Incyclinide

matrix metalloproteinase (MMP)

It has been demonstrated that incyclinide can experimentally stop the growth of melanoma, bladder adenocarcinoma, and malignancies in cultured cells. A final dose of 5–20 μM of incyclinide suppresses the actions of gelatin downstream and casein blocking, inhibits MT1-MMP, prevents pro-MMP-2 activation by MT1-MMP, and lessens the hypotoxicity of HT-1080 fibrosarcoma cells [1]. Mold and filamentous fungus are very resistant to the growth of incyclinide. Most CMT-3's minimum inhibitory concentration (MIC) against filamentous fungus ranges from 0.25 to 8 μg/mL, and cyclocycline typically inhibits these fungi's activity to a 90% degree [2].

Reduction of osteoclasts on the strained side may be the mechanism of tooth movement with incyclinide suspension. This could be brought on by decreased osteoclast migration or stimulated osteoclast fluorescence. The decrease in MMP activity caused by incyclinide may also directly prevent opportunistic bone matrix breakdown [3].

Screening of CMTs as antifungal agents. Strains of four fungal species (which are usually found as clinical isolates), i.e., a Penicillium sp., A. fumigatus (ATCC 1022), a Rhizopus sp., and C. albicans, were cultured on PDA slants aerobically at 35°C for 48 h. A sterile cotton-tipped applicator was moistened with sterile 0.9% saline and rolled over the surface of each PDA slant of a fungus demonstrating copious conidiogenesis. The conidia were suspended in 0.9% saline, and the turbidity was adjusted to match a 0.5 MacFarland standard, which is equivalent to approximately 1.5 × 108 cells/ml. C. albicans was suspended in saline and adjusted to a 0.5 MacFarland standard in a similar manner. These suspensions were diluted 1:100 in sterile 0.9% saline. SABHI agar at pH 7.0 was prepared in 100-ml aliquots and sterilized at 121°C for 15 min. The sterile SABHI agar was allowed to cool to 50°C, at which time 10 ml of each of the CMTs tested (prepared in 10% dimethyl sulfoxide [DMSO] at a concentration of 250 μg/ml) was added to produce a final concentration of 25 μg of CMT/ml incorporated into the agar base. After mixing, 20 ml of the SABHI agar solution with or without CMT was poured into a petri dish and allowed to solidify. A plate with 1% DMSO served as a control. Ten microliters of each of the conidial suspensions and 10 μl of C. albicans suspension were inoculated onto the agar plates, which were then incubated aerobically at 35°C overnight. The fungal growth on plates containing the different CMTs was compared to the control (containing 1% DMSO alone) as follows: growth of the fungus at levels observed in the control cultures was scored as +4; no detectable fungal growth was scored as 0; and +3, +2, and +1 represented 75, 50, and 25% the fungal growth observed in the control cultures, respectively[2].

