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Cladribine is a purine nucleoside found to enhance toxic amyloid protein and cause memory impairment. Patients following chemotherapy treatment commonly suffer from cognitive deficits more prevalent in the elderly than adults. A previous research study revealed that cladribine has a high affinity to the brain, increases the level of amyloid precursor protein, and results in learning deficits. The study was designed to validate an animal model of cladribine administration to rats through mitochondrial oxidative stress, inflammation, apoptosis, tau phosphorylation, and amyloid-β (1-42) accumulation. In this study, all rats were orally given cladribine (0.5 and 1 mg/kg) for 28 days, resulting in impaired spatial memory confirmed by behavioural activity. On day 29, all rats were euthanized, and the hippocampal tissues were isolated and used for the estimation of neuroinflammatory markers, biochemicals parameters (glutathione, catalase, lipid peroxidation, and nitrite), amyloid-β (1-42) level, neurotransmitters, and nuclear factor kappa B analysis. Cladribine administration significantly elevated cytokines release, dysbalanced neurotransmitter concentration, and promoted the Aβ accumulation and hyperphosphorylation of tau protein. Our study outcome confirmed that cladribine produces cognitive impairment via activation of Nuclear factor kappa B, mitochondrial oxidative stress and dysbalanced of the endogenous antioxidant defence system., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cladribine (2-chlorodeoxyadenosine, 2CdA) is one of the most effective disease-modifying drugs for multiple sclerosis (MS). Cladribine is a synthetic purine nucleoside analog that induces cell death of lymphocytes and oral cladribine treatment leads to a long-lasting disease stabilization, potentially attributable to immune reconstitution. In addition to its effects on lymphocytes, cladribine has been shown to have immunomodulatory effects on innate immune cells, including dendritic cells and monocytes, which could also contribute to its therapeutic efficacy. However, whether cladribine can modulate human macrophage/microglial activation or monocyte differentiation is currently unknown. The aim of this study was to determine the immunomodulatory effects of cladribine upon monocytes, monocyte-derived macrophages (MDMs) and microglia. We analyzed the phenotype and differentiation of monocytes from MS patients receiving their first course of oral cladribine both before and three weeks after the start of treatment. Flow cytometric analysis of monocytes from MS patients undergoing cladribine treatment revealed that the number and composition of CD14/CD16 monocyte subsets remained unchanged after treatment. Furthermore, after differentiation with M-CSF, such MDMs from treated MS patients showed no difference in gene expression of the inflammatory markers compared to baseline. We further investigated the direct effects of cladribine in vitro using human adult primary MDMs and microglia. GM-CSF-derived MDMs were more sensitive to cell death than M-CSF-derived MDMs. In addition, MDMs treated with cladribine showed increased expression of costimulatory molecules CD80 and CD40, as well as expression of anti-inflammatory, pro-trophic genes IL10 and MERTK, depending on the differentiation condition. Cladribine treatment in vitro did not modulate the expression of activation markers in human microglia. Our study shows that cladribine treatment in vitro affects the differentiation of monocytes into macrophages by modulating the expression of activation markers, which might occur similarly in tissue after their infiltration in the CNS during MS., Competing Interests: This investigator-sponsored study was funded by Merck Australia, which had the opportunity to comment on the content of the manuscript, but the decision to publish was made independently by the authors., (Copyright © 2022 Medeiros-Furquim, Ayoub, Johnson, Aprico, Nwoke, Binder and Kilpatrick.)

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2'-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT ( Ld NDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiG PEI 25000 - Ld NDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5-9) and temperature (30-60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiG PEI 25000 - Ld NDT-Alg was successfully employed in the green production of cladribine at mg scale.

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides.

Cladribine (2-chloro-2'-deoxy-β-d-adenosine) is a 2'-deoxyadenosine analogue, approved by the FDA for the treatment of hairy cell leukemia and more recently has been proved for therapeutic against many autoimmune diseases as multiple sclerosis. The biosynthesis of this compound using Thermomonospora alba CECT 3324 as biocatalyst is herein reported. This thermophilic microorganism was successfully entrapped in polyacrylamide gel supplemented with nanoclays such as bentonite. The immobilized biocatalyst (T. alba-Ac-Bent 1.00 %), was able to biosynthesize cladribine with a conversion of 89 % in 1 h of reaction and retains its activity for more than 270 reuses without significantly activity loss, showing better operational stability and mechanical properties than the natural matrix. A microscale assay using the developed system, could allow the production of at least 181 mg of cladribine in successive bioprocesses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A stable biocatalyst with magnetic properties based on immobilized Lactobacillus animalis ATCC 35,046 to obtain 2-chloroadenine-2'-deoxyriboside, known as cladribine, is reported for the first time. This nucleoside analogue is an antitumor agent used in the treatment of a wide variety of types of leukemia. In this study, an eco-compatible and alternative bioprocess to obtain cladribine was developed. Product conversion was close to 90% at 2 h in optimized nonconventional reaction media. The microscale biosynthesis of the compound of interest afforded a total productivity close to 370 mg/L/h in the presence of DMSO, and it was stable at least for 30 days in storage conditions.

Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy-cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled-up reaching a productivity of 138 mg L -1 hr -1 . Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions., (© 2019 American Institute of Chemical Engineers.)

