apoptosis; oxidative stress and p53 pathways

Gallium maltolate (GaM), a next generation of gallium compound, displays greater cytotoxicity compared with GaN against a panel of lymphoma cell lines, including lymphoma cells resistant to growth inhibition by GaN. In contrast to GaN, which is a simple metal salt, GaM consists of three maltolate ligands bidentately bound to a central gallium atom in a propeller-like arrangement. Gallium’s anti-inflammatory properties may be relevant to CTCLs, as our studies have shown that CTCL develops in a background of inflammatory changes in the skin [1].

In human CTCL xenograft models, peritumoral injection of GaM limited the growth of CTCL cells, shown by fewer tumor formations, smaller tumor sizes, and decreased neovascularization in tumor microenvironment [1].

Flow cytometric analysis and apoptosis detection[1]
In order to detect surface markers, cells were washed once in PBS, once in FACS buffer, and resuspended in FACS buffer at 5 × 105 ml-1. Cells were then incubated with antibodies at 4 °C for 30 min and washed with FACS buffer. Cell preparation was analyzed on a BD FACSCalibur flow cytometer (San Jose, CA). For apoptosis detection, GAM-treated/untreated cells were washed once in PBS, once in 1x Binding Buffer, and resuspended in 1x Binding Buffer at 5 × 106 ml-1. PE-conjugated Annexin V (5 μl) was added to 100 μl of the cell suspension and incubated for 10–15 minutes at room temperature. Cells were washed in 1x Binding Buffer and resuspended in 200 μl of 1x Binding Buffer. A volume of 5 μl of 7-AAD Viability staining solution was added, and the cells were immediately analyzed by flow cytometry.

Mitochondrial oxygen consumption [1]
The effects of GaM on mitochondrial function was assessed by measuring the oxygen consumption rate in intact HuT78 cells using a Seahorse XF24 Extracellular Flux Analyzer according to the manufacturer’s recommendations. Cells were incubated in tissue culture with increasing concentrations of GaM for 8 hours and then collected, washed in Dulbecco's PBS, and resuspended in unbuffered RPMI-1640 assay media containing 2 mM L-glutamine, 5 mM sodium pyruvate, pH 7.4, without sodium bicarbonate (Life Technologies; Grand Island, NY). Cells (750,000 cells per well) were plated in Seahorse Bioscience V7 tissue culture plates. One hundred microliters of cell suspension in non-buffered assay media was added per well to be measured. Prior to seeding, the assay plates were coated with Cell-Tak (BD Biosciences; San Jose, CA). Cells were centrifuged to the bottom of the plate at 250 × g for 5 minutes prior to the addition of an additional 400 μl of assay media.

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Western blot [1]
CTCL cells treated with and without GaM were collected, lysed, and centrifuged to remove cellular debris. The protein content of supernatants was measured by the bicinchoninic acid protein assay. Samples were resolved by NuPAGE Bis-Tris precast gel and transferred into a nitrocellulose membrane using a Transblot system. Membranes were incubated with specific primary antibody to p38, phosphorylated p38, HO-1, and glyceraldehyde-3-phosphate dehydrogenase. Washed membranes were incubated in appropriate secondary antibody conjugated to horseradish peroxidase, immersed in Pierce ECL chemiluminescence detection solution (Pierce), and recorded by imaging with a Molecular Imager Gel Doc XR+ system with Image Lab Software.

Establishment of tumor mouse models and GaM treatment[1]
Mice were sedated with IP injection of ketamine/xylazine solution. PBS–washed CTCL cells (Hut78 or HH) (1 × 106 per mouse) were injected into the SC space in the center of the shaved skin using a 28 g needle. Mice were monitored during recovery from anesthesia and moved back to normal housing. Tumors were visually examined twice a week until the end point in the 4th week after implantation. For in vivo GaM treatment, daily injections were performed for 5 days. Depending on the initiation of GaM administration, three treatment regimens were used: 1st week, 2nd week, and 3rd week treatment, which started on day 2, 8, and 15, respectively, after tumor cell implantation on day 1, were performed. All GaM treatments involved careful injection of GaM (five consecutive daily injection, 400 μg per injection) into the SC tissue around the site of tumor implantation (peritumoral injection) while avoiding the tumor mass itself. For the control group, equal volumes of PBS (200 μl) were injected in similar manner.[1]

