Chemically Modified Tetracycline-3 Research Articles

CMT-3 targets different \u03b1-synuclein aggregates mitigating their toxic and inflammogenic effects

Parkinson's disease (PD) is a neurodegenerative disorder for which only symptomatic treatments are available. Repurposing drugs that target α-synuclein aggregation, considered one of the main drivers of PD progression, could accelerate the development of disease-modifying therapies. In this work, we focused on chemically modified tetracycline 3 (CMT-3), a derivative with reduced antibiotic activity that crosses the blood–brain barrier and is pharmacologically safe. We found that CMT-3 inhibited α-synuclein amyloid aggregation and led to the formation of non-toxic molecular species, unlike minocycline. Furthermore, CMT-3 disassembled preformed α-synuclein amyloid fibrils into smaller fragments that were unable to seed in subsequent aggregation reactions. Most interestingly, disaggregated species were non-toxic and less inflammogenic on brain microglial cells. Finally, we modelled the interactions between CMT-3 and α-synuclein aggregates by molecular simulations. In this way, we propose a mechanism for fibril disassembly. Our results place CMT-3 as a potential disease modifier for PD and possibly other synucleinopathies.

A Novel Analysis via Micro-CT Imaging Indicates That Chemically Modified Tetracycline-3 (CMT-3) Inhibits Tooth Relapse after Orthodontic Movement: A Pilot Experimental Study.

Objective To evaluate the effect of chemically modified tetracycline-3 (CMT-3) and simvastatin on tooth relapse after orthodontic movement in rats using a novel analysis method employing high-resolution micro-CT (Micro-CT) images. In addition, the correlation between bone density and orthodontic relapse was also evaluated for each experimental group. Methods Forty adult male Wistar rats had stainless steel springs installed on their left upper first molars in order to generate tooth movement for 18 days. After this initial period, the animals were divided into three groups: (1) 30 mg/kg of CMT-3; (2) 5 mg/kg of simvastatin; and (3) 0.5% carboxymethylcellulose, and each group was treated for 20 days. Micro-CT images were analyzed (conventional method and 3D reconstruction) on the 7th and 18th days following spring fixation and finally, 20 days after treatment either with CMT-3 or simvastatin (38th day). Bone mineral density (BMD) of the mesial and distal roots of the upper first molar was also analyzed. Results The difference was statistically significant between the groups as to recurrence (p=0.048), and the post hoc test identified the value of p=0.007 between the control group and the CMT-3 group. Simvastatin was not able to inhibit tooth relapse. The bone mineral densities of both the mesial and distal roots were different between the three groups, after the 20th day of drug use (p=0001 and p < 0001). Conclusion Our findings support the initial evidence that CMT-3 is able to prevent relapse after tooth movement. Future trials in humans should evaluate such treatment as a promising approach to preventing this common phenomenon. Clinical Relevance Considering the results obtained, CMT-3 can be used to avoid relapse after tooth movement.

Microglial Cells Impact Gut Microbiota and Gut Pathology in Angiotensin II-Induced Hypertension.

