cytidine deaminase (CDA)[1]

Tetrahydrouridine, or THU, is a particular cytidine deaminase (CDA) inhibitor that suppresses deamination during the catabolism of analogues of cytotoxic deoxycytidine, such as gemcitabine and ara-C. A combination therapy is used to examine the impact of Tetrahydrouridine on the Gemcitabine-mediated anti-neoplastic activity on lung cancer and pancreatic cells. As anticipated, following a 100 µM Tetrahydrouridine treatment, increased Gemcitabine sensitivity is brought about by elevated CDA expression in BxPC-3 and H441. BxPC-3 and H441 cell lines exhibit a 2.1 and 4.4 times increase in sensitivity, respectively. Conversely, H1299 and MIAPaCa-2 cells unexpectedly exhibit reduced CDA expression and increased gemcitabine sensitivity. The IC50 changes by 2.2 and 2.3 fold in MIAPaCa-2 and H1299 cells, respectively. However, there are no appreciable differences in the drug sensitivity of Panc-1 and H322 cells. These findings suggested that, independent of CDA expression levels, tetrahydrouridine can sensitize some lung and pancreatic cancer cells to gemcitabine-induced cell death. Without causing apoptosis, tetrahydrouridine suppresses S-phase[1].

---

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells. [1]

One male and eight female animals die when 167 mg/kg of Tetrahydrouridine (THU) and 1.0 mg/kg of DAC are administered. Animals who survive until their scheduled termination are usually asymptomatic, and for treatments up to 1.0 mg/kg of DAC combined with 167 mg/kg of Tetrahydrouridine in animals, no treatment-related changes have been seen in body weights, food consumption, clinical chemistry, or urinalysis[2].

---

Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts.[2]

Ki-67 staining [1]
To perform Immunocytochemistry (ICC), cells were seeded in chamber slides at 20–30% confluency. After 12 hrs of incubation, 100 µM tetrahydrouridine (THU) was added for four days. The ICC procedures were performed after fixing the cell samples with 4% paraformaldehyde in PBS for 15 min. For permeabilization, samples were incubated for 10 min with PBS containing 0.3% Triton X-100. Cells were then treated with a blocking solution containing 1% BSA in PBS for 60 min. Samples were incubated for 90 min with the Ki-67 antibody in blocking buffer before applying the secondary goat anti-rabbit antibody for 60 min. Slides were treated with the streptavidin–biotin complex reagent for 30 min and developed with 0.4% diaminobenzidine (DAB)/H2O2. All stages of the staining process were performed at room temperature and the slides were washed in PBS. The slides were mounted and analyzed using light microscopy after stopping the DAB reaction. The evaluation of ICC staining was performed by counting all positive reactions in 1000 cells at a magnification of 400× in the whole cell area.

---

Transient transfections of cytidine deaminase -siRNA [1]
To knockdown CDA expression in MIAPaCa-2, H441, and H1299 cells, all cells were seeded at 70–80% confluency in 6-well plates. Two cytidine deaminase and a negative control siRNAs were purchased commercially. All siRNAs were individually transfected in each cell line at a final concentration of 60 nM per well with Lipofectamine 2000. To measure mRNA levels, cells were incubated for 48 hrs and then harvested for real-time PCR analysis. 12 hrs after transfecting, cell growth was determined following the procedure outline above. We also tested whether CDA inhibition plus tetrahydrouridine (THU) treatment surpasses the efficiency of THU alone using a proliferation assay. Transfected cells were seeded in a 96-well plate overnight and THU was added at 100 µM. As prescribed above, the OD was measured at Day0, 2 and 4.

