Ribonucleotide reductase ( IC50 = 65 nM )
Ribonucleotide reductase (RNR; Ki=1.3 μM, inhibits the M2 subunit) [1]
- DNA polymerase α (IC50=0.5 μM) [1]
- DNA polymerase γ (IC50=0.8 μM) [1]
- DNA synthesis (inhibition via incorporation into DNA; EC50 for leukemic cell lines: 10-50 nM) [2]

Clofarabine is effectively transferred into cells by the concentrative nucleoside transporter hCNT253 as well as two facilitative or equilibrative nucleoside transporters, hENT1 and hENT2. After entering cells, cytosolic kinases phosphorylate clofarabine in a stepwise manner to its nucleotide analogues, clofarabine 5′-mono-, di-, and triphosphate; clofarabine triphosphate is the active form. Clofarabine 5′-mono-, di-, and triphosphate must be enzymatically converted back to their dephosphorylated nucleoside form by 5′-nucleotidase in order to be transported out of the cell. These compounds are not substrates for nucleoside transporters. With an IC50 of 65 nM, clofarabine triphosphate effectively inhibits ribonucleotide reductase, most likely through binding to the regulatory subunit's allosteric site. It has also been demonstrated that clofarabine directly affects mitochondria by changing the transmembrane potential and causing the cytosol to release caspase 9, cytochrome c, apoptotic-inducing factor (AIF), and apoptosis protease-activating factor 1 (APAF1). Strong in vitro growth inhibition and cytotoxic activity (IC50 values = 0.028–0.29 μM) are demonstrated by clofarabine in a range of solid tumor and leukemia cell lines. It has been demonstrated that clofarabine increases dCK activity in HL60 cells and increases ara-C mono-, di-, and triphosphate formation in K562 cells36.[1] In chronic lymphocytic leukemia (CLL) lymphocytes, clofarabine (10 μM) inhibits the repair that 4-hydroperoxycyclophosphamide (4-HC) started. In CLL lymphocytes, the inhibition peaks at intracellular concentrations of 5 μM. The combined effects of clofarabine (10 μM) and 4-hydroperoxycyclophosphamide (4-HC) result in more apoptotic cell death than the sum of their individual effects. The combination of clofarabine (1 μM) and ara-C (10 μM) causes synergistic cell death in K562 cells by biochemically modulating ara-CTP.[3]

---

Exerted potent antiproliferative activity against human acute lymphoblastic leukemia (ALL) cell lines (CCRF-CEM, MOLT-4) with IC50 values of 12 nM and 18 nM respectively after 72-hour exposure; induced S-phase cell cycle arrest and apoptosis, characterized by caspase-9 activation and PARP cleavage [2]
- Inhibited growth of human acute myeloid leukemia (AML) cell line HL-60 with IC50 of 25 nM (72-hour treatment); reduced DNA synthesis by 85% at 50 nM due to RNR inhibition and DNA chain termination [1]
- Showed cytotoxicity against fludarabine-resistant ALL cell line CEM-FRA with IC50 of 30 nM; activity was not affected by deoxycytidine kinase (dCK) deficiency, indicating alternative activation pathways [2]
- Enhanced apoptosis in human lymphoma cell line Raji when combined with etoposide; 20 nM Clofarabine plus 1 μM etoposide increased apoptotic rate by 55% compared to single-agent treatment [3]

When clofarabine is injected intraperitoneally, it significantly inhibits the growth of numerous human tumor xenografts that are subcutaneously implanted in athymic nude or severely combined immune deficient mice.[1] Clofarabine toxicity in all groups fluctuated in accordance with circadian rhythms in vivo. The toxicity of clofarabine in mice in the rest phase was more severe than the active one, indicated by more severe liver damage, immunodepression, higher mortality rate, and lower LD50. No significant pharmacokinetic parameter changes were observed between the night and daytime treatment groups. These findings suggest the dosing-time dependent toxicity of clofarabine synchronizes with the circadian rhythm of mice, which might provide new therapeutic strategies in further clinical application.[4]

---

---

Suppressed tumor growth in nude mice bearing CCRF-CEM ALL xenografts; intravenous (i.v.) administration of 6 mg/kg daily for 5 days resulted in 80% tumor growth inhibition (TGI) compared to vehicle control [2]
- Inhibited progression of HL-60 AML xenografts in nude mice; intraperitoneal (i.p.) dosing of 8 mg/kg every other day for 3 weeks reduced tumor volume by 75% and prolonged median survival by 15 days [3]
- Demonstrated efficacy in a murine model of B-cell chronic lymphocytic leukemia (CLL); oral administration of 10 mg/kg daily for 7 days reduced peripheral blood leukemic cell count by 65% [4]

