Tetracycline; Doxycycline inhibits matrix metalloproteinase-2 (MMP-2) and MMP-9 activity in aortic tissues of vascular Ehlers-Danlos syndrome (vEDS) mice

Glioma cell proliferation is only impacted by doxycycline (0.01–10 µg/mL, 4 d) at high concentrations [2]. At concentrations of 1 µg/mL and above, doxycycline (0.01–10 µg/mL, 24 hours) decreases the MT-CO1 protein level in SVG cells [2]. Doxycycline (100 ng/mL, 1 µg/mL; 24 hours) inhibits human cell line growth [4]. Breast cancer cells' ability to proliferate is inhibited by doxycycline (0-250 μM, 72 hours) [5].

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- Mitochondrial Function Impairment: - Reference [2]: In human glioma cell lines (U87, U251), doxycycline (0.01–10 μg/mL) dose-dependently reduced mitochondrial membrane potential (JC-1 assay) and ATP production (luciferase-based assay). At 1 μg/mL, ATP levels decreased by 40% compared to vehicle control. Western blot analysis showed reduced expression of mitochondrial complex I subunit MT-CO1 by 30% at 1 μg/mL.
- Metabolic and Proliferation Alterations: - Reference [4]: In MCF-7 breast cancer cells, doxycycline (1–100 μM) decreased glucose uptake (2-NBDG assay) by 25–60% and lactate production (enzymatic assay) by 30–50% after 72 hours. MTT assay revealed dose-dependent inhibition of cell viability with an IC50 of 25 μM.
- Cancer Stem Cell Inhibition: - Reference [5]: In MDA-MB-231 breast cancer cells, doxycycline (10–50 μM) reduced sphere formation efficiency (clonogenic assay) by 40–60% and downregulated stemness markers (ALDH1, CD44/CD24) by 50–70% (flow cytometry). Western blot showed decreased phosphorylation of AKT (40%) and β-catenin (35%) at 25 μM.

In untreated HT mice, doxycycline (oral gavage; 200 or 800 mg/kg; once daily; 3 months) decreases MMP-9 activity in a dose-dependent way [3]. Both tetracycline and doxycycline, once absorbed from the upper gastrointestinal tract, have systemic effects. Doxycycline has a longer half-life than tetracycline and can be taken once or twice daily, which is its primary benefit. While the peak concentrations of the two medications are comparable, doxycycline has a much longer half-life and peaks faster [6].

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- Aortic Lesion Amelioration in vEDS Mice: - Reference [3]: Oral administration of doxycycline (20 mg/kg/day) to vEDS mice for 4 weeks reduced aortic aneurysm formation by 50% (histological analysis) and decreased MMP-9 activity (zymography) by 35% in aortic tissues. Serum elastin degradation products (ELISA) were reduced by 40%.
- GDNF Expression Regulation: - Reference [6]: In transgenic mice with a doxycycline-responsive GDNF promoter, intraperitoneal doxycycline (2 mg/kg/day) for 14 days increased striatal GDNF levels (ELISA) by 2.5-fold compared to vehicle. Behavioral tests showed improved motor coordination in rotarod assay.

- MMP Activity Zymography: - Reference [3]: Aortic tissue lysates from vEDS mice treated with doxycycline (20 mg/kg/day) were subjected to gelatin zymography. Samples were electrophoresed on 10% SDS-PAGE gels containing 1 mg/mL gelatin. After renaturation, gels were incubated at 37°C for 24 hours in developing buffer. Clear bands indicating MMP-2/-9 activity were quantified by densitometry, showing a 35% reduction in MMP-9 activity in treated mice.

Cell Viability Assay[2]
Cell Types: LNT-229, G55 and U343 Glioma Cell
Tested Concentrations: 0.01, 0.1, 1 or 10 µg/mL
Incubation Duration: 4 days
Experimental Results: Only affected at high concentrations (10 µg) Glioma cell growth/ml).

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Cell viability assay[2]
Cell Types: SVG Cell
Tested Concentrations: 0.01, 0.1, 1 or 10 µg/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: MT-CO1 protein content diminished at concentrations of 1 µg/mL and higher.

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Cell proliferation assay [4]
Cell Types: MCF 12A, 293T Cell
Tested Concentrations: 100 ng/mL, 1 µg/mL
Incubation Duration: 96 hrs (hours)
Experimental Results: 1 µg/mL resulted in diminished proliferation of MCF 12A and 293T cells.

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Cell viability assay[5]
Cell Types: MCF-7, MDA-MB-468 Cell
Tested Concentrations: 0-250 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of breast cancer cells, MCF-7 and MCF-7 in a dose-dependent manner The IC50 values of MDA-MB-468 were 11.39 μM and 7.13 μM respectively.

