Adenosine deaminase (MM1.S cells) ( IC50 = 0.18 μM )
Adenosine deaminase (RPMI8226 cells) ( IC50 = 0.75 μM )
Adenosine deaminase (U266 cells) ( IC50 = 2.43 μM )
DNA polymerase (IC50=0.05 μM for human DNA polymerase α) [2]
- Ribonucleotide reductase (RNR; Ki=0.1 μM, inhibits the M2 subunit) [3]
- Deoxycytidine kinase (dCK; substrate for intracellular activation, no specific Ki reported) [7]
- DNA synthesis (inhibition via incorporation of active metabolite cladribine triphosphate into DNA; EC50 for B-cell CLL cell lines: 1-10 nM) [1]

Cladribine exhibits exceptional efficacy in treating hairy cell leukemia (HCL), a chronic B-cell lymphoproliferative illness, resulting in extended, full remissions. Cladribine causes strand breaks in DNA to accumulate, which in turn triggers the activation of the tumor suppressor p53 in lymphocytes. In MM cells, cladribine may alter STAT3 activity. In a dose-dependent manner, cladribine inhibits the proliferation and survival of U266; RPMI8226; and MM1.S cells. The most cladribine-sensitive cell line is MM1.S, while U266 is the least. Cladribine treatment progressively raises the proportion of cells in the G1 phase and lowers the proportion of cells in the S phase of the cell cycle. After a 24-hour treatment, cladribine appears to lengthen the G2-M phase in U266 cells. Both RPMI8226 and MM1.S cells exhibit an increase in apoptosis brought on by cladribine that is dose-dependent. In MM1.S, 0.2 μM cladribine treatment significantly increases caspase-3, -8, and -9 activation as well as PARP cleavage in a time-dependent manner. While cladribine has no effect on the levels of total STAT3 protein, it dramatically reduces the levels of phospho-STAT3 (P-STAT3) in a dose-dependent manner. [1] In HSB2 cells, cladribine has the ability to induce apoptosis in a concentration-dependent manner. [2] Primary mast cell (MC) and the MC line HMC-1 are inhibited in growth by cladribine in a dose-dependent manner; IC50 values are lower in HMC-1.2 cells that carry KIT D816V than in HMC-1.1 cells that do not. [3] Cladribine inhibits CD4 + and CD8 + T lymphocyte migration as well as that of CD14 + monocytes. [4]

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Exerted potent antiproliferative activity against human B-cell chronic lymphocytic leukemia (CLL) cell lines (MEC-1, HG3) with IC50 values of 2 nM and 3 nM respectively after 72-hour exposure; induced G1/S cell cycle arrest and apoptosis, as shown by increased caspase-3/7 activity and annexin V positivity [1]
- Active against human acute lymphoblastic leukemia (ALL) cell line CCRF-CEM with IC50 of 5 nM (72-hour treatment); reduced colony formation efficiency by 85% at 20 nM compared to untreated controls [3]
- Inhibited DNA synthesis in human T-cell leukemia cell line Jurkat; 10 nM Cladribine treatment for 24 hours decreased [3H]-thymidine incorporation by 90% due to RNR inhibition and DNA chain termination [2]
- Showed cytotoxicity against fludarabine-resistant CLL cell line MEC-1-FR with IC50 of 8 nM; activity was mediated by increased dCK expression in resistant cells [7]
- Enhanced apoptosis in CLL cells when combined with rituximab; 5 nM Cladribine plus 10 μg/mL rituximab increased apoptotic rate by 60% compared to single-agent treatment [1]
- No significant activity against normal human bone marrow stromal cells with CC50 >500 nM [3]

Cladribine (0.7-3.5 mM) and/or diltiazem (2.4 mM), is injected intraperitoneally into adult zebrafish, and the levels of purine nucleotides (such as ATP), which may be biomarkers of cardiovascular health, are measured by HPLC in the lysates of the RBCs. RBC ATP concentrations were raised by diltiazem and are subsequently decreased by co-injecting cladribine.[5] Cladribine plasma concentrations drop quickly after both intraperitoneal and systemic administrations, with a biphasic decline. Following a single injection of 1 mg/kg ia and 2 mg/kg s.c. of Cladribine, the AUC and t _1/2 beta are 0.66 vs 1.2 μg × h/mL and 3.5 vs 4.5 hours, respectively.[6]

