cell, that is, conversion of analog to the corresponding 5=-monophosphate is the rate-limiting step. Analog triphosphate competes with normal natural congener (a deoxynucleoside triphosphate) for incorporation into DNA by DNA polymerases.7 The incorporation of the analog into DNA blocks or slows further extension of the nascent strand and causes stalling of replication forks. Once sensed at the molecular level, the stalled replication forks lead to activation of cell-cycle checkpoints, signaling for DNA repair responses, and either resistance to the genotoxic insult or cell death.18 Overall, the actions are directed toward DNA replication and hence these agents are Sphase specific, which leads to toxicity to tissues with a large proliferative fraction that include, but are not limited to, hematologic and gastrointestinal toxicities. 401
Editorials continued
In an attempt to design superior analogs, 3 approaches have been taken. Given that membrane penetration is a potentially physiologic as well as physical barrier, the first strategy was creation of analogs that are more cell membrane permeable. Nucleoside analogs as well as nucleobases are hydrophilic and require specific nucleoside transporters.19,20 Chemical modifications such as fatty acid ester derivatization have been synthesized for cytarabine and gemcitabine. These analogs with 5=-fatty acid chain were easily transported in the cells.21 Second, the design of analogs that do not require biotransformation to monophosphates by nucleoside kinases was employed as a strategy to circumvent limited nucleoside kinase expression and poor substrate characteristics of the analog. In fact, in most cases, these modified nucleoside analogs are phosphorylated analogs. Because phosphorylated nucleosides are subject to dephosphorylation by plasma enzymes, and significant quantities do not enter cells owing to negative charges, additional modifications are needed to permit entry into the cell. Examples of these include acyclic nucleoside phosphonate analogs that do not require the rate-limiting first phosphorylation step to produce the cytotoxic triphosphate.15 The guanine derivative of this modification, PMEG [9-(2phosphonylmethoxyethyl)guanine], has been tested in vitro in whole cells and in clinic.22 Similarly, bis[(pivaloyloxy)methyl]2=-deoxy-5-fluorouridine 5=-monophosphate was synthesized for passive diffusion entry in the cell followed by intracellular removal of 2 pivaloylomethoxy group and release of 2=-deoxy-5-fluorouridine 5=-monophosphate.23 Masking of negatively charged groups on the monophosphate has recently been used in the protide approach, which has created several molecules that have entered in the clinic as antiviral drugs.24 A third approach has been taken to synthesize analogs that are directed toward cancer cells while avoiding the normal, healthy cells. Gene therapy tactics that introduce unique metabolic enzymes such as viral thymidine kinase in the target cancer cell have demonstrated preclinical success when ACV was used as a substrate. This analog is phosphorylated to the monophosphate only by the herpes kinase and not by cellular thymidine kinase.15,16 To encompass all 3 desired characteristics, Polvani et al25 in this issue of GASTROENTEROLOGY have synthesized acycloguanosyl 5=-thymidyltriphosphate (ACV-TP-T). Their strategy was that the analog would enter pancreatic cell lines and would avoid the presence of specific transporters. It would then be metabolized to acycloguanosyl diphosphate in cells by the enzyme telomerase and then to triphosphate by cellular diphosphokinases, which are present in abundance. If effective, this would circumvent the need for phosphorylation by nucleoside kinases, in this case viral thymidine kinase, which is the rate-limiting step. Finally, because the expression of telomerase is 402
much higher in the target tumor tissue, the authors postulate that their analog will be more selective to tumor cells, thereby sparing normal cells. A parallel approach, albeit with the use of virus infected cells, has been demonstrated previously.17 ACV-TP-T–treated cells show growth inhibition measured by BrdU incorporation assay and clonogenicity procedures in variety of pancreatic cell lines. This seems to be associated with S-phase cell cycle arrest; BrdU incorporation inhibition was much more pronounced than caspase-3 activation. It is not clear whether the biological activity is comparable with that observed with ACV in viral thymidine kinase–infected cells. Additionally, no data are provided with gemcitabine for similar comparison. Hence, although these results provide some evidence