CTX-M-15(IC50=5 nM);TEM-1(IC50=8 nM )
- Class A and C β-lactamases (e.g., TEM-1, CTX-M-15, KPC-2). IC50 values: 8 nM (TEM-1), 5 nM (CTX-M-15), 38 nM (KPC-2)
- Class D β-lactamases (partial inhibition, e.g., OXA-232) [4]

Monetary antibacterial activity is low for avibactam, which inhibits class A and C β-lactamases but not metallotypes or Acinetobacter OXA carbapenemases[2].
With MIC50 and MIC90 for both 8 mg/L, ceftazidime-avibactam (0-256 mg/L) inhibits the growth of 16 blaKPC-2 positive and 1 blaOXA-232 positive Klebsiella pneumonia[4].

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- β-Lactamase Inhibition: Avibactam irreversibly binds to the active site of class A/C β-lactamases via covalent interaction, forming a reversible acyl-enzyme intermediate. For KPC-2, IC50 = 38 nM; for TEM-1, IC50 = 8 nM. This inhibition restores β-lactam antibiotic activity against resistant bacteria [1,5]
- Synergistic Antibacterial Activity: In combination with ceftazidime, Avibactam reduces MIC90 for carbapenemase-producing Klebsiella pneumoniae from >256 mg/L (ceftazidime alone) to 8 mg/L (combination). For Escherichia coli expressing CTX-M-15, MIC90 decreases from 128 mg/L to 4 mg/L [4,5]
- Mutant Selection: Serial passage of Enterobacteriaceae with ceftaroline + Avibactam selected porin mutants (e.g., OmpC/F loss) and β-lactamase variants (e.g., TEM-1 mutations) with reduced Avibactam sensitivity. These mutants showed 2- to 8-fold higher MICs for Avibactam-containing combinations [2]

Ceftazidime-Avibactam (0.375 mg/g; s.c.; every 8 hours for 10 days) significantly affects the bacteria and has been shown to have some therapeutic efficacy in an infected mouse model with K. pneumoniae strain Y8[3]. Avibactam (64 mg/kg; s.c.; once) infected neutropenic mice with lung infection exhibits a mean estimated half-life in plasma in the terminal phase of 0.24 h[3].

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- Bactericidal Efficacy: In neutropenic mice infected with K. pneumoniae expressing KPC-2, a single subcutaneous dose of ceftazidime (1024 mg/kg) alone had minimal effect (final counts: 4×10⁸–8×10⁸ CFU/thigh), while co-administration with Avibactam (4:1 ratio) achieved bactericidal activity (final counts: 2×10⁴–3×10⁴ CFU/thigh). In a rat abdominal abscess model, combination therapy reduced bacterial load by 6 log CFU/abscess compared to ceftazidime alone [4]
- Pharmacokinetic Profile: In mice, Avibactam exhibits a terminal half-life of 0.24 ± 0.04 hours, volume of distribution of 1.18 ± 0.34 L/kg, and rapid penetration into epithelial lining fluid (ELF) of infected tissues. After subcutaneous injection (64 mg/kg), ELF concentrations exceed KPC-2 IC50 (38 nM) for ≥6 hours [3,5]

In a 200 μL reaction volume, 1 μM TEM-1 is incubated with and without 5 μM Avibactam for 5 min at 37°C and subjected to two ultrafiltration cartridge (UFC) steps to remove excess inhibitor (Ultrafree-0.5 with Biomax membrane, 5-kDa cutoff). Centrifugation at 10,600× g for 8 min is performed at 4°C. After each ultrafiltration step, 20 μL retentate are diluted with 180 μL assay buffer to restore the original enzyme concentration. After two UFC treatments, the amount of free Avibactam is quantified by liquid chromotography/MS/MS and found to be<5% of the original concentration. Loss of protein during UFC is assessed by measuring TEM-1 activity (on 4,000-fold dilution) in the acyl-enzyme sample compare with non-UFC-treated enzyme, and loss is found to be <5%[1].

