Cytotoxic Nucleoside Analogues Research Articles

FNC (4'-azido-2'-deoxy-2'-fluoro(arbino)cytidine) as an Effective Therapeutic Agent for NHL: ROS Generation, Cell Cycle Arrest, and Mitochondrial-Mediated Apoptosis.

Cytotoxic nucleoside analogs (NAs) hold great promise in cancer therapeutics by mimicking endogenous nucleosides and interfering with crucial cellular processes. Here, we investigate the potential of the novel cytidine analog, 4'-azido-2'-deoxy-2'-fluoro(arbino)cytidine (FNC), as a therapeutic agent for Non-Hodgkin lymphoma (NHL) using Dalton's lymphoma (DL) as a T-cell lymphoma model. FNC demonstrated dose- and time-dependent inhibition of DL cell growth and proliferation. IC-50 values of FNC were measured at 1 µM, 0.5 µM, and 0.1 µM after 24, 48, and 72 h, respectively. Further elucidation of FNC's mechanism of action uncovers its role in inducing apoptosis in DL cells. Notable DNA fragmentation and nuclear condensation point to activated apoptotic pathways. FNC-induced apoptosis was concomitant with changes in cellular membranes, characterized by membrane rupture and altered morphology. The robust anticancer effects of FNC are linked to its capacity to induce reactive oxygen species (ROS) production, prompting oxidative stress-mediated apoptosis. Additionally, FNC disrupted mitochondrial membrane potential (MMP), leading to mitochondrial dysfunction, further promoting apoptosis. Dysregulation of apoptotic genes, with upregulation of Bax and downregulation of Bcl-2 and Bcl-xl, implicates the mitochondrial-mediated apoptosis pathway. Furthermore, FNC-induced G2/M phase cell cycle arrest was mediated through modulation of the cell cycle inhibitor p21. Overall, this study highlights the potential of FNC as a promising therapeutic agent for NHL.

Bisantrene in Combination with Fludarabine and Clofarabine As Salvage Therapy for Adult Patients with Relapsed or Refractory Acute Myeloid Leukemia (AML)- an Open-Label, Phase II, Study