---

Immunoblotting analysis [1]
In order to evaluate whether tetrahydrouridine (THU) affects the cell cycle related factor at the protein level, western blotting was performed. Four days after adding 100 µM THU or PBS as a control, protein was extracted from the cell pellet using RIPA buffer with protease inhibitor. Protein concentration was measured using Bio-Rad protein assay solution. Equal amounts of total protein were separated using SDS-PAGE gels under reducing conditions. The protein was then transferred to a polyvinylidene difluoride (PVDF) membrane before being blocked with 5% non-fat milk in TBS-Tween. The membrane was then incubated with primary antibodies against E2F1 and β-actin at a dilution of 1∶1,000. The appropriate primary antibodies were followed by horseradish peroxidase (HRP)-conjugated secondary antibodies at a dilution of 1∶5,000. Visualization was achieved using SuperSignal West chemiluminescent solution.

Gemcitabine chemo-sensitivity with tetrahydrouridine (THU) [1]
A drug sensitivity assay was performed essentially as described in a previous report. Cells were seeded in 96-well plates at 2,500∼5,000 cells/well in triplicate. After incubating for 12 hrs, cell viability was determined by treating cells with stepwise 4-fold serial dilutions of gemcitabine (from 100 µM), and incubating at 37°C for 96 hrs with or without THU. To evaluate cell viability, all cells were fixed with 25% glutaraldehyde for 30 min at room temperature and then stained with 200 µl of 0.05% methylene blue for 20 min. The dye was eluted with 0.33 M HCl for 20 min with agitation. Absorbance was measured in a microplate reader at 598 nm. The 50% inhibitory concentration for cell growth (IC50) was calculated.

---

Cell growth assay [1]
Cell growth for pancreatic and lung carcinoma cell lines was carried out using the colorimetric methylene blue assay in 96-well plates at a density of 5,000 cells/well. Cells were either exposed or not exposed to tetrahydrouridine (THU), counting the first 12 hrs as Day 0. Mean values were calculated from three different wells in triplicates for four days.

---

Cell cycle analysis by flow cytometry [1]
For cell cycle analysis by flow cytometry, cells were seeded in a 10 cm dish at 60% confluency. After 12 hrs, 100 µM tetrahydrouridine (THU) was added to each dish for three days. Cells were then dissociated using trypsin-EDTA (trypsin 0.25%; EDTA 0.02%) for 2 to 5 min at 37°C, transferred to a 15 mL tube, and washed three times with PBS. Cells were then fixed overnight in 70% ethanol at 4°C before being washed again with PBS and resuspended in PBS-triton-X100 (0.1%). The cells were concomitantly treated with RNaseA and stained with propidium iodide. Cell cycle status was determined using a FACS calibur flow cytometer and analyzed using FlowJo-887 software.

Experimental Design [2] Mice were assigned to four dose groups and a vehicle control group as shown in Table 1. Animals were gavaged with Decitabine (5-aza-2'-deoxycytidine; DAC) or its vehicle 1 hour ± 5 minutes after administration of tetrahydrouridine (THU) or its vehicle at a dose volume of 10 mL/kg. The DAC doses were selected based on the range finding study in which the mice tolerated six oral doses (2x/week) of 0.1, 0.2 and 0.4 mg/kg DAC in combination with a fixed dose of 167 mg/kg tetrahydrouridine (THU). A fixed THU dose (500 mg/m2) and the optimal timing between THU and DAC administration (60 min) were selected based on previous studies11. Conversion of milligrams per body surface area dose in mice into milligrams per kilogram body weight dose estimation was based on Michaelis constant (km) values for mice obtained from US Food and Drug Administration published guidelines. In brief, the mouse dose in milligrams per body surface area (500 mg/m2) was divided by the km of 3 to convert the dose to milligrams per kilogram body weight (167 mg/kg). The working body weight range of mice in the guideline is 11-34 gram; the body weight range of mice used in this study was 24-38 gram.

