Background
Tetracyclines are an old class of antibiotics that have been used for specific infections such as brucellosis, Lyme disease, rickettsiosis (e.g. Rocky Mountain spotted fever), genital and ocular infections from Chlamydia), severe acne vulgaris (caused by Propionibacterium acnes), diarrhea from Vibrio (cholera) and pneumonia from Mycoplasma pneumoniae and Chlamydophila pneumoniae. Their availability is quite good and there are derivatives that have a pharmacokinetics different from the original molecules (tetracycline and aureomycin). Absorption occurs mainly in the upper small intestine. Bioavailability varies: for tetracycline we are about 60-80%, for doxycycline and minocycline over 90%; minocycline crosses the blood-brain barrier better than other tetracyclines.
These antibiotics have the characteristic of accumulating in the bones and liver: minocycline is more lipophilic than other tetracyclines, which facilitates penetration into tissues, including the blood-brain barrier. This property is important for them to reach the nervous system for their anti-infective but also molecular actions not related to infections. In fact, tetracyclines, in addition to their antibiotic activity, can influence various molecular processes in eukaryotic cells, including signal transduction pathways. These effects are often linked to the properties of tetracyclines to chelate metal ions, modulate enzymatic activities and influence gene transcription.
Molecular actions of tetracyclines on eukaryotic cells
- Inhibition of Matrix Metalloproteinases (MMPs)
Tetracyclines, such as doxycycline, inhibit matrix metalloproteinases (MMPs) by chelating zinc and calcium ions, which are essential for the activity of these enzymes. This inhibition has significant effects in modulating the extracellular matrix, reducing the degradation of collagen and other structural proteins and is relevant in pathologies such as rheumatoid arthritis, tumor metastases and aortic aneurysms. It is strange how two different classes of antibiotics can have opposite actions on this phenomenon: while tetracyclines can inhibit the expression and function of MMPs, fluoroquinolones (ciprofloxacin, levofloxacin and similar), on the other hand, induce the expression of MMPs and have been incriminated for the appearance of phenomena such as rupture of the Achilles tendon and worsening of abdominal aortic aneurysm.
- Interference with gene transcription
Tetracyclines can affect signaling pathways linked to specific kinases, such as MAPK and p38, and those regulating the transcription factor NF-ÎşB (IKK-alpha, NIK), often involved in inflammatory responses and cell proliferation. For example, minocycline has been shown to inhibit the activation of NF-ÎşB, reducing the transcription of pro-inflammatory genes. Tetracyclines can interfere with transcription factors regulated by extracellular signals, such as STAT (Signal Transducer and Activator of Transcription). Studies suggest that minocycline inhibits the phosphorylation of STAT1 and STAT3, the main effectors of the JAK-STAT pathway, in models of neuroinflammation. In a context of chronic inflammation, the reduction of STAT3 activity is associated with a decrease in the expression of inflammatory genes. Tetracyclines reduce the expression of cytokines such as IL-1 and IL-6, which in turn directly activate the JAK-STAT pathway. This anti-inflammatory effect is particularly evident in experimental models of neurodegeneration and autoimmune diseases.
- Effects on oxidative stress and cell death
Tetracyclines act as free radical scavengers and chelate iron ions, reducing oxidative stress. They have effects on ROS-mediated signal transduction, which affects pathways such as p53 and HIF-1α (Hypoxia-Inducible Factor 1-alpha). Through this mechanism, minocycline and doxycycline can modulate signal transduction by regulating the expression of proteins involved in apoptosis, such as Bcl-2 and Bax. The modulation of these pathways is important in neuroprotection, in conditions such as stroke and neurodegenerative diseases. In this case, as mentioned above, their penetration through the blood-brain barrier allows this effect to be achieved. In addition, tetracyclines affect mitochondrial translation, which can alter ATP production and activate signals related to cellular bioenergetics. These effects can modulate cell survival pathways such as AMPK kinase, which controls the cellular energy status (availability of intermediates).
Clinical applications
Inflammatory Diseases
The suppression of NF-κB and the reduction of inflammatory mediators (e.g. TNF-α, IL-6) make tetracyclines useful in chronic inflammatory diseases. There are data indicating a beneficial effect in the context of rheumatoid arthritis. This condition presents the activation of multiple cellular activation pathways, such as those of stress kinases (p38), NF-kB transcription factors, JAK-STAT life, Wingless pathway (Wnt-CAT) and proliferation-associated tyrosine kinases (c-Syk, c-Btk) of pathological synovial fibroblasts (FLS). Tetracyclines can target at least half of these cellular pathways, which would make them ideal both in the treatment of this condition per se, and in the context of the emergence of infections linked to the immunosuppressive treatment of the disease. Another inflammatory disease for which there is evidence of the anti-inflammatory action of tetracyclines is multiple sclerosis.
