Cyclopentenyl Cytosine Research Articles

Enhanced Reendothelialization and Thrombosis Prevention with a New Drug-Eluting Stent.

The objective of the study is to test the efficacy of cyclopentenyl cytosine (CPEC)-coated stents on blocking artery stenosis, promoting reendothelialization, and reducing thrombosis. Scanning electron microscopy was employed to observe the morphological characteristics of stents coated with a mixture of CPEC and poly(lactic-co-glycolic acid) (PLGA) copolymer. PLGA has been used in various Food and Drug Administration (FDA)-approved therapeutic devices. In vitro release of CPEC was tested to measure the dynamic drug elution. Comparison between CPEC- and everolimus-coated stents on neointimal formation and thrombosis formation was conducted after being implanted into the human internal mammary artery and grafted to the mouse aorta. Optimization in stent coating resulted in uniform and consistent coating with minimal variation. In vitro drug release tests demonstrated a gradual and progressive discharge of CPEC. CPEC- or everolimus-coated stents caused much less stenosis than bare-metal stents. However, CPEC stent-implanted arteries exhibited enhanced reendothelialization compared to everolimus stents. Mechanistically, CPEC-coated stents reduced the proliferation of vascular smooth muscle cells while simultaneously promoting reendothelialization. More significantly, unlike everolimus-coated stents, CPEC-coated stents showed a significant reduction in thrombosis formation even in the absence of ongoing anticoagulant treatment. The study establishes CPEC-coated stent as a promising new device for cardiovascular interventions. By enhancing reendothelialization and preventing thrombosis, CPEC offers advantages over conventional approaches, including the elimination of the need for anti-clogging drugs, which pave the way for improved therapeutic outcomes and management of atherosclerosis-related medical procedures.

Cyclopentenylcytosine (CPE-C): In Vitro and In Vivo Evaluation as an Antiviral against Adenoviral Ocular Infections.

Adenoviruses are the major cause of ocular viral infections worldwide. Currently, there is no approved antiviral treatment for these eye infections. Cyclopentenylcytosine (CPE-C) is an antiviral that has demonstrated activity against more than 20 viruses. The goals of the current study were to determine the in vitro and in vivo antiviral activity of CPE-C as well as its ocular toxicity. Antiviral activity was evaluated in vitro using standard plaque reduction assays to determine the 50% effective concentrations (EC50s) and in vivo in the Ad5/NZW rabbit ocular replication model. Ocular toxicity was determined in uninfected rabbit eyes following topical ocular application. The in vitro EC50s for CPE-C ranged from 0.03 to 0.059 μg/mL for nine adenovirus types that commonly infect the eye. Ocular toxicity testing determined CPE-C to be non-irritating or practically non-irritating by Draize scoring. In vivo, 3% CPE-C topically administered 4X or 2X daily for 7 days to adenovirus-infected eyes demonstrated effective antiviral activity compared with the negative control and comparable antiviral activity to the positive control, 0.5% cidofovir, topically administered twice daily for 7 days. We conclude CPE-C was relatively non-toxic to rabbit eyes and demonstrated potent anti-adenoviral activity in vitro and in vivo.

Abstract 1267: Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117)