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Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model[2]
Antimicrobial efficacy against Lawsonia intracellularis is difficult to evaluate in vitro, thus, the effects of gallium maltolate's (GaM) were investigated in a rabbit model for equine proliferative enteropathy (EPE). Juvenile (5-6-week-old) does were infected with 3.0 × 10(8) L. intracellularis/rabbit and allocated into three groups (n = 8). One week postinfection, one group was treated with GaM, 50 mg/kg; one, with doxycycline, 5 mg/kg; and one with a sham-treatment (control). Feces and blood were collected daily and weekly, respectively, to verify presence of L. intracellularis fecal shedding using qPCR, and seroconversion using immunoperoxidase monolayer assay. Rabbits were sacrificed after 1 week of treatment to collect intestinal tissues focusing on EPE-affected sections. Intestinal lesions were confirmed via immunohistochemistry. No difference was noted between treatments regarding EPE-lesions in jejunum (P = 0.51), ileum (P = 0.74), and cecum (P = 0.35), or in L. intracellularis fecal shedding (P = 0.64). GaM and doxycycline appear to have similar efficacy against EPE in infected rabbits.

[1]. Gallium maltolate inhibits human cutaneous T-cell lymphoma tumor development in mice. J Invest Dermatol. 2015 Mar;135(3):877-884.

[2]. Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model. J Vet Pharmacol Ther. 2014 Dec;37(6):571-8.

Gallium maltolate is Titan’s novel oral agent in development for the potential treatment of chronic bacterial infections, bone disease and cancer.
Gallium Maltolate is an orally bioavailable form of the element gallium (Ga) composed of a trivalent gallium cation (Ga3+) coordinated to three maltolate ligands, with anti-inflammatory, anti-proliferative, antineoplastic, analgesic, antiresorptive and antibacterial activities. Upon administration of gallium maltolate, Ga3+, which is structurally similar to the ferric ion (Fe3+), competes with and replaces Fe3+ in many vital Fe 3+-mediated biological reactions.. Unlike Fe3+, Ga3+ cannot be reduced, cannot participate in redox reactions and cannot mimic Fe3+ functions. In rapidly proliferating cells, such as cancer cells, high amounts of iron are needed for DNA synthesis. The incorporation of Ga3+ inactivates the Fe3+-dependent enzyme ribonucleotide reductase (RR), an enzyme essential for DNA synthesis, leading to an inhibition of DNA synthesis and induction of cell death in rapidly proliferating cells. Gallium similarly reduces bacterial cell growth. In addition, Ga3+ is able to suppress inflammation through the down-regulation of pro-inflammatory cells and the inhibition of pro-inflammatory cytokine secretion. Gallium also exerts analgesic effects due to the inhibition of Fe3+-dependent enzymes involved in inflammation and may interfere with the activity of certain metalloproteinases and neuropeptides that are implicated in pain. Also, gallium is able to inhibit bone resorption by osteoclasts, may inhibit metastasis to bone and may prevent the destruction of bone by tumors.

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Drug Indication
Investigated for use/treatment in bladder cancer, lymphoma (unspecified), multiple myeloma, paget's disease, and prostate cancer.

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Mechanism of Action
Gallium maltolate is a novel orally active formulation of gallium, a semi-metallic element that is a potent inhibitor of ribonucleotide reductase, an enzyme that promotes tumor growth.Gallium is also known to concentrate in malignant tumors and sites of infection, and appears to favorably impact calcium deposition, making bones more resistant to degradation caused by cancer metastasis. Titan's novel product, gallium maltolate, may significantly expand the therapeutic potential of gallium by providing the advantages of enhanced bioavailablity, a potentially improved therapeutic profile and ease of administration.

443.997

C, 48.58; H, 3.40; Ga, 15.67; O, 32.36

108560-70-9

9846339

Off-white to light yellow solid powder

0

9

0

28

200

0

ASYYOZSDALANRF-UHFFFAOYSA-K

InChI=1S/3C6H6O3.Ga/c3*1-4-6(8)5(7)2-3-9-4;/h3*2-3,8H,1H3;/q;;;+3/p-3

gallium tris(2-methyl-4-oxo-4H-pyran-3-olate)

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)

H2O: ~2 mg/mL (4.5 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.

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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.)