Background: Bisantrene (Bis), is a topoisomerase-II inhibitor with anthracycline-like activity, lower cardiotoxicity, and activity in patients (pts) with relapsed (rel)/primary refractory (PR) acute myeloid leukemia (AML) (Am J Hematol, 2021). Bis was also found to synergize with the cytotoxic purine nucleoside analogs clofarabine (Clo) and fludarabine (Flu) in vitro (J Clin Exp Oncol, 2021). We now conducted a phase II study combining Bis with Clo and Flu in pts with Rel/PR AML (NCT04989335). Methods: This was a phase II open-label single-center study with 2 stages: a stage with 3+3 dose escalation to assess the safety and provide phase 2 dose (RP2D, 4 vs 5 days) of infusion (i.v.) with up to 12 pts and an expansion phase with up to 17 pts (Simon 2-stage design 9+8) to assess the primary efficacy and confirm the safety of f Bis/Clo/Flu combination pts with Rel/PR AML. Flu (10 mg/m 2) was administered i.v. over 1 hour (h), followed by a 1-h infusion of Clo (30 mg/m 2), and then a 2-h infusion of Bis at 250 mg/m 2, with a 1-hour break for 4 days. The primary endpoint was achieving a partial response (PR). Efficacy was assessed by bone marrow (BM) examination 21 - 30 days post-therapy. Safety included treatment-emergent adverse events (TEAEs), all-cause mortality, and cardiac monitoring with ECG and troponins Toxicity was assessed as per Common Terminology Criteria for Adverse Events (CTCAE v5.0). Interim analysis was planned after the first 12 pts. Results: 20 pts were included from August 2021. The median age was 48 (19-69) years and 55% were male.14 pts had de novo AML and 5 had secondary AML, 11 pts were in rel, while 9 had PR disease. Median lines of therapy were 4(3-9), 12 with ≤ 4 and 8 >4 lines of therapy. All pts were refractory to the last line of therapy.15 pts (75%) were in relapse post allogeneic transplantation (HSCT). 5 (25%) pts had active extramedullary disease (EMD, 1 with CNS involvement). Median bone marrow blasts pre-Bis/Clo/Flu treatment was 50%. Cytogenetic was normal in 10 (50%) of the pts, 5 had a complex karyotype, 2 MLL rearrangements, 1 t8; 21 and 2 pts had other chromosomal abnormalities. The maximum tolerated dose of the Bis/Clo/Flu combination was 4 days of infusion due to grade 3 liver toxicity in 2 pts and 1 pt that died from sepsis in the first stage of the study. Overall 10 pts developed liver toxicity with elevated liver enzymes and bilirubin in 9 of them (grade 1-4 pts; grade 2-3 pts and grade 3-3 pts). Liver toxicity was transient and resolved in all pts. Two pts had treatment interruption due to liver toxicity. 4 pts had grade I renal toxicity, 7 had mild fluid retention, and 18 developed neutropenic fever of which 10 pts had verified bacteremia. Five pts had pneumonia, 4 invasive fungal infections, and 2 sepsis (1/2 died).1 additional pt died from cerebral venous sinus thrombosis. Overall 5 pts died early and could not be evaluated for response. Clinical-relevant cardiac toxicity was not observed in any of the pts and no one developed ECG changes Transient grade 1 elevation of troponin levels was observed in 4 pts. Six pts responded (CR-5, PR-1) 3 with EMD, however, responses were short-lived and lasted up to 3 months. Five pts (responding-4, active disease-1, being the second transplant in 4) underwent HSCT 1-3 months post-Bis/Clo/Flu with reduced /intermediate intensity conditioning. Stem cell donors were haploidentical-2, matched unrelated -1, and matched sibling-2, respectively. Three pts died: one from graft versus host disease and the other from relapse within 4 months post-transplant and the third from sepsis 2 years post-HSCT. Two of the transplanted pts are in complete remission with a 2-month follow-up. Conclusions : In this Phase II study in a very advanced group of relapsed refractory AML ptsresistant to multiple previous lines of chemotherapy including transplantation and with a median of 50% blasts at study initiation, Bis/Clo/Flu combination therapy was found to be safe and well tolerated without cardiac toxicity or tumor lysis syndrome. The maximum length of Bis/Clo/Flu administration was 4 days due to rapidly reversible liver toxicity, and transaminitis. As expected in this highly pretreated population, the infection rates were high. Six /15 evaluable pts (40%) respond, enabling an HSCT in 5 of them. These rather impressive results in such a heavily pretreated population support further studies of Bisantrene-based combinations, including those with venetoclax or hypomethylating agents.

Therapeutic Potential and Biological Applications of Cordycepin and Metabolic Mechanisms in Cordycepin-Producing Fungi

Cordycepin (3′-deoxyadenosine), a cytotoxic nucleoside analogue found in Cordyceps militaris, has attracted much attention due to its therapeutic potential and biological value. Cordycepin interacts with multiple medicinal targets associated with cancer, tumor, inflammation, oxidant, polyadenylation of mRNA, etc. The investigation of the medicinal drug actions supports the discovery of novel targets and the development of new drugs to enhance the therapeutic potency and reduce toxicity. Cordycepin may be of great value owing to its medicinal potential as an external drug, such as in cosmeceutical, traumatic, antalgic and muscle strain applications. In addition, the biological application of cordycepin, for example, as a ligand, has been used to uncover molecular structures. Notably, studies that investigated the metabolic mechanisms of cordycepin-producing fungi have yielded significant information related to the biosynthesis of high levels of cordycepin. Here, we summarized the medicinal targets, biological applications, cytotoxicity, delivery carriers, stability, and pros/cons of cordycepin in clinical applications, as well as described the metabolic mechanisms of cordycepin in cordycepin-producing fungi. We posit that new approaches, including single-cell analysis, have the potential to enhance medicinal potency and unravel all facets of metabolic mechanisms of cordycepin in Cordyceps militaris.

