Chemically Modified Tetracyclines Research Articles

In Vitro Study of the Effects of Five Chemically Modified Tetracycline (CMT) Analogs on Human Epidermal Melanogenesis: Potential as Novel Anti-Melanogenic Agents

Treatment of hyperpigmented skin disorders by novel drug candidates without side effects remains an ongoing area of research. Chemically modified tetracyclines (CMTs) are a group of nonantimicrobial tetracycline drugs that have been shown to possess multiple pharmacological activities. We have previously documented the anti-melanogenic effects of CMT-3 and its 9-amino derivative, CMT-308. Herein, we have extended our analysis to evaluate other CMT analogs, namely CMT-1, CMT-4, CMT-5, CMT-6, and CMT-8, for their impact on melanogenesis using primary human epidermal melanocytes (HEMn-DP cells). CMT analogs were screened using a tetrazolium-based assay to identify nontoxic concentration ranges that were further used to analyze the effects of CMTs on cellular melanin content and morphology (via quantitation of dendricity). Cellular tyrosinase (TYR) activity and levels of melanogenesis proteins, TYR, and microphthalmia transcription factor (MITF) were also evaluated to elucidate the mechanisms underlying their effects on melanogenesis. The findings demonstrated that exposure to CMT-8 resulted in notable cytotoxic effects at concentrations >10 µM; hence, all five analogs were further evaluated and compared at 10 µM. None of the five CMT analogs exhibited any impact on intracellular melanin in HEMn-DP cells at the concentration of 10 µM. However, CMT-1, CMT-4, and CMT-8 robustly suppressed dendricity parameters in HEMn-DP cells, while CMT-5 and CMT-6 showed no effect, suggesting that only a subset of CMT analogs can attenuate melanocyte dendricity. Moreover, the analog CMT-5, which has β-diketone blocked, was ineffective, thus confirming the role of this moiety in suppressing dendrite formation. CMT-1 and CMT-8 did not affect cellular tyrosinase activity, while CMT-4 suppressed TYR activity at 10 µM. The capacity of CMT-4 and CMT-8 to suppress dendricity was partly associated with their ability to downregulate MITF protein levels, while CMT-1 had no effect on MITF but suppressed TYR protein levels. The results of this study indicate that CMT-1, CMT-4, and CMT-8 merit further investigation using in vivo studies as potential drug candidates for the treatment of hyperpigmentation disorders.

Bullet for periodontal disease in future: Chemically modified tetracyclines

Chronic periodontitis is nowadays popularly regarded as Dysbiosis, [1] which causes destruction of tissues rich in collagen like periodontal ligament, alveolar bone and gingival connective tissue. The oral biofilm comprises many periodontal pathogens better regarded as ‘triggers’ in causing chronic periodontitis. Since, not everyone will be affected in the same manner due to periodontal pathogens. Some might not elicit a host response while, the others might have exaggerated response. So, host modulation therapy came into existence to counteract the exaggerated host response. The chemically modified tetracyclines (CMTs) have emerged to inhibit the inflammatory response or to reduce the collagenolytic activity of host. Though a derivative of tetracyclines, it still lacks an antimicrobial action and hence, can be used for periodontitis for longer duration with no adverse effects of gastrointestinal toxicity which parent tetracyclines have.

Comparative study of doxycycline, sancycline, and 4-dedimethylamino sancycline (CMT-3) on epidermal melanogenesis.

