Accumulation Of Triphosphate Research Articles

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

A phase II study of biweekly gemcitabine at fixed dose rate infusion plus S1 as first-line chemotherapy in patients with locally advanced or metastatic pancreatic cancer.

357 Background: The combination regimen of gemcitabine plus S1 (GS) as first-line chemotherapy has shown good efficacy in advanced pancreatic cancer. However, it is inconvenient to deliver gemcitabine on days 1 and 8 within one treatment cycle. In addition, previous studies have demonstrated that the gemcitabine administration by fixed dose rate infusion of 10 mg/m2/min should maximize the rate of intracellular accumulation of gemcitabine triphosphate and might improve clinical efficacy. The aim of this study was to evaluate the efficacy and safety of biweekly gemcitabine at fixed dose rate infusion combined with S1 (FGS) in chemonaive patients with locally advanced or metastatic pancreatic cancer. Methods: Forty-three eligible patients with locally advanced or metastatic pancreatic cancer received gemcitabine at a fixed dose of 10 mg/m2/min intravenously over a 2-h period on day 1, together with oral S1 40mg/m2twice daily on days 1 to 10, every 2 weeks. Tumor assessment was performed for every 3 cycles of chemotherapy. Results: All patients were evaluable for toxicity and 42 patients for efficacy. The overall response rate was 35.7% with 1 complete response, 14 partial responses, 17 stable diseases, and 10 progressions. Median time to progression was 5.9 months and median overall survival was 9.6 months. The main grade 3/4 toxicities included neutropenia (30.2%), thrombocytopenia (6.9%), anemia (2.3%), nausea (4.6%), diarrhea (2.3%). Conclusions: The biweekly FGS regimen is active, well tolerated and conveniently delivered as first-line chemotherapy for patients with locally advanced or metastatic pancreatic cancer.

Plasma and intracellular exposure to ganciclovir in adult renal transplant recipients: is there an association with haematological toxicity?

Ganciclovir is the most widely used treatment for cytomegalovirus infections. However, neutropenia is a frequent associated adverse effect leading to a decrease in the ganciclovir dose or discontinuation of the therapy, thereby favouring viral resistance. In the present study, the objectives were: (i) to describe the pharmacokinetics of blood and intracellular ganciclovir and its metabolites; and (ii) to explore the relationship between exposure to ganciclovir and/or its metabolites and evolution of the neutrophil count under treatment. Pharmacokinetic profiles (pre-dose and 1, 2, 3 and 5 h after dosing) of ganciclovir and its metabolites were measured in 22 adult renal transplant patients and further modelled by a non-parametric approach (PMetrics(®)). The relationship between exposure indices to ganciclovir and the slope of the neutrophil count was investigated using multiple linear regression. A four-compartment open model was able to accurately describe ganciclovir and its intracellular forms. A significant association was found between intracellular ganciclovir triphosphate concentrations (AUC0-5) and the decrease in neutrophil count over the first 3 months of treatment (β= -0.0019 ± 5 × 10(-4); P < 0.01). In this population of renal transplant patients, the decrease in neutrophil count, used as a surrogate marker of haematological toxicity, was associated with ganciclovir triphosphate accumulation in blood cells. Further studies are needed to test this biomarker as a predictive factor for toxicity.

Vesicular expression and release of ATP from dopaminergic neurons of the mouse retina and midbrain.

Vesicular nucleotide transporter (VNUT) is required for active accumulation of adenosine tri-phosphate (ATP) into vesicles for purinergic neurotransmission, however, the cell types that express VNUT in the central nervous system remain unknown. This study characterized VNUT expression within the mammalian retina and brain and assessed a possible functional role in purinergic signaling. Two native isoforms of VNUT were detected in mouse retina and brain based on RNA transcript and protein analysis. Using immunohistochemistry, VNUT was found to co-localize with tyrosine hydroxylase (TH) positive, dopaminergic (DA) neurons of the substantia nigra and ventral tegmental area, however, VNUT expression in extranigral non-DA neurons was also observed. In the retina, VNUT labeling was found to co-localize solely with TH-positive DA-cells. In the outer retina, VNUT-positive interplexiform cell processes were in close contact with horizontal cells and cone photoreceptor terminals, which are known to express P2 purinergic-receptors. In order to assess function, dissociated retinal neurons were loaded with fluorescent ATP markers (Quinacrine or Mant-ATP) and the DA marker FFN102, co-labeled with a VNUT antibody and imaged in real time. Fluorescent ATP markers and FFN102 puncta were found to co-localize in VNUT positive neurons and upon stimulation with high potassium, ATP marker fluorescence at the cell membrane was reduced. This response was blocked in the presence of cadmium. These data suggest DA neurons co-release ATP via calcium dependent exocytosis and in the retina this may modulate the visual response by activating purine receptors on closely associated neurons.

