Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade.

Arabinoside and derived nucleoside analogs, a family of nucleoside analogs, exhibit diverse typically biological activities and are widely used as antibacterial, antiviral, anti-inflammatory, antitumor, and other drugs in clinical and preclinical trials. Although with a long and rich history in the field of medicinal chemistry, the biosynthesis of arabinoside has only been sporadically designed and studied, and it remains a challenge. Here, we constructed an in vitro multi-enzymatic cascade for the biosynthesis of arabinosides. This artificial biosystem was systematically optimized, involving an exquisite pathway design, NADP+ regeneration, meticulous enzyme selection, optimization of the key enzyme dosage, and the concentration of inorganic phosphate. Under the optimized conditions, we achieved 0.37 mM of vidarabine from 5 mM of sucrose and 2 mM of adenine, representing 18.7% of the theoretical yield. Furthermore, this biosystem also has the capability to produce other arabinosides, such as spongouridine, arabinofuranosylguanine, hypoxanthine arabinofuranoside, fludarabine, and 2-methoxyadenine arabinofuranoside, from sucrose, and corresponding nucleobase by introducing different nucleoside phosphorylases. Overall, our biosynthesis approach provides a pathway for the biosynthesis of arabinose-derived nucleoside analogs, offering potential applications in the pharmaceutical industry.

Non-Invasive Assessment of Locally Overexpressed Human Adenosine 2A Receptors in the Heart of Transgenic Mice.

A2A adenosine receptors (A2A-AR) have a cardio-protective function upon ischemia and reperfusion, but on the other hand, their stimulation could lead to arrhythmias. Our aim was to investigate the potential use of the PET radiotracer [18F]FLUDA to non-invasively determine the A2A-AR availability for diagnosis of the A2AR status. Therefore, we compared mice with cardiomyocyte-specific overexpression of the human A2A-AR (A2A-AR TG) with the respective wild type (WT). We determined: (1) the functional impact of the selective A2AR ligand FLUDA on the contractile function of atrial mouse samples, (2) the binding parameters (Bmax and KD) of [18F]FLUDA on mouse and human atrial tissue samples by autoradiographic studies, and (3) investigated the in vivo uptake of the radiotracer by dynamic PET imaging in A2A-AR TG and WT. After A2A-AR stimulation by the A2A-AR agonist CGS 21680 in isolated atrial preparations, antagonistic effects of FLUDA were found in A2A-AR-TG animals but not in WT. Radiolabelled [18F]FLUDA exhibited a KD of 5.9 ± 1.6 nM and a Bmax of 455 ± 78 fmol/mg protein in cardiac samples of A2A-AR TG, whereas in WT, as well as in human atrial preparations, only low specific binding was found. Dynamic PET studies revealed a significantly higher initial uptake of [18F]FLUDA into the myocardium of A2A-AR TG compared to WT. The hA2A-AR-specific binding of [18F]FLUDA in vivo was verified by pre-administration of the highly affine A2AAR-specific antagonist istradefylline. Conclusion: [18F]FLUDA is a promising PET probe for the non-invasive assessment of the A2A-AR as a marker for pathologies linked to an increased A2A-AR density in the heart, as shown in patients with heart failure.

Synthesis and antiviral effect of phosphamide modified vidarabine for treating HSV 1 infections.

Vidarabine (ARA) was one of the earliest marine-related compounds to be used clinically for antiviral therapy, however, its fast metabolism is the main defect of this drug. To overcome this, we designed and synthesized a group of phosphamide-modified ARA compounds using ProTide technology. With a phosphamide modification, these compounds could become the substrate of specific phospholipase enzymes expressed in the liver. Among all 16 synthesized compounds, most showed stronger activity against herpes simplex virus type 1 (HSV-1) than ARA (EC50 of approximately 10 µM). The top three compounds were compound 2 (EC50 = 0.52 ± 0.04 µM), compound 6 (EC50 = 1.05 ± 0.09 µM) and compound 15 (EC50 = 1.18 ± 0.08 µM) (about 2 times higher than Sp type compound 2). This study provides evidence for use of the phosphamide modification, which could give ARA higher activity and liver cell targeting.