Melanogenesis is regulated by melanocytes, which synthesize the pigment melanin inside melanosomes; these melanosomes are exported through dendritic extensions to adjacent keratinocytes and result in skin coloration. Chemically modified tetracyclines (CMTs) are nonantimicrobial tetracyclines that retain the capacity to inhibit matrix metalloproteinases (MMPs) and have shown several biological benefits; in particular, CMT-3 [(4-dedimethylamino sancycline (SAN)] has emerged as a candidate for therapeutic benefits in our previous studies. However, to date, studies of the effects of CMT-3 or SAN on melanogenesis are lacking. We have previously reported the anti-melanogenic activity of CMT-308 (the 9-amino derivative of CMT-3). Herein, we have compared the three tetracycline analogs, doxycycline (DOX), SAN, and CMT-3, for their effects on melanogenesis using B16F10 mouse melanoma cells and have validated results in primary human melanocytes (HEMn-DP). DOX did not show any significant effects on intracellular melanin or melanosome export in DP cells while SAN was cytotoxic at high doses but without effects on melanogenesis at lower doses. However, CMT-3 showed a robust suppression of dendricity parameters (dendrite number, dendrite length, and proportion of dendritic cells) in DP cells which was associated, at least in part, with a significant reduction of intracellular tyrosinase activity. In spite of its inhibition of tyrosinase activity, CMT-3 had no significant effects on intracellular melanin levels, suggesting that it selectively targets melanosome export. Our results demonstrate a unique structure-activity relationship (SAR) for the effects of these compounds on melanogenesis and support the conclusion that removal of the 4-dimethylamino moiety confers the selective capacity to suppress melanosome export. Collectively, these results indicate that CMT-3 might be a candidate for diminishing hyperpigmentation skin disorders.

Effect of chemically modified tetracycline-8 (CMT-8) on hematology, blood chemistry, cytokines and peripheral blood lymphocyte subsets of healthy dogs

Tetracyclines are antibiotics widely used in human and veterinary medicine. Effects on the immune system and inflammatory response, including effects on blood leukocytes proliferation and function and in cytokines synthesis, have been described. Chemically modified tetracyclines (CMT) have lost their antimicrobial activity, but maintain these other properties. This study analyzes the effect of chemically modified tetracycline-8 (CMT-8) on the evolution of complete blood count, blood chemistry, the mRNA expression of selected cytokines and peripheral blood lymphocyte subpopulations distribution in healthy dogs. CMT-8 at a dose of 10 mg/kg once daily was administered per os to six healthy dogs. A control group of five healthy dogs, living in the same conditions than dogs treated with CMT-8, received placebo with an identical therapeutic regimen. When given at the doses used in this study, no side effects of CMT-8 were detected, suggesting a good tolerance and a limited toxicity of the drug. Dogs treated with CMT-8 showed a gradual increase in mean corpuscular hemoglobin. The administration of CMT-8 in healthy dogs did not affect blood mRNA expression of IFN-γ, TNFα, IL-4, IL-6, IL-10, IL-12 p40 and IL-13. However, the lymphocytes expressing class II MHC on their surface decreased during the first two weeks of CMT-8 treatment and subsequently increased for the next three months. Considering the absence of antimicrobial properties of the drug, the effects of CMT-8 detected in this study seem to be unrelated to the classical antimicrobial activity attributed to tetracyclines.

The Inhibitory Effects of Tetracycline on Mastocytosis

Mastocytosis is a rare disease that causes its victims pains in the body, anaphylactic shocks and in some cases death. There is currently no cure for this disease but there are some medications taken to manage the symptoms of this disease. The cause of mastocytosis is the overabundance of mast cells in the blood stream, body tissues, and bone marrow. These mast cells are well meaning hematopoietic cells which are members of the body’s response to an allergy. It has been established that tetracyclines possess anti-bacterial properties, but recent development has also shown that it also possesses anti-inflammatory properties as well as apoptotic properties which can be applied in the treatment of mastocytosis. This paper focuses on the use of the principle of “Creative synthesis” to formulate a possible treatment to mastocytosis using tetracycline and polyphenols as well as chemically modified tetracyclines (CMTs). In addition, investigations will be carried out to proffer a possible combination which will help in inhibiting the effects of mastocytosis. Based on the synthesis, an economic analysis will be carried out to show the economic potential of this proffered treatment.

Role of Chemically Modified Tetracyclines in the Management of Periodontal Diseases: A Review.