Oral cladribine is a novel treatment for relapsing multiple sclerosis (MS). This appears to be a semi-selective immune-reconstitution therapy that induces long-term therapy from short treatment cycles. It has a relatively good safety profile that currently does not require extensive monitoring associated with some continuous immunosuppressive and relatively non-selective immune reconstitution therapies. The efficacy and safety of cladribine relates to its particular physicochemical properties, the function of the lymphocyte subsets that are selectively targeted by the drug and the repopulation kinetics of these subsets. As such, there is marked and long-term depletion of memory B cell subsets, which probably relates to the therapeutic efficacy. This is also coupled with a more limited, but likewise long-term, depletion of CD4 T subsets. There is limited depletion of cells of the innate immune system and modest effects on CD8 and probably plasma cells, which provide immediate and durable protection from infection. Targeting of CD4 T regulatory cells, CD8 T suppressor cells and regulatory B cell subsets appears more limited as these populations recover rapidly and so repopulating pathogenic cells re-emerge into a regulatory environment. This appears to lead to re-establishment of immune-tolerance that produces long-term control of MS. Although this hypothesis contains a number of unknown details, it is based on knowledge about the biology of cladribine, basic immunology and the effects of other high-efficacy B and T cell depleting agents that exhibit stereotyped repopulation behaviours. These concepts are relatively simple to interrogate, and can be modified as new knowledge about the durability of disease control and safety with cladribine emerges., (Copyright © 2019. Published by Elsevier B.V.)

Introduction: Multiple sclerosis (MS) is an immune-mediated and neurodegenerative disease with an unpredictable outcome. Immune-modulatory treatment aims at decreasing long-term disability. With the increasing number of treatment options, it is essential to fully digest the possible side effects of the available therapeutics and to monitor patients is essential., Areas Covered: All approved disease-modifying drugs (DMD) for MS are discussed in this review. Mode of action, adverse effects, reported risks for infections and malignancies, and pregnancy related issues are discussed in the review. The authors also provide suggestions for monitoring therapy. For all approved DMDs the pivotal studies have been included for possible side effects, as well as reports by health authorities. For this manuscript, PubMed was checked for reports on side effects for various drugs., Expert Opinion: Treatment options in MS are manifold, each carrying different risks. The safety-risk profile for approved agents is favorable. Knowing and monitoring these possible side effects is essential to minimize risks associated with treatment. Presently, the long-term experience for some of these therapies is missing and this must be addressed.

S-adenosyl-l-methionine (SAM) is universal in biology, serving as the second most common cofactor in a variety of enzymatic reactions. One of the main roles of SAM is the methylation of nucleic acids, proteins, and metabolites. Methylation often imparts regulatory control to DNA and proteins, and leads to an increase in the activity of specialized metabolites such as those developed as pharmaceuticals. There has been increased interest in using SAM analogs in methyltransferase-catalyzed modification of biomolecules. However, SAM and its analogs are expensive and unstable, degrading rapidly under physiological conditions. Thus, the availability of methods to prepare SAM in situ is desirable. In addition, synthetic methods to generate SAM analogs suffer from low yields and poor diastereoselectivity. The chlorinase SalL from the marine bacterium Salinispora tropica catalyzes the reversible, nucleophilic attack of chloride at the C5' ribosyl carbon of SAM leading to the formation of 5'-chloro-5'-deoxyadenosine (ClDA) with concomitant displacement of l-methionine. It has been demonstrated that the in vitro equilibrium of the SalL-catalyzed reaction favors the synthesis of SAM. In this chapter, we describe methods for the preparation of SalL, and the chemoenzymatic synthesis of SAM and SAM analogs from ClDA and l-methionine congeners using SalL. In addition, we describe procedures for the in situ chemoenzymatic synthesis of SAM coupled to DNA, peptide, and metabolite methylation, and to the incorporation of isotopes into alkylated products., (© 2018 Elsevier Inc. All rights reserved.)

Chlorinase SalL halogenate S-adenosyl-l-methionine (SAM) reacts with chloride to generate 5'-chloro-5'-deoxyadenosine and l-methionine through a nucleophilic substitution mechanism. Although it is known that chlorinase enhances the rate of reaction by a factor of 1.2 × 10 17 fold, it is not entirely clear how this is accomplished. The search for the origin of the catalysis of chlorinase and other enzymes has led to a desolvation hypothesis. In the present work, we have used well defined computational simulations in order to evaluate the origin of the catalytic efficiency of chlorinase. The results demonstrate that the catalytic effect of chlorinase is associated with the fact that Cl - is "solvated" by the protein more than by the reference solution reaction, which is not in accordance with proposed catalysis by desolvation. It is found that chlorinase SalL active sites provide electrostatic stabilization of the transition state which is the origin of its catalytic effect.

Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin -1 . Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Molecular determinants of FlA1 fluorinase specificity were probed using 5'-chloro-5'-deoxyadenosine (5'-ClDA) analogs as substrates and FlA1 active site mutants. Modifications at F213 or A279 residues are beneficial towards these modified substrates, including 5'-chloro-5'-deoxy-2-ethynyladenosine, ClDEA (>10-fold activity improvement), and conferred novel activity towards substrates not readily accepted by wild-type FlA1.

A mycoplasma-encoded purine nucleoside phosphorylase (designated PNPHyor) has been cloned and characterized for the first time. Efficient phosphorolysis of natural 6-oxopurine and 6-aminopurine nucleosides was observed, with adenosine the preferred natural substrate (Km = 61 µM). Several cytostatic purine nucleoside analogs proved to be susceptible to PNPHyor-mediated phosphorolysis, and a markedly decreased or increased cytostatic activity was observed in Mycoplasma hyorhinis-infected human breast carcinoma MCF-7 cell cultures (MCF-7.Hyor), depending on the properties of the released purine base. We demonstrated an ∼10-fold loss of cytostatic activity of cladribine in MCF-7.Hyor cells and observed a rapid and complete phosphorolysis of this drug when it was exposed to the supernatant of mycoplasma-infected cells. This conversion (inactivation) could be prevented by a specific PNP inhibitor. These findings correlated well with the high efficiency of PNPHyor-catalyzed phosphorolysis of cladribine to its less toxic base 2-chloroadenine (Km = 80 µM). In contrast, the cytostatic activity of nucleoside analogs carrying a highly toxic purine base and being a substrate for PNPHyor, but not human PNP, was substantially increased in MCF-7.Hyor cells (∼130-fold for fludarabine and ∼45-fold for 6-methylpurine-2'-deoxyriboside). Elimination of the mycoplasma from the tumor cell cultures or selective inhibition of PNPHyor by a PNP inhibitor restored the cytostatic activity of the purine-based nucleoside drugs. Since several studies suggest a high and preferential colonization or association of tumor tissue in cancer patients with different prokaryotes (including mycoplasmas), the data presented here may be of relevance for the optimization of purine nucleoside-based anticancer drug treatment.