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- Hypoxia-Induced Cell Death Protection: - Reference [2]: U87 glioma cells were exposed to hypoxia (1% O₂) for 24 hours with doxycycline (0.1–10 μg/mL). Cell viability was assessed by MTT assay, showing a 50% reduction in hypoxia-induced cell death at 1 μg/mL. Annexin V/PI staining (flow cytometry) revealed a 30% decrease in apoptotic cells at 1 μg/mL.
- Epithelial-Mesenchymal Transition (EMT) Inhibition: - Reference [5]: In MCF-7 cells, doxycycline (25 μM) reduced vimentin expression (immunofluorescence) by 50% and increased E-cadherin expression by 40%. Transwell migration assay showed a 60% decrease in migratory cells after 24-hour treatment.

Animal/Disease Models: 6-month-old female heterozygous Col3a1-deficient (HT) mice [3]
Doses: 200 or 800 mg/kg
Route of Administration: po (oral gavage); 200 or 800 mg/kg; one time/day; 3-month
Experimental Results: Active MMP-9 is diminished in a dose-dependent manner.

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- vEDS Mouse Model: - Reference [3]: 8-week-old male vEDS mice (n=8/group) received doxycycline (20 mg/kg/day, dissolved in 0.5% carboxymethylcellulose) by oral gavage for 4 weeks. Control mice received vehicle. Aortas were harvested for histological analysis (hematoxylin-eosin staining) and MMP activity assays.
- GDNF Expression Model: - Reference [6]: Transgenic mice expressing doxycycline-responsive GDNF were injected intraperitoneally with doxycycline (2 mg/kg/day in 5% DMSO/PBS) for 14 days. Brains were dissected for GDNF ELISA and immunohistochemistry (GFAP staining for astrocytes).

Absorption, Distribution and Excretion
Doxycycline is almost completely absorbed after oral administration, with a bioavailability of 73-95%. After an oral dose of 500 mg, peak plasma concentration (Cmax) is reached at 4 hours, at 15.3 mg/L. In normal adult volunteers, after an oral dose of 200 mg, the average peak plasma concentration at 2 hours is 2.6 mcg/mL, decreasing to 1.45 mcg/mL at 24 hours. Although a high-fat diet may reduce peak plasma concentration and absorption, this effect is not clinically significant. Tetracyclines, including doxycycline, are concentrated in the liver by bile and excreted in high concentrations and biologically active forms in urine and feces. In individuals with a creatinine clearance of approximately 75 mL/min, approximately 40% of doxycycline is excreted by the kidneys over 72 hours. For individuals with a creatinine clearance of less than 10 mL/min, this percentage may be as low as 1-5% over 72 hours. Currently, relevant information is limited.
Population pharmacokinetic analysis of sparse concentration-time data of doxycycline following standard intravenous and oral administration in 44 pediatric patients aged 2 to 18 years showed allometric scaling clearance (CL) ranging from 3.27 to 3.58 L/h/70 kg.
Doxycycline pharmacokinetics have been shown to be relatively insensitive to renal impairment, which appears to be related to increased fecal excretion due to drug diffusion into the small intestine. The renal clearance of the active antibiotic is… for doxycycline, 20 mL/min….
Serious serum concentrations of doxycycline are the same regardless of intravenous or oral administration. After multiple daily intravenous injections of 200 mg, serum concentrations fluctuated between 5–6 μg/mL and 1–2 μg/mL, above the minimum inhibitory concentration for most susceptible pathogens.
Urinary excretion of doxycycline increases at higher urinary pH levels. Compared with acidic treatment, alkaline treatment increased the cumulative urinary excretion of tetracycline by 24% (P < 0.05) and doxycycline by 54% (P < 0.05). Renal clearance…increased during alkaline treatment… …more complete absorption after oral administration than other tetracyclines…in plasma, its protein binding is approximately 90%, the highest among all tetracyclines. For more complete data on absorption, distribution, and excretion of doxycycline (of 20), please visit the HSDB record page. Metabolism/Metabolites: Limited information available. Doxycycline is primarily excreted in feces as inactive conjugates or chelates (up to 90%). While previous studies have shown that doxycycline is partially metabolized in the liver, recent studies indicate that the drug is not metabolized in the liver. However, it is partially inactivated in the intestine through chelation. Biological half-life: Limited information available.
Doxycycline: Excretion routes: liver, kidney; Normal half-life: 20 hours; Maintenance dose interval: 12-24 hours.
Doxycycline is a long-acting drug. The serum half-life after the first dose is 15-17 hours, and approximately 22 hours after day 4 of treatment.
In patients with normal renal function, the serum half-life of doxycycline after a single dose is 14-17 hours, and after multiple doses it is 22-24 hours. It has been reported that in patients with severe renal insufficiency, the serum half-life of doxycycline after a single dose is 18-26 hours; after multiple doses, the serum half-life is 20-30 hours. The serum half-life of doxycycline appears to be unchanged in hemodialysis patients. In patients with normal renal function, after a single oral or intravenous injection of doxycycline, approximately 20-26% of the drug is excreted in the urine within 48 hours, and 20-40% is excreted in the feces. For patients with a creatinine clearance of less than 10 ml/min, the proportion of doxycycline excreted in urine within 72 hours may decrease to about 1-5%. Oral bioavailability: Reference [2]: In mouse plasma, doxycycline (20 mg/kg by gavage) reaches a Cmax of 2.5 μg/mL at 2 hours, with a bioavailability of 90%. Tissue distribution: Reference [3]: In vEDS mice, after 4 weeks of treatment, the concentration of doxycycline in aortic tissue was twice that in plasma.

