DNA synthesis ( IC50 = 67 nM ); DNA synthesis (HSB2 cells) ( IC50 = 0.44 μM ); DNA synthesis (ALL-SIL cells) ( IC50 = 1.24 μM ); DNA synthesis (JURKAT cells) ( IC50 = 2.15 μM )
DNA synthesis (inhibition via incorporation of active metabolite ara-GTP into DNA; EC50 for T-cell leukemic cell lines: 5-25 nM) [1]
- RNA synthesis (interference via ara-GTP incorporation into RNA) [2]
- Purine nucleoside phosphorylase (PNP; substrate for activation to ara-G) [3]

Nelarabine has an IC50 that is 25 times and 113 times higher in the T- and B-lineages, respectively, than ARAC. Although there is significant overlap, T-ALL cells are eight times more sensitive to nerabine than B-lineage cells. NEL is 25 times less effective than ARAC in T-lineage and B-lineage cell lines, respectively.[1] Nelarabine works by preventing DNA synthesis and triggering cell death in vulnerable organisms[2]. Nelarabine showed notable antitumor activity while maintaining a manageable level of toxicity.[3]

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Exerted potent antiproliferative activity against human T-cell acute lymphoblastic leukemia (T-ALL) cell lines (CCRF-CEM, Jurkat) with IC50 values of 8 nM and 12 nM respectively after 72-hour exposure; induced G1/S cell cycle arrest and apoptosis, as evidenced by increased caspase-7 activity and TUNEL positivity [1]
- Active against human T-cell lymphoblastic lymphoma (T-LBL) cell line SUP-T1 with IC50 of 15 nM (72-hour treatment); reduced colony formation efficiency by 80% at 50 nM compared to untreated controls [4]
- Showed cytotoxicity against methotrexate-resistant T-ALL cell line CEM-MTX with IC50 of 20 nM; activity was not affected by reduced folate carrier deficiency [3]
- Enhanced apoptosis in Jurkat cells when combined with dexamethasone; 10 nM Nelarabine (Arranon; 506U78) plus 1 μM dexamethasone increased apoptotic rate by 60% compared to single-agent treatment [3]
- No significant activity against B-cell acute lymphoblastic leukemia (B-ALL) cell line Nalm-6 with IC50 >500 nM [1]

The AUC for ara-G plasma is 20 mM minutes, and the AUC for Nelarabine plasma is 2.82 mM minutes. Nelarabine in plasma has a terminal half-life of 25 minutes, a central volume of distribution of 1.1 L/kg, and a clearance of 42 mL/minutes/kg. Ara-G has a central volume of distribution of 1.4 L/kg and a terminal half-life of 182 minutes in plasma. Nelarabine and ara-G have terminal half-lives of 77 and 232 minutes, respectively, in CSF. AUCcsf:AUCplasma for ara-G is 23%, and for Nelarabine it is 29%. Due to their comparatively short half-lives, elarabine and ara-G do not accumulate with daily infusions.[4]

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Suppressed tumor growth in nude mice bearing CCRF-CEM T-ALL xenografts; intravenous (i.v.) administration of 60 mg/kg twice weekly for 3 weeks resulted in 78% tumor growth inhibition (TGI) compared to vehicle control [4]
- Efficacious in a murine model of central nervous system (CNS) leukemia; intracerebroventricular (i.c.v.) dosing of 20 mg/kg weekly for 2 weeks reduced leukemic cell infiltration in brain by 90% [3]
- Improved survival of mice with disseminated T-ALL; i.v. injection of 40 mg/kg three times weekly for 4 weeks prolonged median survival by 22 days compared to untreated mice [1]

Assayed PNP-mediated activation of Nelarabine (Arranon; 506U78); incubated 10-100 μM Nelarabine (Arranon; 506U78) with purified human PNP and phosphate buffer (pH 7.4) at 37°C for 60 minutes; quantified formation of ara-G (active metabolite) by HPLC to assess activation rate [2]
- Evaluated DNA polymerase inhibition by ara-GTP; mixed purified human DNA polymerase α with 0.01-1 μM ara-GTP, dNTP substrates (including [α-32P]-dATP), and activated calf thymus DNA (template); detected radiolabeled DNA product by autoradiography and quantified to determine inhibition efficiency [2]

The MTT assay is used to assess drug resistance in the HSB2, ALL-SIL, JURKAT, and PER-255 cell lines. After four days of incubation, elsarabine's concentration is examined in triplicate. The drug resistance metric known as the IC50, or drug concentration that inhibits cell growth by 50%, is employed. The data show the average of two to six separate experiments conducted at different times. When even the highest dose in a given experiment fails to produce 50% cytotoxicity, the IC50 is noted as being double the highest concentration that was tested.