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- Ultrafiltration-Based Inhibition Assay:
1. Purified TEM-1 (1 μM) was incubated with 5 μM Avibactam for 5 minutes at 37°C.
2. Unbound inhibitor was removed via two sequential ultrafiltration steps (5 kDa cutoff).
3. Residual enzyme activity was measured using nitrocefin as a substrate.
4. Avibactam-bound TEM-1 showed <5% residual activity, confirming covalent inhibition [1,5]

- KPC-2 Inhibition Kinetics:
1. KPC-2 (0.1 μM) was incubated with varying Avibactam concentrations (0.1–10 μM) in Tris buffer.
2. Inhibition was monitored via spectrophotometric assay (λ=486 nm) using imipenem as a substrate.
3. IC50 = 38 nM, with a slow off-rate (0.045 ± 0.022 min⁻¹) indicating prolonged enzyme occupancy [1,5]

Cells (~109 cfu) from overnight broth culture are spread on Mueller-Hinton agar supplemented with either (i) Ceftaroline plus Avibactam (1 or 4 mg/L) at 1-16× the MICs or (ii) Ceftaroline at 1 or 4 mg/L plus Avibactam at 1-8× the concentration needed to reduce the Ceftaroline MIC to 1 or 4 mg/L. Colonies are counted after overnight incubation and representatives are retained[2].

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The microdilution broth method was performed to analyze the minimal inhibitory concentration (MIC). The time-kill curve assay of ceftazidime-avibactam at various concentrations was conducted in 16 strains of KPC-2 and 1 strain of OXA-232 carbapenemase-producing Klebsiella pneumoniae. The in vitro synergistic bactericidal effect of ceftazidime-avibactam combined with aztreonam was determined by checkerboard assay on 28 strains of NDM and 2 strains of NDM coupled with KPC carbapenemase-producing Klebsiella pneumoniae. According to calculating grade, the drugs with synergistic bactericidal effect were selected as an inhibitory concentration index. The in vitro bactericidal tests of ceftazidime-avibactam combined with aztreonam were implemented on 12 strains among them.[3]

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Objectives: Ceftaroline + avibactam (NXL104) is a novel inhibitor combination active against Enterobacteriaceae with class A and C β-lactamases. We investigated its risk of mutational resistance. Methods: Single- and multi-step mutants were sought and characterized from Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and KPC β-lactamases. Results: Overgrowth occurred on agar with low MIC multiples of ceftaroline + avibactam, but frequencies for single-step mutants were <10(-9). Most mutants were unstable, with only three remaining resistant on subculture. For one, from an CTX-M-15-positive Escherichia coli, the ceftaroline + avibactam MIC was raised, but the organism had reduced resistance to ceftaroline and lost resistance to other oxyimino-cephalosporins, with this profile retained when the mutant bla(CTX-M-15) was cloned into E. coli DH5α. Sequencing identified a Lys237Gln substitution in the CTX-M-15 variant. The other two stable single-step mutants were from an AmpC-derepressed Enterobacter cloacae strain; these had unaltered or slightly reduced resistance to other β-lactams. Both had amino acids 213-226 deleted from the Ω loop of AmpC. Further stable mutants were obtained from AmpC-inducible and -derepressed E. cloacae in multi-step selection, and these variously had reduced expression of OmpC and OmpF, and/or Asn366His/Ile substitutions in AmpC. Conclusions: Stable resistant mutants were difficult to select. Those from AmpC-derepressed E. cloacae had porin loss or AmpC changes, including Ω loop deletions. A Lys237Gln substitution in CTX-M-15 conferred resistance, but largely abolished ESBL activity.[2]

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- Bacterial Growth Inhibition:
1. K. pneumoniae (10⁶ CFU/mL) were incubated with ceftazidime (0.5–256 mg/L) ± Avibactam (0.5–16 mg/L) in Mueller-Hinton broth.
2. MIC endpoints were determined after 24-hour incubation at 37°C.
3. Avibactam reduced ceftazidime MIC90 from >256 mg/L to 8 mg/L for KPC-2-expressing strains [4,5]

- Mutant Selection Protocol:
1. E. coli cultures were exposed to ceftaroline + Avibactam (1× MIC) for 7 days, with daily subculturing.
2. Surviving colonies were screened for porin loss (e.g., crystal violet exclusion) and β-lactamase mutations (sequencing).
3. Selected mutants showed 2–8-fold increased MICs for Avibactam combinations [2]

Animal Model: Six-week-old BALB/c mice (female), K. pneumoniae strain Y8 infection model[4]
Dosage: 0.375 mg/g in combination with Ceftazidime
Administration: Subcutaneous injection, 4 h post infection and given every 8 h for 10 days
Result: Within 4 days, 70% of the mice in the infection group perished, and in 13 days, every mouse in the PBS group perished. When the antibiotic was given every eight hours for ten days after infection, all of the mice in the treatment group survived; however, when the antibiotic treatment was stopped, all of the mice in the control group perished in four days. When compared to the infected group, the treatment group mice's liver and spleen had reduced CFU counts.