Researchers have found that Chemically Modified Tetracyclines (CMTs) act through multiple mechanisms, affecting several parameters of osteoclast function and consequently inhibit bone resorption by altering intracellular calcium concentration and interacting with the putative calcium receptor; decreasing ruffled border area; diminishing acid production; diminishing the secretion of lysosomal cysteine proteinases (cathepsins); inducing cell retraction by affecting podosomes; inhibiting osteoclast gelatinase activity; selectively inhibiting osteoclast ontogeny or development; and inducing apoptosis or programmed cell death of osteoclasts. Thus TCs/CMTs, as anti-resorptive drugs, may act similarly to bisphosphonates and primarily affect osteoclast function. Researchers have evaluated the influence of various chemically modified tetracyclines from CMT-1 to CMT-10 on collagenases and gelatinases through in vitro or animal studies and concluded that all the CMTs except CMT-5 inhibited periodontal breakdown through MMP inhibition in the following order of efficacy: CMT-8>CMT-1>CMT-3>CMT-4>CMT-7. Thus the non-antimicrobial actions of the chemically modified analogues of tetracyclines have shown remarkably better mechanisms to those of agents with established anti-inflammatory/antioxidant potential. These findings clarify the multi-faceted actions of tetracyclines which are unique amongst antimicrobials, with therapeutic applications in periodontal and metabolic diseases. Hence, the present review describes the role of chemically modified tetracyclines in the management of periodontal diseases.

Chemically modified tetracyclines: The novel host modulating agents

Periodontal pathogens and destructive host responses are involved in the initiation and progression of periodontitis. The emergence of host response modulation as a treatment concept has resulted from our improved understanding of the pathogenesis of periodontal disease. A variety of drugs have been evaluated as host modulation agents (HMA), including Non Steroidal Anti Inflammatory Drugs (NSAIDS), bisphosphonates, tetracyclines, enamel matrix proteins and bone morphogenetic proteins. Chemically modified tetracyclines (CMTs) are one such group of drugs which have been viewed as potential host modulating agents by their anticollagenolytic property. The CMTs are designed to be more potent inhibitors of pro inflammatory mediators and can increase the levels of anti inflammatory mediators.

Anti-inflammatory Actions of Adjunctive Tetracyclines and Other Agents in Periodontitis and Associated Comorbidities.

The non-antimicrobial properties of tetracyclines such as anti-inflammatory, proanabolic and anti-catabolic actions make them effective pharmaceuticals for the adjunctive management of chronic inflammatory diseases. An over-exuberant inflammatory response to an antigenic trigger in periodontitis and other chronic inflammatory diseases could contribute to an autoimmune element in disease progression. Their adjunctive use in managing periodontitis could have beneficial effects in curbing excessive inflammatory loading from commonly associated comorbidities such as CHD, DM and arthritis. Actions of tetracyclines and their derivatives include interactions with MMPs, tissue inhibitors of MMPs, growth factors and cytokines. They affect the sequence of inflammation with implications on immunomodulation, cell proliferation and angiogenesis; these actions enhance their scope, in treating a range of disease entities. Non-antimicrobial chemically modified tetracyclines (CMTs) sustain their diverse actions in organ systems which include anti-inflammatory, anti-apoptotic, anti-proteolytic actions, inhibition of angiogenesis and tumor metastasis. A spectrum of biological actions in dermatitis, periodontitis, atherosclerosis, diabetes, arthritis, inflammatory bowel disease, malignancy and prevention of bone resorption is particularly relevant to minocycline. Experimental models of ischemia indicate their specific beneficial effects. Parallel molecules with similar functions, improved Zn binding and solubility have been developed for reducing excessive MMP activity. Curbing excessive MMP activity is particularly relevant to periodontitis, and comorbidities addressed here, where specificity is paramount. Unique actions of tetracyclines in a milieu of excessive inflammatory stimuli make them effective therapeutic adjuncts in the management of chronic inflammatory disorders. These beneficial actions of tetracyclines are relevant to the adjunctive management of periodontitis subjects presenting with commonly prevalent comorbidities addressed here.