An experimental investigation in 2014 (Hou et al.) had preliminarily seen that by associating minocycline with interferon-beta, EAE mice (an experimental model of the disease) had improvements superior to treatment with IFN-beta alone. A previous clinical trial in 2009 had seen that there was also synergy with glatiramer acetate in subjects with relapsing-remitting multiple sclerosis, while an experimental investigation in 2017 (Floriou et al.) saw that tetracycline negatively interferes with the production of IL-17 and IFN-gamma in nerve cells derived from the disease. Finally, Faissner et al. (2017), found that minocycline and the immunomodulatory drug Plaquenil (hydroxychloroquine; traditionally used in rheumatoid arthritis), have additive effects in suppressing disease activity.
By inhibiting pathways related to apoptosis and oxidative damage, tetracyclines have been investigated for the treatment of neurodegenerative diseases such as ALS and Alzheimer’s disease. Doxycycline and minocycline are also inhibitors of matrix metalloproteinase 9 (MMP-9), which is required to induce synaptic plasticity necessary for memory consolidation. Therefore, it has been suggested that tetracyclines may negatively affect memory processes, which has been demonstrated in four clinical trials with human subjects. This could potentially limit their chronic use in the management of diseases such as dementia and Parkinson’s disease. In this case, in particular, very recent data have shown that minocycline can prevent the pathological aggregation of alpha-synuclein.
However, scientific data seem to support the fact that this could happen when there is no participation of microglia, the brain’s immune cells. Tetracyclines, in fact, seem to prefer to act against these cells, which are responsible for the neuroinflammation present in these clinical conditions. Microglia, in fact, produce many inflammatory cytokines and chemokines, including TNF-alpha. This activates the NADPH oxidase of white blood cells and microglia; minocycline can interfere with this activation mechanism, interacting directly with its molecular component p47phox. In this way it can prevent neuronal death induced by oxidative stress dependent on stimulation with cytokines.
Human cancers
The modulation of MMPs and cell proliferation-related pathways (e.g. MAPK) has suggested a potential role for tetracyclines as anti-tumor agents, especially in blocking metastatic progression. In an experimental model of prostate cancer, the derivative CMT-3 or lymecycline was able to induce mitochondrial damage, generation of oxidative stress and kill malignant cells both by necrosis and apoptosis. The tumor mass slowed both its growth and its ability to release metastases. MMPs have been shown to be important mediators of bone metastasis formation, contributing largely to the morbidity of breast and prostate cancer patients. The natural osteotropism of tetracyclines would allow them to be highly effective in inhibiting MMPs produced by osteoclasts or tumor cells in bone.
This hypothesis was confirmed about two decades ago by experimental evidence showing that doxycycline reduces tumor burden in a mouse model of osteolytic bone metastases from breast cancer. Fibrosing diseases By reducing MMP activity and modulating inflammatory signaling, tetracyclines have demonstrated anti-fibrotic effects in lung and cardiac diseases. Idiopathic pulmonary fibrosis (IPF) is an incurable condition with very few therapeutic options, the main ones being pirfenidone and nintedanib. IPF is characterized by progressive fibrosis and a poor prognosis. Alveolar epithelial cells are considered important in the release of growth factors and matrix metalloproteinases.
A preliminary study from 2011 investigated the effect of doxycycline on the production of MMPs and fibrotic growth factors (TGF-beta) in lung alveolar cells treated with bleomycin (which induces fibrosis). The antibiotic reduced fibrosis scores and the production of type I collagen, connective tissue growth factor (CTGF) and TGF-beta in in vitro models, inhibited the mRNA expression of MMP-2, MPP-9, CTGF and type I collagen, as well as the production of MMP-2 and platelet-derived growth factor (PDGF-AA) induced in A549 cells by TGF-beta. Conclusion These studies and information support the idea that tetracyclines, especially minocycline, can affect various cellular signaling pathways, making them attractive for the treatment of inflammatory and neurodegenerative diseases.
Their approval would not encounter major obstacles, considering that they are old molecules whose pharmacology, pharmacokinetics and clinical aspects are well known.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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