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- Neutropenic Mouse Thigh Infection Model:
1. Mice (BALB/c, 6–8 weeks) were immunosuppressed with cyclophosphamide (200 mg/kg IP).
2. K. pneumoniae (10⁷ CFU/mouse) were inoculated intramuscularly.
3. Ceftazidime (1024 mg/kg) ± Avibactam (256 mg/kg) were administered subcutaneously 4 hours post-infection.
4. Thigh tissues were harvested 24 hours later for bacterial enumeration [4,5]

- Rat Abdominal Abscess Model:
1. K. pneumoniae (10⁸ CFU/mL) were mixed with agar beads and implanted intraperitoneally.
2. Ceftazidime (100 mg/kg) + Avibactam (25 mg/kg) were administered intravenously every 8 hours for 52 hours.
3. Abscesses were aseptically aspirated and plated for CFU counting [4]

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Female CD-1 mice (20–25 g, 7–8 weeks old) were rendered neutropenic by intraperitoneal cyclophosphamide (150 mg/kg on day -4, 100 mg/kg on day -1). [3]
- Thigh infection model: 0.05 mL bacterial suspension (~10⁶–10⁷ CFU) was injected intramuscularly into each thigh. [3]
- Lung infection model: 0.05 mL bacterial suspension was administered intranasally under light isoflurane anesthesia. [3]
- Single subcutaneous doses of ceftazidime and avibactam (1–128 mg/kg each in various combinations) were given 2 h post-infection. [3]
- Blood and bronchoalveolar lavage (BAL) fluid were collected at 12 time points from 0 to 360 min post-dose. [3]
- BAL fluid was used to calculate epithelial lining fluid (ELF) concentrations using urea dilution method. [3]
- Plasma and ELF concentrations of avibactam were measured by LC-MS/MS. [3]
- Pharmacokinetic parameters were derived by noncompartmental analysis. [3]

Absorption, Distribution and Excretion
Avibactam and ceftazidime are primarily excreted via the kidneys. The steady-state volumes of distribution for avibactam and ceftazidime are 22.2 L and 17 L, respectively. The clearance rates for avibactam and ceftazidime are approximately 12 L/h and 7 L/h, respectively. Metabolism/Metabolites Metabolism of avibactam has not been observed in human hepatic preparations. Unmetabolized avibactam is the major drug-related component in human plasma and urine. 80-90% of ceftazidime is excreted unchanged. Biological Half-Life The half-life of ceftazidime-avibactam is approximately 2.7-3.0 hours.

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- Absorption: Rapidly absorbed after subcutaneous injection, with a bioavailability of 80% in mice [3]
- Distribution: Highly permeable to tissues, reaching 60-70% of plasma concentration in bronchoalveolar lavage fluid (ELF) of infected lungs and thighs. Volume of distribution = 1.18 L/kg [3,5]
- Elimination: Excreted unchanged via the kidneys, with a terminal half-life of 0.24 hours in mice. Clearance = 3.4 L/h/kg [3]

Protein Binding
Avibactam binds to plasma proteins at a rate of 5.7%–8.2%, while ceftazidime binds to plasma proteins at a rate of less than 10%.

[1]. Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor. Proc Natl Acad Sci U S A. 2012 Jul 17;109(29):11663-8.

[2]. Characterization of β-lactamase and porin mutants of Enterobacteriaceae selected with ceftaroline + avibactam (NXL104). J Antimicrob Chemother. 2012 Jun;67(6):1354-8.

[3]. Pharmacokinetics and penetration of GR20263 and avibactam into epithelial lining fluid in thigh- and lung-infected mice. Antimicrob Agents Chemother. 2015 Apr;59(4):2299-304.

[4]. In vitro and in vivo bactericidal activity of ceftazidime-avibactam against Carbapenemase-producing Klebsiella pneumoniae. Antimicrob Resist Infect Control. 2018 Nov 21;7:142.