Reduction of Phytate in Soy Drink by Fermentation with Lactobacillus casei Expressing Phytases From Bifidobacteria.

Plant-based food products can be modified by fermentation to improve flavour and the concentration of some biologically active compounds, but also to increase the mineral availability by eliminating anti-nutrient substances such as phytates. The objective of this study was to develop a fermented soybean drink with improved nutritional quality and source of probiotic bacteria by including as starter for fermentation Lactobacillus casei strains modified to produce phytase enzymes from bifidobacteria. The L. casei strains showed a good adaptation to develop in the soy drink but they needed the addition of external carbohydrates to give rise to an efficient acidification. The strain expressing the Bifidobacterium pseudocatenulatum phytase was able to degrade more than 90 % phytate during product fermentation, whereas expression of Bifidobacterium longum spp. infantis phytase only led to 65 % hydrolysis. In both cases, accumulation of myo-inositol triphosphates was observed. In addition, the hydrolysis of phytate in soy drink fermented with the L. casei strain expressing the B. pseudocatenulatum phytase resulted in phytate/mineral ratios for Fe (0.35) and Zn (2.4), which were below the critical values for reduced mineral bioavailability in humans. This investigation showed the ability of modified L. casei to produce enzymes with technological relevance in the design of new functional foods.

ATP promotes extracellular matrix biosynthesis of intervertebral disc cells.

We have recently found a high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation might regulate biological activities in the IVD. The objective of this study was therefore to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: the annulus fibrosus (AF) and nucleus pulposus (NP). ATP treatment significantly promotes ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in three-dimensional agarose culture. A significant increase in ECM accumulation has been found in AF cells at a lower ATP treatment level (20 μM) compared with NP cells (100 μM), indicating that AF cells are more sensitive to extracellular ATP than NP cells. NP cells also exhibit higher ECM accumulation and intracellular ATP than AF cells under control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, ATP treatment also augments the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via a molecular pathway, but also increases energy supply to fuel that process.

Final results of a multicenter phase II study of the purine nucleoside phosphorylase (PNP) inhibitor forodesine in patients with advanced cutaneous t-cell lymphomas (CTCL) (Mycosis fungoides and Sézary syndrome)

Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP) that leads to intracellular accumulation of deoxyguanosine triphosphate (dGTP) in T and B cells, resulting in apoptosis. Forodesine has demonstrated impressive antitumor activity in early phase clinical trials in cutaneous T-cell lymphoma (CTCL). In this phase II study, patients with CTCL who had already failed three or more systemic therapies were recruited. We investigated the response rate, safety and tolerability of oral forodesine treatment in subjects with cutaneous manifestations of CTCL, stages IB, IIA, IIB, III and IVA. The safety population encompassing all stages was used for analysis of accountability, demographics and safety. The efficacy population differed from the safety population by exclusion of stage IB and IIA patients. All 144 patients had performance status 0-2. The median duration of CTCL from diagnosis was 53 months (5-516 months). The median number of pretreatments was 4 (range: 3-15). No complete remissions were observed. In the efficacy group of patients, 11% achieved partial remission and 50% had stable disease. The median time to response was 56 days and the median duration of response was 191 days. A total of 96% of all treated patients reported one or more adverse events (AEs) and 33% reported a serious AE. The majority of AEs were classified as mild or moderate in severity. The most commonly reported AEs (>10%) were peripheral edema, fatigue, insomnia, pruritus, diarrhea, headache and nausea. Overall eight patients died during the study: five due to sepsis and infections, one due to a second malignancy (esophageal cancer), one due to disease progression and one due to liver failure. Oral forodesine at a dose of 200 mg daily is feasible and shows partial efficacy in this highly selected CTCL population and some durable responses.

P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH

Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1β/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1β release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.