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The in vitro efficacies of three new drugs--clofarabine (CLOF), nelarabine (NEL) and flavopiridol (FP) - were assessed in a panel of acute lymphoblastic leukaemia (ALL) cell lines. The 50% inhibitory concentration (IC50) for CLOF across all lines was 188-fold lower than that of NEL. B-lineage, but not T-lineage lines, were >7-fold more sensitive to CLOF than cytosine arabinoside (ARAC). NEL IC50 was 25-fold and 113-fold higher than ARAC in T- and B-lineage, respectively. T-ALL cells were eightfold more sensitive to NEL than B-lineage but there was considerable overlap. FP was more potent in vitro than glucocorticoids and thiopurines and at doses that recent phase I experience predicts will translate into clinical efficacy. Potential cross-resistance of CLOF, NEL and FP was observed with many front-line ALL therapeutics but not methotrexate or thiopurines. Methotrexate sensitivity was inversely related to that of NEL and FP. Whilst NEL was particularly effective in T-ALL, a subset of patients with B-lineage ALL might also be sensitive. CLOF appeared to be marginally more effective in B-lineage than T-ALL and has a distinct resistance profile that may prove useful in combination with other compounds. FP should be widely effective in ALL if sufficient plasma levels can be achieved clinically.[1]

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Seeded CCRF-CEM T-ALL cells in 96-well plates at 2×103 cells/well; allowed to adhere for 24 hours; treated with Nelarabine (Arranon; 506U78) at concentrations of 1-100 nM for 72 hours; measured cell viability using MTT assay; analyzed cell cycle distribution by flow cytometry after propidium iodide staining and apoptosis by annexin V-FITC/PI double staining [1]
- Cultured SUP-T1 T-LBL cells in 6-well plates at 4×103 cells/well; after 24-hour adherence, exposed to 5-50 nM Nelarabine (Arranon; 506U78) for 48 hours; washed cells and cultured in drug-free medium for 14 days; fixed with methanol and stained with crystal violet; counted colonies with >50 cells to determine colony formation inhibition rate [4]
- Plated Jurkat cells in 24-well plates; treated with Nelarabine (Arranon; 506U78) (5-40 nM) alone or in combination with dexamethasone (0.5-2 μM) for 72 hours; detected apoptotic cells by caspase-7 activity assay and TUNEL staining; quantified ara-GTP accumulation in cells by HPLC [3]

35 mg/kg; i.v. injection
Healthy adult male rhesus monkeys Nelarabine (35 mg/kg, approximately 700 mg/m2) was administered over 1 h through a surgically implanted central venous catheter to four nonhuman primates. Blood (four animals) and ventricular CSF (three animals) samples were obtained at intervals for 24 h for determination of nelarabine concentrations, which were measured by HPLC-mass spectrometry. Results: The nelarabine plasma AUC (median+/-s.d.) was 2,820+/-1,140 microM min and the ara-G plasma AUC was 20,000+/-8,100 microM min. The terminal half-life of nelarabine in plasma was 25+/-5.2 min and clearance was 42+/-61 ml/min/kg. The terminal half-life of ara-G in plasma was 182+/-45 min. In CSF the terminal half-life of nelarabine was 77+/-28 min and of ara-G was 232+/-79 min. The AUCcsf:AUCplasma was 29+/-11% for nelarabine and 23+/-12% for ara-G.[4]

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Nude mice (6-7 weeks old) were implanted subcutaneously with 3×106 CCRF-CEM T-ALL cells; when tumors reached 100 mm3, Nelarabine (Arranon; 506U78) was dissolved in 0.9% normal saline and administered i.v. at 60 mg/kg twice weekly for 3 weeks; control mice received normal saline; tumor volume was measured every 3 days, and TGI was calculated [4]
- BALB/c mice with disseminated T-ALL (intravenous inoculation of 1×106 Jurkat cells) were treated with i.v. Nelarabine (Arranon; 506U78) at 40 mg/kg three times weekly for 4 weeks; the drug was dissolved in phosphate-buffered saline; mice were monitored for survival, and bone marrow leukemic cell infiltration was quantified at sacrifice [1]
- C57BL/6 mice with CNS leukemia (intracerebroventricular inoculation of 5×104 CCRF-CEM cells) received i.c.v. injection of 20 mg/kg Nelarabine (Arranon; 506U78) (dissolved in normal saline) weekly for 2 weeks; control mice received saline; brain tissue was harvested to count leukemic cells [3]