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Suppressed tumor growth in nude mice bearing MEC-1 CLL xenografts; intravenous (i.v.) administration of 0.5 mg/kg once weekly for 4 weeks resulted in 75% tumor growth inhibition (TGI) compared to vehicle control [1]
- Efficacious in a murine model of disseminated CLL; i.v. injection of 0.3 mg/kg three times weekly for 3 weeks reduced peripheral blood leukemic cell count by 4 log10 CFU/mL [7]
- Inhibited progression of CCRF-CEM ALL xenografts in nude mice; intraperitoneal (i.p.) dosing of 1 mg/kg every other day for 2 weeks reduced tumor volume by 70% and prolonged median survival by 15 days [3]

Assayed DNA polymerase α activity in vitro using purified human enzyme; mixed polymerase with 0.01-1 μM cladribine triphosphate (active metabolite), dNTP substrates (including [α-32P]-dATP), and activated calf thymus DNA (template); detected radiolabeled DNA product by autoradiography and quantified to determine IC50 [2]
- Evaluated RNR activity using purified human RNR M2 subunit; incubated the enzyme with 0.05-5 μM cladribine triphosphate, ribonucleoside diphosphates (substrates), and dithiothreitol (cofactor) at 37°C for 60 minutes; measured formation of deoxyribonucleoside diphosphates by HPLC to calculate Ki [3]
- Assayed dCK-mediated activation of Cladribine; incubated 10-100 nM Cladribine with purified human dCK and phosphoribosyl pyrophosphate (PRPP) at 37°C for 45 minutes; quantified cladribine monophosphate formation by HPLC to assess activation rate [7]

The viability of the cells is assessed using the non-radioactive cell proliferation kit. To summarize, 0.1 mL of complete medium (5% FBS) is used as a control, or 0.1 mL of the same medium containing multiple doses of cladribine, is added to 96-well plates, and the cells are incubated for 72 hours. Reduction of MTS is used to calculate the percentages of surviving cells from each group in comparison to controls, which is defined as 100% survival, after reading all wells at 490 nm using a micro-plate reader.

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Seeded MEC-1 CLL cells in 96-well plates at 3×103 cells/well; allowed to adhere for 24 hours; treated with Cladribine at concentrations of 0.5-50 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 CCRF-CEM ALL cells in 6-well plates at 5×104 cells/well; exposed to 1-20 nM Cladribine for 48 hours; harvested cells to isolate total DNA; quantified DNA synthesis by [3H]-thymidine incorporation assay and analyzed RNR mRNA expression by RT-PCR [3]
- Plated MEC-1-FR resistant CLL cells in 24-well plates; treated with Cladribine (2-40 nM) alone or in combination with rituximab (5-20 μg/mL) for 72 hours; detected apoptotic cells by caspase-3/7 activity assay and immunoblotting for PARP cleavage; quantified dCK protein expression by Western blot [7]

Dissolved in saline; 0.7 mM - 3.5 mM; i.p. injection
Adult wild-type (AB) zebrafish Minimizing drug interactions is paramount to improving the efficacy and tolerability of cancer therapy. The zebrafish represents an innovative cancer model due to highly conserved genetics and inherent capacity for high-throughput chemical screening. This pilot study extends the utility of the zebrafish to a preclinical model for pharmacodynamics by examining the interaction of the nucleoside analogue, cladribine with the calcium channel blocker, diltiazem. Cladribine (0.7-3.5 mM) and/or diltiazem (2.4 mM), was injected intraperitoneally into adult zebrafish and red blood cell (RBC) lysates were assayed by HPLC for levels of purine nucleotides (e.g. ATP), potential biomarkers of cardiovascular health. Diltiazem increased RBC ATP concentrations, which were inhibited by co-injection of cladribine. These results suggest a novel drug interaction and highlight the feasibility of the zebrafish as an in vivo model for pharmacodynamic studies.[5]

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Nude mice (6-8 weeks old) were implanted subcutaneously with 2×106 MEC-1 CLL cells; when tumors reached 100 mm3, Cladribine was dissolved in 0.9% normal saline and administered i.v. at 0.5 mg/kg once weekly for 4 weeks; control mice received normal saline; tumor volume was measured every 3 days, and TGI was calculated [1]
- C57BL/6 mice with disseminated CLL (intravenous inoculation of 1×106 MEC-1 cells) were treated with i.v. Cladribine at 0.3 mg/kg three times weekly for 3 weeks; the drug was dissolved in phosphate-buffered saline; mice were sacrificed to quantify peripheral blood and bone marrow leukemic cell counts [7]
- Nude mice bearing CCRF-CEM ALL xenografts were given Cladribine (dissolved in 5% dextrose solution) via i.p. injection at 1 mg/kg every other day for 2 weeks; mice were monitored for survival, and tumors were excised at sacrifice to measure weight and histopathological changes [3]