Increased microglial activation and neuroinflammation within autonomic brain regions have been implicated in sustained hypertension, and their inhibition by minocycline-an anti-inflammatory antibiotic-produces beneficial effects. These observations led us to propose a dysfunctional brain-gut communication hypothesis for hypertension. However, it has been difficult to reconcile whether an anti-inflammatory or antimicrobial action is the primary beneficial effect of minocycline in hypertension. Accordingly, we utilized chemically modified tetracycline-3 (CMT-3)-a derivative of tetracycline that has potent anti-inflammatory activity-to address this question. Test the hypothesis that central administration of CMT-3 would inhibit microglial activation, attenuate neuroinflammation, alter selective gut microbial communities, protect the gut wall from developing hypertension-associated pathology, and attenuate hypertension. Rats were implanted with radiotelemetry devices for recording mean arterial pressure. Ang II (angiotensin II) was infused subcutaneously using osmotic mini-pumps to induce hypertension. Another osmotic mini-pump was surgically implanted to infuse CMT-3 intracerebroventricularly. Intracerebroventricular CMT- 3 infusion was also investigated in SHR (spontaneously hypertensive rats). Physiological, pathological, immunohistological parameters, and fecal microbiota were analyzed. Intracerebroventricular CMT-3 significantly inhibited Ang II-induced increases in number of microglia, their activation, and proinflammatory cytokines in the paraventricular nucleus of hypothalamus. Further, intracerebroventricular CMT-3 attenuated increased mean arterial pressure, normalized sympathetic activity, and left ventricular hypertrophy in Ang II rats, as well as in the SHR. Finally, CMT-3 beneficially restored certain gut microbial communities altered by Ang II and attenuated pathological alterations in gut wall. These observations demonstrate that inhibition of microglial activation alone was sufficient to induce significant antihypertensive effects. This was associated with unique changes in gut microbial communities and profound attenuation of gut pathology. They suggest, for the first time, a link between microglia and certain microbial communities that may have implications for treatment of hypertension.

COL‑3 enhances the anti‑proliferative and pro‑apoptotic effects of paclitaxel in breast cancer cells.

Paclitaxel, a chemotherapeutic agent used in the treatment of breast cancer and other solid tumor types, including ovarian and lung, causes a dose‑dependent neuropathic pain, which limits its use. Chemically modified tetracycline‑3 (COL‑3) has anticancer properties and was previously reported to inhibit neuroinflammation and protect against paclitaxel‑induced neuropathic pain (PINP) in mice models. However, it is not known whether it affects the anticancer activities of paclitaxel. Thus, the aim of the present study was to evaluate the effect of COL‑3 on the anticancer activity of paclitaxel on the breast cancer cell lines MCF‑7 (estrogen receptor‑positive), pII [estrogen receptor‑negative (ER‑ve)] and MDA‑MB‑231 (ER‑ve). Cell proliferation, apoptosis and cell cycle stage were determined using an MTT assay, AnnexinV/7‑aminoactinomycinD and flow cytometry. The expression of various signaling molecules was determined with ELISA‑based proteome profiling and western blotting. Additionally, the degree of cell invasion was determined with a Matrigel assay and caspase‑3 activity was determined with a colorimetric assay. Treatment with paclitaxel or COL‑3 alone inhibited cell proliferation in a concentration‑dependent manner in all cell lines. The anti‑proliferative effects of paclitaxel and COL‑3 in combination varied from synergism against MDA‑MB‑231 and pII cells to notably additive and slight antagonism against MCF‑7 cells. In the highly proliferative and invasive pII cells, the observed synergistic anti‑proliferative effect was partially through the induction of apoptosis via modulation of caspase‑3 levels and activity, and P70S6K phosphorylation, but not cell cycle arrest. COL‑3 inhibited the invasion of pII cells in a concentration‑dependent manner partially through inhibiting total matrix metalloproteinase activity. The combination regimen significantly inhibited the expression of two proteases, ADAM metallopeptidase with thrombospondin type1 motif1 and proteinase3. In conclusion, the combination of paclitaxel and COL‑3 indicated additive to synergistic anti‑proliferative effects on breast cancer cells mediated partially via the induction of apoptosis. The combination regimen could further inhibit invasion and metastasis. Thus, COL‑3 could be a beneficial adjunct to a paclitaxel‑based anticancer regimen to improve therapeutic outcome and reduce the adverse effects of paclitaxel, primarily PINP.

