To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis.[1]

Treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels.[1]

Cell viability assays[1]
Exponentially growing cells were plated into 96-well plates (5 × 103 in 150 μl/well), and 18 h later treated with increasing concentrations of TLN-4601 for 72 h. At the end of the treatment, 20 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well and the plates were incubated for an additional 4 h at 37°C. Following this, the medium was removed and replaced with 200 μl of dimethylsulfoxide (DMSO). Experiments were done in quadruplicate and repeated two to three times. The absorbance at 570 nm was measured by plate reader.[1]
Contact-independent growth analysis was performed according to previous protocols [24]. Briefly, log phase growing cells were trypsinized, and triplicates of 3 × 103 cells per well were suspended in enriched medium (supplemented with an additional 10% fetal calf serum) mixed with 1.5% sterile agar and plated onto dense agar coated six-well plates. One ml of standard medium was added to the top of the gelled matrix and colonies were grown for 21 days. Stock solutions of TLN-4601 were dissolved in DMSO and added to both the agar containing the cells and the feeding medium. After 21 days in culture, live colonies were counted in five random three-dimensional fields per well, stained with MTT, and photographed.[1]

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GTP pull-down assays[1]
MIA PaCa-2 cells were cultured in DMEM medium supplemented with 10% FBS for 18 h, starved for 9 h in DMEM 0.1% FBS and then treated for 18 h with increasing concentrations of TLN-4601. At the end of the treatment, cells were stimulated with EGF (100 mg/ml) for 5 min. HPNE-KRAS cells were grown in M3:5 growth medium supplemented with 5% FBS and treated for 18 hours with increasing concentrations of TLN-4601. Ras GTP levels were determined by a Ras activation assay kit according to the manufacturer's directions, or by previously published protocols [24,51]. Lysates (1 mg of total cell protein in each sample) were incubated with 10 μg Raf-1-RBD for 45 min at 4°C and centrifuged for 15 sec at 14000 × g to pellet the agarose beads. After discarding the supernatant, agarose beads were washed three times with 500 μl of lysis buffer and the pellets were resuspended in 2× Laemmli sample buffer containing DTT, boiled for 5 min, and centrifuged at 14000 × g. The supernatant was collected and cellular proteins resolved by 12% SDS-PAGE and analyzed by western blotting using a K-Ras specific antibody.[1]

Animal studies[1]
The xenograft MIA PaCa-2 pancreatic carcinoma donor tumor was generated by injecting 2 × 107 MIA PaCa-2 cells into the right flanks of female Swiss nude mice (total of 10 mice). Those tumors were excised and small fragments (~1 mm3) were implanted s.c. into the right flanks of female Swiss nude mice (8-9 weeks old). When tumor volumes reached 50-60 mm3, mice were randomized into groups of 10-15 mice and treated. The study involved a negative control group (vehicle-treated), a gemcitabine-treated group (60 mg/kg i.p., once a day, twice per week for four weeks), and a TLN-4601 treated group (30 mg/kg s.c., once a day Monday through Friday for 3 consecutive weeks).Tumor growth was followed twice a week by measuring tumor length (L) and width (W) using a caliper. Measurements were converted to tumor volumes (TV; mm3) using the standard formula, TV = (L × W)2/2. Animals were sacrificed when tumors in the control group reached a predetermined endpoint TV of ~1400 mm3 (Day 49). Compound efficacy was assessed by percentage of treated vs control (%T/C) defined as the (median treated tumor volume/median control tumor volume × 100). Tumor growth reduction was calculated by subtracting the % T/C from 100.[1]
Evaluation of Raf-1 tumor levels was performed in a subset of five mice each from the control and TLN-4601 treated groups after the first five days of treatment. Tumor lysates were prepared by sonication in lysis buffer and samples were processed as described above. Differences were considered to be significant at p < 0.05.

[1]. TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling. J Mol Signal. 2010 Nov 2;5:18.

Diazepinomicin is a dibenzodiazepine with a farnesyl sidechain synthesized by Micromonospora sp.. It has a potent activity against a broad spectrum of tumor cell lines. It has a role as a cathepsin L (EC 3.4.22.15) inhibitor, an antioxidant and an antineoplastic agent. It is a dibenzodiazepine, a secondary amine, a farnesane sesquiterpenoid, a triol and an olefinic compound. It contains a 2-trans,6-trans-farnesyl group.
Diazepinomicin has been used in trials studying the treatment of Glioblastoma Multiforme. It is a proprietary first-in-class small molecule with the potential to treat multiple solid tumours like the well known chemotherapeutics, doxorubicin and mitomycin C. Diazepinomicin is a natural product derived from a non-pathogenic micro-organism. Discovered using Thallion’s DECIPHER technology, diazepinomicin has completed preclinical studies conducted by the National Cancer Institute and Thallion to establish safety and efficacy in animal and in vitro models.
Diazepinomicin has been reported in Micromonospora with data available.
Diazepinomicin is a potent inhibitor of the RAS/RAF/MAPK signaling pathway with potential antineoplastic activity. Diazepinomicin binds to and inhibits Ras kinase, thereby preventing the phosphorylation and activation of proteins downstream of the Ras signal transduction pathway, including serine/threonine kinase RAF (BRAF) and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK-2), that play a crucial role in regulating cell growth and survival. Diazepinomicin also selectively binds to the peripheral benzodiazepine receptor (BZRP), a receptor highly expressed in certain cancer cells, thus inducing cell cycle arrest and apoptosis in BZRP-expressing cells. In addition, diazepinomicin is able to cross the blood-brain barrier, thereby reaching therapeutic concentrations in the brain.

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Mechanism of Action
Diazepinomicin demonstrates broad in vitro cytotoxic activity across a diverse panel of tumour cell lines and in vivo efficacy in a number of xenograft tumour models. Preclinical data suggest that diazepinomicin is a targeted anti-cancer agent with dual activity: selective binding to the peripheral benzodiazepine receptor (PBR) and inhibition of the Ras-MAPK pathway.

C28H34N2O4

462.58056

462.252

733035-26-2

9868980

Off-white to gray solid powder

6.581

4

5

8

34

785

0

CC(=CCC/C(=C/CC/C(=C/CN1C2=C(C(=CC(=C2)O)O)NC3=C(C1=O)C=CC=C3O)/C)/C)C

SALVHVNECODMJP-GNUCVDFRSA-N

InChI=1S/C28H34N2O4/c1-18(2)8-5-9-19(3)10-6-11-20(4)14-15-30-23-16-21(31)17-25(33)27(23)29-26-22(28(30)34)12-7-13-24(26)32/h7-8,10,12-14,16-17,29,31-33H,5-6,9,11,15H2,1-4H3/b19-10+,20-14+

4,6,8-trihydroxy-10-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-5H-dibenzo[b,e][1,4]diazepin-11(10H)-one

ECO 4601; ECO-4601;ECO4601;ECO 04601;TLN4601; TLN 4601; TLN-4601; BU 4664L; BU4664L; BU-4664L; Diazepinomicin.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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