9-aminoacridine (9-AA) interacts with bacterial DNA (predicted binding energy: -4.98 kcal/mol to PDB 4U8A) and disrupts the proton motive force (PMF) in Klebsiella pneumoniae. [2]

9-amino acid, with a MIC of 8–16 μg/mL, exhibits a remarkable antibacterial activity against Klebsiella pneumoniae[1]. When applied as an adjuvant with RIF, 9-amino acids further reduces their toxicity [1]. However, 9-amino acids (0-64 μg/mL) demonstrated considerable cytotoxicity against normal (LO2, HK2, HMC3) and malignant (HepG2, 786-O, U251) human cell lines.

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9-aminoacridine (9-AA) exhibits antimicrobial activity against Klebsiella pneumoniae with MICs ranging from 8 to 16 μg/mL and MBCs from 16 to 64 μg/mL. It shows synergistic effects with rifampin (RIF) against drug-sensitive, extensively drug-resistant (XDR), and pan-drug-resistant (PDR) K. pneumoniae strains, with fractional inhibitory concentration index (FICI) values of 0.313-0.5. Time-kill assays demonstrate that the combination of sub-MIC 9-AA and RIF results in significant bactericidal activity. 9-AA specifically targets bacterial DNA, as shown by confocal microscopy colocalization with DNA dye PI, displacement of DNA-binding probe SYTO 9, and inhibition of its antimicrobial activity by exogenous DNA. It disrupts PMF by dissipating the membrane potential (ΔΨ) and increasing the transmembrane proton gradient (ΔpH), as measured using fluorescent probes DISC₃(5) and BCECF-AM. 9-AA also increases intracellular reactive oxygen species (ROS) and ATP levels in bacteria. It shows partial synergy with tetracycline (driven by ΔpH) and antagonism with FeCl₃ (which increases ΔΨ). 9-AA inhibits bacterial surface motility. [2]

9-Aminoacidine (Aminacrine; 15 mg/kg; subcutaneous injection; single dosage) decreases skin abscesses in a model of subcutaneous abscesses [2].

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In a mouse wound infection model, topical application of an ointment containing 0.5% (wt/wt) 9-aminoacridine (9-AA) combined with 0.5% RIF significantly reduced bacterial load by 3.18 log₁₀ CFU compared to the vehicle. In a mouse subcutaneous abscess model, a single subcutaneous dose of 15 mg/kg 9-AA combined with 20 mg/kg RIF reduced bacterial load by 3.15 log₁₀ CFU/abscess compared to monotherapy. Liposomal formulations of 9-AA [9-AA(L)] alone (15 mg/kg) or in combination with RIF(L) (7.5 mg/kg each or 15 mg/kg each) also significantly reduced bacterial loads in abscesses. Histopathological analysis (H&E staining) showed reduced inflammatory infiltration in combination therapy groups. [2]

Molecular docking was performed to simulate the binding of 9-aminoacridine (9-AA) to DNA. The 3D structure of 9-AA was obtained from PubChem and optimized. DNA structures (T7, CG, G10) were constructed and optimized, and the crystal structure of double-stranded DNA (PDB 4U8A) was downloaded. Docking simulations were carried out using AutoDock Tools. The binding affinity was predicted based on binding energy, intermolecular energy, electrostatic energy, and internal energy. The best binding was observed with PDB 4U8A (predicted binding energy: -4.98 kcal/mol). [2]