The Effect of Chemically Modified Tetracycline-3 on the Progression of Dental Caries in Rats

Objective: Matrix metalloproteinases (MMPs) exist in human saliva and dentin and play an important role in the degradation of organic matrix in teeth. Chemically modified tetracycline-3 (CMT-3) is an inhibitor of MMPs. CMT-3 has been used experimentally to treat caries since 1999, but no distinction between dental caries prevalence and dentin caries prevalence has been described. Methods: A total of 65 Sprague-Dawley rats were randomly divided into three groups. The positive control group (25 rats) was inoculated with Streptococcus mutans (ATCC700610) and fed the cariogenic feed of improved Keyes Diet 2000. The CMT-3 group (25 rats) was also inoculated with S. mutans and fed the cariogenic feed of improved Keyes Diet 2000; the surfaces of rats’ molars were daily treated with 0.02% CMT-3. The negative control group (15 rats) was only fed the standard rodent chow. At the end of the 10th week, the dental caries prevalence and dentin caries prevalence of each group were calculated, and the regions of caries were assessed. Results: No caries was found in the negative control group. The dental caries prevalence of the CMT-3 and the positive control group was 75.0 and 83.3%, respectively (p > 0.05, Table 2). The dentin caries prevalence of the CMT-3 and the positive control group was 33.3 and 70.8%, respectively (p < 0.05, Table 2). The Keyes scoring of dentin caries in the CMT-3 group was significantly lower than that in the positive control group (p < 0.05, Table 3). Conclusions: CMT-3 had no effect on the prevalence of dental caries, but could lower the prevalence and slow down the progression of dentin caries.

CMT‐3, an anti‐inflammatory, non‐antibiotic tetracycline, attenuates AngII‐induced hypertension

BackgroundOur previous studies have established that activation of microglial cells and neuro‐inflammation in autonomic brain regions, particularly in the paraventricular nucleus (PVN) of the hypothalamus, plays a critical role in hypertension (HTN). This conclusion is primarily based on the evidence that the tetracycline antibiotic minocycline (Mino) inhibits microglial activation and attenuates HTN. Since Mino has both anti‐inflammatory and antibiotic properties, we sought to determine the property responsible for its antihypertensive effects using the chemically modified tetracycline‐3 (CMT‐3), that has no antibiotic activity.ObjectiveWe hypothesized that central administration of CMT‐3, would inhibit microglial activation, neuro‐inflammation and attenuate HTN.MethodsSix‐week‐old male SD rats (n=8–12/group) were implanted with telemetry transducers. Ten days after surgery, 2 osmotic minipumps were implanted to subcutaneously infuse AngII (200ng/kg/min,) or vehicle and intracerebroventricularly infuse CMT‐3 (3.5μg/hr) or vehicle. Mean arterial pressure (MAP) was recorded weekly; after 4 weeks of AngII treatment, spectral analysis of was performed for autonomic nerve activity and anti‐Iba1 immunostaining was carried out to quantitate activated microglia in the brain.ResultsCMT‐3 significantly attenuated the AngII‐induced increase in MAP both in light: (Con:102±3, CMT‐3:101±4, AngII:158±23, AngII+CMT‐3:125±20 mmHg) and dark cycles (Con:102±2, CMT‐3: 102±3, AngII: 174±20, AngII+CMT‐3:145±28 mmHg). The heart to body weight ratio was similarly decreased (Con:2.9±0.3, CMT‐3:2.9±0.1, AngII:3.9±0.4, AngII+CMT‐3:3.3±0.2 mg/g; p&lt;0.05) with CMT‐3. CMT‐3 normalized AngII perturbed autonomic activity, as measured by LF/HF of SBP (Con:1.8±0.7, CMT‐3:2.0±1.6, AngII:5.4±4, AngII+CMT‐3:2.9±1.9; p&lt;0.001)). Furthermore, AngII increased activated microglial cells ~2.5‐fold, predominantly in the PVN, and CMT‐3 significantly attenuated this increase (p&lt;0.01).ConclusionThese data indicate that inhibition of neuro‐inflammation is key in correction of autonomic dysregulation and attenuation of HTN. They suggest that the anti‐inflammatory, rather than the antibiotic property, of Mino mediates these effects. Thus, CMT‐3 may be a better therapeutic candidate for neurogenic resistant hypertension in humans as it circumvents the problems associated with continuous antibiotic administration.Support or Funding InformationThis work is supported by NIH grant R01HL033610

Neutrophil elastase ameliorates matrix metalloproteinase-9 to promote lipopolysaccharide-induced acute lung injury in mice 1.