Abstract RX-3117 is a novel cytidine analog showing encouraging results in ongoing Phase 2a studies with gemcitabine-resistant pancreatic ductal adenocarcinoma and advanced urothelial bladder cancer. Cellular uptake of RX-3117 is mediated by the equilibrative nucleoside transporter 1 (hENT1). RX-3117 is activated by uridine-cytidine kinase-2 (UCK2) and is not a substrate for cytidine deaminase (CDA), and shows a good oral bioavailability. In an earlier study accumulation of RX-3117 nucleotides was associated with sensitivity to the drug. RX-3117 is incorporated into RNA and DNA and inhibits DNA methyltransferase 1 (DNMT1). RX-3117 is active against gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines. In this study we aimed to elucidate potential resistance mechanisms against RX-3117. For that purpose the NSCLC cell lines H460, A549 and SW1573 (including its gemcitabine resistant variant SW-G) were exposed to increasing concentrations of RX-3117. For A549 and SW1573 two variants were obtained: A549/RX1, A549/RX2, SW1573/RX1 and SW1573/RX2. Resistance varied from 10 (H460; SW-G) to >100-fold for the other cell lines. Resistance was stable except for H460 cells. Cross-resistance in the same range was observed for the cytidine analogs ethynyl-cytidine (ETC), cyclopentenylcytosine (CPEC) and aza-cytidine (aza-CR), which are all activated by UCK2; no cross-resistance was observed for gemcitabine and aza-deoxycytidine which are activated by deoxycytidine kinase. No change was observed in the expression of DNMT1, while expression of ribonucleotide reductase 1 was decreased. However, analysis of UCK2 at the enzyme activity level, protein and gene expression did not reveal a decrease in UCK2 while for ETC, aza-CR and CPEC resistance was reported to be mediated by a UCK2 deficiency. No change was found in the other activating enzymes UMP-kinase, CMP-kinase and NME1/NDKA. Despite this lack of change, accumulation of RX-3117 and RX-3117 nucleotides (mono-, di- and triphosphate) was 2-5-fold lower in the resistant cells after incubation with 10 or 100 μM RX-3117 for 4-24 hr. No evidence for accumulation of RX-3117 deoxynucleotides was found in both the parent and the resistant cell lines. Since no change in hENT1 was found, cell lines were further investigated by using RNA-seq, and western blotting focusing on nucleoside/nucleotide degradation enzymes. CDA was decreased in resistant cells, but the cytosolic pyrimidine nucleotidase NT5C3 was increased in the resistant cell lines as well as DCTPP1, which can degrade deoxynucleoside triphosphates. Moreover several DNA repair enzymes SAMHD1, MTH1 and TDP1 were increased. However, inhibition of MTH1 and DCTPP1 did not increase sensitivity to RX-3117. In conclusion, induction of resistance to the novel cytidine analog RX-3117 led to a decreased accumulation of RX-3117 ribonucleotides, which might be associated with an increased degradation. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Btissame El Hassouni, Richard J. Honeywell, Joris R. Julsing, Beatrice Balboni, Daniel J. De Klerk, Safet Zekanovic, Kees Smid, Elisa Giovannetti, Young B. Lee, Deog J. Kim. Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1267.

Smooth Muscle Cell Proangiogenic Phenotype Induced by Cyclopentenyl Cytosine Promotes Endothelial Cell Proliferation and Migration

Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) are in close contact with blood vessels. SMC phenotypes can be altered during pathological vascular remodeling. However, how SMC phenotypes affect EC properties remains largely unknown. In this study, we found that PDGF-BB-induced synthetic SMCs suppressed EC proliferation and migration while exhibiting increased expression of anti-angiogenic factors, such as endostatin, and decreased pro-angiogenic factors, including CXC motif ligand 1 (CXCL1). Cyclopentenyl cytosine (CPEC), a CTP synthase inhibitor that has been reported previously to inhibit SMC proliferation and injury-induced neointima formation, induced SMC redifferentiation. Interestingly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration because of an increase in CXCL1 along with decreased endostatin production in SMCs. Addition of recombinant endostatin protein or blockade of CXCL1 with a neutralizing antibody suppressed the EC proliferation and migration induced by CPEC-conditioned SMC medium. Mechanistically, CPEC functions as a cytosine derivate to stimulate adenosine receptors A1 and A2a, which further activate downstream cAMP and Akt signaling, leading to the phosphorylation of cAMP response element binding protein and, consequently, SMC redifferentiation. These data provided proof of a novel concept that synthetic SMC exhibits an anti-angiogenic SMC phenotype, whereas contractile SMC shows a pro-angiogenic phenotype. CPEC appears to be a potent stimulator for switching the anti-angiogenic SMC phenotype to the pro-angiogenic phenotype, which may be essential for CPEC to accelerate re-endothelialization for vascular repair during injury-induced vascular wall remodeling.