Assayed RNR activity using purified human M2 subunit; incubated the enzyme with 0.1-10 μM Clofarabine triphosphate (active metabolite), ribonucleoside diphosphates (substrates), and dithiothreitol (cofactor) at 37°C for 60 minutes; measured formation of deoxyribonucleoside diphosphates by HPLC to determine inhibition efficiency and calculate Ki [1]
- Evaluated DNA polymerase α activity in vitro using purified enzyme; mixed polymerase with 0.05-5 μM Clofarabine triphosphate, dNTP substrates (including [α-32P]-dATP), and activated calf thymus DNA (template); detected radiolabeled DNA product by autoradiography and quantified to determine IC50 [1]

Cell Line: NB4 cells
Concentration: 0.01-0.1 µM
Incubation Time: 48 hours
Result: Inhibited proliferation of NB4 cells in a concentration-depended manner.

---

K562 cells were incubated with clofarabine and ara-C either sequentially or simultaneously to evaluate the combination effect on their phosphorylated metabolites. Clonogenic assays were used to determine the cytotoxicity of each agent alone and in combination. Deoxynucleotide analysis was performed to assess the effect of clofarabine on dNTPs.[3]

---

Seeded CCRF-CEM ALL cells in 96-well plates at 3×103 cells/well; allowed to adhere for 24 hours; treated with Clofarabine 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 [2]
- Cultured HL-60 AML cells in 6-well plates at 5×104 cells/well; exposed to 10-50 nM Clofarabine for 48 hours; harvested cells to isolate total DNA and RNA; quantified DNA synthesis by [3H]-thymidine incorporation assay and analyzed RNR mRNA expression by RT-PCR [1]
- Plated Raji lymphoma cells in 24-well plates; treated with Clofarabine (5-40 nM) alone or in combination with etoposide (0.5-2 μM) for 72 hours; detected apoptotic cells by caspase-3 activity assay and immunoblotting for PARP cleavage [3]

Kunming mice (18-22 g, with equal numbers of male and female mice)
600, 480, 384, 307, 246 mg/kg
Injected intraperitoneally at 8:00 am, 12:00 noon, 8:00 pm and 12:00 midnight; 7 days continuous administration

---

To evaluate the time- and dose-dependent toxicity of clofarabine in mice and to further define the chronotherapy strategy of it in leukemia, we compared the mortality rates, LD50s, biochemical parameters, histological changes and organ indexes of mice treated with clofarabine at various doses and time points. Plasma clofarabine levels and pharmacokinetic parameters were monitored continuously for up to 8 hours after the single intravenous administration of 20 mg/kg at 12:00 noon and 12:00 midnight by high performance liquid chromatography (HPLC)-UV method. Clofarabine toxicity in all groups fluctuated in accordance with circadian rhythms in vivo. The toxicity of clofarabine in mice in the rest phase was more severe than the active one, indicated by more severe liver damage, immunodepression, higher mortality rate, and lower LD50. No significant pharmacokinetic parameter changes were observed between the night and daytime treatment groups. These findings suggest the dosing-time dependent toxicity of clofarabine synchronizes with the circadian rhythm of mice, which might provide new therapeutic strategies in further clinical application.[4]

---

Nude mice (6-7 weeks old) were implanted subcutaneously with 2×106 CCRF-CEM ALL cells; when tumors reached 100 mm3, Clofarabine was dissolved in 0.9% normal saline and administered i.v. at 6 mg/kg daily for 5 days; control mice received normal saline; tumor volume was measured every 2 days, and TGI was calculated [2]
- Nude mice bearing HL-60 AML xenografts were treated with Clofarabine (dissolved in 5% dextrose solution) via i.p. injection at 8 mg/kg every other day for 3 weeks; mice were monitored for survival, and tumors were excised at sacrifice to measure weight and histopathological changes [3]
- C57BL/6 mice with murine B-cell CLL were given Clofarabine via oral gavage at 10 mg/kg daily for 7 days; the drug was suspended in 0.5% carboxymethylcellulose sodium; peripheral blood was collected before and after treatment to count leukemic cells [4]

Absorption, Distribution and Excretion
Based on 24-hour urine collection results from pediatric studies, 49-60% of the dose is excreted unchanged in the urine. 172 L/m² 28.8 L/h/m² [In children (2-19 years old) with relapsed or refractory acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) treated with a dose of 52 mg/m²] Metabolism/Metabolites Cladribine is metabolized intracellularly by deoxycytidine kinase, monophosphate kinase, and diphosphate kinase sequentially to a 5'-monophosphate metabolite, and finally to an active 5'-triphosphate metabolite. Clofarabine has a high affinity for the phosphorylase deoxycytidine kinase, which is equal to or higher than that of the natural substrate deoxycytidine. Biological Half-Life The terminal half-life is estimated to be 5.2 hours.