that ACV-TP-T is effective in pancreatic cancer cell lines, they do not demonstrate that ACV-TP-T is equivalent or better than ACV (with viral TK) or gemcitabine. The authors present some data to elucidate their hypothesis that ACV-TP-T is converted to ACV-diphosphate by cellular telomerase. For example, there was a relationship between the level of endogenous telomerase in cell lines and cytotoxicity to ACV-TP-T. Additionally, knocking down this enzyme resulted in relative resistance to this analog. Conversely, over-expression of the enzyme made cells sensitive to this analog. Finally, ACV-TP-dCyd was not effective in cell lines because this was not the substrate of telomerase enzyme, which incorporates only T, A, and Gs. These data are correlative and suggestive of a role of telomerase, but do not provide a direct evidence to demonstrate intracellular conversion of ACV-TP-T to ACV-diphosphate by telomerases. At a cellular level, proof of concept could have been achieved by using dual-labeled [3H]ACV and [32P] alpha and beta phosphates of the ACV-TP-T. Metabolism of nucleosides could be defined by use of radioactive material. Double labeling with [32P] and [3H] has been cleverly used to identify and trace metabolites of nucleoside analogs.26 At a biochemical level, evidence could have been obtained by doing in vitro assays using telomerase with ACV-TP-T. At the structural level, molecular modeling could have been explored with the known crystal structure of telomerase27,28 and ACV-TP-T as substrate. The cytotoxic effect of the ACV-TP-T is presumed to be due to incorporation of ACV-TP in the nuclear DNA during DNA replication. This postulate could have been tested using radiolabeled ACV-TP-T and quantitating its incorporation into 3=-terminus of DNA as it is a de facto chain terminator. Again, this could have been tested by digesting the DNA with incorporated ACV moieties.29 Importantly, the authors extend their work in vivo in nude mice with xenografts from sensitive or resistant pancreatic cell lines. In both cases, there was reduction in
Editorials continued
tumor volume with minimal toxicity. Histochemical analyses show growth inhibition and apoptosis of the tumor tissue. Unfortunately, again comparison with established agents is not provided. In conclusion, the authors provide an intriguing and impressive new concept in analog synthesis that tackles transport into cells, bypassing the nucleoside kinase requirement, and selectivity to cancer cells. More work is required to provide a proof of principle for each of these 3 properties. If all 3 characteristics are achieved, this may provide yet another and tumor-targeted nucleoside analog for this insidious disease. Because pancreatic carcinoma is surrounded by desmoplastic stroma and sparse vasculature, pharmacologic manipulation of the microenvironment by interfering with Hedgehog signaling may be employed for maximal analog delivery to target tumor tissue.30 Because nucleoside analogs are used for varieties of malignancies, the unique mechanism-based approach used by the authors could benefit several diseases.
VARSHA GANDHI Department of Experimental Therapeutics and Department of Leukemia The University of Texas MD Anderson Cancer Center Houston, Texas References 1. Rustgi AK. The molecular pathogenesis of pancreatic cancer: clarifying a complex circuitry. Genes Dev 2006;20:3049 –3053. 2. Li D, Abbruzzese JL. New strategies in pancreatic cancer: emerging epidemiologic and therapeutic concepts. Clin Cancer Res 2010;16:4313– 4138. 3. Philip PA, Mooney M, Jaffe D, et al. Consensus report of the national cancer institute clinical trials planning meeting on pancreas cancer treatment. J Clin Oncol 2009;27:5660 –5669. 4. Hingorani SR, Petricoin EF, Maitra A, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 2003;4:437– 450. 5. Hingorani SR, Wang L, Multani AS, et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 2005;7:469 – 483. 6. Elion GB. The purine path to chemotherapy. Science 1989;244: 41– 47. 7. Plunkett W, Huang P, Searcy CE, et al. Gemcitabine: preclinical pharmacology and mechanisms of action. Semin Oncol 1996;23: 3–15. 8. Burris HA, 3rd, Moore MJ, Andersen J, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997;15:2403–2413. 9. Neoptolemos JP, Stocken DD, Bassi C, et al. Adjuvant chemotherapy with fluorouracil plus folinic acid vs gemcitabine following pancreatic cancer resection: a randomized controlled trial. JAMA 2010;304:1073–1081. 10. Van Cutsem E, Vervenne WL, Bennouna J, et al. Phase III trial of bevacizumab in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer. J Clin Oncol 2009; 27:2231–2237.