Lead optimization and biological evaluation of fragment-based cN-II inhibitors

The development of cytosolic 5′-nucleotidase II (cN-II) inhibitors is essential to validate cN-II as a potential target for the reversion of resistance to cytotoxic nucleoside analogues. We previously reported a fragment-based approach combined with molecular modelling, herein, the selected hit-fragments were used again in another computational approach based on the Ilib-diverse (a software enabling to build virtual molecule libraries through fragment based de novo design) program to generate a focused library of potential inhibitors. A molecular scaffold related to a previously identified compound was selected and led to a novel series of compounds. Ten out of nineteen derivatives showed 50–75% inhibition on the purified recombinant protein at 200 μM and among them three derivatives (12, 13 and 18) exhibited Ki in the sub-millimolar range (0.84, 2.4 and 0.58 mM, respectively). Despite their only modest potency, the cN-II inhibitors showed synergistic effects when used in combination with cytotoxic purine nucleoside analogues on cancer cells. Therefore, these derivatives represent a family of non-nucleos(t)idic cN-II inhibitors with potential usefulness to overcome cancer drug resistance especially in hematological malignancies in which cN-II activity has been described as an important parameter.

Efficacy of lentivirus‑mediated Drosophilamelanogaster deoxyribonucleoside kinase combined with (E)‑5‑(2‑bromovinyl)‑2'‑deoxyuridine or 1‑β‑D‑arabinofuranosylthymine therapy in human keloid fibroblasts.

Keloid scarring is a type of fibroproliferative disease with a high recurrence rate. However, no effective treatment is currently available. Combined therapy with recombinant lentivirus‑mediated Drosophilamelanogaster deoxyribonucleoside kinase (Dm‑dNK) and prodrug has been widely studied and used for cancer treatment. Due to the similarities between keloid scars and tumors, the aim of the present study was to investigate the efficacy of a Dm‑dNK/nucleoside analog system for the treatment of keloid scars. Recombinant lentivirus expression of the Dm‑dNK suicide gene was assessed. Western blotting was used to examine the protein expression of lentivirus mediated Dm‑dNK in keloid fibroblasts. Enzyme activity assays were conducted using [3H]‑labeled substrates. Furthermore, cytotoxicity and bystander effects were evaluated using MTT assays. The expression of green fluorescent protein was observed using fluorescence microscope and results indicated that there was no notable difference in lentivirus infectivity between the multiplicity of infection (MOI) of 1 and 10 in cells. Notably, western blotting revealed that Dm‑dNK was stably expressed in keloid fibroblasts and the enzymatic activity assays revealed that the enzyme was activated following introduction into the keloid fibroblasts via the lentivirus. The cytotoxicity and bystander effects of Dm‑dNK combined with cytotoxic nucleoside analogs were both observed in Dm‑dNK+ keloid fibroblasts. These results demonstrated that the lentivirus‑mediated Dm‑dNK therapy may be effective in treating keloid fibroblasts, which provides some evidence for the use of Dm‑dNK/prodrug therapy for keloid treatment invivo in the future.

Efficacy and Toxicity Profile of Clofarabine in Patients with Recurrent/Refractory Langerhans Cell Histiocytosis and Other Histiocytic Disorders