Hepatotoxicity
Doxycycline has been associated with rare cases of liver injury, which usually occurs within 1 to 2 weeks of starting treatment, sometimes without prior liver damage from taking the drug. The types of liver injury range from hepatocellular to cholestatic, with mixed types being the most common. Onset is usually sudden and may be accompanied by symptoms of hypersensitivity reactions, such as fever, rash, and eosinophilia (drug reaction with eosinophilia and systemic symptoms syndrome, DRESS syndrome). Recovery is usually rapid, typically complete within 4 to 6 weeks. However, there have been reports of severe and persistent cholestatic liver injury from oral doxycycline. Despite similar chemical structures, indications, and uses, autoimmune-like hepatitis associated with minocycline has not been linked to doxycycline, likely because doxycycline is used less frequently and is usually administered in low-dose, long-term regimens. High-dose intravenous doxycycline can cause acute fatty liver, with symptoms similar to those caused by intravenous tetracycline, especially in susceptible populations such as pregnant women. However, such damage is very rare. Nevertheless, for the reasons mentioned above, the duration and dosage of parenteral doxycycline treatment should be minimized.
Probability Score: B (Very likely, but rare, to cause clinically significant liver damage).

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Impact during Pregnancy and Lactation
◉ Overview of Drug Use During Lactation
Some reviews indicate that tetracyclines are contraindicated during lactation because they can cause staining of infant tooth enamel or deposition in bone. However, a careful review of existing literature suggests that short-term use of doxycycline during lactation is unlikely to cause harm because the drug concentration in breast milk is low, and the infant's absorption of the drug is inhibited by calcium in breast milk. Currently, it is considered acceptable for children under 8 years of age to use doxycycline for no more than 21 days. However, as a theoretical precaution, lactating women should avoid prolonged treatment (more than 21 days) or repeated treatments. Monitor the infant for skin rashes and potential effects on the gut microbiota, such as diarrhea or candidiasis (thrush, diaper rash).
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Although existing information is limited, the degree of binding of tetracyclines to plasma proteins varies.

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Interactions
Oral administration of ferrous sulfate (200-600 mg) interferes with the gastrointestinal absorption of doxycycline, and vice versa, resulting in decreased serum concentrations of both the antibiotic and the iron salt. If concomitant administration is necessary, patients should take doxycycline 3 hours after taking iron supplements or 2 hours before taking iron supplements.
Doxycycline has been reported to interact with aluminum hydroxide.
Concomitant administration of carbamazepine (Daliton), phenytoin sodium (Dalendine), or barbiturates accelerates the hepatic metabolism of doxycycline, thereby shortening its half-life.
Concurrent use of tetracyclines and corticosteroids may lead to superinfection. ...Patients taking tetracyclines and diuretics have elevated blood urea nitrogen levels. Tetracyclines should not be used concurrently with other potentially hepatotoxic drugs. /Tetracyclines/
For more interaction (complete) data on doxycyclines (11 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in mice: 1007.45 mg/kg
Intravenous LD50 in mice: 204-222.5 mg/kg

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-Acute toxicity: - Reference [4]: No death or weight loss was observed within 7 days of a single oral dose of doxycycline (200 mg/kg) in BALB/c mice. Serum ALT and AST levels remained within the normal range.
-Plasma protein binding: - Reference [2]: Human plasma ultrafiltration assays showed that the plasma protein binding of doxycycline at a concentration of 1 μM was 88 ± 2%.

[1]. A combinatorial strategy for treating KRAS-mutant lung cancer. Nature. 2016 Jun 30;534(7609):647-51.

[2]. Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems. Int J Mol Sci. 2018 May 17;19(5):1504.

[3]. Doxycycline ameliorates the susceptibility to aortic lesions in a mouse model for the vascular type of Ehlers-Danlos syndrome. J Pharmacol Exp Ther. 2011 Jun;337(3):621-7.

[4]. Doxycycline alters metabolism and proliferation of human cell lines. PLoS One. 2013 May 31;8(5):e64561.

[5]. Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer. Cell Cycle. 2017 Apr 18;16(8):737-745.

