TNF-α

Roquinimex inhibited the growth and migration of capillary endothelial cells stimulated by vascular endothelial growth factor (VEGF). [2]
Roquinimex inhibited the ability of rat macrophages to secrete the pro-angiogenic cytokine TNF-α. [2]

The carboxamide-quinoline LS 2616 is a novel immunomodulator augmenting natural killer (NK) cell activity and T-lymphocyte related effector functions. inoculation of B16-F10 cells significantly reduced tumor take. Continuous treatment of mice with LS 2616 initiated 4 days prior to i.v. injection of tumor cells reduced the number of pulmonary metastases by 85%. When treatment with LS 2616 was started 4 days after i.v. injection of tumor cells, a time when established tumor foci were readily detectable in the lungs, a significant reduction in the number of pulmonary metastases resulted. LS 2616 significantly reduced the number of spontaneous pulmonary metastases developing from a B16-F10 tumor growing in the footpad. When treatment with LS 2616 was initiated after the establishment of grossly visible spontaneous pulmonary metastases, no significant effect on the number of metastases was found after 2 weeks of treatment. However, combined treatment with a dose of cyclophosphamide which in itself was ineffective resulted in a statistically significant 70% reduction in the number of remaining pulmonary metastases. Injection of antibodies to asialomonoganglioside which strongly reduce NK cell activity in various organs was used as a probe for the involvement of NK cells in the effects of LS 2616 on the B16-F10 tumor. The therapeutic efficiency of LS 2616 on tumor take when given from the time of s.c. inoculation, on the number of i.v. induced pulmonary metastases when treatment was started before tumor cell injection, as well as the spontaneous development of pulmonary metastases during exposure to the substance was abrogated by simultaneous injection with antibodies to asialomonoganglioside. In contrast, the beneficial effects of LS 2616 on already established i.v. produced or spontaneous pulmonary metastases were unaltered in mice made NK cell deficient by injection of anti-asialomonoganglioside antibodies. In conclusion, LS 2616 has potent antitumor activities mediated by NK cells as well as non-NK cell related defense mechanisms.[1]

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Daily oral roquinimex treatment stimulates the immune system and has anticancer efficacy in multiple animal models, this raised the possibility that combining daily oral roquinimex with intermittent cycles of cytotoxic chemotherapy might be a more curative approach for prostate cancer.[2]
The anticancer and antimetastatic responses to roquinimex in rats are associated with a decrease in tumor-infiltrating macrophages and that roquinimex inhibits the ability of rat macrophages to secrete TNF-α, a known angiogenic stimulator, confirming earlier mouse studies. These studies also documented that antitumor, antimetastatic, and antimacrophage effects of roquinimex are unaffected by NK cell depletion in vivo . That human prostate cancer cells growing as xenografts in nude mice are sensitive to apoptotic death induced in vivo by the antiangiogenic effects of roquinimex .[2]

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Daily oral Roquinimex treatment inhibited the growth and metastasis of dimethylbenzanthracene (DMBA)-induced autonomous breast cancers in rats and restored suppressed delayed-type hypersensitivity (DTH) in these tumor-bearing rats. [2]
In rats inoculated with syngeneic Lewis lung carcinoma cells, daily oral Roquinimex inhibited the development of lung metastasis by 50%. [2]
In mice bearing B16 melanoma, daily oral Roquinimex inhibited tumor growth and metastasis. This effect was mediated by both Natural Killer (NK) cell-dependent and independent mechanisms. [2]
In a series of rodent prostate cancer models, daily oral Roquinimex robustly and consistently inhibited in vivo growth and metastasis without suppressing the immune system or decreasing blood cell counts. This antitumor effect was also observed in T-cell-deficient nude rats and was not prevented by depletion of NK cells. [2]
The anticancer mechanism of Roquinimex was identified as inhibition of tumor angiogenesis, leading to decreased tumor blood flow. This antiangiogenic effect was confirmed in a hamster model and in the Lewis lung carcinoma model in mice, demonstrating it was not species-specific. [2]
Roquinimex did not inhibit wound healing in a rat model; instead, it stimulated the healing process. [2]
Roquinimex inhibited tumor-infiltrating macrophages and their secretion of TNF-α in rat prostate cancer models, effects which were also unaffected by NK cell depletion. [2]
Roquinimex induced apoptotic death in human prostate cancer cells growing as xenografts in nude mice, mediated by its antiangiogenic effects. [2]
Roquinimex synergistically inhibited the growth of rat prostate cancers when combined with androgen ablation therapy. This synergy was attributed to androgen ablation suppressing VEGF secretion by cancer cells, while Roquinimex inhibited the angiogenic response to the remaining VEGF. [2]
Roquinimex suppressed the in vivo development of prostate and breast cancers in rodent chemoprevention models. [2]
Roquinimex inhibited VEGF-induced angiogenesis and the growth of human breast cancer cells engineered to overexpress VEGF when inoculated as xenografts in nude mice. [2]
Roquinimex was therapeutic in a mouse chronic relapsing experimental autoimmune encephalomyelitis (EAE) model, an animal model for multiple sclerosis (MS). [2]
Roquinimex prevented death in four different experimental mouse models of septic shock, an effect linked to its inhibition of macrophage TNF-α secretion. [2]

The effect of Roquinimex on endothelial cell function was assessed in vitro. Capillary endothelial cells were stimulated with vascular endothelial growth factor (VEGF) to induce growth and migration. Roquinimex was added to the culture, and its ability to inhibit these VEGF-stimulated processes was measured. [2]
To study its effect on immune cells, the ability of Roquinimex to inhibit cytokine secretion was evaluated. Rat macrophages were treated with Roquinimex, and the level of TNF-α secreted into the culture medium was measured. [2]