Introduction: Langerhans Cell Histiocytosis (LCH) is an inflammatory myeloid neoplastic disorder. Patients with recurrent or refractory LCH are at increased risk of mortality and long-term morbidity. The standard of care for high-risk LCH patients who are refractory to vinblastine/prednisone or who relapse is very high dose While mostly cytarabine/cladribine. While effective, this strategy also has very high treatment-related mortality. Clofarabine is a nucleoside analog with efficacy in other myeloid malignancies. Case reports have suggest that is has activity in LCH and other histiocytic disorders at moderate doses. The purpose of this study was to report the efficacy and toxicity profile of a restrospective cohort of patients with histiocytic disorders treated with clofarabine.Methods: Medical records were retrospectively reviewed for 26 pediatric patients with histiocytic disorders who were treated with clofarabine. Twenty-one of these patients had LCH and had failed at least one prior systemic therapy, while the remaining 5 patients had other histiocytic disorders (JXG, Rosai-Dorfman disease or mixed histiocytic disease). Patients were treated for a minimum of 6 months of with clofarabine (25 mg/m2/day x 5 days every 28 days), were reassessed for response at the end of therapy and monitored for relapse or progression post-treatment.Results: All patients in this series started treatment under the age of 18 years old (median=1.5 years; range: 0.3-16.3). Patients with LCH had received a median of 3 prior treatments (range:1-5). A majority of the patients (n = 17, 65%) were received all clofarabine infusions in an outpatient clinic. The most common adverse event was fever requiring hospital admission (n = 18; 69%), followed by Grade 3 neutropenia (n = 12; 46%), and intractable nausea/vomiting (n = 7; 27%). Additional adverse events included Grade 3 anemia, Grade 2 dehydration, Grade 3 cytopenias, and Grade 3 infection. Overall survival was 100%. LCH patients who completd 6-months of clofarabine had an 85% overall response (33% complete response, 52% partial response); 10% progressed on therapy and 5% had stable disease. Relapse occurred in 5 (28%) patients with LCH after completion of treatment (median time to relapse=22 months; range: 1-42). The odds of relapse was approximately 4-times greater in LCH patients with CNS involvement (n=16) compared to those without (n=4), and also in those with high risk disease (n=5) relative to standard risk (n=10). One of the patients who experienced disease recurrence received an additional 12 cycles of clofarabine and is currently doing well with no active disease.Conclusions: Clofarabine may be an effective and relatively safe salvage therapy for LCH and other histiocytic disorders. Future prospective trials for patients with refractory histiocytic disorders will directly compare efficacy and toxicity relative to other current salvage strategies with cytotoxic nucleoside analogs or targeted inhibitors. DisclosuresNo relevant conflicts of interest to declare.

The pivotal role of uridine-cytidine kinases in pyrimidine metabolism and activation of cytotoxic nucleoside analogues in neuroblastoma

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine as well as the pharmacological activation of several cytotoxic pyrimidine ribonucleoside analogues. In this study, we investigated the functional role of two isoforms of UCK in neuroblastoma cell lines. Analysis of mRNA coding for UCK1 and UCK2 showed that UCK2 is the most abundantly expressed UCK in a panel of neuroblastoma cell lines. Transient and stable overexpression of UCK2 in neuroblastoma cells increased the metabolism of uridine and cytidine as well as the cytotoxicity of 3-deazauridine. Knockdown of endogenous UCK2 as well as overexpression of UCK1 resulted in decreased metabolism of uridine and cytidine and protected the neuroblastoma cells from 3-deazauridine-induced toxicity. Subcellular localization studies showed that UCK1-GFP and UCK2-GFP were localized in the cell nucleus and cytosol, respectively. However, co-expression of UCK1 with UCK2 resulted in a nuclear localization of UCK2 instead of its normal cytosolic localization, thereby impairing its normal function. The physical association of UCK1 and UCK2 was further demonstrated through pull-down analysis using his-tagged UCK. The discovery that UCK2 is highly expressed in neuroblastoma opens the possibility for selectively targeting neuroblastoma cells using UCK2-dependent pyrimidine analogues, while sparing normal tissues.

Tanshinone IIA enhances bystander cell killing of cancer cells expressing Drosophila melanogaster deoxyribonucleoside kinase in nuclei and mitochondria.

Heterologous expression of the Drosophila melanogaster multi-substrate deoxyribonucleoside kinase (Dm-dNK) increases the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Thus, it may be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize this potential suicide gene, we constructed two retroviral vectors that enabled the expression of Dm-dNK in cancer cells. One vector harbored the wild‑type enzyme that localized to the nucleus. The other vector harbored a mitochondrial localized mutant enzyme that was constructed by deleting the nuclear localization signal and fusing it to a mitochondrial import signal of cytochrome c oxidase. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant viruses. The sensitivity and bystander cell killing in the presence of pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)‑2'‑deoxyuridine and 1-β-D-arabinofuranosylthymine were investigated. TanshinoneIIA is a constituent of Danshen; a traditional Chinese medicine used in the treatment of cardiovascular diseases. This study also looked at the influence of TanshinoneIIA on the bystander effect and the underlying mechanisms. We showed that sensitivity of the osteosarcoma cell line to the nucleoside analogs and the efficiency of bystander cell killing were independent of the subcellular localization of Dm-dNK. The enhanced effect of tanshinone IIA on the bystander effect was related to the increased expression of Cx43 and Cx26.

