DNA synthesis; antimetabolite

Bromodeoxyuridine (300 mg/kg, i.p. or 0.8 mg/ml, p.o.) markedly slows the progression of the tumor in the rat glioma RG2 tumor model.[1]

Telomere Length[1]
To determine whether the effects of BrdU are related to changes in telomere length, we performed a TeloTAGGG assay. Briefly, genomic DNA was isolated and digested with Hinf1 and Rsa1 enzymes. After digestion, the DNA fragments were separated by gel electrophoresis and transferred to a nylon membrane for Southern blot analysis. The blotted DNA fragments were hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats and incubated with a DIG-specific antibody covalently coupled to alkaline phosphatase. Finally, the immobilized telomere probe was visualized by virtue of alkaline phosphatase-metabolizing CDP-Star, a highly sensitive chemiluminescence substrate.

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Telomerase Activity[1]
We used the TRAPeze ELISA kit assay to determine levels of telomerase activity in our control and BrdU-treated cells. Briefly, the sample cells' telomerase adds a number of telomeric repeats (GGTTAG) onto the 3′ end of the biotinylated telomerase substrate oligonucleotide (b-TS), and the extended products are then amplified by polymerase chain reaction. The extension/amplification was performed with biotinylated primer andDNP-labeled dCTP. Thus, the telomeric repeat amplification protocol (TRAP) products are tagged with biotin and DNP residues, and the labeled products can be immobilized onto streptavidin-coated microtiter plates through biotin-streptavidin interaction, and then detected by anti-DNP antibody conjugated to horseradish peroxidase (HRP). The amount of TRAP products was determined by means of the HRP activity using substrate TMB and subsequent color development.

Initially plated at 2000 cells/cm², cultures are measured using a Z2 Coulter Counter. After treating RG2 rat glioma cells once for 24 hours with 0, 1, 10, or 50 µM BrdU, cumulative growth curves were measured over a period of 18 days. After five, twelve, and eighteen days of treatment, control and treated cells are counted and replated at equal densities.

Subcutaneous Tumors and In Vivo BrdU Administration[1]
A bolus of either untreated or pretreated RG2 glioma cells (1 x 106 cells in 250 µl of PBS) was injected subcutaneously between the scapulae of anesthetized adult male Fisher 344 rats as previously described. Pretreated RG2 cells were treated with 50 µM BrdU for 24 hours before implantation. Tumors were measured every other day in two dimensions with digital calipers, and tumor volume was calculated [(π/6) x W2 x L (W = shortest dimension and L = longest dimension)]. The experimental end point was defined as a tumor volume ≥3000 mm3. At end point, euthanasia was performed by transcardial perfusion with 200 ml of 4% paraformaldehyde in PBS under deep sodium pentobarbital anesthesia (150 mg/kg, i.p.).[1]
BrdU administration, i.p. Untreated RG2 cells were implanted into 10 animals as described previously. The BrdU regimen was initiated when palpable tumors had reached a volume of 200 mm3. Half of the animals received three i.p. injections of BrdU (300 mg/kg) per day for 2 days, whereas the other half served as controls and received an equal number and volume of sterile saline injections.[1]
BrdU administration, oral. Again, untreated RG2 cells were implanted subcutaneously into 20 animals as described. Immediately after implantation, half of the animals were provided with drinking water containing BrdU (0.8 mg/ml), and half received normal drinking water. All animals were provided with freshly prepared water (either with or without BrdU) each day for 7 days, ad libitum. On the eighth day after implantation, all animals were placed on normal drinking water for the duration of the experiment.[1]
A dose of 300 mg/kg corresponds to a clinical dose of 1800 mg/m2. The rats received three of these doses per day for 2 days, thus receiving a total of 10,800 mg/m2. The drinking water dose (based on the standard 20-ml/day consumption by adult rats) is 640 mg/m2 per day for 7 days, or a total of 4480 mg/m2. By way of comparison, previous clinical trials (e.g., Kinsella et al.) included BrdU as a radiosensitizer as part of a multimodal therapy-treated patients with 350 mg/m2 for continuous 12-hour infusions every day for 14 days or 4900 mg/m2 total. Thus, the treatment range in our study is generally in accord with previous human clinical applications, because, although our injected BrdU was theoretically approximately twice what humans received, it is known that BrdU is active in plasma only for approximately 2 hours. Therefore, the continuous infusion used in the human trials likely resulted in more widespread BrdU incorporation than our injection paradigm.[1]

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300 mg/kg, i.p. or 0.8 mg/ml, p.o.

Rat glioma RG2 tumor model

[1]. Neoplasia . 2008 Aug;10(8):804-16.

[2]. Curr Protoc Cytom . 2007 Apr:Chapter 7:Unit7.31.

5-bromo-2'-deoxyuridine is a pyrimidine 2'-deoxyribonucleoside compound having 5-bromouracil as the nucleobase. It has a role as an antineoplastic agent and an antimetabolite.
Broxuridine has been used in trials studying the treatment of Leukemia, Stage I Prostate Cancer, Stage IIB Prostate Cancer, and Stage IIA Prostate Cancer.
Broxuridine is a halogenated thymidine analogue with potential antineoplastic and radiosensitizing activities. Bromodeoxyuridine competes with thymidine for incorporation into DNA, resulting in DNA mutation and the inhibition of cell proliferation. As a radiosensitizer, this agent is associated with the inhibition of repair of radiation-induced DNA double-strand breaks.
Bromodeoxyuridine is an organobromide compound and a synthetic nucleoside that is an analogue of thymidine. It is brominated derivative of deoxyuridine that acts as an antimetabolite or base analog, substituting for thymidine in DNA. It can induce DNA mutations in the same way as 2-aminopurine It is used in the detection of proliferating cells in living tissues, as it can be incorporated into the newly synthesized DNA of replicating cells, then detected using antibodies.
A nucleoside that substitutes for thymidine in DNA and thus acts as an antimetabolite. It causes breaks in chromosomes and has been proposed as an antiviral and antineoplastic agent. It has been given orphan drug status for use in the treatment of primary brain tumors.

C9H11BRN2O5

307.1

305.99

C, 35.20; H, 3.61; Br, 26.02; N, 9.12; O, 26.05

59-14-3

6035

White to off-white solid powder

1.9±0.1 g/cm3

191-194 °C

1.652

-0.81

104.55

C1[C@@H]([C@H](O[C@H]1N2C=C(C(=O)NC2=O)Br)CO)O

WOVKYSAHUYNSMH-RRKCRQDMSA-N

InChI=1S/C9H11BrN2O5/c10-4-2-12(9(16)11-8(4)15)7-1-5(14)6(3-13)17-7/h2,5-7,13-14H,1,3H2,(H,11,15,16)/t5-,6+,7+/m0/s1

5-bromo-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dione

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: ≥ 2.08 mg/mL (6.77 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.77 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.77 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.

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Solubility in Formulation 4: 14.29 mg/mL (46.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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