Cyclopentenyl cytosine promotes endothelial protection by inducing smooth muscle re‐differentiation in vascular remodeling

Vascular remodeling due to endothelium denudation followed by pathological smooth muscle cell (SMC) proliferation is a major medical challenge in cardiovascular intervention. Our previous studies h...

Animal Models in Studies of Cardiotoxicity Side Effects from Antiblastic Drugs in Patients and Occupational Exposed Workers

Cardiotoxicity is an important side effect of cytotoxic drugs and may be a risk factor of long-term morbidity for both patients during therapy and also for staff exposed during the phases of manipulation of antiblastic drugs. The mechanism of cardiotoxicity studied in vitro and in vivo essentially concerns the formation of free radicals leading to oxidative stress, with apoptosis of cardiac cells or immunologic reactions, but other mechanisms may play a role in antiblastic-induced cardiotoxicity. Actually, some new cytotoxic drugs like trastuzumab and cyclopentenyl cytosine show cardiotoxic effects. In this report we discuss the different mechanisms of cardiotoxicity induced by antiblastic drugs assessed using animal models.

Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117; TV-1360)

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

CTP Synthase 1, a Smooth Muscle–Sensitive Therapeutic Target for Effective Vascular Repair

Vascular remodeling as a result of smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, antineointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, because of their nonspecific effect on endothelial cells (ECs). The objective of this study is to identify a therapeutic target that differentially regulates SMC and EC proliferation. Using both rat balloon injury and mouse wire injury models, we identified CTP synthase 1 (CTPS1) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization as a result of induction of CTP synthesis salvage pathway enzymes nucleoside-diphosphate kinase A and B in ECs. Diphosphate kinase B seemed to preserve EC proliferation via use of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelialization in vivo. Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair after injury.

Is there an anti‐adenoviral drug on the horizon?

AbstractAdenovirus ocular infections (epidemic keratoconjunctivitis [EKC], follicular conjunctivitis, and pharyngeal conjunctival fever) are the most common ocular viral infections worldwide, including more than 1,000,000 cases annually in Japan. To date, there is no US FDA or European Medicines Agency approved antiviral therapy for these infections. Little progress on anti‐adenoviral development was made until our group developed the first reliable animal model for ocular anti‐adenoviral testing 20 years ago. Using that animal model, our group was instrumental in the preclinical development of the first topical antiviral drug for the treatment of adenoviral conjunctivitis tested in clinical trials, the nucleoside analog cidofovir. However, toxicity concerns, not efficacy, caused the sponsor to discontinue development. Since then, there has been considerable progress made regarding the discovery and development of antivirals by both academia and the pharmaceutical industry. Preclinical in vitro and in vivo studies have identified several new antiviral candidates such as 2,3‐dideoxycytidine (ddC), cyclopentenylcytosine (CPE‐C), intravenous immune globulin (IVIG), NVC‐422, and FST‐100. These preclinical studies have led to industry sponsored clinical trials which have been initiated with several agents NVC‐422 (NovaBay), Zirgan (0.15% Ganciclovir gel, Bausch + Lomb), and FST‐100 (0.4% povidone‐iodine/0.1% dexamethasone, Foresight Biotherapeutics). The first antiviral to treat adenovirus ocular infections will hopefully be available in the not too distant future. Commercial interest

Identification of critical host mitochondrion-associated genes during Ehrlichia chaffeensis infections.

