Colchicine metabolite; tubulin

Effective scavenging of reactive radicals and low reactivity of generated secondary antioxidant radicals towards vital intracellular components are two critical requirements for a chain-breaking antioxidant. Tubulin-binding properties aside, colchicine metabolites remain largely untested for other possible biological activities, including antioxidant activity. Mourelle et al. [Life Sci. 45 (1989) 891] proposed that colchiceine (EIN) acts as an antioxidant and protective agent against lipid peroxidation in a rat model of liver injury. Since EIN as well as two other colchicine metabolites, 2-demethylcolchicine (2DM) and 3-demethylcolchicine (3DM), possess a hydroxy-group on their carbon ring that could participate in radical scavenging, we tested whether they can act as chain-breaking antioxidants. Using our fluorescence-HPLC assay with metabolically incorporated oxidation-sensitive cis-parinaric acid (PnA) we studied the effects of colchicine metabolites on peroxidation of different classes of membrane phospholipids in HL-60 cells. None of the colchicine metabolites in concentrations ranging from 10(-6) to 10(-4) M was able to protect phospholipids against peroxidation induced by either azo-initiators of peroxyl radicals or via myeloperoxidase (MPO)-catalyzed reactions in the presence of hydrogen peroxide. However, the metabolites did exhibit dose-dependent depletion of glutathione, resembling the behavior of etoposide, a hindered phenol with antioxidant properties against lipid peroxidation. Electron spin resonance (ESR) experiments demonstrated that in a catalytic system containing horseradish peroxidase (HRP)/H(2)O(2), colchicine metabolites undergo one-electron oxidation to form phenoxyl radicals that, in turn, cause ESR-detectable ascorbate radicals by oxidation of ascorbate. Phenoxyl radicals of colchicine metabolites formed through MPO-catalyzed H(2)O(2)-dependent reactions in HL-60 cells and via HRP/H(2)O(2) in model systems can also oxidize GSH. Thus, colchicine metabolites possess the prerequisites of many antioxidants, i.e. a nucleophilic hydroxy-group on a carbon ring and the ability to scavenge reactive radicals and form a secondary radical. However, the latter retain high reactivity towards critical biomolecules in cells such as lipids, thiols, ascorbate, thereby, rendering colchicine metabolites effective radical scavengers but not effective chain-breaking antioxidants. [1]

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The effects of colchicine and its analogs on the carrageenin-induced footpad edema in rats were investigated. The anti-inflammatory effects of colchicine analogs were measured at 3 and 5 hr after the carrageenin injection. Colchicine, 1-demethylcolchicine and 3-demethylcolchicine markedly inhibited the carrageenin edema whereas 2-demethylcolchicine was much less active. Thiocolchicinoids, having a thiomethyl group at C-10 instead of a methoxy group, were considerably less potent. These results suggest that the presence of methoxy groups at C-2 and C-10 in colchicine is necessary to maintain anti-inflammatory activity. Inactivity of deacetylcolchicine indicates that substitution of the amino group at C-7 with electron withdrawing groups is also important. Significant inhibition of carrageenin edema and strong binding to tubulin in vitro were manifested by colchicine, 3-demethylcolchicine, N-butyryldeacetylcolchicine and colchifoline. On the other hand, N-carbethoxydeacetylcolchicine which did bind well to tubulin, did not show much effect on the carrageenin edema. These results suggest that the anti-inflammatory action of colchicinoids may not be regulated through the microtubule system [2].

Lipid peroxidation experiments [1]
Two milliliter of cell suspension (1×106 cells per ml) were transferred to glass tubes with screw-caps and were pre-incubated for 15 min with one of the following agents: PMC, phenol, etoposide, 2DM, 3-demethylcolchicine (3DM), COL, EIN, or NDE. Concentration of colchicine and its metabolites varied from 10−6 to 10−4 M. Control samples were treated with the vehicle DMSO. Pre-incubated cells were exposed to three different oxidants: a lipid-soluble azo-initiator of peroxyl radicals, AMVN (0.5 mM for 2 h at 37 °C), a water-soluble azo-initiators, AAPH (50 mM for 30 min at 37 °C), and H2O2 (25 μM, added every 15 min during 1 h incubation at 37 °C).