Endothelial Lipase Modulates Pressure Overload–Induced Heart Failure Through Alternative Pathway for Fatty Acid Uptake

Lipoprotein lipase has been considered as the only enzyme capable of generating lipid-derived fatty acids for cardiac energy. Endothelial lipase is another member of the triglyceride lipase family and hydrolyzes high-density lipoproteins. Although endothelial lipase is expressed in the heart, its function remains unclear. We assessed the role of endothelial lipase in the genesis of heart failure. Pressure overload-induced cardiac hypertrophy was generated in endothelial lipase(-/-) and wild-type mice by ascending aortic banding. Endothelial lipase expression in cardiac tissues was markedly elevated in the early phase of cardiac hypertrophy in wild-type mice, whereas lipoprotein lipase expression was significantly reduced. Endothelial lipase(-/-) mice showed more severe systolic dysfunction with left-ventricular dilatation compared with wild-type mice in response to pressure overload. The expression of mitochondrial fatty acid oxidation-related genes, such as carnitine palmitoyltransferase-1 and medium-chain acyl coenzyme A dehydrogenase, was significantly lower in the heart of endothelial lipase(-/-) mice than in wild-type mice. Also, endothelial lipase(-/-) mice had lower myocardial adenosine triphosphate levels than wild-type mice after aortic banding. In cultured cardiomyocytes, endothelial lipase was upregulated by inflammatory stimuli, whereas lipoprotein lipase was downregulated. Endothelial lipase-overexpression in cardiomyocytes resulted in an upregulation of fatty acid oxidation-related enzymes and intracellular adenosine triphosphate accumulation in the presence of high-density lipoprotein. Endothelial lipase may act as an alternative candidate to provide fatty acids to the heart and regulate cardiac function. This effect seemed relevant particularly in the diseased heart, where lipoprotein lipase action is downregulated.

Serum 25-dihydroxyvitamin D levels correlate with CD4+Foxp3+ T-cell numbers in moderate/severe asthma

impact of B-cell immunodeficiency in cases of PNP deficiency and is probably explained by the complete lack of PNP enzyme activity due to the reported nonsense mutation. Numerous symptomatic mutations, mostly missense, of the PNP gene have been identified. These mutations produce proteins with variable degrees of enzymatic activity, which correlate with the accumulation of nucleoside substrates and, to some degree, with the clinical course. It is the progressive accumulation of deoxyguanosine triphosphate in PNP deficiency that leads to progressive disease. Interestingly, similar to the progressive T-cells toxicity caused by PNP deficiency during the first years of life, the more severe B-cell defect observed in our older patient also suggests accumulating effects of PNP deficiency on B cells. Our results concur with several recent in vitro and in vivo studies describing B-cell death following PNP inhibition with excess deoxyguanosine/deoxyguanosine triphosphate concentrations, albeit at a reduced frequency compared with T-cell apoptosis. Additional studies, including longitudinal studies, are still required to determine whether B-cell function, like T-cell function, deteriorates over time in PNP-deficient patients. In normal peripheral blood B cells, the addition of deoxyguanosine leads to an inhibition of proliferation and differentiation. This effect was found to be independent of deoxyguanosine accumulation. Complete lack of PNP (as observed in our patients) triggers accumulation of deoxyguanosine, thereby disrupting B-cell development, the consequence of which is more profound with time, as indeed was found in the older sister. Another line supporting the hypothesis that the accumulation of deoxyguanosine results in global cell toxicity and that it is not restricted to T cells alone came from the use of purine and pyrimidine nucleoside analogs to treat certain malignancies, including B-cell malignancies. For example, forodesine, a PNP inhibitor, converts deoxyguanine to deoxyguanosine triphosphate and causes general apoptotic cell effect, similar to what is seen in PNP deficiency. We conclude that the variable effect on B-cell function that is observed in PNP deficiencymay be an independent event of T-cell dysfunction and that it is subject to the severity of the deficiency and the duration of the disease. Raz Somech, MD, PhD Atar Lev, MSc Amos J. Simon, PhD Suhair Hanna, MD Amos Etzioni, MD

Effects of cocaine self‐administration and extinction on D2‐like and A2A receptor recognition and D2‐like/Gi protein coupling in rat striatum