---

Toxicokinetics [2] Sample collection tubes were prepared prior to each collection day by adding 10 μL/tube of a 10 mg/mL tetrahydrouridine (THU) solution. Blood samples (~0.5 mL) were collected via intra-cardiac puncture from non-fasted, anesthetized toxicokinetic animals on study day 1 (Groups 2 to 5) and 58 (Groups 2 to 5 with the exception of Group 4 females) at 15, 30, 60, 90, 120 and 180 minutes after administration of Decitabine (5-aza-2'-deoxycytidine; DAC) from 3 animals/sex/group at each time point. Due to mortality in the Group 4 females, the first 5 surviving animals were necropsied on study day 38 and blood samples were collected from three females per time point at 15, 30, and 120 minutes following administration of DAC. All samples were collected within 5 minutes of the target time.

dog LD unreported >1 gm/kg Pharmaceutical Chemistry Journal, 10(423), 1976
monkey LD unreported >1 gm/kg Pharmaceutical Chemistry Journal, 10(423), 1976
dog LD intravenous >1 gm/kg National Technical Information Service., PB211-590

[1]. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels. PLoS One. 2012;7(5):e37424.

[2]. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85.

Tetrahydrouridine has been used in trials studying the treatment of Neoplasms, Lung Neoplasms, Breast Neoplasms, Sickle Cell Disease, and Head and Neck Cancer, among others.
Tetrahydrouridine is a synthetic pyrimidine nucleoside analogue with biomodulating activity. Tetrahydrouridine increases the efficacy of the radiosensitizer cytochlor (5-chloro-2'-deoxycytidine) by inhibiting the enzyme deoxycytidine monophosphate (dCMP) deaminase and preventing the premature deamination of the cytochlor metabolite 5-chloro-2'-deoxycytidine monophosphate (CldCMP) to 5-chloro-2'-deoxyuridine monophosphate (CldUMP); in turn, this increases tumor concentrations of CldUMP which is then further anabolized and incorporated selectively into tumor DNA as CldU (5-chloro-2'-deoxyuridine). (NCI04)
An inhibitor of nucleotide metabolism.

---

Finally, a second chemotherapy regimen for advanced pancreatic carcinoma is a clinically relevant issue to be resolved urgently. In this study, we demonstrated that tetrahydrouridine (THU) acts on some tumor cell lines to be both A) independently cytotoxic and B) to sensitize gemcitabine cytotoxicity through E2F1 in both CDA-high and CDA-low expressing cells. Consequently, tetrahydrouridine (THU) could improve gemcitabine sensitivity for at least some gemcitabine resistant cells regardless of CDA expression. Importantly, tetrahydrouridine (THU) has been clinically available, has been established as safe, and is more reasonably priced than newly developed molecular targeted drugs. Further research is necessary to discover which cell types are appropriate for THU treatment to control proliferation. With improved THU stability in vivo, a combination treatment could be considered a more feasible and effective therapeutic approach than existing ones for advanced pancreatic carcinoma. [1]

---

In summary, the combination therapy resulted in increased sensitivity to DAC-induced toxicity as compared to the DAC treatment alone, correlating with DAC plasma levels. DAC Cmax was in a range expected to cause cytotoxicity (> 0.5 μM) in all DAC treatment groups. Female mice had higher Cmax and are more sensitive compared to males. Furthermore, hematologic effects appeared to be the most sensitive safety biomarkers, suitable for monitoring in clinical trials. treatment-related effects resolved fully or showed a trend towards resolution by the end of the recovery period. An oral route of administration for this combination therapy appears to be promising for clinical evaluation in sickle cell disease and β-thalassemia. Thus, the current study in CD-1 mice yielded important toxicity parameters pertinent to potential therapeutic application of tetrahydrouridine (THU) and DAC combination oral therapy in human settings.[2]

C9H20N2O8

Tetrahydrouridine;18771-50-1

Typically exists as solid at room temperature

-1.85

UCKYOOZPSJFJIZ-XVKVHKPRSA-N

InChI=1S/C9H16N2O6/c12-3-4-6(14)7(15)8(17-4)11-2-1-5(13)10-9(11)16/h4-8,12-15H,1-3H2,(H,10,16)/t4-,5?,6-,7-,8-/m1/s1

1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-hydroxytetrahydropyrimidin-2(1H)-one dihydrate

THU dihydrate; NSC-112907 dihydrate

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)

DMSO :≥ 100 mg/mL (~351.79 mM)
H2O :~50 mg/mL (~175.90 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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