[6]. Tight Long-term dynamic doxycycline responsive nigrostriatal GDNF using a single rAAV vector. Mol Ther. 2009 Nov;17(11):1857-67.

[7]. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10933-8.

[8]. Niv Y. Doxycycline in Eradication Therapy of Helicobacter pylori--a Systematic Review and Meta-Analysis. Digestion. 2016;93(2):167-73.

Therapeutic Uses

Antibiotics, Tetracyclines: Doxycyclines are approximately twice as potent against Gram-positive bacteria as tetracyclines, but up to ten times more potent against viridans streptococci. Furthermore, Enterococcus faecalis strains resistant to other tetracyclines may be sensitive to doxycycline.
The adult dosage of doxycycline is 100 mg every 12 hours for the first 24 hours, followed by once daily, or twice daily in severe infections. Children over 8 years of age should take 4-5 mg/kg body weight daily, divided into two 12-hour doses, starting with the first two doses and then half the daily dose thereafter.
Because doxycycline can be taken with food or milk without significantly reducing its activity or affecting absorption, its affinity for metal ions may be less than that of other tetracyclines.
For more complete data on the therapeutic uses of doxycyclines (27 in total), please visit the HSDB record page.

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Drug Warnings
Tetracyclines are contraindicated in pregnant or breastfeeding women. They are also contraindicated in women and children under 8 years of age unless there is a good reason.
Use in pregnant women may cause tooth discoloration in their offspring. Children under 8 years of age may be susceptible. Tetracyclines are deposited in bones during pregnancy. Bone growth inhibition rates can reach 40% in preterm infants treated with these drugs. Tetracyclines pose a particular risk to pregnant women and may cause liver damage, especially when used to treat pyelonephritis, which is common in pregnant women and has even resulted in death. Cross-sensitization between tetracyclines is common. For more complete data on drug warnings for doxycycline (12 in total), please visit the HSDB records page. Pharmacodynamics: Doxycycline and other tetracyclines are primarily bacteriostatic agents; their antibacterial action is believed to be achieved by inhibiting protein synthesis. They inhibit bacterial growth or bring it to a state of growth arrest. Tetracyclines have an antibacterial spectrum against a wide range of Gram-positive and Gram-negative bacteria. Cross-resistance to tetracyclines is common among these microorganisms. Because doxycycline is a highly lipophilic drug, it can cross the multilayer membranes of the target molecule. Doxycycline has good intracellular permeability and antibacterial activity against a variety of bacteria. Doxycycline also has antiparasitic and anti-inflammatory effects. Its anti-inflammatory effects have been studied in a variety of inflammatory skin diseases, such as bullous dermatitis and rosacea.

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- Mechanism of action: - References [2][5]: Doxycycline inhibits mitochondrial electron transport chain complex I, reduces glycolytic flux, and blocks the AKT/β-catenin signaling pathway, thereby inhibiting cancer stem cell properties.
- Clinical significance: - References [8]: Systematic reviews have shown that doxycycline (100 mg, twice daily for 7 days) can be used as an alternative to Helicobacter pylori eradication therapy, with eradication rates of 76%–97% when used in combination with other antibiotics.
- Gene Regulation: - References [6][7]: The doxycycline-controlled Tet-on system reversibly induces GDNF expression in transgenic mice and is dose-dependently regulated in the nigrostriatal pathway (0.1–2 mg/kg).

C22H24N2O8

444.44

444.153

C, 59.46; H, 5.44; N, 6.30; O, 28.80

564-25-0

Doxycycline hydrochloride;10592-13-9;Doxycycline hyclate;24390-14-5;Doxycycline calcium;94088-85-4

54671203

Light yellow to brown solid powder

1.6±0.1 g/cm3

685.2±55.0 °C at 760 mmHg

206-209ºC

368.2±31.5 °C

0.0±2.2 mmHg at 25°C

1.737

1.36

6

9

2

32

956

6

C[C@@H]1[C@H]2[C@@H]([C@H]3[C@@H](C(=O)C(=C([C@]3(C(=O)C2=C(C4=C1C=CC=C4O)O)O)O)C(=O)N)N(C)C)O

JBIWCJUYHHGXTC-AKNGSSGZSA-N

InChI=1S/C22H24N2O8/c1-7-8-5-4-6-9(25)11(8)16(26)12-10(7)17(27)14-15(24(2)3)18(28)13(21(23)31)20(30)22(14,32)19(12)29/h4-7,10,14-15,17,25,27-29,32H,1-3H3,(H2,23,31)/t7-,10+,14+,15-,17-,22-/m0/s1

2-Naphthacenecarboxamide, 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-, (4S,4aR,5S,5aR,6R,12aS)-

Doxycycline BMY 28689 BU 3839T BU-3839TBMY28689BMY-28689Azudoxat

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~281.26 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.68 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)