Structural and dynamics analysis of matrix metalloproteinases MMP-2 complexed with chemically modified tetracyclines (CMTs)

Matrix metalloproteinases (MMPs) play a critical role in physiological processes and pathological conditions such tumor invasion and metastasis. In recent years, a number of MMP inhibitors have been proposed, including the chemically modified tetracyclines (CMTs), which have been evaluated in preclinical cancer models showing promising results. This work provides insights into the structure and dynamics of the MMP-2 catalytic domain complexed with seven CMT (CMT-n), based on the analysis of molecular dynamics trajectories in solution. The comparative analysis of various relevant molecular aspects of the different complexes of MMP-2 and CMT-n derivatives was performed aiming to elucidate the effect of ligands on the enzyme structure. These include the radial distribution function of the water molecules around the catalytic zinc, the solvent accessible surface area for the inhibitors and the root-mean-square fluctuation for all amino acid residues. The results help to understand the differences in the binding modes of related compounds and, therefore, add to further design of novel tetracycline-based inhibitors for MMP enzymes.

Inhibitory impacts of chemically modified tetracycline-3 and underlying mechanism in human cervical cancer cells

Chemically modified tetracyclines (CMTs) have been rationally designed from tetracyclines. The CMTs that show the antimicrobial properties are eliminated, whereas matrix metalloproteinase inhibitory properties are retained. Interestingly, CMT-3 (COL-3, by eliminating the dimethylamino, methyl, and hydroxyl functionalities on the basic tetracycline structure), one of the CMTs, has shown strong anticancer activity. In this study, we found that CMT-3 showed dose-dependent and time-dependent cytotoxicity in HeLa and Siha cells, two human cervical cancer cell lines. HeLa cells were more sensitive to CMT-3 compared with Siha cells. The antiproliferation potential of CMT-3 was associated with the mitochondrial apoptosis pathway, increasing reactive oxygen species level, and proapoptosis protein (e.g. caspase-3) expression, but decreasing antiapoptosis protein expression (e.g. Bcl-2). N-acetylcysteine (a reactive oxygen species inhibitor) and Z-LEHD-FMK significantly reduced or blocked the apoptosis event resulting from cytotoxic effect of CMT-3. CMT-3 also induced G0/G1 phase arrest with the reduction of cell cycle regulatory protein cyclin E and the translocation of NF-κB from the cytoplasm to the nucleus. Our findings provide the important foundation for further investigation of the underlying mechanism for the anticancer activity of CMT-3 and the potential application of CMT-3 as a new therapeutic candidate for clinical cervical cancer therapy.

Host Modulatory Therapy: A Novel Approach in Periodontal Therapy

Although bacterial plaque is primary etiologic factor in the initiation and progression of periodontal diseases, the host’s reactions to the presence of the bacteria are the one which mediates the periodontal tissue destruction. The periodontal tissue destruction is believed to be due to the host response, it is logical to consider therapeutic approaches that modulate the host response in addition to antibacterial and analgesic approaches in the management of periodontal diseases. A number of host modulatory agents like chemically modified tetracyclines (CMT), non-steroidal anti-inflammatory drugs, and bisphosphonates are effective in treating periodontal diseases in adjunct to the nonsurgical and surgical periodontal therapy. This paper reviews the Host Modulatory Therapy which is the effective means when used as an adjunct to mechanical periodontal therapy in treating periodontal diseases.

