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].

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CMT-3 exhibited antifungal activity against a wide range of fungal strains. The MICs of CMT-3 against 47 fungal strains were determined, generally ranging between 0.25 and 8.00 µg/ml.[2]
CMT-3 was particularly effective against filamentous fungi. For example, MICs were 0.5 µg/ml against Epidermophyton floccosum, Seedosporium apiospermum, and a Tricothecium sp., 0.25 µg/ml against Pseudallescheria boydii, and 1.0 µg/ml against Microsporum gypsum and Trichophyton rubrum. Some filamentous fungi like a Cunninghamella sp. had an MIC of 8.0 µg/ml.[2]
Against yeast species, the potency of CMT-3 varied. Candida albicans strains showed MICs ranging from 0.25 to >8 µg/ml. Some strains of Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis were not inhibited by CMT-3 at concentrations up to 8 µg/ml.[2]
In a turbidity assay using C. albicans (isolate 2730) in potato dextrose broth, the MIC of CMT-3 was 2 µg/ml, and the 50% inhibitory concentration was approximately 1 µg/ml.[2]
A fungal viability assay (measuring the ability of fungal cells to grow after drug removal) was performed on 39 strains. At 10 µg/ml, CMT-3 reduced viability by more than 50% for 33 out of 39 strains (85%). It showed potent fungicidal activity against filamentous fungi, with 84% (16 of 19) of tested filamentous fungi showing >90% inhibition of viability. In contrast, CMT-3 was less fungicidal against yeasts compared to amphotericin B.[2]
A time-course study showed that exposure to CMT-3 (10 µg/ml) for 4 hours and 12 hours was required to kill 90% of Aspergillus fumigatus conidia and C. albicans cells, respectively.[2]
Confocal laser scanning microscopy revealed that after incubation with CMT-3, fluorescence from the drug was clearly observed inside both C. albicans and a Penicillium sp. cells, indicating widespread intracellular distribution, with some fluorescence also detected in the cell wall region.[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].

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In a rat orthodontic tooth movement model, daily oral administration of CMT-3 significantly inhibited tooth displacement in a dose-dependent manner over 14 days. The molars moved 1.72 ± 0.53 mm (0 mg/kg group), 1.45 ± 0.18 mm (6 mg/kg group), and 1.13 ± 0.13 mm (30 mg/kg group). Statistical analysis showed a significant inhibitory effect (p = 0.03).[1]
CMT-3 (30 mg/kg) significantly reduced the number of osteoclasts on the compression side (mesial side) of the experimentally moved teeth compared to the control (0 mg/kg) and low-dose (6 mg/kg) groups (p < 0.05).[1]
Immunohistochemical staining suggested that osteoclasts in the 30 mg/kg CMT-3 group appeared to contain less MMP-9 compared to those in the 0 mg/kg group, although this was not quantified.[1]

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]

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Initial Screening of Antifungal Activity: Four fungal species (a Penicillium sp., A. fumigatus, a Rhizopus sp., and C. albicans) were cultured on agar slants. Conidia or yeast cells were suspended in saline to a 0.5 McFarland standard and diluted. Different CMT-3 solutions (final concentration 25 µg/ml in agar) were incorporated into SABHI agar plates. Fungal suspensions were spotted onto the plates, incubated aerobically at 35°C overnight, and growth was scored visually compared to a DMSO control.[2]
MIC Determination (PDA Method): Filamentous fungi and yeasts were cultured, and suspensions were prepared. Potato dextrose agar was sterilized, cooled, and mixed with CMT-3 solutions to achieve final concentrations (e.g., 0, 0.06, 0.12, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 µg/ml) in the agar. The agar-drug mixture was poured into multi-well plates. After solidification, fungal suspensions were inoculated into each well. Plates were incubated aerobically at 35°C for 48 hours or until control growth was apparent. The MIC was determined as the lowest drug concentration that completely inhibited fungal growth visually.[2]
Fungal Viability Assay: Fungal strains were grown, and conidia/cells were collected and suspended in Tris-NaCl buffer. Suspensions were pre-incubated with CMT-3 (10 µg/ml) at 35°C for 12 hours. After incubation, the suspensions were diluted to render the drug concentration ineffective and plated onto potato dextrose agar plates. Plates were incubated at 35°C for 48 hours, colonies were counted, and viability was calculated as a percentage of the colony count from DMSO-treated control cultures.[2]
Time-Course Viability Study: A. fumigatus conidia and C. albicans cells were suspended in buffer. CMT-3 was added to a final concentration of 10 µg/ml. Control received DMSO. Suspensions were incubated at 35°C for 0, 4, 8, 12, and 24 hours. At each time point, suspensions were diluted 1:1000 and plated onto agar plates. After incubation, colonies were counted to determine the fungicidal effect over time.[2]
Growth Inhibition (Turbidity) Assay: A series of tubes containing potato dextrose broth with different concentrations of CMT-3 (0, 0.125, 0.25, 0.5, 1.0, 2.0 µg/ml) were inoculated with a late-log-phase suspension of C. albicans. Tubes were incubated aerobically at 35°C. Turbidity (optical density at 600 nm) was measured spectrophotometrically at time points 0, 1, 2, 4, 6, 12, and 24 hours to monitor growth inhibition.[2]
Confocal Laser Scanning Microscopy (Intracellular Localization): C. albicans and a Penicillium sp. were grown, and cells/conidia were collected, washed, and resuspended in buffer. Aliquots were incubated with different concentrations of CMT-3 (0.5, 5, 10 µg/ml) at 35°C for various times (0, 1, 6, 12 h). After incubation, cells were washed to remove extracellular drug, resuspended, and placed on glass slides with an antifade reagent. Samples were examined using confocal laser scanning microscopy with excitation at 380 nm and emission at 520 nm to detect the intrinsic fluorescence of CMT-3.[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]