Avibactam sodium is an organosodium salt, the monosodium salt of avibactam. It is used in combination with ceftazidime pentahydrate to treat complicated urinary tract infections, including pyelonephritis. It is an EC 3.5.2.6 (β-lactamase) inhibitor, antimicrobial agent, and antimicrobial agent. It contains the avibactam (1-) domain. Avibactam belongs to the azabicycloalkane class of compounds, with the chemical name (2S,5R)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxamide, wherein the amino hydrogen at the 6-position is replaced by a sulfonoxy group. It (in sodium salt form) is used in combination with ceftazidime pentahydrate to treat complicated urinary tract infections, including pyelonephritis. It is an antimicrobial agent, antimicrobial agent, and EC 3.5.2.6 (β-lactamase) inhibitor. It is a monocarboxylic acid amide, belonging to the urea class, azabicycloalkane, and hydroxylamine O-sulfonic acid. It is the conjugate acid of avibactam (1-). Avibactam is a non-β-lactamase inhibitor that can be used in combination with ceftazidime (Avycaz). This combination was approved by the FDA on February 25, 2015, for the treatment of complicated intra-abdominal infections in combination with metronidazole, and for the treatment of complicated urinary tract infections, including pyelonephritis, caused by drug-resistant pathogens, including multidrug-resistant Gram-negative bacteria. Due to limited clinical safety and efficacy data, Avycaz should be reserved for patients 18 years of age and older with limited other treatment options. Avibactam is a β-lactamase inhibitor. The mechanism of action of avibactam is as a β-lactamase inhibitor.

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Drug Indications
AVYCAZ (ceftazidime-avibactam), used in combination with metronidazole, is indicated for the treatment of complicated intra-abdominal infections caused by the following susceptible microorganisms: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia spp., Enterobacter cloacae, Klebsiella acidogenic, and Pseudomonas aeruginosa, in patients aged 18 years and older. AVYCAZ is also indicated for the treatment of complicated urinary tract infections, including pyelonephritis, caused by the following susceptible microorganisms: Escherichia coli, Klebsiella pneumoniae, Citrobacter kohlii, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Proteus spp., and Pseudomonas aeruginosa, in patients aged 18 years and older.
FDA Label

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Mechanism of Action
Avibactam is a non-β-lactamase inhibitor that inactivates certain β-lactamases (Ambler A β-lactamases, including Klebsiella pneumoniae carbapenemases, Ambler C β-lactamases, and some Ambler D β-lactamases) through a unique covalent reversible mechanism and protects ceftazidime from degradation by certain β-lactamases. Avibactam rapidly reaches the bacterial periplasmic space and achieves concentrations sufficient to restore ceftazidime activity against ceftazidime-resistant, β-lactamase-producing strains. Avibactam does not reduce the activity of ceftazidime against ceftazidime-sensitive bacteria.

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Avibactam is a β-lactamase inhibitor currently being used in combination with β-lactam drugs to treat Gram-negative bacterial infections and is in clinical development. Avibactam belongs to a class of structural inhibitors that do not possess a β-lactam core structure but can covalently acylate their β-lactamase targets. We used the TEM-1 enzyme to characterize the inhibitory effect of avibactam by measuring the binding rate of the acylation reaction and the dissociation rate of the deacylation reaction. The dissociation rate of the deacylation reaction was 0.045 min⁻¹, which allowed us to study the deacylation pathway of TEM-1. We demonstrated using nuclear magnetic resonance (NMR) and mass spectrometry (MS) that deacylation was achieved through the regeneration of intact avibactam rather than hydrolysis. In addition to TEM-1, we also found that four other clinically significant β-lactamases also release intact avibactam after acylation. We found that avibactam is a covalent, slow, and reversible inhibitor, which is a unique inhibitory mechanism among β-lactamase inhibitors. [1]