New insight into the in vitro and in vivo metabolism of Cladribine (2-chloro-2'-deoxyadenosine, [2-CdA]) are presented. Following incubation of [(14)C]-2-CdA in mouse, rat, rabbit, dog, monkey and human hepatocyte cultures, variable turnover was observed with oxidations and direct glucuronidation pathways. The oxidative cleavage to 2-chloroadenine (2-CA, M1) was only observed in rabbit and rat. Following incubation of [(14)C]-2-CdA in whole blood from mouse, monkey and human, a significant turnover was observed. The main metabolites in monkey and human were 2-chlorodeoxyinosine (M11, 16% of total radioactivity) and 2-chlorodeoxyinosine (M12, 43%). In mouse, 2-CA was the major metabolite (2-CA; M1, 73%). After single intravenous and oral administration of [(14)C]-2-CdA to mice, 2-chlorodeoxyinosine (M11) was confirmed in plasma, while 2-chlorohypoxanthine (M12) and 2-CA (M1) were found in urine. Overall, the use of [(14)C]-2-CdA both in vitro (incubations in mouse, monkey and human whole blood) and in vivo (mouse) has confirmed the existence of an additional metabolism pathway leading to the formation of 2-chlorodeoxyinosine (M11) and 2-chlorohypoxanthine (M12). Formation of these two metabolites demonstrates that Cladribine as free form is not fully resistant to adenosine deaminase as suggested earlier, an enzyme involved in its mode of action.

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fludarabine, clofarabine, and cladribine are anticancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3)). Radioligand binding studies utilizing Chinese hamster ovary cells, stably transfected with adenosine A(1), A(2A), or A(3) receptor subtype, were used to assess the binding affinities of these compounds, whereas adenylyl cyclase activity was used to assess the binding to A(2B) receptors. Clofarabine and cladribine both bound to the A(2A) receptor with a K (i) of 17 and 15 μM, respectively. Clofarabine was the only adenosine analogue to bind to the A(3) receptor with a K (i) of 10 μM, and none of these compounds bound to the A(2B) receptor. Results show that clofarabine, cladribine, and fludarabine bind to the A(1) receptor. In addition, clofarabine, cladribine, and fludarabine were A(1) agonists (IC(50) 3.1, 30, and 30 μM, respectively). Neither pyrimidine nucleoside analogues gemcitabine nor cytarabine associated with any of the adenosine receptor subtypes (K (i) > 100μM). This is the first report of an interaction between all adenosine receptor subtypes and chemotherapeutic nucleoside analogues commonly used in the treatment of cancer. Therefore, activation of these receptors may be at least one mechanism through which fludarabine-associated toxicity occurs.

We have previously shown that a cocktail-containing phosphodiesterase inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP promoted specific protein tyrosine phosphorylation in ram spermatozoa during incubation in capacitating conditions. Here, we show, for the first time, that this cocktail induced a progressive time-dependent increase in the capacitated-sperm subpopulation. The addition of either the analogue of adenosine, 2-chloro-2'-deoxyadenosine (Cl-Ado) or caffeine provided a significant increase in the proportion of capacitated spermatozoa and total tyrosine phosphorylation. Computer-assisted semen analysis was used to identify hyperactivated spermatozoa by setting maximum threshold for linearity (< or =45%) and minimum for amplitude of lateral head displacement (> or =3.5 microm). Our results showed that ram spermatozoa can be capacitated in vitro without displaying hyperactivated movement. Among the above-mentioned compounds, only caffeine was able to induce hyperactivation that achieved the maximal response at 8 min of incubation, with a significant increase in hyperactivated spermatozoa of 44.4 +/- 5.6% related to control samples. Flow cytometry analyses showed that caffeine induced a significant increase in the content of calcium in viable spermatozoa during the time-course of incubation in capacitating conditions. BAPTA-AM, a cell-permeable calcium chelator, did not suppress the caffeine-dependent hyperactivation. Quantitative analysis revealed that the addition of caffeine or Cl-Ado accounted for an increase in intracellular cAMP level. However, this increase in cAMP does not seem to be responsible for the caffeine-induced hyperactivation because the cAMP-elevating agents (cocktail) did not promote hyperactivation either, although they greatly induced capacitation and protein tyrosine phosphorylation. The inhibition of PKA with H89 reduced both capacitation and protein tyrosine phosphorylation although hyperactivation increased. These results suggest that calcium from internal stores would be enough to initiate the hyperactivated movement, and that protein tyrosine phosphorylation implicated in ram sperm hyperactivation would be regulated by calcium rather than by PKA-dependent cAMP.