In Silico Exploration of Potential Natural Inhibitors against SARS-Cov-2 nsp10.

In continuation of our previous effort, different in silico selection methods were applied to 310 naturally isolated metabolites that exhibited antiviral potentialities before. The applied selection methods aimed to pick the most relevant inhibitor of SARS-CoV-2 nsp10. At first, a structural similarity study against the co-crystallized ligand, S-Adenosyl Methionine (SAM), of SARS-CoV-2 nonstructural protein (nsp10) (PDB ID: 6W4H) was carried out. The similarity analysis culled 30 candidates. Secondly, a fingerprint study against SAM preferred compounds 44, 48, 85, 102, 105, 182, 220, 221, 282, 284, 285, 301, and 302. The docking studies picked 48, 182, 220, 221, and 284. While the ADMET analysis expected the likeness of the five candidates to be drugs, the toxicity study preferred compounds 48 and 182. Finally, a density-functional theory (DFT) study suggested vidarabine (182) to be the most relevant SARS-Cov-2 nsp10 inhibitor.

Fludarabine Inhibits Infection of Zika Virus, SFTS Phlebovirus, and Enterovirus A71.

Viral infections are one of the leading causes in human mortality and disease. Broad-spectrum antiviral drugs are a powerful weapon against new and re-emerging viruses. However, viral resistance to existing broad-spectrum antivirals remains a challenge, which demands development of new broad-spectrum therapeutics. In this report, we showed that fludarabine, a fluorinated purine analogue, effectively inhibited infection of RNA viruses, including Zika virus, Severe fever with thrombocytopenia syndrome virus, and Enterovirus A71, with all IC50 values below 1 µM in Vero, BHK21, U251 MG, and HMC3 cells. We observed that fludarabine has shown cytotoxicity to these cells only at high doses indicating it could be safe for future clinical use if approved. In conclusion, this study suggests that fludarabine could be developed as a potential broad-spectrum anti-RNA virus therapeutic agent.

An Enzymatic Flow-Based Preparative Route to Vidarabine.

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

The interaction mechanism between fludarabine and human serum albumin researched by comprehensive spectroscopic methods and molecular docking technique.

Fludarabine (Flu) is widely used to treat B-cell chronic lymphocytic leukemia. HSA is of the essence to human, especially in blood circulation system. The interaction mechanism between Flu and HSA was studied by comprehensive spectroscopic methods and molecular docking technique. UV-vis and FL spectrum results indicated that Flu bond with HSA, and there was a new complex produced at the binding site I in subdomain IIA. Association constants at 298 K were 1.637 × 104 M-1 and 1.552 × 104 M-1 at 310 K, respectively. The negative enthalpy (ΔH) and positive entropy (ΔS) values for the interaction revealed that the binding behavior was driven by hydrophobic forces and hydrogen bonds. The results obtained from UV, RLS spectra, 3D fluorescence and CD spectrum illustrated that Flu could change the secondary structure of HSA. According to molecule docking result, the binding energy of interaction is -11.15 kcal/mol.

Elucidation of binding interactions and mechanism of Fludarabine with dsDNA via multispectroscopic and molecular docking studies.

Fludarabine is a purine derivative, anti-neoplastic drug and is still being used in the treatments of chronic lymphocytic leukemia, small lymphocytic lymphoma, acute myeloid leukemia, Non-Hodgkin's lymphoma. It achieves its function by interacting with DNA. Therefore, the binding interactions of such drugs with deoxyribonucleic acid (DNA) is an important subject for pharmaceutical and biochemical studies aiming at designing better DNA binding drugs. Although DNA binding mode of some of the anti-neoplastic drugs has been studied, DNA interaction of Fludarabine has not been explored yet. For this reason, this work has been dedicated to deciphering the experimental and theoretical investigation of Fludarabine binding mechanism via multispectroscopic techniques including UV absorption spectroscopy, thermal denaturation, fluorescence and FTIR spectroscopy, electrochemical and viscosity measurement methods as well as with molecular docking studies under physiological conditions. We observed in the lowest energy docking poses that Fludarabine binds to DNA via major groove binding mode. The nonplanar and extended structure and hydrogen bonding interactions of Fludarabine with the Adenine-Thymine base-pair played a very decisive role in the binding mode as supported by the experimental results.