Antimicrobial Susceptibility Testing: MICs and MBCs of 9-aminoacridine (9-AA) were determined using a standard broth microdilution method according to CLSI guidelines. Log-phase bacteria were diluted to ~1×10⁶ CFU/mL in Mueller-Hinton (MH) broth, mixed with 2-fold serially diluted 9-AA in a 96-well plate, incubated at 37°C for 16-18 hours, and bacterial growth (OD₆₃₀) was measured. MBC was determined by plating aliquots from MIC wells onto agar plates. [2]
Checkerboard Assay: Synergy between 9-AA and other antibiotics (e.g., RIF) was assessed. Two-fold serial dilutions of both compounds were prepared in MH broth in a 96-well plate. Bacterial suspension was added to each well (final density: 5×10⁵ CFU/mL). After incubation at 37°C for 16-18 hours, OD₆₃₀ was measured. FICI was calculated. [2]
Time-Kill Assay: Bacteria were exposed to 9-AA and/or RIF in MH broth at 37°C with shaking. Viable counts were determined by agar plate counting at 0, 2, 4, 8, 16, and 24 hours. [2]
Confocal Laser Scanning Microscopy (CLSM): K. pneumoniae or mammalian 293T cells were treated with 9-AA (32 μg/mL) for 60 minutes, then stained with propidium iodide (PI, nuclear dye) and FM4-64 (membrane dye). Cells were washed, mounted, and imaged using CLSM to observe localization of 9-AA fluorescence. [2]
SYTO 9 Displacement Assay: Log-phase bacteria were incubated with the DNA-binding fluorescent dye SYTO 9, then treated with increasing concentrations of 9-AA. Fluorescence intensity was measured to assess competitive displacement of SYTO 9 from DNA. [2]
Exogenous DNA Competition Assay: Bacteria were treated with 9-AA in the presence or absence of exogenous DNA (0.2 μg/mL). Bacterial growth (OD₆₃₀) and viable counts (CFU) were measured after 24 hours to assess inhibition of 9-AA's antimicrobial activity by DNA. [2]
Proton Motive Force (PMF) Detection: ΔpH was measured using the pH-sensitive fluorescent probe BCECF-AM. Bacteria were incubated with 9-AA and BCECF-AM, and fluorescence was measured. ΔΨ was measured using the membrane potential-sensitive dye DISC₃(5). Bacteria were incubated with DISC₃(5) and then treated with 9-AA, and fluorescence was measured. [2]
Intracellular ATP Measurement: Bacteria were treated with 9-AA, lysed, and intracellular ATP levels were measured using a commercial ATP determination kit based on luminescence. [2]
Reactive Oxygen Species (ROS) Measurement: Bacteria were incubated with the ROS-sensitive fluorescent probe DCFH-DA, then treated with 9-AA. Fluorescence intensity was measured to assess intracellular ROS levels. [2]
Hemolysis Assay: Human red blood cells (RBCs) were incubated with various concentrations of 9-AA. After centrifugation, supernatant absorbance at 570 nm was measured to determine hemolysis rate. RBC morphology was also observed under microscopy. [2]
Cytotoxicity Assay (CCK-8): Various human cell lines (LO2, HepG2, HK2, 786-O, HMC3, U251) were seeded in 96-well plates and treated with 9-AA or its liposomal formulation [9-AA(L)] for 24 hours. CCK-8 reagent was added, and after incubation, absorbance at 450 nm was measured to calculate cell viability and IC₅₀ values. [2]

Animal/Disease Models: 7weeks old ICR female mouse skin subcutaneousabscess model [2]
Doses: 15 mg/kg
Route of Administration: subcutaneous injection
Experimental Results: A single dose of 15 mg/kg 9-AA diminished the abscess area. A single dose of 15 mg/kg 9-AA or 20 mg/kg rifampicin (RIF) did not Dramatically reduce the viable bacterial load in abscesses. The combination of drugs Dramatically diminished bacterial load by 3.15 log10 CFU/abscess.

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Wound Infection Model:** Female ICR mice were anesthetized, and a 6-mm diameter wound was created on the shaved dorsal skin. The wound was infected with 50 μL of *K. pneumoniae* suspension (3×10⁸ CFU/mL). Two hours post-infection, an ointment containing 0.5% (wt/wt) 9-aminoacridine (9-AA) and/or 0.5% RIF in a Glaxal Base moisturizing cream was applied topically to the wound. The ointment was replaced at 24 hours. At 48 hours post-infection, mice were sacrificed, wound skin was excised, homogenized, and bacterial loads were quantified by plate counting. [2]
**Subcutaneous Abscess Model:** Female ICR mice were anesthetized, and 50 μL of *K. pneumoniae* suspension (9×10⁸ CFU/mL) was injected subcutaneously into the dorsal skin. One hour post-inoculation, drugs were administered via subcutaneous injection at the infection site. For 9-AA alone, a dose of 15 mg/kg was used. For combination therapy, 15 mg/kg 9-AA with 20 mg/kg RIF was used. Liposomal formulations [9-AA(L) or 9-AA plus RIF(L)] were administered at 15 mg/kg (9-AA equivalent) or 7.5/15 mg/kg combinations. Mice were euthanized 24 hours post-treatment, abscesses were excised, homogenized, and bacterial loads were quantified. Skin tissues were also collected for H&E staining. [2]
**In Vivo Toxicology:** Female ICR mice were administered a single subcutaneous dose of 15 mg/kg 9-AA or 15 mg/kg 9-AA(L). Control mice received 1% DMSO. After 24 hours, blood was collected for hematological and biochemical analysis (liver, renal, myocardial function biomarkers). Major organs (heart, liver, spleen, lung, kidney) were excised for H&E staining and histopathological examination. [2]