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

We have studied the potential contribution of ABCG2 (breast cancer resistance protein) to resistance to nucleoside analogues. In cells transfected with DNA constructs resulting in overexpression of human or mouse ABCG2, we found resistance against cladribine, clofarabine, fludarabine, 6-mercaptopurine, and 6-mercaptopurine riboside in both MDCKII and HEK293 cells and against gemcitabine only in HEK293 cells. With Transwell studies in MDCK cells and transport experiments with vesicles from Sf9 and HEK293 cells, we show that ABCG2 is able to transport not only the nucleotide CdAMP, like several other ATP-binding cassette transporters of the ABCC (multidrug resistance protein) family, but also the nucleoside cladribine itself. Expression of ABCG2 in cells results in a substantial decrease of intracellular CdATP, explaining the resistance against cladribine. The high transport rate of cladribine and clofarabine by ABCG2 deduced from Transwell experiments raises the possibility that this transporter could affect the disposition of nucleoside analogues in patients or cause resistance in tumors.

Cladribine (2-chloro-2'-deoxyadenosine) was synthesized using intact cells of the recombinant Escherichia coli strain producing Geobacillus stearothermophilus B-2194 thermostable purine-nucleoside phosphorylase II (EC 2.4.2.1). Use of the cells containing this thermostable enzyme allowed the process to be conducted at a temperature of 70 degrees C, which provided the maximal concentrations of sparingly soluble substrates. The best results were obtained with 2-chloroadenine as a modified base. The highest yield of the target 2-chloro-2'-deoxyadenosine (up to 95% in the case of deoxyguanosine) was reached when using 2'-deoxypurines as donors of deoxyribose. Use of thymidine for these purposes required its considerable molar excess over 2-chloroadenine (up to 6 : 1), which is connected with a nonoptimal amount of endogenous thymidine phosphorylase, necessary for synthesis of deoxyribose-1-phosphate, in the transglycosylation reaction.

2-Chlorodeoxyadenosine (CldAdo, Cladribine), a nucleoside analog used in the treatment of hairy cell leukemia, is phosphorylated and incorporated into DNA, but is not an absolute chain terminator. We hypothesized that the presence of a chlorine molecule projecting into the DNA minor groove would affect DNA:protein-binding interactions. Here, we investigated recognition of and binding to double-stranded CldAMP-substituted TATA promoter sequences by human TATA-binding protein (TBP) using mobility shift assays. Depending on the site, CldAMP in place of dAMP within a TATA sequence decreased in vitro TBP binding by approximately 30% to 55% compared to control sites. When bound to a CldAMP-substituted TATA box, however, the TBP complex was more resistant to polyanions, suggesting enhanced stability. Limited exposure of the TBP:DNA complex to proteases indicated that TBP conformation was altered on CldAMP-substituted DNA compared to control. Further, binding of transcription factor IIB to TBP was diminished on analog-containing TATA sequences. These results suggest normal TBP-binding interactions--specifically recognition, stability, and conformation-are disrupted by CldAMP insertion into eukaryotic promoter sequences.

Members of the concentrative nucleoside transporter (CNT) family (SLC28) mediate the transport of naturally-occurring nucleosides, and nucleoside analog drugs across the plasma membrane of epithelial cells. Each of the three CNT family members has a distinct specificity for naturally occurring nucleosides, and residues that contribute to the specificity of each transporter have been identified. In contrast, the molecular determinants of specificity for synthetic nucleoside analogs are not known. In this study, we take advantage of the large species difference that exists between human and rat CNT2 (hCNT2 and rCNT2) in their ability to transport the nucleoside analog drug cladribine, 2CdA, (rCNT2 > > > hCNT2) to identify the critical domains and amino acid residues that contribute to the observed difference in specificity between CNT2 orthologs. Using chimeric proteins of human and rat CNT2, we determined that the C-terminal half of CNT2 contained the determinants of 2CdA selectivity. We replaced key residues in the C terminus of hCNT2 with the equivalent residue in rCNT2. One residue in the C-terminal portion of CNT2 was found to significantly contribute to 2CdA selectivity: hCNT2-S354A. This mutant caused an increase of 5-6-fold over hCNT2. The 2-chloro pharmacophore, rather than the 2'-deoxyribose was responsible for the reduced 2CdA uptake by hCNT2. Our data are consistent with a model in which an increased capability for hydrogen bonding in critical amino acids that reside in the C terminus of rCNT2 contributes to its enhanced selectivity for 2CdA.

2-Chloro-9-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine) and 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.

This report presents a quantitative analysis of the synergistic interaction of arabinosylcytosine (araC) and cladribine (CdA) in human H9-lymphoid cell lines sensitive and resistant to araC (H9-araC cells). H9-araC cells obtained by cultivation of H9 cells in the presence of 0.5 microM arabinosylcytosine (araC) had lower deoxycytidine kinase (dCK) than the parental cell line. The IC50 values of araC and CdA calculated by using median-effect analysis and CalcuSyn software were: 0.55 microM and 1.16 microM for CdA and 0.0058 microM and 3.5 microM for araC in H9 and H9-araC cells, respectively. These values were reduced to 0.10 microM and 0.38 microM for CdA and to 0.004 microM and to 0.77 microM for araC when the drugs were used in combination. Computerized simulation of dose reduction index (DRI) indicated that at 50-99% growth inhibition levels, the doses of araC could be reduced by 2.0 to 11.9-fold and 2.9 to 5.3-fold and the doses of CdA by 5.9 and 183.7-fold and 3.1 to 164.8-fold in H9 and H9-araC cells, respectively, when the drugs are used in combination. Assessment by combination index (CI) analysis showed that the combination exhibited moderate to strong synergistic lympho-cytotoxic effects. CdA metabolic studies (influx and activation) in the presence of deoxyadenosine, deoxycytidine, or araC suggested that CdA enters cells by a deoxyadenosine-inhibitable transport system, which is different than that of araC and deoxycytidine transport system. Thus, in addition to the known mechanisms, other mechanisms might be involved in the metabolism of CdA. The demonstration that araC and CdA combinations exert synergistic cytotoxicity even in the resistant cells raises hope that such a combination may be useful in tumors that were found resistant to these drugs.

