Cladribine induces apoptosis, neuroinflammation, mitochondrial oxidative stress, tau phosphorylation and Aß (1-42) pathway in the hippocampus: An in vivo approach.

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-ß (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-ß (1-42) level, neurotransmitters, and nuclear factor kappa B analysis. Cladribine administration significantly elevated cytokines release, dysbalanced neurotransmitter concentration, and promoted the Aß 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.

Cladribine Treatment for MS Preserves the Differentiative Capacity of Subsequently Generated Monocytes, Whereas Its Administration In Vitro Acutely Influences Monocyte Differentiation but Not Microglial Activation.

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.

Green Production of Cladribine by Using Immobilized 2'-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology.

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 (LdNDT) 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, SiGPEI 25000-LdNDT-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, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.

A Novel One-Pot Enzyme Cascade for the Biosynthesis of Cladribine Triphosphate.

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.

Biotransformation of cladribine by a nanostabilized extremophilic biocatalyst.

Cladribine (2-chloro-2'-deoxy-ß-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.

Biotransformation of cladribine by a magnetic immobilizated biocatalyst of Lactobacillus animalis.

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.

Biotransformation of cladribine using a stabilized biocatalyst in calcium alginate beads.

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.

Potential mechanisms of action related to the efficacy and safety of cladribine.

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.

Preparation, Assay, and Application of Chlorinase SalL for the Chemoenzymatic Synthesis of S-Adenosyl-l-Methionine and Analogs.

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.

Managing the side effects of multiple sclerosis therapy: pharmacotherapy options for patients.

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.

Catalysis by solvation rather than the desolvation effect: exploring the catalytic efficiency of SAM-dependent chlorinase.

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 × 1017 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.

Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells.

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.

Probing the molecular determinants of fluorinase specificity.

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.

Mycoplasma hyorhinis-encoded purine nucleoside phosphorylase: kinetic properties and its effect on the cytostatic potential of purine-based anticancer drugs.

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.

Comparison of the in vitro and in vivo metabolism of Cladribine (Leustatin, Movectro) in animals and human.

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.

Phosphorylation of deoxycytidine kinase on Ser-74: impact on kinetic properties and nucleoside analog activation in cancer cells.

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.

Cytotoxic purine nucleoside analogues bind to A1, A2A, and A3 adenosine receptors.

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.

Caffeine induces ram sperm hyperactivation independent of cAMP-dependent protein kinase.

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.

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine.

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.

Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides.

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.