Structural Basis for Inhibition of Human Primase by Arabinofuranosyl Nucleoside Analogues Fludarabine and Vidarabine.

Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin's lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity toward primase, possibly leading to less toxic and more effective therapeutic agents.

Fludarabine-Specific Molecular Interactions with Maltose-Modified Poly(propyleneimine) Dendrimer Enable Effective Cell Entry of the Active Drug Form: Comparison with Clofarabine.

Fludarabine is an anticancer antimetabolite essential for modern chemotherapy, but its efficacy is limited due to the complex pharmacokinetics. We demonstrated the potential use of maltose-modified poly(propyleneimine) dendrimer as drug delivery agent to improve the efficiency of therapy with fludarabine. In this study, we elaborated a novel synthesis technique for radioactively labeled fludarabine triphosphate to prove for the first time the direct ability of nucleotide-glycodendrimer complex to enter and kill leukemic cells, without the involvement of membrane nucleoside transporters and intracellular kinases. This will potentially allow to bypass the most common drug resistance mechanisms observed in the clinical setting. Further, we applied surface plasmon resonance and molecular modeling to elucidate the properties of the drug-dendrimer complexes. We showed that clofarabine, a more toxic nucleoside analogue drug, is characterized by significantly different molecular interactions with poly(propyleneimine) dendrimers than fludarabine, leading to different cellular outcomes (decreased rather than increased treatment efficiency). The most probable mechanistic explanation of uniquely dendrimer-enhanced fludarabine toxicity points to a crucial role of both an alternative cellular uptake pathway and the avoidance of intracellular phosphorylation of nucleoside drug form.

Selectively Targeted Anti-Neoplastic Cytotoxicity of Three Immunopharmaceuticals with Covalently Bound Fludarabine, Gemcitabine and Dexamethasone Moieties Synthesized Utilizing Organic Chemistry Reactions in a Multi-Stage Regimen.

BACKGROUND: Unintentional passive diffusion of conventional small molecular weight pharmaceuticals across intact membranes of normal healthy cells in tissues and organ systems induces sequelae that limit therapeutic dosage and duration of administration. Selective "targeted" delivery of pharmaceuticals is a molecular strategy that can potentially provide heightened margins-of-safety with greater potency and improved efficacy. MATERIALS AND METHODS: Monophosphate analogs of fludarabine, gemcitabine, and dexamethasone were combined with a carbodiimide reagent in the presence of imidazole to produce reactive intermediates that were subsequently covalently bound to monoclonal anti-IGF-1R or anti-EGFR IgG-immunoglobulin. The resulting covalent immunopharmaceutical end-products, fludarabine-(5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'- phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti-EGFR] were evaluated by SDS-PAGE/chemiluminescent autoradiography (fragmentation/polymerization detection), UV spectrophotometric absorbance (purity; molar-incorporation-index), cell-ELISA (retained selective binding-avidity), and cell vitality-viability (selectively "targeted" anti-neoplastic cytotoxicity). RESULTS: Maximum selectively "targeted" anti-neoplastic cytotoxicity of fludarabine-(5'-phosphoramidate)-[anti- IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-(C21-phosphoramidate)-[anti- EGFR] was detected at the pharmaceutical-equivalent concentrations of 10-5 M (94.7%), 10-7 M (93.1%), and 10-7 M (64.9%) respectively. DISCUSSION: Organic chemistry reactions were optimized in a template multi-stage synthesis regimen for fludarabine-( 5'-phosphoramidate)-[anti-IGF-1R], gemcitabine-(5'-phosphoramidate)-[anti-IGF-1R], and dexamethasone-( C21-phosphoramidate)-[anti-EGFR]. Attributes of the synthesis regimen include; [-i-] covalent bonding of pharmaceutical moeities at high molar incorporation indexes, [-ii-] implementation of organic chemistry reactions in a non-dedicated synthesis regimen allowing component substitution and [-iii-] optional preservation of presynthesized amine-reactive pharmaceutical intermediates for on-demand immunopharmaceutical synthesis. Attributes of the covalent immunopharmaceuticals are; absence of any synthetically introduced chemical groups, retained IgG-immunoglobulin binding-avidity and potent selective "targeted" anti-neoplastic cytotoxic potency. Under in-vivo conditions, supplemental anti-neoplastic cytotoxicity is realized through trophic receptor inhibition and activation of multiple cytotoxic host immune responses.

Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

While working with G418-resistant stably transfected cells, we realized the neomycin resistance (NeoR) gene, which encodes the aminoglycoside-3'-phosphotransferase-IIa [APH(3')-IIa], also confers resistance to the nucleoside analog fludarabine. Fludarabine is a cytostatic drug widely used in the treatment of hematologic and solid tumors, as well as in the conditioning of patients before transplantation of hematopoietic progenitors. We present evidence that NeoR-transfected cells do not incorporate fludarabine, thus avoiding DNA damage caused by the drug, evidenced by a lack of FANCD2 monoubiquitination and impaired apoptosis. A screening of other nucleoside analogs revealed that APH(3')-IIa only protects against ATP purine analogs. Moreover, APH(3')-IIa ATPase activity is inhibited by fludarabine monophosphate, suggesting that APH(3')-IIa blocks fludarabine incorporation into DNA by dephosphorylating its active fludarabine triphosphate form. Furthermore, overexpression of the catalytic subunit of the eukaryotic kinase PKA, which is structurally related to APHs, also provides resistance to fludarabine, anticipating its putative utility as a response marker to the drug. Our results preclude the use of Neo marker plasmids in the study of purine analogs and unveils a new resistance mechanism against these chemotherapeuticals.-Sánchez-Carrera, D., Bravo-Navas, S., Cabezón, E., Arechaga, I., Cabezas, M., Yáñez, L., Pipaón, C. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

An Unusual Protector-Protégé Strategy for the Biosynthesis of Purine Nucleoside Antibiotics.

Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that the biosynthesis of PTN and Ara-A employs an unusual protector-protégé strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions as a reversible oxidoreductase for the final step of PTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the host adenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.

Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2ß2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.

Synthesis, characterization and bioevaluation of drug-collagen hybrid materials for biomedical applications.

This work presents a study based on the preparation and characterization of drug-collagen hybrid materials. Materials used for obtaining drug-collagen hybrids were collagen type I (Coll) as matrix and fludarabine (F) and epirubicin (E) as hydrophilic active substances. After incorporation of drugs into Coll in different ratios, the obtained hybrid materials (Coll/F and Coll/E) could be used according to our results as potential drug delivery systems in medicine for the topical (local) treatment of cancerous tissues (e.g. the treatment of breast, stomach, lung, colorectal or advanced ovarian cancer). The materials were characterized considering their composition (by XRD, FT-IR and DTA-TG) and their morphology (by SEM). The delivery of drug was assessed by UV-vis. The in vitro citotoxicity demonstrates an antitumoral activity of the obtained hybrid materials and their potential use for biomedical applications as drug delivery systems in tumoral treatments.

Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.

The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.

Simultaneous determination of fludarabine and clofarabine in human plasma by LC-MS/MS.