Cytotoxic nucleoside analogues are widely used in treatment of patients with hematological malignancies as well as for some solid tumors. Resistance developed against these molecules limit their clinical use. Many studies on cell models and clinical samples have identified cellular mechanisms involved in this phenomenon. Here, we describe the available data concerning the proteins involved in the metabolism and the mechanism of action of nucleoside analogues, as well as the clinical studies showing their implication in the resistance to these drugs.

The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction--presumably by supplying dATP or its analogues for the apoptosome formation--deserves consideration. Two-hour exposure of human tonsillar lymphocytes to 2-chloro-deoxyadenosine (CdA) led to a two-fold activation of dCK. This activation process was inhibited by pifithrin-alpha, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide-treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.

A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5'-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 microg/ml in the assay. An amount of 8 x 10(3) cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.

1. The in vivo metabolism of the antineoplastic and immunosuppressive drug 2-CdA (Leustatin) was investigated in mice, monkeys and humans after a single subcutaneous dose of cladribine 60 mg kg(-1) to eight male and eight female mice and 10 mg kg(-1) to one male and one female monkey, and an intravenous infusion dose of cladribine 22-45 mg(-1) per subject to 12 male patients. 2. Plasma (1 h), red blood cells (1 h) and faecal samples (0-24 h) were obtained from mice and monkeys, and urine samples (0-24 h) were obtained from these species and humans. 3. Unchanged cladribine (urine: 47% of the sample in human; 60% of the sample in mouse; 73% of the sample in monkey) and 10 metabolites, consisting of four phase I metabolites (M1-3, M7) and six phase II metabolites -- five glucuronides (M4, M6, M8-10) and one sulfate (M5) -- were profiled, characterized and tentatively identified in plasma, red blood cells, and faecal and urine samples on the basis of API ionspray-mass spectrometry (MS) and MS/MS data. 4. Metabolites were formed via the following three metabolic pathways: oxidative cleavage at the adenosine and deoxyribose linkage (A); oxidation at adenosine/deoxyribose (B); and conjugation (C). 5. Pathways A and B appear to be major steps, forming four oxidative/cleavage metabolites (M1-3, M7) (each 3-20% of the sample). 6. Pathway C along or in conjunction with pathways A and B produced cladribine glucuronide, cladribine sulfate and four glucuronides of oxidative/cleavage metabolites in minor/trace quantities (each < or = 5% of the sample). 7. In addition, the in vitro metabolism of cladribine was conducted using rat and human liver microsomal fractions in the presence of an beta-nicotinamide adenine dinucleotide phosphate-generating system. Unchanged cladribine (> or = 90% of the sample) plus three minor metabolites, M1-3 (each < 8% of the sample), were profiled and tentatively identified by thin-layer chromatography and MS data. 8. Cladribine is not extensively metabolized in vitro and in vivo in all species. However, humans appear to metabolize cladribine to a greater extent than other animals.

The nucleoside analog 2-chloro-2'-deoxyadenosine (CldAdo; cladribine) is effective in the treatment of hairy cell leukemia and chronic lymphocytic leukemia. CldAdo is phosphorylated and incorporated into cellular DNA but is not an absolute chain terminator. We demonstrated by in vitro gel-shift assays that binding interactions of the human TATA box-binding protein (TBP) were disrupted on 2-chlorodeoxyadenosine monophosphate (CldAMP)-substituted TATA box consensus sequences. We hypothesized that human RNA polymerase II (pol II) transcriptional processes would therefore be affected by 2-chlorodeoxyadenosine triphosphate (CldATP) incorporation into a promoter TATA element. Double-stranded DNA templates containing the adenovirus major late promoter and coding sequences were enzymatically synthesized as control or with site-specific CldAMP residues, incubated with HeLa extract, and the synthesis of radiolabeled 44-base transcripts was assessed. With increasing amounts of HeLa extract, CldAMP substitution for dAMP within the TATA box decreased in vitro pol II transcription by approximately 35% compared with control substrates. Time-course studies showed that transcript production increased in a linear fashion on control substrates. In contrast, transcription on CldAMP-substituted TATA sequences reached a plateau after 20 min. Furthermore, CldAMP-substituted promoter sequences trapped or sequestered TBP, preventing its dissociation from DNA and subsequent binding to additional TATA elements to reinitiate transcription. CldAdo thus represents the first example of a nucleoside analog that acts as a transcriptional antagonist. CldATP incorporation into gene regulatory sequences may provide a novel strategy to modulate specific protein/DNA interactions.

The inherent or acquired resistance of leukemic cells to cytostatic agents is a major clinical challenge. The purpose of this review was to elucidate and analyse the available data concerning mechanisms of resistance of cladribine with emphasis on recent advances in the characterization of activating and inactivating enzymes in the induction of resistance to cladribine. All available in vitro and clinical data on cladribine was undertaken. Cladribine, unlike many other drugs, is toxic to both dividing and indolent lymphoid malignancies. Cladribine is a prodrug and must be phosphorylated intracellularly to cladribine-monophosphate (MP) by the nuclear/cystosol enzyme deoxycytidine kinase (dCK) and the mitochondrial enzyme deoxyguanosine kinase. The cytotoxicity mainly depends on the accumulation of cladribine-triphosphates (TP) after phosphorylation of cladribine-MP by nucleoside monophosphate kinase and nucleoside diphosphate kinase. 5'-Nucleotidase (5'-NT) dephosphorylates cladribine-MP and the accumulation of cladribine-TP depends on the ratio of dCK and 5'-NT in the cells. The mechanisms underlying cladribine resistance are multifactorial, e.g. decreased nucleoside transport, decreased activity or deficiency of dCK, altered intracellular pools of competing nucleotides, altered regulation of ribonucleotide reductase and increased drug inactivation by 5'-NT. Finally, cladribine resistance may be a consequence of a defective induction of apoptosis. In spite of the fact that more than one mechanism can contribute to a cladribine resistance phenotype, a reduction in dCK activity is probably the major determinant of cladribine resistance. Insight into the mechanism of action and resistance to cladribine is crucial for its optimal use as well as for the development of newer analogues.