To investigate the regulatory roles of neutrophil elastase (NE) and matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. To construct LPS-induced ALI mouse models, wild-type C57BL/6 mice were administered 5.0 mg/kg of LPS through endotracheal, and/or 1.0 mg/kg of ONO-5046, and/or 20.0 mg/kg of chemically modified tetracycline-3 (CMT-3) by gavage. The levels of MMP-9, tissue inhibitor of metalloprotease-1, interleukin (IL)-6 were detected by real time RT-PCR at 6 h, 24 h and 48 h, and tumor necrosis factor (TNF), lung wet-dry weight ratio, white blood cell (WBC) count and polymorphonuclear (PMN) count in bronchoalveolar lavage fluid (BALF) were tested at 48 h after administration. The 5-day survival analysis of the ALI mice was also performed. Both ONO-5046 and CMT-3, regardless of being used individually or combined, significantly reduced the levels of MMP-9, IL-6, and TNF in lung tissue as well as in BALF, and the WBC and PMN count in BALF. Combined treatment with ONO-5046 and CMT-3 remarkably improved the survival rate of ALI mice. Neutrophil elastase synergizes with matrix metalloproteinase-9 to promote and regulate the release of inflammatory mediators and the infiltration of inflammatory cells, consequently affecting the survival of lipopolysaccharide-induced acute lung injury mice.

Cytotoxic effects of tetracycline analogues (doxycycline, minocycline and COL-3) in acute myeloid leukemia HL-60 cells.

Tetracycline analogues (TCNAs) have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY), minocycline (MINO) and chemically modified tetracycline-3 (COL-3) were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5–100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm), caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.

Novel mechanisms involving chemically modified tetracycline 3 cytotoxicity.

Chemically modified tetracycline 3 (CMT-3) is a potential anticancer drug because of its retained matrix metalloproteinases inhibitory property. In the present study,we showed that CMT-3 significantly inhibited the growth and proliferation of human hepatocellular carcinoma HepG2 cells. Novel mechanisms including increased intracellular autophagy level and high-mobility group box 1 (HMGB1)release were involved. In addition, a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS),significantly increased the cytotoxic effects of CMT-3 in HepG2 cells. Combining CMT-3 with TSN-SS led to enhanced accumulation of endogenous LC3-II, but reduced HMGB1 cytoplasmic translocation. Altogether, these findings suggest that autophagy and HMGB1 release may play important roles in the anticancer effect of CMT-3. As an ovel candidate for cancer therapy, CMT-3 may be used in combination with TSN-SS, which possibly facilitates the execution of a death signal (e.g. autophagy) and prevents the survival of an inducer (e.g. HMGB1 cytoplasmic translocation), thus improving its therapeutic effect.

COL-3, a Chemically Modified Tetracycline, Inhibits Lipopolysaccharide-Induced Microglia Activation and Cytokine Expression in the Brain

Microglia activation results in release of proinflammatory molecules including cytokines, which contribute to neuronal damage in the central nervous system (CNS) if not controlled. Tetracycline antibiotics such as minocycline inhibit microglial activation and cytokine expression during CNS inflammation. In the present study we found that administration of chemically modified tetracycline-3 (COL-3), inhibits lipopolysaccharide (LPS)-induced microglial and p38 MAPK activation, as well as the increase in TNF-α, but not IL-1β expression, in the brains of BALB/c mice. COL-3 has been described to have no antibacterial activity. We observed that COL-3 had no activity against a Gram-negative bacteria, Escherichia coli; however surprisingly, COL-3 had antibacterial activity against a Gram-positive bacteria Staphylococcus aureus, with a minimum inhibitory concentration of 1 mg/ml. Our data show that COL-3 has some antibacterial activity against S. aureus, inhibits LPS-induced neuroinflammation, and displays potential as a therapeutic agent for treatment of conditions involving CNS inflammation.