Preparation and characterization of 4-dedimethylamino sancycline (CMT-3) loaded nanostructured lipid carrier (CMT-3/NLC) formulations

Chemically modified tetracyclines (CMTs) have been reported to strongly inhibit proliferation and metastasis of various cancers, but their efficacy is restricted by poor water solubility. In the present study, a hydrophilic 4-dedimethylamino sancycline (CMT-3) loaded nanostructured lipid carrier (CMT-3/NLC) was produced by high pressure homogenization (HPH). The physical properties of CMT-3/NLC formulations were characterized by dynamic light scattering (DLS), high efficiency liquid chromatography (HPLC), atomic force microscopy (AFM), scanning electron microscopy (SEM), small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS) and wide-angle X-ray powder diffraction (XRD). The lipid and surfactant ingredients, as well as drug/lipid concentrations (m/m) were optimized to produce stable and sustained NLC formulations. In vitro cytotoxicity of CMT-3/NLC against HeLa cells was evaluated by MTT assay. The diameter of CMT-3/NLC was found to increase from 153.1±3.0 nm to a maximum of 168.5±2.0 nm after 30 days of storage, while the entrapment efficiency remained constant at >90%. CMT-3/NLC demonstrated a burst-sustained release profile in release media with different pH, a property attributed to the 3-dimensional structure of CMT-3/NLC. Cell uptake and localization studies indicated that NLC reached the cytoplasm and could thereby facilitate CMT-3 entry into HeLa cells.

Chemically Modified Tetracyclines as Inhibitors of MMP-2 Matrix Metalloproteinase: A Molecular and Structural Study

The present study focuses on the direct interaction of chemically modified tetracyclines (CMTs) with the active site of the matrix metalloproteinase 2 (MMP-2). Molecular docking, molecular dynamics (MD) simulations, and free energy calculations were accomplished for seven CMT derivatives. New sets of parameters are proposed for structural and catalytic zinc atoms in order to study MMPs and their complexes by means of the AMBER force field. Our computational results show that six CMTs studied bind to the catalytic zinc of the MMP-2 enzyme at the O11-O12 site as proposed experimentally. The exception was the CMT-3 analogue that is found embedding within the active site, enhancing the van der Waals and hydrophobic contacts with the hydrophobic S1' pocket in the MMP-2 enzyme. The binding energy calculated in solution predicts the CMT-3 complexes as the most favorable, followed by the CMT-7 and CMT-8 analogues, respectively, which is in line with experimental findings. This work is the first step toward understanding the mechanism of CMTs as MMP inhibitors at a molecular level.

Chemically modified tetracyclines: Novel therapeutic agents in the management of chronic periodontitis

Chronic periodontitis is a complex infection initiated by gram-negative bacteria which destroy the supporting structures of the tooth. Recently, it has been recognized that it is the host response to bacterial infection which causes greater destruction of the connective tissue elements, periodontal ligament and alveolar bone in periodontitis. This has led to the development of various host modulating approaches to target cells and their destructive mediators involved in tissue degradation. Chemically modified tetracyclines (CMTs) are derivatives of tetracycline group of drugs which lack antimicrobial action but have potent host modulating affects. They inhibit pathologically elevated matrix metal loproteinases, pro-inflammtory cytokines and other destructive mediators. Bone resorption is also suppressed due to their combined anti-proteinase and apoptotic affects on osteoblasts and osteoclasts, respectively. Development of resistant bacteria and gastrointestinal toxicity seen with parent tetracyclines is not produced by CMTs. Hence, CMTs are viewed as potential therapeutic agents in the management of chronic diseases like periodontitis that involve destruction of connective tissue and bone.

Effect of Tetracyclines on IgE Allergic Responses and Asthma

There are no current therapies that specifically target IgE production in human allergic disease. We found that tetracyclines and chemically modified tetracyclines (CMT) that lack antibiotic activity prevent IgE production, making them ideal candidates for anti-allergy therapy. This is based on our findings that minocycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses. Tetracyclines and CMT also suppress ongoing IgE responses of BPO-KLH sensitized mice in vivo and in vitro and humans IgE responses in vitro. We also found that highly increased levels of phosphorylated p38 MAP kinase, but not phosphorylated JNK or ERK, are expressed by blood T and B cells and monocytes of allergic asthmatic humans. Levels of phosphorylated p38 MAP kinase, but not ERK or JNK, correlated with their IgE. Tetracyclines significantly suppressed expression of phosphorylated p38 MAP kinase by CD4+ and CD8+ T cells but not B cells or monocytes in vivo and in vitro. Our findings open the door to development of new drugs and patents, especially for CMTs that lack antibiotic activity (US 7649113), for treatment of human allergic disease.