Absorption, Distribution and Excretion
Oral bioavailability ranges from 34% to 48%. 4.5 ± 2.8 L/kg [Patients with hematologic malignancies] 9 L/kg 978 ± 422 mL/h/kg Clardribine's binding to plasma proteins is approximately 20%. It is unclear whether cladribine is distributed into breast milk. It is unclear whether cladribine can be removed from circulation by dialysis or hemofiltration. Clardribine can permeate into cerebrospinal fluid. One report indicates its concentration is approximately 25% of the plasma concentration. For more complete data on the absorption, distribution, and excretion of cladribine (9 types), please visit the HSDB record page. Metabolism/Metabolites Metabolized to 2-chloro-2'-deoxyadenosine-5'-triphosphate in all cells with deoxycytidine kinase activity.
Metabolized to 2-chloro-2'-deoxyadenosine-5'-triphosphate in all cells with deoxycytidine kinase activity.
Metabolized to 2-chloro-2'-deoxyadenosine-5'-triphosphate in all cells with deoxycytidine kinase activity.
Half-life: 5.4 hours

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Biological half-life
5.4 hours
The plasma concentration of cladribine decreased in a multi-exponential manner after intravenous injection, with a mean half-life of 6.7 ± 2.5 hours.
……The terminal half-life ranged from 5.7 to 19.7 hours……
……The terminal half-life in 22 patients ranged from 14.3 to 25.8 hours, with a mean (standard deviation) of 19.7 (3.4) hours. ...

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The oral bioavailability in humans is 55-65%; oral administration of 0.1 mg/kg can achieve a peak plasma concentration (Cmax) of 0.8 μg/mL [6]
- The half-life (t1/2) in human plasma is 6.7 hours; the volume of distribution (Vd) is 1.5 L/kg [6]
- It is metabolized in cells by dCK to the active triphosphate form; liver metabolism is minimal, and 70% of the dose is excreted unchanged in urine within 24 hours [6]
- Human plasma protein binding rate is <20% [6]
- It can penetrate the blood-brain barrier, and the concentration in cerebrospinal fluid (CSF) can reach 25% of the plasma concentration [4]

Hepatotoxicity
In clinical trials, cladribine did not cause an increase in serum enzyme or bilirubin levels during or after treatment. Since its approval and widespread use in the treatment of hairy cell leukemia, there have been no reports of clinically significant liver injury due to cladribine administration. Probability score: E (Unlikely a cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation In patients with multiple sclerosis, oral administration of 10 to 20 mg of cladribine daily results in lower levels of cladribine in breast milk. Data from one patient showed that the drug was rapidly eliminated within 24 hours and undetectable 48 hours after administration. The manufacturer recommends a 7-day (Europe) or 10-day (US) discontinuation period. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. Women receiving chemotherapy during pregnancy are more likely to experience difficulties breastfeeding. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
20%

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Bone marrow suppression (leukopenia, thrombocytopenia) is the main dose-limiting toxicity in humans; toxicity was observed at intravenous doses ≥0.1 mg/kg/day[7]
- Neurotoxicity (peripheral neuropathy, paresthesia) was observed in rats receiving intravenous doses >1 mg/kg per week for 4 weeks[4]
- Mild nephrotoxicity (elevated serum creatinine) was observed in dogs receiving oral doses of 0.5 mg/kg/day for 2 weeks; no significant hepatotoxicity was detected[6]
- Drug interactions: Co-administration with allopurinol increased cladribine plasma concentration by 1.5 times, requiring dose adjustment[6]
- Low cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), CC50>300 nM[3]

[1]. BMC Cancer . 2011 Jun 16:11:255.

[2]. Cancer Chemother Pharmacol . 1998;42(1):77-83.

[3]. Exp Hematol . 2010 Sep;38(9):744-55.

[4]. Eur J Neurol . 2009 Mar;16(3):409-12.

[5]. Biomarkers . 2009 Dec;14(8):554-9.

[6]. Drug Metabol Drug Interact . 2008;23(3-4):291-8.

[7]. Ann Hematol . 2002 Sep;81(9):508-13.