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis (HME). To determine what host components are important for bacterial replication, we performed microarray analysis on Drosophila melanogaster S2 cells by comparing host gene transcript levels between permissive and nonpermissive conditions for E. chaffeensis growth. Five-hundred twenty-seven genes had increased transcript levels unique to permissive growth conditions 24 h postinfection. We screened adult flies that were mutants for several of the "permissive" genes for the ability to support Ehrlichia replication. Three additional D. melanogaster fly lines with putative mutations in pyrimidine metabolism were also tested. Ten fly lines carrying mutations in the genes CG6479, separation anxiety, chitinase 11, CG6364 (Uck2), CG6543 (Echs1), withered (whd), CG15881 (Ccdc58), CG14806 (Apop1), CG11875 (Nup37), and dumpy (dp) had increased resistance to infection with Ehrlichia. Analysis of RNA by quantitative real-time reverse transcription-PCR (qRT-PCR) confirmed that the bacterial load was decreased in these mutant flies compared to wild-type infected control flies. Seven of these genes (san, Cht11, Uck2, Echs1, whd, Ccdc58, and Apop1) encoded proteins that had mitochondrial functions or could be associated with proteins with mitochondrial functions. Treatment of THP-1 cells with double-stranded RNA to silence the human UCK2 gene indicates that the disruption of the uridine-cytidine kinase affects E. chaffeensis replication in human macrophages. Experiments with cyclopentenyl cytosine (CPEC), a CTP synthetase inhibitor and cytosine, suggest that the nucleotide salvage pathway is essential for E. chaffeensis replication and that it may be important for the provision of CTP, uridine, and cytidine nucleotides.

Cyclopentenyl Cytosine Induces Senescence in Breast Cancer Cells through the Nucleolar Stress Response and Activation of p53

The induction of senescence has emerged as a potentially important contributor to the effects of chemotherapeutic agents against tumors. We have demonstrated that depletion of CTP induced by cyclopentenyl cytosine (CPEC; NSC 375575), a specific inhibitor of the enzyme CTP synthetase, induces irreversible growth arrest and senescence characterized by altered morphology and expression of senescence-associated β-galactosidase activity in MCF-7 breast cancer cells expressing wild-type p53. In contrast, differentiation in the absence of senescence resulted from CPEC treatment in MDA-MB-231 breast cancer cells that express a mutated p53. Both senescence of MCF-7 cells and differentiation of MDA-MB-231 cells were prevented by repletion of CTP through the cytidine salvage pathway. Senescence in MCF-7 cells was associated with a G(2)- and S-phase arrest, whereas differentiation in MDA-MB-231 cells was associated with arrest in G(1) phase at 5 days. Mechanistic studies revealed that CTP depletion induced a rapid translocation of nucleolar proteins, including nucleostemin and nucleolin into the nucleoplasm. This nucleolar stress response resulted in a sustained elevation of p53 and the p53 target genes, p21 and Mdm2, in cells with wild-type p53. Furthermore, short interfering RNA-induced knockdown of p53 in MCF-7 cells treated with CPEC prevented cellular senescence and increased apoptotic cell death. We conclude that CTP depletion and the resulting nucleolar stress response results in a senescence-like growth arrest through activation of p53, whereas cells with mutated p53 undergo differentiation or apoptotic cell death.

Cyclopentenylcytosine does not enhance cisplatin-induced radiosensitization in human lung tumour cells

The search for agents that enhance the effect of ionizing radiation has been an object of study for decades. In this study, the sensitizing properties of cyclopentenylcytosine (CPEC) on radiation and cisplatin-induced radiosensitization in human squamous lung carcinoma cells were investigated. Human lung tumour SW-1573 cells (SWp, parental; SWg, gemcitabine-resistant) were incubated with CPEC and cisplatin and subsequently irradiated with different doses of γ-rays. Clonogenic survival was determined to measure the effectiveness of the treatments. CPEC (1 or 2 μM) treatment for 4 h decreased the plating efficiency to 75 and 50% in SWp and SWg cells, respectively. In the SWg cells, 0.1 and 1 μM CPEC for 4 h enhanced the cell killing effect of cisplatin. However, an increase was not noted in the SWp cells. Due to the moderate toxicity of 1 μM for 4 h, this CPEC dose was used in the radiosensitization experiments. However, CPEC neither radiosensitized the lung tumour cells nor enhanced the radiosensitizing effect of cisplatin. A 2-h incubation with 4 μM cisplatin also decreased the plating efficiency to 75-80% in the two cell lines. Using this cisplatin dose, radiosensitization was obtained in the two cell lines. Although cisplatin treatment clearly radiosensitized the lung tumour cells, CPEC treatment did not. Cisplatin-induced radiosensitization was also not enhanced by CPEC.