Background and Objectives: Clofarabine (CAFdA), one of the newer nucleoside drugs is undergoing a phase II clinical trial for the treatment of pediatric refractory/relapsed acute myeloid and lymphocytic leukemia. Although CAFdA is structurally similar to the clinically established analogs fludarabine and cladribine (CdA), its metabolism and mechanism of actions are significantly different. The present study investigates the in vitro cytotoxicity of CAFdA and CdA in mononuclear cells isolated from 52 patients with chronic lymphocytic (CLL) and acute myeloid leukemia (AML)., Design and Methods: We incubated the leukemic cells with drugs for 48 hours and cytotoxicity was then evaluated by the MTT dye assay. We also determined the levels of deoxycytidine and deoxyguanosine kinase with radio-chemical substrate-based assays and used a high performance liquid chromatographic method to measure cellular nucleotides in leukemia cells after 2 hours' incubation., Results: Using equimolar concentrations of CAFdA and CdA, the in vitro cytotoxicity for the population was significantly higher with CAFdA than with CdA (median EC50 for CAFdA 0.12 microM and for CdA 0.15 microM, p<0.001). From the individual estimates the difference in cytotoxicity between CAFdA and CdA was more pronounced in cells from CLL patients (median EC50 for CAFdA 0.08 microM and for CdA 0.16 microM p<0.001) than in those from AML patients. We also found that CAFdA was phosphorylated more efficiently than CdA. No correlations were detected in this study between the levels of CdA and CAFdA nucleotides, enzymes levels and the in vitro responses., Interpretation and Conclusions: The greater in vitro cytotoxicity and cell metabolism of CAFdA compared to CdA confirm the high activity of CAFdA and encourage clinical trials with CAFdA in leukemic patients.

Phenylacetic and retinoic acids are carboxyacidic cell differentiating agents displaying anticancer activities. We report on a new class of compounds including the 5'-esters of 2'-deoxyadenosine (dA) or 2-chloro-2'-deoxyadenosine (cladribine, 2CdA) and the aforementioned acids. The rationale behind the synthesis of these esters was that if they are hydrolyzed inside the lymphoid cells, either dA will be removed from the intracellular environment by deamination, or 2CdA will be phosphorylated and accumulated. In either case targetted delivery of the differentiating agent to the lymphoid cells may be envisaged. The said compounds were synthesized by the Mitsunobu procedure employing triphenylphosphine and azadicarboxylic acid esters, and their stability was tested against various esterases. Esters of dA and 2CdA with phenylacetic acids were found to be resistant to enzymatic hydrolysis, whereas those with retinoic acids were efficiently hydrolyzed by commercially available hepatic esterase as well as by esterases present in the blood plasma and in diluted human lymphocyte lysate. Susceptibility to enzymatic hydrolysis was found to be a prerequisite of cytotoxic and/or differentiating activity of these esters in leukemic cell lines.

2-Chloro-2'-deoxyadenosine (CdA) is a deoxyadenosine analogue which targets enzymes involved in DNA synthesis, and hence might interfere with the resynthesis step of DNA repair. We tested this hypothesis in resting B cell chronic lymphocytic leukemia (B-CLL) lymphocytes, after firstly characterizing unscheduled DNA synthesis occurring in these cells. We observed that the spontaneous incorporation of [methyl-3H]thymidine (dThd) into DNA of B-CLL cells was not completely inhibitable by hydroxyurea (HU) which blocks DNA replication. In addition, in the presence of HU, dThd incorporation could be upregulated by UVC radiation or DNA alkylation, without re-entry of the cells into S phase. CdA was found to inhibit both spontaneous and upregulated DNA synthesis in B-CLL cells. Phosphorylation of CdA was essential to exert this effect. We finally observed a strong synergistic cytotoxicity between UV light and CdA, which was correlated with activation of caspase-3 and high molecular weight DNA fragmentation, two markers of apoptosis. Taken together, these observations indicate that in B-CLL cells CdA inhibits unscheduled DNA synthesis which represents the polymerizing step of a repair process responsive to DNA aggression. Inhibition of this process by CdA, together with a combined activation of the apoptotic proteolytic cascade by CdA and UV, may explain their synergistic cytotoxicity.