The intraperitoneal LD50 in mice was 570 ug/kg. (Journal of Medicinal Chemistry, 24(636), 1981)

[1]. Anti-/pro-oxidant effects of phenolic compounds in cells: are colchicine metabolites chain-breaking antioxidants?. Toxicology. 2002;177(1):105-117.

[2]. Separation of tubulin-binding and anti-inflammatory activity in colchicine analogs and congeners. Life Sci. 1987;40(1):35-39.

Colchicine has been reported to exist in Colchicum arenarium, Colchicum autumnale, and other organisms with relevant data. The colchicine metabolites tested contain electron-donating hydroxyl groups, which are readily oxidized by myeloperoxidase (MPO)/hydrogen peroxide (H₂O₂) to the corresponding phenoxy radicals. Our results clearly demonstrate that even at high concentrations (up to 100 μM), colchicine metabolites fail to significantly inhibit MPO/H₂O₂-catalyzed phospholipid peroxidation in HL-60 cells (in contrast to the formation of vitamin E homologues PMC or the phenolic antitumor drug etoposide). Colchicine metabolites also do not enhance phospholipid peroxidation—an effect readily observed in H₂O₂-treated HL-60 cells exposed to phenol. These results suggest that the reactivity of phenoxy radicals generated by colchicine homologues may be lower than that generated by phenol, but sufficient to directly attack phospholipids and lead to their peroxidation. To further confirm the peroxidase-catalyzed formation of phenoxy radicals from colchicine metabolites, we conducted model experiments using an HRP/H₂O₂ catalytic system. We detected the formation of free radicals through the single-electron oxidation of ascorbic acid and monitored the ascorbic acid radicals using ESR. We found that colchicine metabolites containing hydroxyl groups were indeed oxidized to phenoxy radicals, which could then react with ascorbic acid to generate ascorbic acid radicals, thus being detected by ESR. We further confirmed that the phenoxy radicals generated from colchicine homologues by peroxidase (MPO in HL-60 cells or HRP in the model system) could react with thiols, leading to GSH oxidation. The phenoloxy radicals of colchicine metabolites exhibited high reactivity, similar to those of etoposide or phenol. In contrast, the vitamin E homologue PMC protected GSH from peroxidase-catalyzed oxidation, indicating that its phenoloxy radicals were not reactive towards thiols (Kagan et al., 1999). In summary, the results indicate that although all colchicine metabolites can react with more reactive free radicals to generate phenoxy radicals, they still have sufficient reactivity to not only deplete glutathione and ascorbic acid but also lead to phospholipid oxidation. Therefore, they are not chain-terminating antioxidants. This finding excludes the hypothesis that EIN (and other colchicine metabolites) can prevent lipid peroxidation through direct antioxidant action. However, this does not rule out the possibility that they may prevent lipid peroxidation through other pathways, such as regulating inflammation-related neutrophil “oxidative bursts”. [1]

C21H23NO6

385.41

357.158

7336-33-6

299664

White to yellow solid powder

1.31g/cm3

768.3ºC at 760 mmHg

163-166ºC

418.5ºC

1.61

3.157

2

6

4

28

724

1

CC(=O)N[C@H]1CCC2=CC(=C(C(=C2C3=CC=C(C(=O)C=C13)OC)OC)OC)O

JRRUSQGIRBEMRN-HNNXBMFYSA-N

InChI=1S/C21H23NO6/c1-11(23)22-15-7-5-12-9-17(25)20(27-3)21(28-4)19(12)13-6-8-18(26-2)16(24)10-14(13)15/h6,8-10,15,25H,5,7H2,1-4H3,(H,22,23)/t15-/m0/s1

N-[(7S)-3-hydroxy-1,2,10-trimethoxy-9-oxo-6,7-dihydro-5H-benzo[a]heptalen-7-yl]acetamide

3-Desmethylcolchicine; 7336-33-6; 3-Demethylcolchicine; 3-Demethyl Colchicine; (-)-3-demethylcolchicine; O3-Demethylcolchicine; 3-O-Demethylcolchicine; Desmethylcolchicine;

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)

DMSO: 100 mg/mL (259.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.49 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 25.0 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: ≥ 2.5 mg/mL (6.49 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 25.0 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: ≥ 2.5 mg/mL (6.49 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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