The purine analog fludarabine acts as a cytosolic 5′-nucleotidase II inhibitor

For several years the IMP/GMP-preferring cytosolic 5′-nucleotidase II (cN-II) has been considered as a therapeutic target in oncology. Indeed, various reports have indicated associations between cN-II expression level and resistance to anticancer agents in several cancer cell lines and in patients affected with neoplasia, mainly by hematologic malignancies. In this paper we present evidence showing that, among the commonly used cytotoxic nucleoside analogs, fludarabine can act as a cN-II inhibitor. In vitro studies using the wild type recombinant cN-II demonstrated that fludarabine inhibited enzymatic activity in a mixed manner (Ki 0.5mM and Ki′ 9mM), whereas no inhibition was observed with clofarabine and cladribine. Additional experiments with mutant recombinant proteins and an in silico molecular docking indicated that this inhibition is due to an interaction with a regulatory site of cN-II known to interact with adenylic compounds. Moreover, synergy experiments between fludarabine and 6-mercaptopurine in human follicular lymphoma (RL) and human acute promyelocytic leukemia (HL-60) cells transfected with control or cN-II-targeting shRNA-encoding plasmids, showed synergy in control cells and antagonism in cells with decreased cN-II expression. This is in line with the hypothesis that fludarabine acts as a cN-II inhibitor and supports the idea of using cN-II inhibitors in association with other drugs to increase their therapeutic effect and decrease their resistance.

Antitumoral activity of a trichloromethyl pyrimidine analogue: molecular cross-talk between intrinsic and extrinsic apoptosis.

Acute lymphoblastic leukemia (ALL) is a malignant disorder caused by the proliferation of lymphoid progenitor cells and is the most common cancer in children. Cytotoxic nucleoside analogues are important chemotherapeutic agents, which are used in many cancers, including leukemias. In this study, we investigated the effects of the synthetic nucleoside analogue 1-(5,5,5-trichloro-2-methoxy-4-oxopenten-2-yl)-4-trichloromethyl-pyrimidin-2(1H)-one, named compound 3 or C3, on leukemia cell lines. The compound stimulated cell death by apoptosis, evidenced by DNA fragmentation, phosphatidylserine externalization, and caspase-3 activation. Compound 3 seemed to trigger several cell death pathways. The mitochondrial pathway was evidenced through a disturbance of mitochondrial membrane potential, strong cytochrome c liberation, decrease of antiapoptotic Bcl-2 protein expression, and caspase-9 activation. The C3 also induced caspase-8 and -12 activation, an increase in the intracellular calcium level, and an overproduction of reactive oxygen species. Increased caspase 8 activity suggests that the extrinsic pathway was activated and that the ROS production and enzyme activity alteration (glutathione S-transferase, glutathione peroxidase, catalase, and glutathione reductase) might be related to oxidative stress. Finally, the increase in calcium release, CHOP expression, and caspase-12 activity might characterize endoplasmic reticulum stress. Compound 3 was likewise cytotoxic to leukemic and melanoma human cell lines. Taken together, the results contribute to further understanding the new pyrimidine analogue as a potential chemotherapeutic drug or lead molecule.

Structure–activity relationships of β-hydroxyphosphonate nucleoside analogues as cytosolic 5′-nucleotidase II potential inhibitors: Synthesis, in vitro evaluation and molecular modeling studies

The cytosolic 5′-nucleotidase II (cN-II) has been proposed as an attractive molecular target for the development of novel drugs circumventing resistance to cytotoxic nucleoside analogues currently used for treating leukemia and other malignant hemopathies. In the present work, synthesis of β-hydroxyphosphonate nucleoside analogues incorporating modifications either on the sugar residue or the nucleobase, and their in vitro evaluation towards the purified enzyme were carried out in order to determine their potency towards the inhibition of cN-II. In addition to the biochemical investigations, molecular modeling studies revealed important structural features for binding affinities towards the target enzyme.