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Eighteen young adult male Wistar rats (290–330 g) were used. A standardized orthodontic appliance delivering a constant 10 cN mesial force was placed on the right maxillary molars under general anesthesia.[1]
Rats were divided into three groups (n=6). Group 1 received 1 mL of vehicle (2% carboxymethylcellulose in saline) daily by oral gavage for 14 days. Groups 2 and 3 received the same volume of vehicle containing 6 mg/kg or 30 mg/kg body weight of CMT-3, respectively.[1]
Orthodontic tooth displacement was measured under general anesthesia at days 0, 3, 7, 10, and 14 using a digital caliper. Measurements were taken between a fixed point on the maxillary molar unit and the cemento-enamel junction of the ipsilateral incisor at the gingival level, on both the experimental (appliance) and control (contralateral) sides. The net displacement was calculated from the difference between sides to account for physiological growth and drift.[1]
On day 14, rats were perfused with 4% paraformaldehyde. The maxillae were dissected, fixed, decalcified in EDTA, embedded in paraffin, and sectioned (7 μm). Sections were stained with hematoxylin-eosin for general observation.[1]
For immunohistochemistry, deparaffinized sections were treated for antigen retrieval and endogenous peroxidase inactivation. Osteoclasts and mononuclear phagocyte lineage cells were identified using a primary ED-1 monoclonal antibody, followed by a biotinylated secondary antibody and ABC-peroxidase detection with DAB. MMP-9 was detected using a specific monoclonal antibody with a similar immunohistochemical procedure.[1]
Cell counting was performed on three selected sections per tooth. Osteoclasts were defined as multinucleated, ED-1-positive cells located on or near the bone surface. Mono- and multinuclear ED-1-positive cells within the periodontal ligament (PDL) but not contacting bone/root surfaces were also counted separately.[1]

CMT-3 was described as lacking antibacterial activity, a modification of traditional tetracyclines. [1]
The weight gain was similar in the three experimental groups (0, 6, and 30 mg/kg CMT-3), indicating that no significant systemic toxicity was observed at the tested doses and durations. [1]

[1]. 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]. CMT-3 inhibits orthodontic tooth displacement in the rat. Arch Oral Biol. 2007 Jun;52(6):571-8.

Incyclinide has been used in clinical trials to investigate the treatment of HIV infection, AIDS-related Kaposi's sarcoma, brain and central nervous system tumors, and unidentified types of adult solid tumors under certain protocols. Incyclinide is a chemically modified tetracycline with potential antitumor activity. Incyclinide inhibits matrix metalloproteinases (MMPs), thereby inducing extracellular matrix degradation and inhibiting angiogenesis, tumor growth, invasion, and metastasis. The drug also causes mitochondrial depolarization in tumor cells and induces apoptosis and tissue necrosis. CMT-3 is a chemically modified tetracycline developed to inhibit matrix metalloproteinases (MMPs) without antibiotic activity. [1] Mechanisms of inhibiting orthodontic tooth movement include direct inhibition of MMP activity (which may impair the degradation of bone organic matrix) and reduction of osteoclasts on the pressure side. [1]
Possible mechanisms for reduced osteoclast numbers include: specific induction of activated osteoclast apoptosis, impaired osteoclast adhesion leading to detachment, inhibition of osteoclast migration (possibly through reduced MMP-9), or impaired monocyte precursor differentiation. The number of ED-1 positive cells in the periodontal ligament remained unchanged across groups, suggesting that their recruitment may be unaffected, thus facilitating their action on active osteoclasts. [1]
This study suggests that, since orthodontic relapse involves a similar tissue remodeling process, CMT-3 may help prevent relapse after orthodontic treatment. [1]

C19H17NO7

371.34

371.101

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

15866-90-7

54678924

Light yellow to yellow solid powder

1.28

5

7

1

27

813

3

O=C(C(C1=O)=C(O)C[C@]2([H])C[C@]3([H])CC4=C(C(C3=C(O)[C@@]21O)=O)C(O)=CC=C4)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.)