Matrix Metalloproteinase Inhibitor COL-3 Prevents the Development of Paclitaxel-Induced Hyperalgesia in Mice

Objective: To study the potential of chemically modified tetracycline-3 (COL-3), a potent matrix metalloproteinase (MMP) inhibitor, to protect against the development of paclitaxel-induced painful neuropathy and its immunomodulatory effects. Materials and Methods: The reaction latency to thermal stimuli (hot plate test) of female BALB/c mice was recorded before and after treatment with paclitaxel (2 mg/kg i.p.), paclitaxel plus COL-3 (4, 20 or 40 mg/kg p.o.) or their vehicles for 5 consecutive days. Gene transcripts of CD11b (marker for microglia), 5 cytokines (IFN-γ, IL-1β, IL-6, IL-10 and TNF-α) and 3 chemokines (CCL2, CXCL10 and CX3CL1) were quantified by real-time PCR in the brains, spinal cords and spleens of mice sacrificed on day 7 after treatment. Results: Treatment with paclitaxel reduced the reaction latency time to thermal stimuli (thermal hyperalgesia) for 4 weeks, with maximum effect on days 7 and 10. The coadministration of paclitaxel with COL-3 40 mg/kg, but not lower doses, prevented the development of paclitaxel-induced thermal hyperalgesia. Treatment with paclitaxel alone or coadministration with COL-3 increased CD11b transcript levels in the brain but not in the spinal cord. Treatment with paclitaxel reduced IL-6 transcript levels in the spinal cord but did not alter the transcript levels of other cytokines or chemokines in the brain, spinal cord or spleen. The coadministration of COL-3 with paclitaxel significantly increased the transcript levels of IL-6 in the spleen and decreased CX3CL1 transcripts in the brain in comparison to treatment with paclitaxel alone. Conclusion: Our results indicate that the MMP inhibitor COL-3 protected against paclitaxel-induced thermal hyperalgesia and, thus, could be useful in the prevention of chemotherapy-induced painful neuropathy.

Chemically Modified Tetracycline 3 Prevents Acute Respiratory Distress Syndrome in a Porcine Model of Sepsis + Ischemia/Reperfusion–Induced Lung Injury

Experimental pharmacotherapies for the acute respiratory distress syndrome (ARDS) have not met with success in the clinical realm. We hypothesized that chemically modified tetracycline 3 (CMT-3), an anti-inflammatory agent that blocks multiple proteases and cytokines, would prevent ARDS and injury in other organs in a clinically applicable, porcine model of inflammation-induced lung injury. Pigs (n = 15) were anesthetized and instrumented for monitoring. A "2-hit" injury was induced: (a) peritoneal sepsis-by placement of a fecal clot in the peritoneum, and (b) ischemia/reperfusion-by clamping the superior mesenteric artery for 30 min. Animals were randomized into two groups: CMT-3 group (n = 7) received CMT-3 (200 mg/kg); placebo group (n = 9) received the same dose of a CMT-3 vehicle (carboxymethylcellulose). Experiment duration was 48 h or until early mortality. Animals in both groups developed polymicrobial bacteremia. Chemically modified tetracycline 3 treatment prevented ARDS as indicated by PaO(2)/FIO(2) ratio, static compliance, and plateau airway pressure (P < 0.05 vs. placebo). It improved all histological lesions of ARDS (P < 0.05 vs. placebo). The placebo group developed severe ARDS, coagulopathy, and histological injury to the bowel. Chemically modified tetracycline 3 treatment prevented coagulopathy and protected against bowel injury. It significantly lowered plasma concentrations of interleukin 1β (IL-1β), tumor necrosis factor α, IL-6, IL-8, and IL-10. This study presents a clinically relevant model of lung injury in which CMT-3 treatment prevented the development of ARDS due in part to reduction of multiple plasma cytokines. Treatment of sepsis patients with CMT-3 could significantly reduce progression from sepsis into ARDS.