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Wound Infection Model: Female ICR mice were anesthetized, and a 6-mm diameter wound was created on the shaved dorsal skin. The wound was infected with 50 μL of K. pneumoniae suspension (3×10⁸ CFU/mL). Two hours post-infection, an ointment containing 0.5% (wt/wt) 9-aminoacridine (9-AA) and/or 0.5% RIF in a Glaxal Base moisturizing cream was applied topically to the wound. The ointment was replaced at 24 hours. At 48 hours post-infection, mice were sacrificed, wound skin was excised, homogenized, and bacterial loads were quantified by plate counting. [2]
Subcutaneous Abscess Model: Female ICR mice were anesthetized, and 50 μL of K. pneumoniae suspension (9×10⁸ CFU/mL) was injected subcutaneously into the dorsal skin. One hour post-inoculation, drugs were administered via subcutaneous injection at the infection site. For 9-AA alone, a dose of 15 mg/kg was used. For combination therapy, 15 mg/kg 9-AA with 20 mg/kg RIF was used. Liposomal formulations [9-AA(L) or 9-AA plus RIF(L)] were administered at 15 mg/kg (9-AA equivalent) or 7.5/15 mg/kg combinations. Mice were euthanized 24 hours post-treatment, abscesses were excised, homogenized, and bacterial loads were quantified. Skin tissues were also collected for H&E staining. [2]
In Vivo Toxicology: Female ICR mice were administered a single subcutaneous dose of 15 mg/kg 9-AA or 15 mg/kg 9-AA(L). Control mice received 1% DMSO. After 24 hours, blood was collected for hematological and biochemical analysis (liver, renal, myocardial function biomarkers). Major organs (heart, liver, spleen, lung, kidney) were excised for H&E staining and histopathological examination. [2]

In vitro, 9-aminoacridine (9-AA) showed moderate cytotoxicity to mammalian cells with IC₅₀ values ranging from approximately 5.62 to 42.58 μM across different human cell lines (LO2, HepG2, HK2, 786-O, HMC3, U251). Its liposomal formulation [9-AA(L)] significantly reduced cytotoxicity, increasing IC₅₀ values. 9-AA did not cause hemolysis of human red blood cells at concentrations up to 64 μg/mL. In vivo, a single subcutaneous dose of 15 mg/kg 9-AA or 9-AA(L) in mice showed no significant changes in hematological parameters or organ function biomarkers compared to the vehicle control, and no pathological changes were observed in major organs via H&E staining. [2]

[1]. The 'delta pH'-probe 9-aminoacridine: response time, binding behaviour and dimerization at the membrane. Biochim Biophys Acta. 1988 Mar 3;938(3):411-24.

[2]. Repurposing 9-Aminoacridine as an Adjuvant Enhances the Antimicrobial Effects of Rifampin against Multidrug-Resistant Klebsiella pneumonia. Microbiol Spectr. 2023 Jun 15;11(3):e0447422.

[3]. 9-Aminoacridine inhibition of HIV-1 Tat dependent transcription. Virol J. 2009 Jul 24:6:114.

9-Aminoacridine is a yellow needle-like crystal, readily soluble in ethanol. 9-Aminoacridine is an aminoacridine with a structure in which the hydrogen atom at the 9-position of acridine is replaced by an amino group. It is a fluorescent dye and a topical disinfectant, commonly used in eye drops as an acid salt to treat superficial eye infections. It has various uses, including as an anti-infective agent, disinfectant, fluorescent dye, MALDI matrix material, acid-base indicator, and mutagen. It is an aminoacridine compound and also a primary amino compound. It is the conjugate base of 9-aminoacridine (1+). 9-Aminoacridine is a highly fluorescent anti-infective dye, used clinically as a topical disinfectant and experimentally as a mutagen because it interacts with DNA. It can also be used as an intracellular pH indicator.

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9-aminoacridine (9-AA) is an FDA-approved drug repurposed as an antimicrobial agent and adjuvant for rifampin (RIF) against multidrug-resistant Klebsiella pneumoniae. It exhibits a dual mechanism of action: interaction with bacterial DNA and disruption of the proton motive force (PMF). 9-AA reduces the tendency of RIF to induce bacterial resistance. Liposomal encapsulation of 9-AA [9-AA(L)] improves its safety profile by reducing cytotoxicity while maintaining antimicrobial activity. The study highlights the potential of 9-AA, particularly in combination therapy, for treating skin and soft tissue infections caused by drug-resistant K. pneumoniae. [2]

C13H10N2

194.24

194.084

90-45-9

7019

Light yellow to yellow solid powder

1.268g/cm3

413.5ºC at 760 mmHg

241ºC

233.2ºC

1.78

3.551

1

2

0

15

207

0

XJGFWWJLMVZSIG-UHFFFAOYSA-N

InChI=1S/C13H10N2/c14-13-9-5-1-3-7-11(9)15-12-8-4-2-6-10(12)13/h1-8H,(H2,14,15)

acridin-9-amine

Aminoacridine NSC-13000 NSC 13000

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~27.5 mg/mL (~141.58 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (14.16 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 27.5 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: ≥ 2.75 mg/mL (14.16 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 27.5 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.)