Truncated Fluorocyclopentenyl Pyrimidines as S-Adenosylhomocysteine Hydrolase Inhibitors

On the basis of inhibitory activity of truncated cyclopentenyl cytosine against S-adenosylhomocysteine hydrolase (SAH), its fluorocyclopentenyl pyrimidine derivatives were efficiently synthesized from D-ribose via electrophilic fluorination as a key step. The final nucleosides were evaluated for SAH inhibitory activity, among which the uracil derivative 9 showed significant inhibitory activity (IC50 = 8.53 μM). They were also evaluated for cytotoxic effects in several human cancer cell lines such as fibro sarcoma, stomach cancer, leukemia, and colon cancer, but they did not show any cytotoxic effects up to 100 μM, indicating that 4′-hydroxymethyl groups are essential for the anticancer activity.

Cyclopentenyl cytosine has biological and anti-tumour activity, but does not enhance the efficacy of gemcitabine and radiation in two animal tumour models

Cyclopentenyl cytosine (CPEC), targetting the de novo biosynthesis of cytidine triphosphate (CTP), increases the cytotoxicity of gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) alone and in combination with irradiation in several human tumour cells in vitro. We investigated whether CPEC enhances the therapeutic ratio of gemcitabine and irradiation in human pancreatic BxPC-3 xenografts and in rat syngeneic L44 lung tumours. These models were selected because gemcitabine and radiation are used to treat both pancreatic and lung cancer patients and both models differ in growth capacity and in gemcitabine-induced radiosensitisation. A profound dose-dependent CTP-depletion was observed after a single injection of CPEC in both tumour tissue and in normal jejunum. In both models, CPEC alone induced a slight but significant tumour growth delay. The combination of CPEC with gemcitabine, at time intervals that showed CTP-depletion after CPEC, enhanced neither tumour growth delay nor toxicity as compared to gemcitabine alone. In addition, no beneficial effect of CPEC was observed in combination with gemcitabine and radiation. These results suggest that CPEC and gemcitabine alone as well as in combination with radiation target a similar cell population in both tumour models. In conclusion, future clinical development of CPEC as a modulator of gemcitabine combined with radiation is unlikely.

Quantitative analysis of the experimental cytotoxic drug cyclopentenyl cytosine and its metabolite in plasma with HPLC tandem mass spectrometry

The cytotoxic drug cyclopentenyl cytosine (CPEC) is currently being investigated in early clinical trials. Monitoring of plasma levels is required for pharmacokinetic analysis and management of toxicity. This paper describes the analysis of CPEC and cyclopentenyl uracil (CPEU) in plasma by HPLC-electrospray ionization tandem mass spectrometry. The calibration curves for CPEC and the metabolite CPEU were linear up to 10 microm with correlation coefficients of 0.997 (SD = 0.002, n = 10) and 0.997 (SD = 0.004, n = 10), respectively. The detection limit for CPEC was 0.03 and 0.12 microM for CPEU. The intra- and interassay coefficients of variation for CPEC and CPEU were less then 10%. The usefulness of the method was demonstrated by analyzing CPEC and CPEU in plasma of a patient treated with CPEC. HPLC with electrospray ionization tandem mass spectrometry allowed rapid and sensitive determination of CPEC and CPEU levels in plasma.

Human Cytidine Triphosphate Synthetase 1 Interacting Proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.