Inhibition of fungal cell viability by CMT-3 and AMB.
(i) Time course study. In this study, fungal cell viability is described as the ability of fungal cells to grow under the in vitro conditions of the experiment. We measured the viability of fungal cells after they were incubated with a drug, and then the drug was removed or diluted to an ineffective level. The inhibition of fungal cell viability reflects the irreversible growth inhibition of the fungal cells, i.e., the fungicidal activity. To determine the time needed for the inhibition of fungal cell viability by CMT-3 and AMB, freshly collected conidia of A. fumigatus and C. albicans cells were used. Both fungal cells and conidia were suspended individually in Tris-NaCl buffer (75 mM Tris, 140 mM NaCl, 11 mM KCl [pH 7.0]) at a concentration of between 5 × 105 and 2 × 106 CFU/ml. CMT-3 and AMB at 100 times their final concentrations in DMSO were added to the suspensions to yield a final drug concentration of 10 μg/ml. The controls were prepared by adding DMSO to the fungal suspensions to yield a final concentration of 1% (vol/vol). The control and drug-treated fungal suspensions were incubated at 35°C for 0, 1, 4, 8, 12, and 24 h. At the end of the incubation, each fungal suspension was diluted 1:1,000 with the same buffer to yield an ineffective drug concentration (0.01 μg/ml) and a fungal concentration of between 5 × 102 and 2 × 103 CFU/ml. One hundred microliters of each diluted fungal suspension was evenly inoculated onto PDA in a sterilized petri dish (100 by 15 mm) and then incubated at 35°C for 48 h or until the control colonies were clearly visible to be counted. (ii) Viability assay. Thirty-nine fungal strains were grown on PDA slants at 35°C for 2 days or until the conidiogenesis phase. The conidia or cells of each fungus were collected and suspended in Tris-NaCl buffer, preincubated with 10 μg of CMT-3 or AMB/ml at 35°C for 12 h, and then diluted and inoculated onto PDA petri dishes as described above. The viability of each fungal strain after exposure to CMT-3 or AMB was calculated as a percentage of the colony count on the control cultures. Three parallel assays for each fungal strain were carried out to obtain a mean value for percent fungal viability.
Inhibition of C. albicans growth by CMT-3.
Determination of C. albicans growth inhibition by CMT-3 was carried out by a modified turbidity assay. A series of tubes containing PDB (5 ml) and different concentrations of CMT-3 (0, 0.125, 0.25, 0.5, 1.0, and 2.0 μg/ml) were each inoculated with a 100-μl suspension of C. albicans (isolate 2730) in late log phase to yield a final cell concentration of 106/ml. The tubes were aerobically incubated at 35°C, and at each time point (0, 1, 2, 4, 6, 12, and 24 h), the turbidity in each tube was determined spectrophotometrically (Spectronic 70; Bausch & Lomb) at 600 nm.[2]

Rats: Eighteen Wistar rats receive a standardized orthodontic appliance at one side of the maxilla. During 14 days, three groups of six rats receive a daily dose of 0, 6 or 30 mg/kg incyclinide, and tooth displacement is measured. Thereafter, osteoclasts are counted on histological sections using an ED-1 staining. Multi- and mononuclear ED-1-positive cells in the PDL are also counted. In addition, sections are stained for MMP-9. [3]

[1]. Lee HM, et al. CMT-3, a non-antimicrobial tetracycline (TC), inhibits MT1-MMPactivity: relevance to cancer. Curr Med Chem. 2001 Feb;8(3):257-60.
[2]. Liu Y, A chemically modified tetracycline (CMT-3) is a new antifungal agent. Antimicrob Agents Chemother. 2002 May;46(5):1447-54.
[3]. Bildt MM, et al. CMT-3 inhibits orthodontic tooth displacement in the rat. Arch Oral Biol. 2007 Jun;52(6):571-8

C19H17NO7

371.34

371.10

C, 61.45; H, 4.61; N, 3.77; O, 30.16

15866-90-7

Light yellow to yellow solid powder

1

158Ų

O=C(C1=C(O)C[C@@](C[C@@]2([H])C(C(C3=C(O)C=CC=C3C2)=O)=C4O)([H])[C@@]4(O)C1=O)N

ZXFCRFYULUUSDW-OWXODZSWSA-N

InChI=1S/C19H17NO7/c20-18(26)14-11(22)6-9-5-8-4-7-2-1-3-10(21)12(7)15(23)13(8)16(24)19(9,27)17(14)25/h1-3,8-9,21-22,24,27H,4-6H2,(H2,20,26)/t8-,9-,19-/m0/s1

(4aS,5aR,12aS)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

COL3; COL-3; COL 3; CMT-3; CMT 3; CMT3; Incyclinide; 4-dedimethylamino sancycline; Chemically modified tetracycline-3. Trade name: Metastat.

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)

Solubility in Formulation 1: 2.5 mg/mL (6.73 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)