---

After intravenous injection of 6 mg/kg clofarabine in rats, the plasma half-life (t1/2) of clofarabine was 2.8 hours; the volume of distribution (Vd) was 0.7 L/kg [1]
-The oral bioavailability in dogs and humans was 70-80%; after oral administration of 10 mg/kg in dogs, the peak plasma concentration (Cmax) was 2.5 μM [4]
-The protein binding rate in human plasma was 47% [1]
-It is metabolized in cells by dCK phosphorylation to the active triphosphate form; 60% of the parent drug is excreted unchanged in the urine within 24 hours [1]

Hepatotoxicity
In clinical trials, up to 75% of patients with refractory or relapsed acute leukemia receiving cladribine monotherapy experienced elevated serum enzymes. These elevations typically occur within 5 to 10 days of treatment initiation and are generally transient and asymptomatic. These elevations rarely require dose adjustments or treatment delays. Case reports of clinically significant liver injury caused by cladribine have been published, but details are limited, and most patients were concurrently receiving other anticancer chemotherapy drugs. A case report of toxic epidermal necrosis and fulminant hepatic failure in a child with ALL treated with cladribine has been published. High doses of cladribine are associated with a very high incidence of elevated serum enzymes and hyperbilirubinemia, which constitute dose-limiting factors. Case reports of capillary leak syndrome and possible hepatic sinusoidal obstruction syndrome have been reported. Probability score: D (likely a rare cause of clinically significant liver injury). Protein binding 47% is bound to plasma proteins, primarily albumin.

---

In nude mice, bone marrow suppression (leukopenia, thrombocytopenia) was observed at intravenous doses ≥6 mg/kg; the lowest white blood cell count was observed 7 days after treatment [2]
-Mild nephrotoxicity (elevated serum creatinine) was observed in rats treated with 10 mg/kg intravenously for 5 days; no significant hepatotoxicity was detected [1]
-Low cytotoxicity against normal human bone marrow stromal cells in vitro, CC50 >500 nM, indicating a therapeutic window [3]

[1]. Nat Rev Drug Discov . 2006 Oct;5(10):855-63.

[2]. Clin Cancer Res . 2001 Nov;7(11):3580-9.

[3]. Cancer Chemother Pharmacol . 2005 Apr;55(4):361-368.

[4]. Kaohsiung J Med Sci. 2016 May;32(5):227-34.

Pharmacodynamics
Cladribine is a purine nucleoside antimetabolite that differs from other purine nucleoside analogs in that it contains a chlorine atom on its purine ring and a fluorine atom on its ribose moiety. Cladribine appears to interfere with the growth of cancer cells, ultimately leading to cancer cell death. It may also have other effects because cladribine may affect the growth of normal somatic cells. Clofarabine inhibits the growth of cancer cells by interfering with nucleic acid synthesis, preventing cells from synthesizing DNA and RNA. Clofarabine is a purine nucleoside analog with a dual mechanism of action: inhibiting ribonuclease (RNR) and incorporating into DNA to terminate chain elongation [1] - It has been approved by the FDA for the treatment of relapsed or refractory acute lymphoblastic leukemia (ALL) in children aged 1–21 years [1] - It is active on both mitotic and non-mitotic leukemia cells, which distinguishes it from other nucleoside analogs [2] - The active triphosphate metabolite can accumulate in cells for up to 48 hours, thus reducing the frequency of administration [4]

C10H11CLFN5O3

303.68

303.053

C, 39.55; H, 3.65; Cl, 11.67; F, 6.26; N, 23.06; O, 15.81

123318-82-1

119182

White to off-white solid powder

2.1±0.1 g/cm3

599.5±60.0 °C at 760 mmHg

228-2310C

316.4±32.9 °C

0.0±1.8 mmHg at 25°C

1.844

0.24

3

8

2

20

370

4

ClC1=NC(=C2C(=N1)N(C([H])=N2)[C@@]1([H])[C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])F)N([H])[H]

WDDPHFBMKLOVOX-AYQXTPAHSA-N

InChI=1S/C10H11ClFN5O3/c11-10-15-7(13)5-8(16-10)17(2-14-5)9-4(12)6(19)3(1-18)20-9/h2-4,6,9,18-19H,1H2,(H2,13,15,16)/t3-,4+,6-,9-/m1/s1

(2R,3R,4S,5R)-5-(6-amino-2-chloropurin-9-yl)-4-fluoro-2-(hydroxymethyl)oxolan-3-ol

C1 F-Ara-A; C1-F-Ara-A; Clofarabine; C1 F-Ara-A; trade names: Clofarex; Clolar. Abbreviation: CAFdA; 123318-82-1; Evoltra; Clofarex; CAFdA; Cl-F-Ara-A; C1-F-Ara-A;

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: ≥ 2.08 mg/mL (6.85 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 20.8 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (6.85 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (6.85 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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