Cladribine, an adenosine deaminase inhibitor, has been developed and launched by Ortho Biotech in collaboration with The Scripps Research Institute for the treatment of several neoplasms, including acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, cutaneos T-cell lymphoma, hairy-cell leukemia and non-Hodgkin's lymphoma. It was first launched in the US in February 1993. Ortho Biotech and The Scripps Research Institute have since been developing the compound for its potential use in multiple sclerosis (MS). In 1997, Ortho filed air NDA in the US for the use of cladribine in the treatment of relapsing-remitting and secondary progressive MS. An FDA drug advisory committee was planning to meet in January 1999 to discuss the NDA. However, Ortho cancelled the meeting. Following an FDA inspection during December 1998 and January 1999, the Scripps Clinic received a warning letter from the FDA in April 1999 regarding violations in the clinical studies of cladribine for MS, and Ortho withdrew the NDA after concluding that further clinical studies would be necessary. Cladribine has been known since the 1960s as an intermediate for the synthesis of 2-deoxynucleotides and its potential for the treatment of leukemia was disclosed in 1984. The Scripps Research Institute and the Johnson & Johnson group hold several patents claiming preparation methods (US 05208327), and additional indications, such as multiple sclerosis (WO-09316706) and rheumatoid arthritis (US-05310732). The associated patent, WO-09323508, is the only one among those patents that claims the use of unmodified cladribine for the treatment of leukemia, but it focuses particularly on a specific form of the disease, chronic myelogenous leukemia. Analysts at UBS Warburg predicted in October 2001, that the product would make US sales of $50 million in 2004 for its MS indication.

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.

EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro-2'-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2-3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 microM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an IC(50) of 16 microM in EHEB cells and 5 microM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies.

WSU-CLL cells, a fludarabine resistant B-cell chronic lymphocytic leukemia cell line, has been shown to exhibit enhanced sensitivity to 2-chlorodeoxyadenosine (2-CdA) following 48-72 h exposure to bryostatin 1. For 2-CdA to manifest its chemotherapeutic activity, it must first enter the cell through one of several specific nucleoside transporter systems. We present data to show that bryostatin 1-induced enhanced influx of 2-CdA is in part the result of bryostatin 1-induced modulation of nucleoside transporters in WSU-CLL cells. The bi-directional equilibrative NBMPR sensitive transporters in WSU-CLL cells were significantly down-regulated 90 min post-exposure to 1-200 nM bryostatin 1. This down-regulation was evident up to 144 h. In contrast, WSU-CLL cells exhibited a transient increase in Na+-dependent concentrative 2-CdA influx from 48 to 96 h after bryostatin 1 exposure which was evident for a longer duration than that accounted for by the increase in deocycytidine kinase activity. These data may, in part, explain the enhanced efficacy of 2-CdA seen in WSU-CLL cells following 48-72 h exposure to bryostatin 1. It may raise questions as to the importance of the bi-directional transporters in determining the resistance or sensitivity of CLL cells to 2-CdA or other nucleoside analogues.

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)--a fluorinated analogue of cladribine [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability--in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5'-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.

The purine analogues fludarabine and cladribine (CdA) have recently become established to be effective treatment for low-grade non-Hodgkin's lymphoma (NHL). The pyrimidine nucleoside analogue cytarabine (AraC) has an important place in the treatment of acute leukemia, and gemcitabine is a new pyrimidin antimetabolite which has shown clinical activity against solid tumors. We have used the semiautomated fluorometric microculture cytotoxicity assay (FMCA), based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA), to study these drugs. Eighty samples from 60 patients with low-grade NHL were studied. Fifty samples from patients with acute lymphoid leukemia (ALL) and 118 samples from patients with acute myeloid leukemia (AML) were included for comparison. The results indicate that the purine- and pyrimidine nucleoside analogues tested may be as active against low-grade NHL as against acute leukemia. In low-grade NHL, AraC seems to be even more active in comparison to CdA (p=<0.0001) and fludarabine (p=0.001). Untreated patients were more drug sensitive than previously treated patients. Gemcitabine showed the highest correlation with AraC (0.90) whereas CdA showed the highest correlation with fludarabine (0.84). Based on these results we propose that AraC and gemcitabine may have a role in the treatment of low-grade NHL.

Background and Aims: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity., Methods: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites., Results: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times., Conclusion: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.

The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG), and 2',2'-difluorodeoxyguanosine (dFdG) are phosphorylated by human mitochondrial deoxyguanosine kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was approximately 4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.

Purpose: The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na(+)-dependent, purine-selective nucleoside transporter (SPNTint) and to study the interactions of nucleosides and nucleoside analogs with this transporter., Methods: Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNTint. Nucleoside transport activity was measured using [3H]inosine, [3H]uridine, [3H]-dideoxyinosine (ddI), and [3H]-2-chloro-2'-deoxyadenosine (2CdA) as model substrates., Results: Expression of SPNTint was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na(+)-stimulated uptake of [3H]inosine in cells transiently transfected with SPNTint was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na(+)-stimulated uptake of both inosine and uridine was saturable (K(m) = 28.1 +/- 7.1 microM and 20.6 +/- 5.6 microM, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddI (IC50 = 46 microM) and 2CdA (IC50 = 13 microM) also significantly inhibited the Na(+)-stimulated uptake of [3H]inosine. A Na(+)-stimulated uptake of [3H]2CdA was observed suggesting that 2CdA is also a permeant of SPNTint., Conclusions: HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.

Deoxycytidine kinase (dCK) is a salvage pathway enzyme with broad substrate specificity that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. The kinetic behaviour of dCK is complex with saturation curves showing negative cooperativity. In this study, we have expressed and affinity purified recombinant dCK, using the pET 9d vector system with a histidine tag-sequence and a thrombin cleavage site fused to the N-terminus of the dCK coding sequence. The His-tagged protein showed essentially the same kinetic properties as the protease cleaved protein and the purified protein isolated from human spleen. However, addition of 0.2-0.4 M NaCl during the dCK reaction caused a stimulation of 2'-deoxycytidine (dCyd), and the antileukemic analog 2-chlorodeoxyadenosine (CldAdo) phosphorylation, but an inhibition of the 2'-deoxyguanosine (dGuo) phosphorylation, both with His-tagged and protease cleaved dCK. The negative cooperativity observed with dCyd was eliminated by the presence of 0.4 M NaCl so that the Hill coefficient changed from 0.6 to 1.4. In contrast, dGuo phosphorylation that initially followed Michaelis-Menten kinetics showed negative cooperativity after addition of 0.4 NaCl. The alterations in kinetic properties were not accompanied by any apparent changes in subunit structure as revealed by gel filtration. The major form of dCK eluted in the position corresponding to a dimer in the presence or absence of salt, but a minor fraction of dCK, eluting in the position of a tetramer, was diminished in the presence of salt. The mechanism for the effects of 0.4 M NaCl on dCK kinetic behaviour is not known but it is most likely due to alterations in the conformation of the active site of the enzyme. The effects described here also may explain some of the discrepancies reported in the literature on the substrate specificity of this complex enzyme.