Steroid hormones are novel nucleoside transport inhibitors by competition with nucleosides for their transporters

Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

Consistent efficacy and safety of gemcitabine-paclitaxel in patients with metastatic breast cancer: A retrospective comparison of East Asian and global studies

The incidence of breast cancer in China is increasing at a rate greater than the global average. For treatment of metastatic breast cancer, a phase 3 trial of 529 patients (study JHQG) found that the combination of gemcitabine, a cytotoxic nucleoside analog, and paclitaxel, a taxane, significantly improved both overall and progression-free survival compared with paclitaxel monotherapy. As ethnic differences in the safety and efficacy of some anticancer therapies have been reported, this review provides oncologists treating East Asian patients with an evidence base to extrapolate results of the predominately Caucasian phase 3 JHQG study to their patients. Three phase 2 studies in Chinese or Japanese patients with metastatic breast cancer are reviewed with reference to the global study JHQG. The results of pharmacokinetic, efficacy and safety analyses are descriptively compared between the global and Asian studies. Peak and total drug exposure values differed by less than 10% across the studies. Response rate, overall and progression-free survival values were similar, although values from the Asian studies for each of these parameters were slightly higher. Asian patients had higher rates of hematologic toxicities but lower rates of other adverse events. Examination of the data from three studies of the gemcitabine-paclitaxel combination reveals no clinically significant ethnic differences in efficacy and safety between East Asian patients and their global counterparts. Given its demonstrated efficacy and safety profile, the gemcitabine-paclitaxel regimen should be one of the standard treatments for East Asian patients with metastatic breast cancer.

The adenosine transporter, ENT1, in cardiomyocytes is sensitive to inhibition by ethanol in a kinase-dependent manner: implications for ethanol-dependent cardioprotection and nucleoside analog drug cytotoxicity

The adenosine transporter 1 (ENT1) transports nucleosides, such as adenosine, and cytotoxic nucleoside analog drugs. ENT1 is well established to play a role in adenosinergic signaling in the cardiovascular system by modulating adenosine levels. Moderate ethanol consumption is cardioprotective and underlying mechanisms of action are not clear although adenosinergic signaling has been implicated. Here, we show that ethanol (5-200 mM) significantly reduces ENT1-dependent [(3)H] 2-chloroadenosine uptake (by up to 27 %) in the cardiomyocyte cell line, HL-1. Inhibition or absence of ENT1 is known to be cardioprotective, suggesting that the interaction of ethanol with ENT1 may promote adenosinergic cardioprotective pathways in the cardiovasculature.Ethanol sensitivity of adenosine uptake is altered by pharmacological activation of PKA and PKC. Primary cardiomyocytes from PKCε-null mice have significantly greater sensitivity to inhibition (by approximately 37 %) of adenosine uptake by ethanol than controls. These data suggest that the presence of ethanol may compromise ENT1-dependent nucleoside analog drug cytotoxicity, and indeed, ethanol (5 mM) reduces the cytotoxic effects of gemcitabine (2 nM), an anti-cancer drug, in the human cancer cell line, HTB2. Thus, the pharmacological inhibition of ENT1 by ethanol may contribute to ethanol-dependent cardioprotection but compromise gemcitabine cytotoxicity.

Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake

It has long been established that the Trypanosoma brucei TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest Trypanosoma congolense paralogue, TcoAT1, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of TcoAT1 and show that in fact it is the syntenic homologue of another T. brucei gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The T. congolense genome does not seem to contain a syntenic equivalent to TbAT1. Two TcoAT1 alleles, differentiated by three independent SNPs, were expressed in the T. brucei clone B48, a TbAT1-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); TbAT1 was also expressed as a control. The TbAT1 and TcoAT1 transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only TbAT1 increased sensitivity to diamidines and to cymelarsan. Uptake of [3H]-diminazene was detectable only in the B48 cells expressing TbAT1 but not TcoAT1, whereas uptake of [3H]-inosine was increased by both TcoAT1 alleles but not by TbAT1. Uptake of [3H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in T. congolense.