Interaction of chemically modified tetracyclines with catalytic Zn(II) ion in matrix metalloproteinase: evidence for metal coordination sites

Chemically modified tetracyclines (CMTs) have shown promising activity as matrix metalloproteinase (MMP) inhibitors acting as zinc-binding groups. The first step in the design of new and effective drugs is the molecular description of the mechanism of action in chemical and biological environments. In the present study, the structure and stability of [Zn(LH n )(H2O)2]2−x (n = 0, 1, 2 and x = −2, −1, 0) and [Zn(L)(His)3], where L represents five distinct, structurally related CMTs, are discussed. In addition to the effect of the ligand on Zn(II) coordination, the role of the solvent and pH was also determined. The results suggested that O1–Oam (labeled as site II in the present paper) of CMT-1, CMT-4 and CMT-7 was the most stable site in the gas phase and aqueous solution. However, for CMT-3 and CMT-8, coordination at the O11–O12 moiety (site VI) was preferred. This coordination site is an essential binding mode of CMTs with active zinc in the MMP catalytic site; therefore, our results support the singular behavior of CMT-3 and CMT-8 as promising MMP inhibitors.

Introduction and Background

The rationale for this issue, dedicated to the non-antimicrobial activities of tetracyclines (TCs) as matrix metalloproteinase (and cytokine) – inhibitors, and clinical applications of these properties, is addressed in this introduction. From a historical perspective, the author describes two “breakthrough” experiments that opened this field: (1) the discovery of animal collagenase, the first of a series of matrix metalloproteinases (MMPs) which are now known to be essential mediators of collagen-and connective tissue-destruction including bone loss during various diseases; and (2) the discovery by the author and his team of the unexpected ability of TCs to inhibit these MMPs, and by mechanisms unrelated to the antibiotic activity of these drugs. This led to the development of (i) non-antimicrobial formulations of TCs, ie., sub-antimicrobial-dose doxycycline which resulted in two approved drugs, one for the treatment of periodontal disease, the other for a chronic inflammatory skin disease, and (ii) non-antimicrobial compositions of TCs, ie., the chemically-modified TCs or CMTs or COLs—one of which has shown evidence of efficacy as an anti-angiogenesis agent in human clinical trials on a type of cancer. The development of the CMTs also resulted in the identification of the active site of the TC molecule as an MMP-inhibitor, the calcium and zinc binding site at carbon-11 and 12. And finally, the recently recognized importance of not excessively inhibiting the MMPs because basal levels are needed for various physiologic functions, and the therapeutic potential of TCs as inhibitors of intracellular not just extracellular MMP activity, are both introduced.

Inhibition of smooth muscle cell proliferation by a chemically modified tetracycline

Chemically modified tetracyclines (CMTs) are a group of nonantimicrobial derivatives of tetracycline, which exert antiproliferative and anticollagenolytic properties. The molecular mechanisms, however, are poorly understood. The effect of CMT-3 on cultured, subconfluent rat aortic smooth muscle cells (SMCs) was analyzed by [(3)H]-thymidine incorporation, counting cell numbers, and flow cytometry analysis. CMT-3 inhibited the incorporation of [(3)H]-thymidine and reduced the cell number dose-dependently, with approximately 60% inhibition at the maximal CMT-3 concentration used (20 mumol/l). CMT-3 decreased the SMC proportion in S-phase and gradually increased the proportion at G2/M. Initially, the proportion of cells in G1-phase increased and then gradually decreased back to baseline as the CMT-3 concentration increased. CMT-3 treatment of confluent SMCs for 24 h did not induce apoptosis. CMT-3 inhibited SMC proliferation by inducing cell cycle arrest at the G2/M restriction point. Nonetheless, CMT-3 did not induce SMC apoptosis.