Chemically modified tetracycline-3 (CMT-3): A novel inhibitor of the serine proteinase, elastase

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α1-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases.

Clinical applications of non-antimicrobial tetracyclines in dermatology

There are many proposed non-antimicrobial actions of tetracyclines. Pathways affected by these medications are often overexpressed in various dermatologic conditions. Matrix metalloproteinases (MMPs) are enzymes best known for breaking down connective tissue proteins and are upregulated in conditions involving dermal destruction. Inhibition of MMPs by tetracyclines has been emphasized as one major non-antimicrobial action. Other effects of tetracyclines that are important in dermatology include inflammatory cytokine regulation, inhibition of leukocyte chemotaxis and activation, and anti-oxidation. Dermatologists have utilized the non-antimicrobial benefits of using tetracycline, through their success in treating disorders that do not have a primary infectious etiology such as rosacea. Even in acne, there is believed to be overactive inflammation to a normally commensal organism which is inhibited by tetracyclines. These medications have also been reported as successful in cases of less common skin conditions, such as pyoderma gangrenosum and bullous pemphigoid, both of which involve inflammation and dermal destruction which are inhibited by tetracyclines. The pathologic mechanisms of several dermatologic conditions are reviewed, followed by evidence of how tetracyclines and chemically modified tetracyclines (CMTs; structurally altered tetracyclines to remove antimicrobial properties while retaining non-antimicrobial properties) affect these pathways. Clinical testing of sub-antimicrobial doxycycline, in both 20 mg twice daily and 40 mg once daily (controlled release; 30 mg immediate release, 10 mg delayed release) forms, in rosacea and acne is reviewed as evidence that non-antimicrobial actions are valuable for treatment. Chemically modified tetracycline-3 (CMT-3) for Kaposi's sarcoma is highlighted as the only clinical evidence available for CMTs in dermatology. Certain evidence of success using antimicrobial tetracyclines in inflammatory conditions of the skin is reviewed as well, because they are likely working through non-antimicrobial properties. Finally, dermatologic side effects of non-antimicrobial tetracyclines are assessed.

Tetracyclines and Chemically Modified Tetracycline-3 (CMT-3) Modulate Cytokine Secretion by Lipopolysaccharide-Stimulated Whole Blood

In addition to their bacteriostatic effect, tetracyclines, which are often used in the treatment of periodontitis, also present anti-inflammatory properties. In the present study, we investigated the effects of tetracycline (TC), doxycycline (doxy), and chemically modified tetracycline-3 (CMT-3) on the production of pro-inflammatory mediators and matrix metalloproteinases (MMPs) in an ex vivo human whole blood (WB) model stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). WB samples obtained from three periodontitis patients and six healthy subjects were stimulated with P. gingivalis LPS in the absence and presence of TC, doxy, or CMT-3. The secretion of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), MMP-8, and MMP-9 by the WB samples was determined using enzyme-linked immunosorbent assays. P. gingivalis LPS significantly increased the secretion of all cytokines and MMPs tested. While we observed inter-patient variations, TC, doxy, and CMT-3 caused reductions of LPS-induced cytokine secretion to various degrees. TC, doxy, and CMT-3 had no significant effect on MMP-8 and MMP-9 secretion by LPS-stimulated WB samples. In conclusion, we used a human WB model that takes into consideration relevant in vivo immune cell interactions in the presence of plasma proteins to show that TC, doxy, and CMT-3 can reduce the production of pro-inflammatory mediators. This property may contribute to the clinically proven benefits of these molecules in the treatment of periodontitis and other chronic inflammatory diseases.