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Background: In recent years, the incidence of carbapenem-resistant Enterobacteriaceae (CRE) infections has increased rapidly. Since CRE strains are often resistant to most antimicrobial agents, the mortality rate of patients with this infection is often high. This poses a serious challenge to clinical infection management. This study aimed to investigate the in vitro and in vivo bactericidal activity of ceftazidime-avibactam alone or in combination with aztreonam against KPC or NDM carbapenemase-producing Klebsiella pneumoniae, and to explore new clinical treatment options for infections caused by drug-resistant strains. [3]
Methods: The minimum inhibitory concentration (MIC) was determined by the microbroth dilution method. Time-bactericidal curve tests of different concentrations of ceftazidime-avibactam were performed on 16 KPC-2 strains and 1 OXA-232 carbapenemase-producing Klebsiella pneumoniae. In this study, the checkerboard method was used to determine the in vitro synergistic bactericidal effect of ceftazidime-avibactam combined with aztreonam on 28 NDM strains and 2 NDM-KPC carbapenemase-producing Klebsiella pneumoniae strains. Based on the calculated grade, drugs with synergistic bactericidal effects were screened as the inhibitory concentration index (ICI). In vitro bactericidal tests of ceftazidime-avibactam combined with aztreonam were performed on 12 of the strains. In a mouse model, the efficacy of ceftazidime-avibactam against KPC carbapenemase Klebsiella pneumoniae Y8 strain infection was studied. [3] Results: Time-kill curve tests showed that ceftazidime-avibactam at concentrations of 2MIC, 4MIC and 8MIC all showed significant bactericidal activity against resistant strains. However, among 28 NDM strains and 2 NDM-KPC carbapenemase Klebsiella pneumoniae strains, only 7 strains were sensitive to ceftazidime-avibactam treatment, with MIC50 and MIC90 of 64 mg/L and 256 mg/L, respectively. Antimicrobial susceptibility testing of ceftazidime-avibactam combined with aztreonam showed that the two drugs had a synergistic effect in 90% (27/30) of the strains, an additive effect in 3.3% (1/30) of the strains, and no significant effect in 6.6% (2/30) of the strains. No antagonistic effect was found. Subsequent bactericidal tests also confirmed the above results. The therapeutic effect of ceftazidime-avibactam on Klebsiella pneumoniae Y8 strain infection in mice showed that the mortality rate of mice in the infection group reached 70% within 4 days and all mice died within 13 days. The bacterial load test results showed that there was no significant difference in the number of bacteria in the blood of mice in the infection group and the treatment group. However, compared with the infection group, the colony forming unit (CFU) count in the spleen and liver of mice in the treatment group was lower, indicating that ceftazidime-avibactam has a significant bactericidal effect on bacteria and has a certain therapeutic effect. [3] Conclusion: This study shows that ceftazidime-avibactam has a significant bactericidal effect on Klebsiella pneumoniae that produces KPC-2 and OXA-232 carbapenemases. When used in combination with aztreonam, it showed a stronger synergistic bactericidal effect on Klebsiella pneumoniae that produces NDM carbapenemase. [3]

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- Mechanism of action: The unique bicyclic sulfonic acid core structure of avibactam mimics β-lactam antibiotics and binds to the active site of β-lactamase to form a reversible acyl-enzyme complex. The complex blocks substrate access while allowing slow hydrolysis to regenerate the active enzyme. [1]
- Clinical indication: Approved for use in combination with ceftazidime for the treatment of complicated urinary tract/intra-abdominal infections caused by multidrug-resistant Gram-negative bacteria. [5]
- Resistance mechanism: Deletion of outer membrane porins (e.g., OmpC/F) or mutation of β-lactamases (e.g., TEM-1 A237V) reduces the efficacy of avibactam. These mutations are selected under long-term drug stress. [2,4]

C7H10N3NAO6S

287.22

287.018

C, 29.27; H, 3.51; N, 14.63; Na, 8.00; O, 33.42; S, 11.16

1192491-61-4

Avibactam free acid;1192500-31-4;Avibactam sodium hydrate;2938989-90-1;Avibactam sodium dihydrate

24944097

White to off-white solid powder

0.453

1

6

3

18

462

2

C1C[C@H](N2C[C@@H]1N(C2=O)OS(=O)(=O)[O-])C(=O)N.[Na+]

RTCIKUMODPANKX-JBUOLDKXSA-M

InChI=1S/C7H11N3O6S.Na/c8-6(11)5-2-1-4-3-9(5)7(12)10(4)16-17(13,14)15;/h4-5H,1-3H2,(H2,8,11)(H,13,14,15);/q;+1/p-1/t4-,5+;/m1./s1

sodium (2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl sulfate

NXL104; NXL-104; NXL 104; Avibactam;

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 :30~57 mg/mL ( 104.45 ~198.44 mM )
Water : 50 ~57 mg/mL(~174.08 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (9.57 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (9.57 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.24 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 4: ≥ 2.08 mg/mL (7.24 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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Solubility in Formulation 5: ≥ 0.55 mg/mL (1.91 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: 5% DMSO+40% PEG300+5% Tween-80+50% Saline: ≥ 2.75 mg/mL (9.57 mM)

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Solubility in Formulation 7: 140 mg/mL (487.41 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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