Striatal adenosine (A)2 -dopamine (D)2 receptor (R) heteromers exist with antagonistic interactions. We have studied these Rs and their interactions during cocaine self-administration and extinction using a 'yoked' protocol to understand the role of motivational mechanisms behind the adaptive observed. In the ventral striatum, a significant increase in the A2A R density was observed in rats that received 'yoked' cocaine during maintenance phase and following its extinction while this significant increase was only observed after extinction from cocaine self-administration. In the dorsal striatum, a significant increase in the affinity of A2A Rs was determined in the two groups of rats that received cocaine during maintenance. D2 -like Rs were significantly increased in the dorsal striatum of animals that received 'yoked' cocaine during maintenance. In the rat dorsal, but not the ventral, striatum significant reductions in the EC50 values for dopamine and increases in the guanosine5'-([γ]-thio)triphosphate (GTPγS) accumulation were observed following active and passive cocaine injections during maintenance. After 10-day extinction, a significant reduction of the Bmax value of GTPγS accumulation was demonstrated in the dorsal striatum of rats previously self-administered cocaine, while a significant reduction of the EC50 value for dopamine in the ventral striatum was found in the 'yoked' cocaine group. By comparing the cocaine self-administration group with the 'yoked' cocaine group, evidence for the existence of motivational mechanisms that guide adaptive changes in the A2A R and D2 R and in the D2 -Gi coupling differentially developed in the ventral and dorsal striatum during cocaine maintenance and its extinction has been demonstrated.

Decitabine triphosphate levels in peripheral blood mononuclear cells from patients receiving prolonged low-dose decitabine administration: a pilot study

Decitabine is a nucleoside analog used in the treatment for myelodysplastic syndrome. The compound requires intracellular conversion to its triphosphate to become active. Decitabine triphosphate has, however, never been quantified in peripheral blood mononuclear cells (PBMCs) from patients. This article describes a method for the quantitative determination of decitabine triphosphate in PBMCs using liquid chromatography coupled to tandem mass spectrometry. The method was applied to ex vivo incubated whole blood samples and samples from three patients receiving prolonged low-dose decitabine treatment. We successfully quantitated decitabine triphosphate in PBMCs. Considerable levels were detected in PBMCs from two patients that responded well to therapy, whereas only low levels were present in a non-responding patient. Moreover, the data show that, in contrast to plasma decitabine, intracellular decitabine triphosphate accumulates during a treatment cycle of nine infusions at a dose of 15mg/m(2). The results suggest a relationship between decitabine triphosphate levels and response to therapy. Based on the observed accumulation of decitabine triphosphate during a treatment cycle, a less intensive dose scheme could be feasible.

Phase I/II study of gemcitabine as a fixed dose rate infusion and S-1 combination therapy (FGS) in gemcitabine-refractory pancreatic cancer patients

There is no standard regimen for gemcitabine (Gem)-refractory pancreatic cancer (PC) patients. In a previous phase II trial, S-1 was found to exhibit marginal efficacy. Gem administration by fixed dose rate infusion of 10 mg/m(2)/min (FDR-Gem) should maximize the rate of intracellular accumulation of gemcitabine triphosphate and might improve clinical efficacy. We conducted the phase I/II of FDR-Gem and S-1 (FGS) in patients with Gem-refractory PC. The patients received FDR-Gem on day 1 and S-1 orally twice daily on days 1-7. Cycles were repeated every 14 days. Patients were scheduled to receive Gem (mg/m(2)/week) and S-1 (mg/m(2)/day) at four dose levels in the phase I: 800/80 (level 1), 1,000/80 (level 2), 1,200/80 (level 3) and 1,200/100 (level 4). Forty patients were enrolled in the phase II study at recommended dose. The recommended dose was the level 3. In the phase II, a partial response has been confirmed in seven patients (18%). The median overall survival time and median progression-free survival time are 7.0 and 2.8 months, respectively. The common adverse reactions were anorexia, leukocytopenia and neutropenia. This combination regimen of FGS is active and well tolerated in patients with Gem-refractory PC.

Inosine Triphosphate Protects Against Ribavirin-Induced Adenosine Triphosphate Loss by Adenylosuccinate Synthase Function

Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown. We examined whether ITP can be used by adenosine triphosphatase (ATPase) in human erythrocytes or recombinant human adenylosuccinate synthase (ADSS). RBV-induced adenosine triphosphate (ATP) reduction in erythrocytes was compared with the genetically determined low or normal activity of ITPA, leading respectively to high or normal ITP levels. Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR). Interestingly, the alleviation of ATP reduction by the hemolysis protective ITPA genotype was canceled by the ADSS inhibitor 6-mercaptoethanol (6-MP). ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.