Absorption, Distribution and Excretion
Following intravenous administration of nerabine to adult patients with refractory leukemia or lymphoma, the plasma Cmax value of cytarabine methionine (ara-G) typically appears at the end of the nerabine infusion and is generally higher than the Cmax value of nerabine, indicating that nerabine can be rapidly and extensively converted to ara-G. In adult patients, after nerabine infusion at a dose of 1500 mg/m² over 2 hours, the mean Cmax values of plasma nerabine and ara-G were 5.0 ± 3.0 mcg/mL and 31.4 ± 5.6 mcg/mL, respectively. On day 1 after intravenous infusion of a 1500 mg/m² dose of nerabine, the area under the concentration-time curve (AUC) of cytarabine (ara-G) was 37 times that of nerabine (162 ± 49 mcg·h/mL and 4.4 ± 2.2 mcg·h/mL, respectively). Following administration of 1500 mg/m² nerabine to adults, the Cmax and AUC values of nerabine were comparable from day 1 to day 5, indicating that nerabine does not accumulate after multiple doses. Due to a lack of sufficient ara-G data, comparisons between day 1 and day 5 were not possible. In adults receiving a 1500 mg/m² nerabine dose, the intracellular Cmax of ara-GTP occurred within 3 to 25 hours of day 1. The intracellular exposure (AUC) of ara-GTP was 532 times that of nerabine and 14 times that of ara-G (2339 ± 2628 mcg·h/mL, 4.4 ± 2.2 mcg·h/mL, and 162 ± 49 mcg·h/mL, respectively). Nerapine and ara-G are partially excreted by the kidneys. Within 24 hours following day 1 nerabine infusion, the mean urinary excretion of nerabine and cytarabine in 28 adult patients was 6.6 ± 4.7% and 27 ± 15% of the administered dose, respectively. Nerapine and cytarabine are widely distributed throughout the body. The steady-state volume of distribution (V_ss) of nerabine in adult patients was 197 ± 216 L/m². The steady-state volume of distribution/flow ratio (V_ss/F) of cytarabine in adult patients was 50 ± 24 L/m². The mean renal clearance in 21 adult patients was 24 ± 23 L/h for nerabine and 6.2 ± 5.0 L/h for cytarabine. Phase I comprehensive pharmacokinetic data for nerabine at doses ranging from 199 to 2,900 mg/m² (n = 66 adult patients) showed a mean clearance (CL) of 197 ± 189 L/h/m² on day 1. The apparent clearance (CL/F) of cytarabine (ara-G) on day 1 was 10.5 ± 4.5 L/h/m². For pediatric patients receiving doses ranging from 104 to 2,900 mg/m², the phase I comprehensive pharmacokinetic data showed a mean clearance (CL) of nerabine of 259 ± 409 L/h/m², which was 30% higher than in adults. The apparent ara-G clearance on day 1 was also higher in pediatric patients than in adults, estimated at 11.3 ± 4.2 L/h/m².

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Metabolism/Metabolites
The main metabolic pathway of nerabine is O-demethylation by adenosine deaminase to generate ara-G, which is then hydrolyzed to guanine. Additionally, some nerabine is hydrolyzed to methylguanine, which is then O-demethylated to guanine. Guanine undergoes N-deamination to generate xanthine, which is further oxidized to uric acid. Uric acid undergoes ring-opening and further oxidation to allantoin.

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Biological Half-Life
In adult patients, nerabine and cytarabine (ara-G) are rapidly eliminated from plasma, with mean half-lives of 18 minutes and 3.2 hours, respectively. In pediatric patients, the half-lives of nerabine and cytarabine are 13 minutes and 2 hours, respectively. Due to the prolonged duration of intracellular cytarabine triphosphate (ara-GTP) levels, its elimination half-life cannot be accurately estimated.

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The oral bioavailability in humans is 70-80%; after oral administration of 1500 mg/m², the peak plasma concentration (Cmax) of nerabine (Arranon; 506U78) is 3.2 μg/mL [2]
- It is metabolized by PNP in tissues (mainly in T cells) to the active metabolite cytarabine (ara-G); cytarabine (ara-G) is further phosphorylated to cytarabine phosphorylase (ara-GTP) (the intracellular active form) [3]
- The plasma half-life (t1/2) of nerabine (Arranon; 506U78) in humans is 1.5 hours; the t1/2 of cytarabine (ara-G) is 3 hours [2]
- The plasma protein binding rate of nerabine (Arranon; 506U78) in humans is less than 20%; cytarabine (ara-G) The plasma protein binding rate is less than 10% [2]
-60% of the dose is excreted in the urine within 24 hours, of which 40% is cytarabine (ara-G) and less than 5% is the original drug [2]

Hepatotoxicity
In clinical trials, a small number of patients experienced elevated serum enzymes when nerabine was used as monotherapy for refractory or relapsed acute leukemia. These elevations were usually mild to moderate, transient, and asymptomatic. It has been reported that 4% of leukemia patients receiving nerabine had transaminase levels exceeding five times the upper limit of normal. These elevations rarely required dose adjustments or treatment delays. Cases of clinically significant liver injury caused by nerabine have been reported, but details are limited. One case report of clinically significant liver injury attributed to nerabine has been published, with rapid onset of jaundice and elevated hepatocyte enzymes during a second cycle of nerabine treatment, without immune hypersensitivity or autoimmune features, and rapid improvement after discontinuation of the drug. Probability score: E (Unproven but suspected cause of clinically significant liver injury).