11. Moore MJ, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2007;25:1960 –1966. 12. Oettle H, Hilbig A. Does the addition of erlotinib to gemcitabine improve outcome in patients with advanced pancreatic cancer? Nat Clin Pract Oncol 2007;4:686 – 687. 13. Xiong HQ, Carr K, Abbruzzese JL. Cytotoxic chemotherapy for pancreatic cancer: Advances to date and future directions. Drugs 2006;66:1059 –1072. 14. Van Cutsem E, van de Velde H, Karasek P, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430 –1438. 15. De Clercq E. The design of drugs for HIV and HCV. Nat Rev Drug Discov 2007;6:1001–1018. 16. De Clercq E, Field HJ. Antiviral prodrugs—the development of successful prodrug strategies for antiviral chemotherapy. Br J Pharmacol 2006;147:1–11. 17. Shewach DS, Zerbe LK, Hughes TL, et al. Enhanced cytotoxicity of antiviral drugs mediated by adenovirus directed transfer of the herpes simplex virus thymidine kinase gene in rat glioma cells. Cancer Gene Ther 1994;1:107–112. 18. Ewald B, Sampath D, Plunkett W. Nucleoside analogs: molecular mechanisms signaling cell death. Oncogene 2008;27: 6522– 6537. 19. Ko AH, Tempero MA. Personalized medicine for pancreatic cancer: a step in the right direction. Gastroenterology 2009; 136:43– 45. 20. Zhang J, Visser F, King KM, et al. The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs. Cancer Metastasis Rev 2007;26:85–110. 21. Bergman AM, Kuiper CM, Voorn DA, et al. Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines. Biochem Pharmacol 2004;67:503–511. 22. Rose WC, Crosswell AR, Bronson JJ, et al. In vivo antitumor activity of 9-[(2-phosphonylmethoxy)ethyl]-guanine and related phosphonate nucleotide analogues. J Natl Cancer Inst 1990;82: 510 –512. 23. Farquhar D, Khan S, Srivastva DN, et al. Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2’-deoxy-5-fluorouridine 5’monophosphate (FdUMP): a strategy to introduce nucleotides into cells. J Med Chem 1994;37:3902–3909. 24. Mehellou Y, Balzarini J, McGuigan C. Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. ChemMedChem 2009;4: 1779 –1791. 25. Polvani S, Calamante M, Foresta V, et al. Acycloguanosyl 5=thymidyltriphosphate, a thymidine analogue pro-drug for pancreatic cancer therapy. Gastroenterology 2011;140:709 –720. 26. Plunkett W, Lapi L, Ortiz PJ, et al. Penetration of mouse fibroblasts by the 5’-phosphate of 9-beta-D-arabinofuranosyladenine and incorporation of the nucleotide into DNA. Proc Natl Acad Sci U S A 1974;71:73–77. 27. Jacobs SA, Podell ER, Cech TR. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase. Nat Struct Mol Biol 2006;13:218 –225. 28. Mitchell M, Gillis A, Futahashi M, et al. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat Struct Mol Biol 2010;17:513–518. 29. Rodriguez CO, Jr., Stellrecht CM, Gandhi V. Mechanisms for T-cell selective cytotoxicity of arabinosylguanine. Blood 2003;102: 1842–1848. 403
Editorials continued
30. Olive KP, Jacobetz MA, Davidson CJ, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 2009;324:1457–1461.
Reprint requests Address requests for reprints to: Varsha Gandhi, PhD, Department of Experimental Therapeutics and Department of Leukemia, The
404
University of Texas MD Anderson Cancer Center, Houston, Texas 77030. e-mail: [email protected]; fax: 713-794-4316. Conflicts of interest The author discloses no conflicts. © 2011 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.12.018