The pharmacological advantage of prolonged dose rate gemcitabine is restricted to patients with variant alleles of cytidine deaminase c.79A&gt;C

Controversy exists over the optimal dosing for the nucleoside analogue gemcitabine. A pharmacological advantage is achieved by prolonging infusion times but evidence for a clinical benefit has been conflicting. We hypothesized that polymorphisms in genes involved in gemcitabine accumulation, particularly the cytidine deaminase CDA c.79A>C, may influence the optimal dosing regimen in individual patients. DNA was collected from 32 patients participating in a randomized crossover study comparing 30-min with 100-min infusions of gemcitabine. The relationships between seven polymorphisms among three genes (CDA, RRM1 and DCK) and (i) gemcitabine triphosphate accumulation; (ii) gemcitabine-induced toxicity; and (iii) dose delivery were examined for each infusion time and week of administration. There were trends for increased accumulation of gemcitabine-triphosphate (GEM-TP) with the variant alleles of CDA c.79A>C, and RRM1-37C>A and -524T>C but none of these reached statistical significance in a univariate analysis. In a multivariable model there were significant effects of infusion duration and week of administration on GEM-TP accumulation. There were significant interactions between CDA c.79A>C (P=0.01) and RRM1-37C>A (P=0.019) genotypes, infusion time, and arm. More patients with one or two CDA c.79 variant alleles had doses delays (57 vs 13 %, P=0.03) and a pharmacological advantage for prolonged infusion after week 1. It is important to consider both pharmacokinetics and pharmacogenetics in optimizing gemcitabine accumulation. This represents a classical interaction between genes and environment and provides support for the consideration of both CDA genotype and infusion duration in development of an individualized dosing strategy.

Forodesine, a Purine Nucleoside Phosphorylase (PNP) Inhibitor, Shows Clinical Activity In a Phase 2 Trial In Patients with Previously Treated Chronic Lymphocytic Leukemia (CLL) – Interim Analysis

AbstractAbstract 1397 Introduction:Forodesine is a potent, specific transition-state inhibitor of the purine salvage pathway enzyme PNP. It induces apoptosis in primary CLL cells through accumulation of deoxyguanosine triphosphate. Unlike other nucleoside analogues, forodesine is not phosphorylated, is not incorporated into DNA and is not cytotoxic. Previous phase 1 and 2 studies showed clinical activity in T-cell acute lymphoblastic leukemia (i.v. forodesine) and cutaneous T-cell lymphoma (i.v. and oral). In a single institute exploratory study 5 of 13 (38%) CLL patients demonstrated anti-leukemic effects (decrease in peripheral blood malignant lymphocyte counts) with oral forodesine (200mg QD). However none of the patients achieved remission. Forodesine is absorbed via a saturable mechanism, and in a clinical pharmacology study in normal subjects, 200mg twice-daily (BID) dosing was found to partially overcome the exposure limits from once daily dosing; the 200mg BID regimen was used in the current study. Methods and Results:This is a multicenter phase II open label study. Eligible CLL patients had either failed at least 1 prior therapy (progressive disease [PD] or refractory) or were treatment-naïve provided age was >65 or ECOG 2 or 3, or unable to tolerate standard therapy. Among the 26 patients enrolled 1 was deemed ineligible (Waldenstrom's). The median age was 70 (48-92) years, 65% male and 77% had Rai Stage III/IV disease. The median duration from time to CLL diagnosis was 92.7 (8.4-243.5) months. Features of high-risk disease were del (17p) (n=6), ZAP70+ (n=13), and CD38+ (n=15). All patients had relapsed or refractory CLL with a median of 2 (1-21) prior therapies including fludarabine (76%), alkylating agents (72%), anti-CD20 monoclonal antibodies (72%) and experimental agents (36%). Interim analysis was conducted when 13 patients had been observed for ≥ 6 months: maximum duration of continuous BID forodesine therapy was 364 days. At the date of data cut-off, 20 were evaluable for response (5 were not evaluable because they had received < 8 weeks of therapy), and 12 were still on study. The revised NCI-WG (1996) criteria were used for response assessment. Partial remission was noted in 3 (15%) patients. Additionally 12 (48%) patients demonstrated stabilization of disease. PR onset was noted at 2, 6 and 6 months of therapy. Three patients discontinued due to adverse events (AEs). Serious AEs were reported in 18 patients. Individual SAEs reports included 7 cases of febrile neutropenia, 7 cases of non-neutropenic infections, pneumonia in 4, and herpes zoster in 1. Conclusion:Oral forodesine 200mg BID given continuously was generally safe and well-tolerated in previously-treated CLL patients. The overall response rate was 15%. These safety and efficacy results are particularly intriguing given the relapsed / refractory CLL population studied, which included elderly and poor performance status patients. Furthermore, we observed that, despite being a purine analog, forodesine was well tolerated with continuous daily dosing. This is particularly of interest when considering combination regimens with this new agent. Further studies of optimized dosing regimens of PNP inhibitors in CLL are warranted. These studies could include evaluation of forodesine in combination with other antileukemic agents with different mechanisms of action, in patients intolerant of or ineligible for aggressive conventional chemotherapeutic regimens, or maintenance treatment after conventional therapy. Updated results including ≥6 months follow up for all patients in the Phase II study will be presented at the meeting. Disclosures:Lyman:BioCryst Pharmaceuticals, Inc.: Employment, Equity Ownership. Cantey-Kiser:PharPoint Research, Inc.: Employment. Sheridan:BioCryst Pharmaceuticals, Inc.: Employment, Equity Ownership.