For anticancer efficacy studies in rodent models (e.g., rat prostate cancer, mouse melanoma, Lewis lung carcinoma), Roquinimex was administered daily via oral gavage. The specific doses varied by study but were in the range that stimulated immune responses or inhibited tumor growth without causing overt toxicity in these preclinical settings. For example, in some rat studies, it was given daily to achieve maximal anticancer efficacy. [2]
In the wound healing rat model, Roquinimex was administered to assess its effect on the healing process, where it was found to stimulate rather than inhibit healing. [2]
In the mouse septic shock models, Roquinimex was administered to evaluate its protective effect against death. [2]
In the mouse experimental autoimmune encephalomyelitis (EAE) model for MS, Roquinimex was administered to assess its therapeutic effect on disease progression. [2]

Metabolism / Metabolites
Linomede's known human metabolites include: 4,7-dihydroxy-N,1-dimethyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide, 4-hydroxy-N-(4-hydroxyphenyl)-N,1-dimethyl-2-oxo-1,2-dihydroquinoline-3-carboxamide, 4,6-dihydroxy-N,1-dimethyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide, 4-hydroxy-N-methyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide, 4,8-dihydroxy-N,1-dimethyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide, and 4-hydroxy-1-methyl-2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide.

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Roquinoxa is primarily metabolized by the cytochrome P450 enzyme CYP3A4. This metabolic process involves the demethylation of the nitrogen in the formamide side chain, producing a planar metabolite. [2]

In a phase II clinical trial for metastatic renal cell carcinoma, daily oral administration of 10 mg roquinolone was poorly tolerated. Adverse events leading to discontinuation (17% of patients) or dose reduction (23% of patients) included flu-like symptoms such as myalgia, arthralgia, and fatigue. In addition, cases of pericarditis and neuropathy were observed. [2] In a large phase III registration trial for multiple sclerosis (MS), daily oral administration of as low as 1 mg roquinolone caused serious cardiopulmonary toxicity. These unexpected adverse events included pericarditis, pleural effusion, myocardial infarction, and death, leading to early termination of the trial. [2] The pro-inflammatory side effects of roquinolone are attributed to planar metabolites generated by CYP3A4-mediated demethylation. [2]

[1]. PCancer Res . 1986 Jun;46(6):3018-22.

[2]. Expert Opin Investig Drugs . 2010 Oct;19(10):1235-43.

4-Hydroxy-N,1-Dimethyl-2-oxo-N-phenyl-3-quinoline carboxamide is an aromatic amide. Roquinic acid is a quinoline derivative with immunostimulatory properties. Its mechanism of action is believed to enhance NK cell activity and macrophage cytotoxicity. Furthermore, roquinic acid also inhibits angiogenesis and reduces TNF-α synthesis. Roquinic acid is a quinoline-3-carboxamide with potential antitumor activity. Roquinic acid inhibits endothelial cell proliferation, migration, and basement membrane invasion; reduces the secretion of the angiogenic factor tumor necrosis factor-α by tumor-associated macrophages (TAMs); and inhibits angiogenesis. This drug is also an immunomodulatory agent, appearing to alter the cytokine profile and enhance the activity of T cells, natural killer cells, and macrophages. (NCI04)

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Drug Indications
Roquinic acid is being investigated for the treatment of various cancers and autoimmune diseases. In addition, roquinic acid is also being investigated for adjuvant therapy after bone marrow transplantation in acute leukemia. Roquinox (generic name), also known as Linomede, with the research code LS 2616, is a first-generation quinoline-3-carboxamide analog. [2] It was initially discovered in the search for anti-inflammatory drugs, but studies found that it stimulated rather than inhibited carrageenan-induced inflammation. [2] Its early development focused on its immunomodulatory properties, including the ability to enhance natural killer (NK) cell activity. [2] It has shown efficacy in preclinical models of cancer (through anti-angiogenesis) and autoimmune diseases (e.g., multiple sclerosis models). This led to parallel clinical development pathways for oncology and neurology indications. [2] Its clinical development was halted due to dose-limiting cardiopulmonary toxicity observed in a phase III multiple sclerosis trial and it has never been tested in a prostate cancer clinical trial. [2]
Its toxicity prompted the development of second-generation quinoline-3-carboxamide analogs (such as taquinolone and laquinolone) aimed at minimizing the formation of pro-inflammatory metabolites that cause the side effects of roquinolone. [2]

C18H16N2O3

308.33

308.116

C, 70.12; H, 5.23; N, 9.09; O, 15.57

84088-42-6

54676478

White to off-white solid powder

1.4±0.1 g/cm3

436.2±45.0 °C at 760 mmHg

204 °C(dec.)

217.6±28.7 °C

0.0±1.1 mmHg at 25°C

1.686

1.65

1

3

2

23

522

0

O=C(N(C)C1C=CC=CC=1)C1=C(O)C2C(=CC=CC=2)N(C)C1=O

SGOOQMRIPALTEL-UHFFFAOYSA-N

InChI=1S/C18H16N2O3/c1-19(12-8-4-3-5-9-12)17(22)15-16(21)13-10-6-7-11-14(13)20(2)18(15)23/h3-11,21H,1-2H3

4-hydroxy-N,1-dimethyl-2-oxo-N-phenylquinoline-3-carboxamide

Roquinimex; FCF-89; FCF 89; FCF89; LS2616; LS 2616; LS-2616; Linomide. Trade name: Linomide

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)

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.)