Cyclopentenyl cytosine increases gemcitabine radiosensitisation in human pancreatic cancer cells

The deoxycytidine analogue 2′,2′-difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a potent radiosensitiser, but has limited efficacy in combination with radiotherapy in patients with pancreatic cancer due to acute toxicity. We investigated whether cyclopentenyl cytosine (CPEC), targetting the ‘de novo’ biosynthesis of cytidine triphosphate (CTP), could increase dFdC cytotoxicity alone or in combination with irradiation in a panel of human pancreatic cancer cells (Panc-1, Miapaca-2, BxPC-3). To investigate the role of deoxycytidine kinase (dCK), the rate-limiting enzyme in the activation of dFdC, human lung cancer cells without (dFdC-resistant SWg) and with an intact dCK gene (dFdC-sensitive SWp) were included. We found that CPEC (100–1000 nmol l−1) specifically reduced CTP levels in a dose-dependent manner that lasted up to 72 h in all cell lines. Preincubation with CPEC resulted in a dose-dependent increase in dFdC incorporated into the DNA only in dFdC-sensitive cells. Consequently, CPEC increased the effectiveness of dFdC (300 nmol l−1 for 4 h) only in dFdC-sensitive cells, which was accompanied by an increase in apoptosis. We also found that CPEC enhanced the radiosensitivity of cells treated with dFdC (30–300 nmol l−1 for 4 h). These results indicate that CPEC enhances the cytotoxicity of dFdC alone and in combination with irradiation in several human tumour cell lines with an intact dCK gene.

The pyrimidin analogue cyclopentenyl cytosine induces alloantigen-specific non-responsiveness of human T lymphocytes.

Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25(high) FoxP3+ CD4+ and CD103+ CD8+ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro.

Cyclopentenyl Cytosine (CPEC): An Overview of its in vitro and in vivo Activity

The experimental cytotoxic drug cyclopentenyl cytosine (CPEC) is an analogue of cytidine. Besides its antiviral effect, its potential use in the treatment of cancer has become an important area of research. CPEC is activated by intracellular phosphorylation ultimately forming its metabolite CPEC-TP. CPEC-TP is a non competitive inhibitor of cytidine-5'-triphosphate synthetase (CTP-synthetase), an important enzyme in the formation of CTP. Studies have shown that cancer cells have a high CTP synthetase activity, thus making them interesting targets for chemotherapy. CPEC has been preclinically studied in different malignancy models. In vitro results on leukemia show activity in the nanomolar range on several cell lines. However in vivo results are conflicting and the findings vary from increase in life span over 100% to only limited effectiveness. Interesting results have been obtained in colorectal and neuroblastoma cells. In several neuroblastoma cell lines incubation with CPEC in combination with cytarabine or gemcitabine has resulted in increased cell death compared to incubation with with only one of the agents. CPEC has been studied in a phase I trial in patients with solid tumors. In five of 26 patients unexplained cardiotoxicity (extreme hypotension) occurred. The cardiotoxic effects could not be reproduced in animal models. However, precautions should be taken when using this drug in future clinical trials. Low dosage of CPEC seems necessary and intensive cardiac monitoring is advisable. In this manuscript, it is demonstrated that CPEC has an anti-cancer effect in several tumor models: CPEC might be a potentially useful drug in anticancer treatment.

Limited antitumor-effect associated with toxicity of the experimental cytotoxic drug cyclopentenyl cytosine in NOD/scid mice with acute lymphoblastic leukemia

The experimental cytotoxic drug cyclopentenyl cytosine (CPEC) is a non-competitive inhibitor of the enzyme cytidine triphosphate (CTP) synthethase. We evaluated the in vitro and in vivo antitumor activity of CPEC on human acute lymphoblastic leukemia (ALL) cell lines. CPEC displayed anti-leukemic activity with IC50 (after 3 days of incubation) ranging from 6 to 15 nM. Subsequently the in vivo activity of CPEC against primary human ALL was evaluated in a xenogeneic model of human ALL using NOD/scid mice inoculated with primary human ALL cells. In the model, only a marginal anti-leukemic activity was observed at 1.5 mg kg −1 (5 days per week) and 5 mg kg −1 (2 days per week), however, this activity was associated with severe systemic toxicity. The observed toxicity was not specific for the NOD/scid model, as toxicity at comparable treatment intensity was also observed in Balb/c mice. In conclusion, although CPEC showed antitumor activity against human ALL cells in vitro, its activity in the in vivo human leukemia model was only marginal and accompanied by severe toxicity.