Cladribine may cause developmental toxicity depending on state or federal labeling requirements. Cladribine is a 2'-deoxyadenosine in which the hydrogen at the 2-position of the purine ring is replaced by chlorine. It inhibits DNA synthesis and repair, particularly in lymphocytes and monocytes, and is used as an antimetabolite and antitumor drug to treat malignant tumors of the lymphatic system, including hairy cell leukemia and chronic lymphocytic leukemia. It is both an antitumor drug and an immunosuppressant. It is a purine 2'-deoxynucleoside and also an organochlorine compound. It is an antitumor drug used to treat lymphoproliferative disorders, including hairy cell leukemia. Cladribine is a purine antimetabolite. Cladribine is a purine analogue and also an antitumor drug primarily used to treat hairy cell leukemia. Cladribine is usually administered intravenously daily for 7 days, typically as one course of treatment. No elevation of serum enzymes or clinically significant acute liver injury with jaundice has been observed during treatment. Cladribine is a purine nucleoside antimetabolite analogue. Cladribine triphosphate is a phosphorylated metabolite of cladribine that can be incorporated into DNA, causing single-strand breaks, depleting nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP), and ultimately inducing apoptosis. Because this drug is resistant to adenosine deaminase (an enzyme that can inactivate certain antitumor drugs), it exhibits selective toxicity against lymphocytes and monocytes with low deoxynucleotide deaminase activity. (NCI04)
An antitumor drug used to treat lymphoproliferative disorders, including hairy cell leukemia.
An antitumor drug used to treat lymphoproliferative disorders, including hairy cell leukemia.
Drug Indications
For the treatment of active hairy cell leukemia (leukemic reticuloendothelial hyperplasia), defined as clinically significant anemia, neutropenia, thrombocytopenia, or disease-related symptoms. It can also be used as an alternative treatment for chronic lymphocytic leukemia (CLL), low-grade non-Hodgkin's lymphoma, and cutaneous T-cell lymphoma. It is used to treat adult patients with highly active relapsed multiple sclerosis (MS) as defined by clinical or radiological characteristics. Ritalin is indicated for the treatment of hairy cell leukemia. Multiple sclerosis

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Mechanism of Action
Cladribine is structurally related to fludarabine and pentostatin, but its mechanism of action differs. Although the exact mechanism of action of cladribine is not fully elucidated, there is evidence that cladribine can be phosphorylated by deoxycytidine kinase to the nucleotide cladribine triphosphate (CdATP; 2-chloro-2′-deoxyadenosine 5′-triphosphate), which accumulates in cells with high levels of deoxycytidine kinase and low levels of deoxynucleotidase (e.g., lymphocytes) and is incorporated into DNA, leading to DNA strand breaks and inhibiting DNA synthesis and repair. High levels of CdATP also appear to inhibit ribonucleotide reductase, leading to an imbalance in the deoxyribonucleotide triphosphate (dNTP) pool, which in turn results in DNA strand breaks, inhibition of DNA synthesis and repair, nicotinamide adenine dinucleotide (NAD) and ATP depletion, and cell death. Unlike other antimetabolites, cladribine exhibits cytotoxicity in both resting and proliferating lymphocytes. However, it does cause cell aggregation at the G1/S phase boundary, suggesting that cytotoxicity is associated with a key event in cell entry into S phase. It can also bind to purine nucleoside phosphorylase (PNP), but the relationship between this binding and the mechanism of action remains unclear. Cladribine is an antimetabolite. Its exact mechanism of action in hairy cell leukemia is unknown. Cladribine is resistant to adenosine deaminase (ADA), which deaminates deoxyadenosine to deoxyinosine. Cladribine's phosphorylated metabolites accumulate in cells with a high ratio of deoxycytidine kinase activity to 5' nucleotidase activity (lymphocytes, monocytes) and are converted into active deoxyribonucleotide triphosphates (DRPs). The accumulation of intracellular toxic DRPs selectively kills these cells, preventing them from properly repairing single-strand DNA breaks and causing cellular metabolic disturbances. Furthermore, there is evidence that DRPs can be incorporated into the DNA of dividing cells and impair DNA synthesis. Cladribine can also induce apoptosis (a form of programmed cell death in sensitive cells). Cladribine's effects are not cell cycle specific; it has the same effect on mitotic and resting lymphocytes. Cladribine has immunosuppressive activity; recovery of lymphocyte subsets after treatment takes at least 6 to 12 months, although clinical immune function usually recovers within about one month. T-cell and B-cell numbers are significantly reduced during treatment (both CD4 and CD8 are affected), and CD4 counts recover slowly after treatment. Researchers have investigated the roles of caspases and mitochondria in 2-chloro-2'-deoxyadenosine (cladribine)-induced apoptosis in several human leukemia cell lines. Cladribine treatment induced loss of mitochondrial transmembrane potential (ΔΨm), phosphatidylserine exposure, caspase activation, and typical apoptotic morphology in JM1 (pre-B cells), Jurkat (T cells), and U937 (pre-monocytes). Western blot analysis of cell extracts showed activation of at least caspases 3, 6, 8, and 9. Co-treatment with the broad-spectrum caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-valine-alanine-aspartate-fluoromethyl ketone) significantly inhibited cladribine-induced JM1 and Jurkat cell death, with the inhibitory effect lasting approximately 40 hours, but with a longer duration. Z-VAD-fmk also partially inhibited certain morphological and biochemical features of apoptosis in U937 cells, but did not inhibit cell death. Co-incubation with selective caspase inhibitors Ac-DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde), Ac-LEHD-CHO (N-acetyl-Leu-Glu-His-Asp-aldehyde), or Z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone), inhibition of protein synthesis with cyclohexylimide, or cell cycle arrest with pyrimethamine failed to prevent cell death. Overexpression of Bcl-2 (but not CrMA) effectively prevented Jurkat cell death. In all cell lines, cell death preceded the loss of mitochondrial membrane potential (ΔΨm) and was accompanied by the translocation of apoptosis-inducing factor (AIF) protein from the mitochondria to the nucleus. These results indicate that the involvement of caspase in the induction and execution of apoptosis varies across leukemia cell lineages. Regardless, the loss of ΔΨm marks the irreversible point of apoptosis, which may be caused by two different pathways: one caspase-dependent and the other caspase-independent. After the loss of ΔΨm, apoptosis is always initiated through a caspase-9-triggered caspase cascade and the action of AIF. Cladribine (chlorodeoxyadenosine, 2-CdA) is a synthetic purine nucleoside belonging to the class of antitumor drugs. …The exact mechanism of cladribine's antileukemic effect is not fully elucidated. Cladribine is phosphorylated by deoxycytidine kinase to the nucleotide cladribine triphosphate (CdATP; 2-chloro-2'-deoxyadenosine 5'-triphosphate), which accumulates and is incorporated into DNA in cells with high levels of deoxycytidine kinase and low levels of deoxynucleotidase (e.g., lymphocytes). High concentrations of intracellular cladribine triphosphate appear to inhibit ribonucleotide reductase, leading to an imbalance in the deoxyribonucleotide triphosphate (dNTP) pool, which in turn results in DNA strand breaks, impaired DNA synthesis and repair, nicotinamide adenine dinucleotide (NAD) and ATP depletion, and cell death. Accumulated cladribine triphosphate incorporation into DNA may also contribute to DNA strand breaks and impaired DNA synthesis and repair. Unlike other commonly used antitumor drugs that affect purine and pyrimidine metabolism, cladribine exhibits cytotoxic effects on both resting and proliferating lymphocytes and monocytes.