Degradation of DNA produces deoxycytidine. Metabolism of deoxycytidine to dCTP inhibits phosphorylation of cytosine arabinoside (araC), fludarabine (FaraA) and 2-chlorodeoxyadenosine (CdA) by deoxycytidine kinase. This study measured plasma deoxycytidine in healthy adults and two leukemia patients and then determined how clinically relevant deoxycytidine levels would affect drug toxicity in human leukemia and lymphoma cells. Deoxycytidine was well below 0.05 microM in ten healthy persons. In the leukemia patients it was <0.05 and 0.44 microM before chemotherapy, rising to 10.3 and 5.5 microM during treatment. A broad range of clinically relevant deoxycytidine levels were high enough to profoundly decrease araC, FaraA and CdA toxicity in MOLT3, CA46 and HL60 leukemia/lymphoma cells and to change dCTP, DNA synthesis and drug incorporation into DNA in a manner consistent with prior mechanistic studies. Varying deoxycytidine levels could be an important factor influencing leukemia therapy.

Cladribine is a new purine nucleoside analogue with promising activity in low-grade lymphoproliferative disorders, childhood acute myelogenous leukaemia and multiple sclerosis. Reversed phase high performance liquid chromatography and radioimmunoassay have been used for the analysis of the plasma pharmacokinetics of cladribine. The major (inactive) metabolite in plasma, chloroadenine, can only be detected by liquid chromatography. The oral bioavailability of cladribine is 37 to 51%, and that of subcutaneous administration is 100%. The terminal half-life varies from 5.7 to 19.7 hours and the apparent volume of distribution from 54 to 357 L/m2. The concentration in the cerebrospinal fluid is 25% of that in plasma in patients without central nervous system disease; in patients with meningeal disease, the cladribine concentration in the cerebrospinal fluid exceeds that in plasma. Cladribine is a prodrug and needs intracellular phosphorylation to active nucelotides. The intracellular concentration of these metabolites is several hundred-fold higher than that of cladribine in plasma and they are retained in leukaemia cells with half-lives between 9 and > 30 hours depending on diagnosis and sampling schedule. There is no correlation between the plasma concentration of cladribine and that of the intracellular metabolites. The renal clearance of cladribine is 51% of total clearance and 21 to 35% of an intravenously administered dose is excreted unchanged in the urine. Pretreatment with cladribine increases the intracellular accumulation of the active metabolite of cytarabine, cytosine arabinoside 5'-triphosphate, by 36 to 40%.

A specific isocratic ion-pair HPLC method for the quantitation of mono-, di- and triphosphates of 2-chloro-2'-deoxyadenosine (CdA) in leukemic cells from patients is described. The method is based on an extraction of nucleotides from cells with a solution of perchloric acid containing triethylammonium phosphate followed by an isocratic separation on an Ultrasphere ODS column (250 x 4.6 mm, 5 microns) with a mixture of 89% triethylammonium phosphate buffer (0.08 M, pH 6.1) and 11% methanol as the eluent. UV absorbance at 265 nm was used. The limit of detection was 65 nM. Standard curves for the CdA triphosphate (CdATP) were linear within the concentration range of 200 nM to 12 microM. The mean overall recovery of CdATP was 90% within a concentration range of standard curves. The within-day and day-to-day coefficients of variation at concentrations of 1.44 microM and 6.25 microM CdATP were < 10%. The applicability of the method was demonstrated by in vitro studies of the accumulation of CdA mono-, di- and triphosphates in CCRF-CEM cells and by determination of the cellular pharmacokinetics of CdA nucleotides in leukemic cells from a patient treated with CdA.

Deoxycytidine kinase (dCK) phosphorylates a number of nucleoside analogues that are useful in the treatment of various malignancies. Although the level of dCK activity in malignant cells is thought to correlate with chemotherapeutic response, no direct data are available to support this assumption. We have tested this hypothesis by infecting three tumor cell lines, MCF-7, HT-29, and H1437, with the retroviral vector LNPO containing either dCK or LacZ cDNA and measuring the corresponding sensitivity to nucleoside analogues. DCK activity was increased by 1.7-, 2.3-, and 16-fold in MCF-7, HT-29, and H1437 cells, respectively. Northern and Western blots demonstrated a similar increase in mRNA and protein levels. As a result of dCK expression, MCF-7 cells demonstrated a 2.5-fold increase in drug sensitivity to 1-beta-D-arabinofuranosylcytosine (AraC) and 2-chloro-2'-deoxyadenosine (CdA). HT-29 cells had a 7-fold increase in sensitivity to AraC, CdA, and 2-fluoro-9-beta-D-arabinofuranosyladenine, whereas H1437 cells demonstrated a 20- to 106-fold increase. For all three drugs, there was a linear relationship between dCK activity in clonally derived cell lines and IC50s. These data demonstrate a direct effect of dCK activity on drug sensitivity in cell lines. Because many tumors have relatively low levels of dCK, it is possible that dCK gene transfer will be a useful adjunct to the treatment of these malignancies.