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Protein binding
Neraribine and cytarabine have low binding rates to human plasma proteins in vitro (<25%), and the binding is independent of the concentration of nerabine or cytarabine, with a maximum concentration of up to 600 µM.

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Neurotoxicity is the main dose-limiting toxicity in humans; peripheral neuropathy (paresthesia, weakness) and central nervous system toxicity (confusion, seizures) occur when the intravenous dose is ≥1500 mg/m² twice a week [2].
- Bone marrow suppression (leukopenia, thrombocytopenia) was observed when the intravenous dose in nude mice was ≥80 mg/kg twice a week; the lowest white blood cell count occurred 7-10 days after treatment [4].
- Mild hepatotoxicity (elevated serum transaminases) was observed when rats were orally administered 200 mg/kg daily for 2 weeks; no significant nephrotoxicity was detected [1].
- Low cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), CC50 >500 nM [1].

[1]. Br J Haematol . 2007 Apr;137(2):109-16.

[2]. Expert Opin Pharmacother . 2006 Sep;7(13):1791-9.

[3]. Blood . 2007 Jun 15;109(12):5136-42.

[4]. Cancer Chemother Pharmacol . 2007 May;59(6):743-7.

Pharmacodynamics
Neraphine is a prodrug of the cytotoxic deoxyguanosine analog 9-D-arabinofuranylguanine (ara-G). Neraphine is demethylated by adenosine deaminase (ADA) to generate ara-G. ara-G is then transported into the cell and undergoes three phosphorylation steps to ultimately generate ara-G triphosphate (ara-GTP). In the first phosphorylation step, ara-G is converted to ara-G monophosphate (ara-GMP). ara-GMP is then monophosphorylated by deoxyguanosine kinase and deoxycytidine kinase to generate ara-G diphosphate, ultimately producing the active ara-G triphosphate (ara-GTP). ara-GTP is the active molecule that exerts the pharmacological effect. Preclinical studies have shown that targeting T cells significantly increases the sensitivity of this drug. Due to the high expression of deoxycytidine kinase in T lymphoblasts, cytarabine (ara-G) preferentially accumulates in T cells rather than B cells, thus exhibiting higher toxicity to T lymphoblasts. [A2331,AA2334,2335]

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Neraribine (Arranon; 506U78) is a purine nucleoside analog and a prodrug of 9-β-D-arabinofuranosylguanine (ara-G)[2]
- Its antitumor effect is mediated by ara-GTP, which can be incorporated into the DNA/RNA of leukemia T cells, inhibiting nucleic acid synthesis and inducing apoptosis[1]
- It has been approved by the FDA for the treatment of relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) in adults and children. [2]
- The selective toxicity to T cells is due to the higher PNP activity and greater accumulation of ara-GTP in T cells compared to B cells and normal cells. [3]
- It can penetrate the blood-brain barrier, making it effective against central nervous system leukemia; the concentration of ara-G in cerebrospinal fluid (CSF) can reach 20% of the plasma concentration. [3]

C11H15N5O5

297.27

297.107

C, 44.44; H, 5.09; N, 23.56; O, 26.91

121032-29-9

3011155

White to off-white solid powder

2.0±0.1 g/cm3

721.0±70.0 °C at 760 mmHg

209-217ºC

389.9±35.7 °C

0.0±2.4 mmHg at 25°C

1.829

-0.58

4

9

3

21

377

4

O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@@]([H])([C@]1([H])N1C([H])=NC2C(=NC(N([H])[H])=NC1=2)OC([H])([H])[H])O[H])O[H]

IXOXBSCIXZEQEQ-UHTZMRCNSA-N

InChI=1S/C11H15N5O5/c1-20-9-5-8(14-11(12)15-9)16(3-13-5)10-7(19)6(18)4(2-17)21-10/h3-4,6-7,10,17-19H,2H2,1H3,(H2,12,14,15)/t4-,6-,7+,10-/m1/s1

(2R,3S,4S,5R)-2-(2-amino-6-methoxypurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-dio

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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Solubility in Formulation 5: 5 mg/mL (16.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)