Influence of bone marrow stromal microenvironment on forodesine-induced responses in CLL primary cells

AbstractForodesine, a purine nucleoside phosphorylase inhibitor, displays in vitro activity in chronic lymphocytic leukemia (CLL) cells in presence of dGuo, which is the basis for an ongoing clinical trial in patients with fludarabine-refractory CLL. Initial clinical data indicate forodesine has significant activity on circulating CLL cells, but less activity in clearing CLL cells from tissues such as marrow. In tissue microenvironments, lymphocytes interact with accessory stromal cells that provide survival and drug-resistance signals, which may account for residual disease. Therefore, we investigated the impact of marrow stromal cells (MSCs) on forodesine-induced response in CLL lymphocytes. We demonstrate that spontaneous and forodesine-induced apoptosis of CLL cells was significantly inhibited by human and murine MSCs. Forodesine-promoted dGuo triphosphate (dGTP) accumulation and GTP and ATP depletion in CLL cells was inhibited by MSCs, providing a mechanism for resistance. Also, MSCs rescued CLL cells from forodesine-induced RNA- and protein-synthesis inhibition and stabilized and increased Mcl-1 transcript and protein levels. Conversely, MSC viability was not affected by forodesine and dGuo. Collectively, MSC-induced biochemical changes antagonized forodesine-induced CLL cell apoptosis. This provides a biochemical mechanism for MSC-derived resistance to forodesine and emphasizes the need to move toward combinations with agents that interfere with the microenvironment's protective role for improving current therapeutic efforts.

2′–Cyano–2′–Deoxyarabinofuranosylcytosine Is Active in Acute Myeloid Leukaemia and Acts in Synergy with Cytarabine.