Identification and characterization of inhibitors of cytoplasmic 5′-nucleotidase cN-II issued from virtual screening

Clinical and preclinical observations have lead to the hypothesis that 5′-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se or to increase the activity of cytotoxic nucleoside analogs.To identify potential cN-II inhibitors, we performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant cN-II, soaking experiments with crystals of truncated cN-II as well as biological evaluation of selected compounds, alone or in combination with cytotoxic nucleoside analogs, on cancer cells.The top ranked compounds from virtual screening included an anthraquinone derivative (AdiS) that were shown to block the enzyme activity with a Ki of 2.0mM. Soaking experiments performed with crystals of truncated cN-II allowed to obtain crystallographic data at a resolution of 2.9Å and indicating interaction between AdiS and F354/I152 situated in the effector site 1 of cN-II. In addition, this compound exhibited different levels of cytotoxicity in vitro on several cancer cell lines and increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5μM cladribine, 0.05μM clofarabine or 30μM fludarabine. Finally, AdiS showed synergy with cladribine and additivity with clofarabine.This study showed that virtual screening is a useful tool for the identification of potent cN-II inhibitors, and our biological results indicated interesting activity for one lead compound that can be further developed as therapeutics.

Preclinical rationale for synergistic interaction of pemetrexed and cytotoxic nucleoside analogues

Cytotoxic nucleoside analogues are widely used in cancer chemotherapy. We used the cytosine arabinoside (Ara-C)-resistant erythroleukaemia cell line K562 and the Ara-C-sensitive myeloid leukaemia cell line HL60 to examine the differential expression of molecular markers. We found increased expression levels of deoxycytidine kinase (dCK) and human equilibrative nucleoside transporter 1 (hENT1) and decreased levels of multidrug resistance protein 5 (ABCC5) and ribonucleoside reductase subunit M1 (RRM1) expression in Ara-C-sensitive HL60 cells. We previously established the pemetrexed (MTA)-resistant small cell lung cancer cell lines PC6/MTA-0.4 and PC6/MTA-1.6 and found that MTA-resistant cells are more sensitive to gemcitabine (GEM) and Ara-C compared with parental PC-6 cells. We examined the molecular markers for GEM and Ara-C sensitivity in MTA-resistant cells and found increased gene expression of dCK and hENT1. Furthermore, treatment with MTA resulted in increased expression of dCK and hENT1 and decreased expression of ABCC5 and RRM1, concomitant with the alteration of the resistance to Ara-C in Ara-C-resistant K562 cells. These results provide evidence that the chemotherapeutic activity of the combination of MTA and cytotoxic nucleoside analogues is synergistic with regard to the alteration of metabolic molecules.

Elacytarabine has single‐agent activity in patients with advanced acute myeloid leukaemia

Elacytarabine is a novel cytotoxic nucleoside analogue, independent of nucleoside transporters (e.g. human Equilibrative Nucleoside Transporter 1 [hENT1]) for cell uptake, and mechanisms of action similar to those of cytarabine. This Phase II study assessed the efficacy and safety of elacytarabine in patients with advanced stage acute myeloid leukaemia (AML). Patients received 2000 mg/m(2) per d continuously i.v. during days 1-5 every 3 weeks. Patients were matched by six risk factors with historical controls; remission rate (assessed after 1 or 2 cycles) and 6-month survival were compared. Sixty-one patients, median age 58 years, were enrolled; 52% had five or six risk factors. The remission rate was 18% (95% confidence interval: 9-30%) vs. 4% in controls (P < 0·0001), 6-month survival rate was 43%, median overall survival was 5·3 months (vs. 1·5 months); 10 patients (16%) were referred for stem cell transplantation after treatment. Side effects were predictable and manageable. The most common grade 3/4 non-haematological adverse events were febrile neutropenia, hypokalemia, fatigue, hyponatraemia, dyspnoea and pyrexia. Thirty-day all-cause mortality, after start of treatment, was 13% vs. 25% in controls. Elacytarabine has monotherapy activity in patients with advanced AML. This study provides proof-of-concept that lipid esterification of nucleoside analogues is clinically relevant.