Human laminin‐332 degradation by Candida proteinases

Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied. Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis, C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography. Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans, and C. dubliniensis showed 20-70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata, C. krusei, and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55-70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100-130 kDa bands. C. albicans, C. dubliniensis, and C. tropicalis reference and clinical strain's cell-free fractions generated 100 kDa band. Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future.

The effect of chemical inhibition of matrix metalloproteinases on the size of experimentally induced apical periodontitis

To determine the effect of matrix metalloproteinase (MMP) inhibition on periapical lesion formation in a rat model. The pulp chambers of mandibular fist molars of adult SD rats were exposed to be infected by oral microbes. The experimental group was fed 20 mg kg(-1) MMP-inhibitor chemically modified tetracycline-3 (CMT-3) daily in an oral gavage and the controls were fed the vehicle. After 2 and 4 weeks, the mandibles (n = 10 in both groups at both times) were radiographed, decalcified and subjected to histological analysis. Extension of necrosis in first molar distal root canals was measured from the histological sections, and periapical lesion sizes in the same roots were determined from radiographs and histological sections. Mann-Whitney U-test was used for the statistical analysis. There was a statistically significant difference in the extension of necrosis in root canals between 2 and 4 weeks in the control group (P < 0.05), but not with MMP inhibition. Radiographically, MMP inhibition increased the periapical lesion size by 70% and 34% after 2 and 4 weeks respectively (P < 0.05 in after 2 weeks). In histological measurements, lesion size increased with MMP inhibition by 26% and 8% after 2 and 4 weeks respectively. MMP inhibition affects pulpal and periapical inflammation, increasing the rate of spreading of necrosis in root canals and the rate of periapical lesion formation.

Chemically Modified Tetracycline Improves Contractility in Porcine Coronary Ischemia/Reperfusion Injury

Reperfusion of ischemic myocardium has been implicated in extension of infarct size and deleterious clinical outcomes. Anti-inflammatory agents reduce this reperfusion injury. Chemically modified tetracycline-3 (CMT-3) (Collagenex Pharmaceuticals, Newtown, PA, USA) lacks antimicrobial properties yet retains anti-inflammatory activity. We examined infarct size and myocardial function in a porcine coronary artery occlusion/reperfusion model in CMT-3-treated and control animals. Yorkshire pigs (n = 8) underwent median sternotomy, pretreatment with heparin (300 U/kg and 67 U/kg/hr IV) and lidocaine (1 mg/kg IV) and were divided into two groups. Group one (n = 4) had the left anterior descending artery (LAD) occluded for 1 hour, after which it was reperfused for 2 hours. Group two (n = 4) had an identical protocol to group one except CMT-3 (2 mg/kg IV) was administered prior to occlusion of the LAD. Animals receiving CMT-3 had significantly decreased infarct size in relation to the ventricular area-at-risk (AAR) (28 +/- 9% vs. 64 +/- 8%; p < 0.05). Myocardial contractile function was superior in the CMT-3 treatment, indicated by a higher cardiac index (2.9 +/- 0.3 vs. 2.0 +/- 0.3 L/min/m(2); p < 0.05) and stroke volume index (22 +/- 2 vs. 17 +/- 1 L/m(2)/beat; p < 0.05). CMT-3 decreased infarct size in relation to the AAR resulting in relative preservation of contractility, suggesting CMT-3 may improve outcomes during myocardial ischemia reperfusion.

Role of chemically modified tetracycline on TNF-alpha and mitogen-activated protein kinases in sepsis.