Abstract 41532′–Cyano–2′–deoxyarabinofuranosylcytosine (CNDAC) is the metabolic product of sapacitabine following hydrolysis of the palmitoyl sidechain from the pyrimidine analog primarily by plasma, gut and liver amidases. CNDAC is in turn phosphorylated into the active triphosphate form (CNDACTP) by deoxycytidine kinase (dCK). CNDACTP is incorporated into DNA resulting in single stranded DNA breaks during replication and inducing cell cycle arrest. Previously the cytotoxic effects of CNDAC have also been associated with intracellular accumulation of CNDAC triphosphate and chain termination. CNDAC and sapacitabine have overlapping cytotoxic effects. Acute myeloid leukaemia (AML) cell lines NB4 and HL-60 had an LD50 of 0.24μM (± 0.24) for CNDAC and 0.23μM (± 0.21) for cytarabine (AraC) following 24 hours treatment. Primary AML blasts isolated from patients at diagnosis (n = 15) had a higher mean LD50 (25.22μM ± 19.41) for CNDAC and AraC (8.09μM ± 8.93). This is thought to be due to the requirement of cells to be actively cycling in order to be susceptible to these agents. CNDAC induces apoptosis in NB4 and HL-60 cell lines with significant increases in the percentage of cells with increased Annexin V/propidium iodide staining at concentrations of 1.0μM and above (P < 0.04) and significant caspase-3 activation at concentrations of 0.1μM and above (P < 0.05). Treatment with CNDAC also results in a significant concentration-dependent accumulation in the G2 phase of the cell cycle after 24 hours in NB4 and HL-60 cells (P = 0.003 and 0.011 respectively). Synergy was observed in the AML cell lines when CNDAC was combined with AraC at a ratio of 2:1 The mean combination index for CNDAC and AraC was 0.67 (± 0.21). The activity of deoxycytidine kinase (dCK) was blocked by the addition of excess deoxycytidine, under these conditions the effects of CNDAC were abrogated (P < 0.05) in NB4 and HL-60 cells suggesting that CNDAC requires phosphorylation by dCK for its activation in the cells. The nucleoside transporters hENT 1 and 2 and hCNT3 transport a range of nucleoside analogues through the cell membrane into cells, the use of hENT inhibitors led to a 2.5 fold increase in the LD50 for CNDAC (P = 0.028) over 48 hours. This prolonged exposure to CNDAC could have resulted in some passive uptake of CNDAC into the cells potentially explaining why the agent retained some cell killing activity. Equivalent results have been obtained with dCK and hENT inhibitors in other cell lines indicating that there is a general requirement for these enzymes for CNDAC activity. Interestingly, when cells are treated with the parent drug sapacitabine in the presence of excess deoxycytidine the cytotoxicity is reduced, but when cells are treated in the presence of hENT inhibitors, sapacitabine's cytotoxicity is improved. This suggests that the presence of the palmitoyl side-chain allows membrane permeability even in the absence of the traditional nucleoside transporters. Disclosures:Green:Cyclacel Ltd: Employment.

Inhibition of ATP synthase by chlorinated adenosine analogue

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F O intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (α dpβ dp) and loose binding (α tpβ tp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the α tpβ tp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (α dpβ dp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF 3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Pharmacokinetics of Gemcitabine at Fixed-Dose Rate Infusion in Patients with Normal and Impaired Hepatic Function

Gemcitabine (2,2-difluorodeoxycytidine [dFdC]) can be administered in a standard 30-minute infusion or in a fixed-dose-rate (FDR) infusion to maximize the rate of accumulation of triphosphate, its major intracellular metabolite. The standard 30-minute infusion requires dose adjustment in patients with organ dysfunction, especially in patients with elevated baseline serum bilirubin levels. On the other hand, the FDR infusion is burdened by increased haematological toxicity. The primary aim of this study was to evaluate the pharmacokinetics of dFdC and its metabolite difluorodeoxyuridine (dFdU) in patients with normal and impaired hepatic function. In this prospective study, patients with pancreatic or biliary tract carcinoma and normal or impaired hepatic function tests were considered eligible for recruitment. Patients were recruited according to the following criteria: (i) serum bilirubin <1.6 mg/dL and AST and ALT <2 times the upper the limit of normal (ULN) [cohort I]; and (ii) serum bilirubin >1.6 mg/dL and/or AST/ALT >2 times the ULN (cohort II). An FDR infusion of gemcitabine 1000 mg/m2 was administered on days 1, 8 and 15 every 4 weeks. The pharmacokinetic analysis of gemcitabine and dFdU was performed with high-performance liquid chromatography-tandem mass spectrometry assay in cycles 1 and 2. Thirteen patients were enrolled, four in cohort I and nine in cohort II. All patients were assessable for toxicity and pharmacokinetic analysis. The grade and rate of toxicities were similar in both groups, and patients with elevation of bilirubin and/or transaminases did not require dose reduction of gemcitabine. Pharmacokinetic analysis revealed a reduction of the experimental area under the plasma concentration-time curve for gemcitabine and dFdU in patients with hepatic dysfunction when compared with patients with normal hepatic function. All other pharmacokinetic parameters were similar in the two cohorts. No statistical difference was demonstrated for all parameters evaluated between cycle 1 and cycle 2 in the two groups. Gemcitabine 1000 mg/m2 can be administered as an FDR infusion in patients with altered hepatic function without causing additional toxicity compared with patients with normal hepatic function.