A method for quantification of fludarabine (FDB) and clofarabine (CFB) in human plasma was developed with an API5000 LC-MS/MS system. FDB and CFB were extracted from EDTA plasma samples by protein precipitation with trichloroacetic acid. Briefly, 50 µL plasma sample was mixed with 25 µL internal standard (50 ng/mL aqueous 2-Cl-adensosine) and 25 µL 20% trichloroacetic acid, centrifuged at 25,000 × g (20,000 rpm) for 3 min, and then transfered to an autosampler vial. The extracted sample was injected onto an Eclipse extend C18 column (2.1 mm×150 mm, 5 µm) and eluted with 1mM NH4OH (pH 9.6) - acetonitrile in a gradient mode. Electrospray ionization in positive mode (ESI(+)) and multiple reaction monitoring (MRM) were used, and ion pairs 286/134 for FDB, 304/170 for CFB and 302/134 for the internal standard were selected for quantification. The retention times were typically 3.72 min for FDB, 4.34 min for the internal standard, 4.79 min for CFB. Total run time was 10 min per sample. Calibration range was 0.5-80 ng/mL for CFB and 2-800 ng/mL for FDB. The method was applied to a clinical pharmacokinetic study in pediatric patients.

Targeting the proliferative and chemoresistant compartment in chronic lymphocytic leukemia by inhibiting survivin protein.

Chronic lymphocytic leukemia (CLL) cells located in proliferation centers are constantly stimulated by accessory cells, which provide them with survival and proliferative signals and mediate chemotherapy resistance. Herein, we designed an experimental strategy with the aim of mimicking the microenvironment found in the proliferative centers to specifically target actively proliferating CLL cells. For this, we co-cultured CLL cells and bone marrow stromal cells with concomitant CD40 and Toll-like receptor 9 stimulation. This co-culture system induced proliferation, cell-cycle entry and marked resistance to treatment with fludarabine and bendamustine. Proliferating CLL cells clustered together showed a typical morphology of activated B cells and expressed survivin protein, a member of the inhibitor of apoptosis family that is mainly expressed by CLL cells in the proliferation centers. With the aim of specifically targeting actively proliferating and chemoresistant CLL cells, we investigated the effects of treatment with YM155, a small-molecule survivin inhibitor. YM155 treatment suppressed the co-culture-induced survivin expression and that was sufficient to inhibit proliferation and effectively induce apoptosis particularly in the proliferative subset of CLL cells. Interestingly, sensitivity to YM155 was independent from common prognostic markers, including 17p13.1 deletion. Altogether, these findings provide a rationale for clinical development of YM155 in CLL.

Vidarabine is neither a potent nor a selective AC5 inhibitor.

Vidarabine was the first clinically approved antiviral drug, but due to safety and efficacy issues the drug is currently only used topically for herpes virus keratitis. Scientific interest in vidarabine has been recently renewed due to the fact that the compound exhibits beneficial effects in animal models of heart failure and cancer, replicating effects of the knockout of adenylyl cyclase 5 (AC5). Therefore, vidarabine has been suggested to mediate these effects via selective inhibition of AC5. Based on these results, clinical studies with vidarabine in humans for heart failure and cancer have been proposed. Here, evidence is presented that vidarabine is neither a potent nor a selective AC5 inhibitor. Greatest caution should be exerted when proposing new mechanisms of actions and clinical uses for vidarabine.

Simultaneous quantification of idelalisib, fludarabine and lenalidomide in rat plasma by using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry.

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of idelalisib, fludarabine and lenalidomide using tolbutamide as an internal standard. Analytes were recovered by liquid-liquid extraction and separated on a reverse phase C18 column (150mm×4.6mm i.d., 5µm) using methanol:0.1% formic acid buffer (70:30) as mobile phase at a flow rate of 1mL/min in isocratic mode. Selective reaction monitoring was performed using the transitions, i.e. m/z 416.25/176.48, 286.11/154.10, 260.15/149.15, and 271.14/155.06 to quantify idelalisib, fludarabine and lenalidomide and tolbutamide, respectively. The method was validated over the concentration range of 1.15-576.84ng/mL for idelalisib, 0.95-476.25ng/mL for fludarabine and 0.97-486.19ng/mL for lenalidomide. Intra and inter-day accuracy and precision of validated method were within the acceptable limits of <15%. Coefficients of correlation (r(2)) for the calibration curves were >0.998 for all analytes. The method was successfully applied for simultaneous estimation of idelalisib, fludarabine and lenalidomide in a pharmacokinetic study in rats.