Lentivirus-mediated expression of Drosophila melanogaster deoxyribonucleoside kinase driven by the hTERT promoter combined with gemcitabine: A potential strategy for cancer therapy

In contrast to other enzymes, Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) has a broad substrate specificity and high catalytic rate when transferred in human cells. This makes it a promising therapeutic agent when administered together with several cytotoxic nucleoside analogs, such as gemcitabine 2',2'-difluoro-deoxycytidine (dFdC). Therefore, lentiviral vectors, which potentially allow stable long-term transgene expression, are good candidates for gene delivery vehicles. In the present study, we successfully developed a lentivirus-mediated transgene expression system of Dm-dNK under the control of hTERT promoter against the breast cancer cell line (Bcap37), the gastric cancer cell line (SGC7901) and the normal fibroblast cell line (WI-38). Moreover, we also analyzed its targeted cytotoxicity in vitro with treatment of the prodrug dFdC. Bcap37 tumor growth was inhibited in nude mice. Both cancer cell lines exhibited apparent cytotoxicity when infected with recombinant lentivirus constructs expressing Dm-dNK. In contrast, lentivirus-infected WI-38 cells exhibited less cytotoxicity. These data suggested that Dm-dNK was sensitive to dFdC, and it resulted in synergistic growth inhibition and apoptosis induction in vitro. In addition, Lenti-hTERT-dNK/dFdC also suppressed tumor growth in vivo. Our results suggest that the Lenti-hTERT-dNK/dFdC system is a safe and feasible treatment strategy in the development of suicide gene therapy.

Abstract B50: Per oral nanogel conjugates of activated nucleoside analogs are effective in the treatment of drug-resistant tumors

Abstract Purpose: Chemotherapeutic application of cytotoxic nucleoside analogs and bases resulted in the development of specific mechanisms of drug resistance. Numerous attempts to develop phosphorylated prodrugs of nucleoside analogs effective in vivo have not yet yielded clinical drugs. On the other hand, there exist a very limited list of oral anticancer drugs despite of the fact that per oral administration remains a preferred route of administration due to its convenience and better patient compliancy. Recently, we developed novel conjugates of biocompatible polymers and phosphorylated nucleoside analogs, which fold into small nanogel particles in aqueous media and show an enhanced activity against aggressive and drug-resistant tumor cells. These nanogel-drug conjugates are stable in conditions existing in the gastrointestinal tract, but can release activated drug molecules after internalization and enzymatic hydrolysis in cancer cells. Method: Poly(vinyl alcohol), a nontoxic biocompatible polymer, having excellent mucoadhesive properties that are important for gastrointestinal (GI) absorption, was decorated with cholesterol moieties(CPVA) and conjugated with floxuridine(FU) and N-acylated gemcitabine(Gem) via a tetraphosphorylated linker. These CPVA-drug conjugates formed compact nanogels (20-60 nm in diameter) in aqueous media. Drug content in the conjugates was usually 8–12%. We examined cytotoxicity of the nanogel-drug conjugates in human breast, prostate and drug-resistant pancreatic cancer, leukemic and lymphoma cells. Tumor growth inhibition was evaluated in s.c. xenograft animal models following the administration of nanogel-drug conjugates peritumorally or orally, for example, by liquid gavage or with drinking water ad libitum. Results: The hydrophobic modification by cholesterol moieties played a significant part in the intracellular drug accumulation due to higher cytotoxic activity. In most cases, the nanogel-drug conjugates demonstrated a 3- to 30-fold higher efficacy than nucleoside analogs showing both cytotoxic and cytostatic activities. We found selective drug release patterns depending on the environmental pH and tissue-related enzymatic activities. Importantly, these conjugates were stable at low pH and, therefore, could be administered orally. High viscosity of nanogel-drug conjugate solutions is also a positive factor increasing their absorption in GI tract and providing a depot-like pattern of drug release after s.c. injections. Due to the molecular mass of nanogel-drug conjugates (&amp;gt;50 kDa), absorbed drug nanoformulations can potentially be retained in tumors via leaking neovasculature due to the enhanced permeability and retention effect. In all experiments, we observed sustained antitumor effects at relatively low drug doses compared to control groups and no chronic toxicity or weight loss was detected. Conclusion: Nanogel drug conjugates demonstrated advanced accumulation and drug release profiles during the treatment of cancer cells resistant to nucleoside analogs and displayed a strong growth inhibition of drug-resistant tumors in xenograft animal models following per oral administration. Therefore, nanogel drug conjugates constitute a novel efficient formulation for the treatment of drug resistant aggressive cancers. Acknowledgements: The National Cancer Institute's support CA136921 (for S.V.V.) is greatly appreciated.