Chemically modified tetracyclines are orally active inhibitors of multiple proteases and cytokines. In this study, we focused on the regulation of tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinases (MAPKs) in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracycline-3 (CMT-3), which retains their antiinflammatory activity. Sepsis was induced in rats by cecal ligation and puncture (CLP). At 24 h and 1 h before CLP, treated rats received CMT-3 (25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. TNF-alpha was determined by ELISA, and MAPKs were determined by Western blot analysis. A significant activation of p38 MAPK was observed after 0.5 h and 1.5 h of sepsis that appeared to coincide with the increased circulating TNF-alpha level. The activation of p42/44 was increased after 24 h of sepsis, whereas that of SAPK/JNK was unaltered throughout the course of sepsis. CMT-3 pretreatment inhibited the TNF-alpha level as well as p38 MAPK activation seen after 0.5 and 1.5 h of CLP and also suppressed the activation of p42/44 after 24 h post-CLP. These results indicate increased activity of TNF-alpha and MAPK following sepsis and demonstrate the beneficial effect of CMT-3 in preventing the increase in TNF-alpha, p38 MAPK, p42/44 MAPK, and the progression of septic shock.

Metalloproteinase Inhibition Prevents Acute Respiratory Distress Syndrome

Background. The acute respiratory distress syndrome (ARDS) occurs in patients with clearly identifiable risk factors, and its treatment remains merely supportive. We postulated that patients at risk for ARDS can be protected against lung injury by a prophylactic treatment strategy that targets neutrophil-derived proteases. We hypothesized that a chemically modified tetracycline 3 (COL-3), a potent inhibitor of neutrophil matrix metalloproteinases (MMPs) and neutrophil elastase (NE) with minimal toxicity, would prevent ARDS in our porcine endotoxin-induced ARDS model.Methods. Yorkshire pigs were anesthetized, intubated, surgically instrumented for hemodynamic monitoring, and randomized into three groups: (1) control (n = 4), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 4), infusion of Escherichia coli lipopolysaccharide at 100 μg/kg; and (3) COL-3 + LPS (n = 5), ingestion of COL-3 (100 mg/kg) 12 h before LPS infusion. All animals were monitored for 6 h following LPS or sham LPS infusion. Serial bronchoalveolar lavage (BAL) samples were analyzed for MMP concentration by gelatin zymography. Lung tissue was fixed for morphometric assessment at necropsy.Results. LPS infusion was marked by significant (P < 0.05) physiological deterioration as compared with the control group, including increased plateau airway pressure (Pplat) (control = 15.7 ± 0.4 mm Hg, LPS = 23.0 ± 1.5 mm Hg) and a decrement in arterial oxygen partial pressure (PaO2) (LPS = 66 ± 15 mm Hg, Control = 263 ± 25 mm Hg) 6 h following LPS or sham LPS infusion, respectively. Pretreatment with COL-3 reduced the above pathophysiological changes 6 h following LPS infusion (Pplat = 18.5 ± 1.7 mm Hg, PaO2 = 199 ± 35 mm Hg; P = NS vs control). MMP-9 and MMP-2 concentration in BAL fluid was significantly increased between 2 and 4 h post-LPS infusion; COL-3 reduced the increase in MMP-9 and MMP-2 concentration at all time periods. Morphometrically LPS caused a significant sequestration of neutrophils and monocytes into pulmonary tissue. Pretreatment with COL-3 ameliorated this response. The wet/dry lung weight ratio was significantly greater (P < 0.05) in the LPS group (10.1 ± 1.0 ratio) than in either the control (6.4 ± 0.5 ratio) or LPS+COL-3 (7.4 ± 0.6 ratio) group.Conclusions. A single prophylactic treatment with COL-3 prevented lung injury in our model of endotoxin-induced ARDS. The proposed mechanism of COL-3 is a synergistic inhibition of the terminal neutrophil effectors MMPs and NE. Similar to the universal practice of prophylaxis against gastric stress ulceration and deep venous thromboses in trauma patients, chemically modified tetracyclines may likewise be administered to prevent acute lung injury in critically injured patients at risk of developing ARDS.