CMT-3 inhibits orthodontic tooth displacement in the rat

Objective Orthodontic tooth movement requires extensive remodeling of the periodontal ligament (PDL) and the alveolar bone. Osteoclasts resorb bone, allowing teeth to migrate in the direction of the force. Matrix metalloproteinases (MMPs) are able to degrade the extracellular matrix of the periodontal tissues. Chemically modified tetracyclines (CMTs) can inhibit MMPs, but lack antimicrobial activity. We hypothesize that CMT-3 will decrease the rate of orthodontic tooth movement in the rat. Design Eighteen Wistar rats received a standardized orthodontic appliance at one side of the maxilla. During 14 days, three groups of six rats received a daily dose of 0, 6 or 30 mg/kg CMT-3, and tooth displacement was measured. Thereafter, osteoclasts were counted on histological sections using an ED-1 staining. Multi- and mononuclear ED-1-positive cells in the PDL were also counted. In addition, sections were stained for MMP-9. Results CMT-3 significantly inhibited tooth movement ( p = 0.03) and also decreased the number of osteoclasts at the compression sides in the 30 mg/kg group ( p < 0.05). Significantly more mono- than multinuclear ED-1-positive cells were present in the PDL, but no significant differences were found between the dosage groups. Osteoclasts in the 30 mg/kg group seemed to contain less MMP-9 than in the control. Conclusions CMT-3 inhibits tooth movement in the rat, probably by reducing the number of osteoclasts at the compression side. This might be due to induction of apoptosis in activated osteoclasts or reduced osteoclast migration. Reduced MMP activity by CMT-3 might also directly inhibit degradation of the organic bone matrix.

Chemically modified tetracyclines inhibit VEGF secretion by breast cancer cell lines

Chemically Modified Tetracyclines (CMTs) are antiproteolytic agents that have been shown to inhibit tumor invasiveness and metastasis. CMT 300 has shown promise in phase I clinical trials in patients with Kaposi’s Sarcoma, which is characterized by over-production of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF). In this study, we report a novel activity of CMT 308, a 9-amino derivative of CMT 300, on reducing levels of VEGF secreted by breast cancer cell lines. CMT 308, at sub-cytotoxic concentrations, reduced basal levels of secreted VEGF in the poorly invasive MCF-7 cell line as well as the more aggressively invasive MDA-MB-435s cell line in a dose-dependent manner. In addition, CMT 308 also reduced transforming growth factor β (TGFβ)-induced VEGF secretion in both cell lines. While VEGF could be detected in the conditioned media of untreated MCF-7 cells within 4 h, levels of secreted VEGF in CMT 308-treated cells remained undetectable up to 8 h. CMT 308 diminished secretion of VEGF from MCF-7 cells up to 8 h regardless of previous time in culture. CMT 308 did not reduce the levels of basal VEGF mRNA in either cell line, but did reduce pools of total intracellular VEGF protein. Although TGFβ stimulated an increase in VEGF levels in the conditioned media as well as in the cytoplasm, TGFβ treatment did not increase VEGF mRNA levels. Thus, augmented expression of VEGF protein by breast cancer cell lines in the presence of TGFβ appears to involve upregulation at a step beyond transcription. Moreover, the data strongly indicate that in these breast cancer cell lines, CMT 308 reduces VEGF secretion by targeting some post-transcriptional event. The capacity of CMT 308 to diminish levels of a major pro-angiogenic signal makes the nonantimicrobial tetracycline derivative an attractive candidate for anti-angiogenic therapy in management of breast cancer.