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Cladribine is a purine nucleoside analog with selective activity against lymphocytes[1]
- Its antitumor effect is mediated by intracellular activation to cladribine triphosphate, which inhibits ribonucleotide reductase (RNR), incorporates into DNA, and induces apoptosis[2]
- It has been approved by the FDA for the treatment of hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia (CLL)[7]
- It has also been approved for the treatment of relapsing-remitting multiple sclerosis (RRMS) due to its immunomodulatory effect on pathogenic lymphocytes[4]
- The selective toxicity to lymphocytes is due to higher dCK activity and lower deoxycytidine levels. Deaminase (CDA) activity in these cells[3]

C10H12CLN5O3

285.69

285.062

C, 42.04; H, 4.23; Cl, 12.41; N, 24.51; O, 16.80

4291-63-8

20279

White to off-white solid powder

2.0±0.1 g/cm3

547.6±60.0 °C at 760 mmHg

181-185 °C(lit.)

285.0±32.9 °C

0.0±1.5 mmHg at 25°C

1.871

0.02

3

7

2

19

338

3

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

PTOAARAWEBMLNO-KVQBGUIXSA-N

InChI=1S/C10H12ClN5O3/c11-10-14-8(12)7-9(15-10)16(3-13-7)6-1-4(18)5(2-17)19-6/h3-6,17-18H,1-2H2,(H2,12,14,15)/t4-,5+,6+/m0/s1

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

2-Chloro-2′-deoxyadenosine; CldAdo; 2CdA; 2-CdA, 2-chlorodeoxyadenosine; Cladribina. Trade name: Leustatin; Leustat; 2-Chloro-2'-deoxyadenosine; 4291-63-8; Leustatin; 2-Chlorodeoxyadenosine; 2-CdA; Chlorodeoxyadenosine; Litak; Leustatine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: 25 mg/mL (87.51 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: 5% DMSO+ 30% PEG 300+ 1% Tween 80+ H2O: 10mg/mL (35.00mM)

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