β-lactam
β-Lactamases (Richmond types II, III, IV, and V) (inhibitor; Ki for type III β-lactamase with ampicillin is 4.3 μM without preincubation; Ki with preincubation is 8.4 × 10⁻³ μM; Ki for type III β-lactamase with cephaloridine is 0.17 μM) [2]

Clavulanic acid and ampicillin exhibit synergistic antibacterial activity (against β-lactamase-producing organisms)[2].
Ab11 and Ab51 strains are inhibited by clavulanic acid at MICs of 2–8 μg/mL[3].

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Clavulanic acid demonstrated poor antibacterial activity against Staphylococcus aureus (mean MIC 25 μg/ml), Enterobacteriaceae (mean MIC 25 μg/ml), and Pseudomonas aeruginosa (MIC >200 μg/ml). It inhibited 75% of Neisseria gonorrhoeae isolates at 0.1 μg/ml and Haemophilus influenzae at 6.3 μg/ml. [2]
Clavulanic acid acted synergistically with ampicillin, amoxicillin, cephaloridine, cephalothin, and cefamandole against β-lactamase-producing S. aureus and Enterobacteriaceae. Synergy was observed in 100% of S. aureus, E. coli, K. pneumoniae, Enterobacter sp., and Salmonella sp. tested. The combination of clavulanic acid and ampicillin was synergistic over a concentration range of 1:10 to 1:1 (clavulanic acid:ampicillin). [2]
In killing curve studies with an E. coli strain producing a Richmond type III β-lactamase, the combination of clavulanic acid (0.8-1.6 μg/ml) and ampicillin (6.3 μg/ml) resulted in a 2-log reduction in colony-forming units over 6 hours, while the organism grew in the presence of either drug alone. [2]
Clavulanic acid inhibited the hydrolytic activity of β-lactamases from Richmond types II, III, IV, and V. It was a poor inhibitor of Richmond type I β-lactamases. Inhibition was time-dependent and irreversible, as it could not be dialyzed away from the enzyme once bound. [2]
Kinetic studies with purified type III β-lactamase showed that without preincubation, the inhibition was competitive. The Km of ampicillin was 0.118 mM; with clavulanic acid, it was 0.1818 mM, giving a Ki of 4.3 μM. For cephaloridine, the Km was 0.5 mM; with clavulanic acid, it was 4 mM, giving a Ki of 0.17 μM. With 10 minutes of preincubation, the Ki for ampicillin was 8.4 × 10⁻³ μM. For comparison, the Ki of cloxacillin for this enzyme was 18.7 μM. [2]

An A. baumannii-infected C57BL/6 mouse pneumonia model's lung bacterial load is reduced by clavulanic acid (13 mg/kg, i.p.)[3]. Clavulanic acid (13 mg/kg, i.p.) in the pneumonia model of Ab51-infected C57BL/6 mice exhibits a t1/2 of 6.69 h and an AUC of 4.03 mg·h/L[3].
Rat models of paw edema induced by carrageenan (HY-125474) exhibit anti-inflammatory effects when treated with clavulanic acid (100–300 mg/kg, i.p.)[4].

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In a murine model of A. baumannii pneumonia, treatment with clavulanic acid (13 mg/kg i.p., with a dosing interval calculated to maintain serum levels above the MIC for 40% of the interval) significantly reduced the bacterial load in the lungs of infected mice. For strain Ab11, the lung bacterial count was reduced by 1.78 log₁₀ CFU/g (P < 0.001) compared to untreated controls. For strain Ab51, the reduction was 2.41 log₁₀ CFU/g (P < 0.001). [3]
Clavulanic acid treatment increased the rate of sterile blood cultures in infected mice. For strain Ab11, 60% of blood cultures were sterile in the treated group versus 26.7% in controls (P = 0.07, not significant). For strain Ab51, 93.3% of blood cultures were sterile in the treated group versus 20% in controls (P < 0.005). [3]
Clavulanic acid treatment reduced mortality in mice infected with the more virulent strain Ab51. Mortality was 33.3% in the treated group compared to 93.3% in the untreated control group. For strain Ab11, mortality was 26.7% in the treated group versus 40% in controls. [3]
The efficacy of clavulanic acid was comparable to that of imipenem (30 mg/kg i.m.) in reducing lung bacterial loads. For strain Ab11, imipenem reduced the lung count by 2.47 log₁₀ CFU/g (vs. 1.78 for CLA); for strain Ab51, imipenem reduced it by 2.28 log₁₀ CFU/g (vs. 2.41 for CLA). [3]

β-lactamases were prepared from bacterial cultures. Organisms were grown, induced with appropriate β-lactam antibiotics (e.g., cephalothin for Klebsiella, cephaloridine for Citrobacter and Serratia, penicillin G for Pseudomonas) where necessary, and then harvested. Cells were disrupted by sonication, and cellular debris was removed by centrifugation. The supernatant, containing the enzyme, was dialyzed. Some β-lactamases were purified using osmotic shock and column chromatography. [2]
β-lactamase activity was measured using a spectrophotometric method (tracking optical density change at 255 nm for cephalosporin substrates) or a microiodometric method. To test inhibition, β-lactamase was incubated with various concentrations of clavulanic acid (e.g., 0.5-10 μg/ml) for different preincubation times (0-20 minutes) before adding the substrate (e.g., ampicillin or cephaloridine at 0.1-0.2 mM). The residual hydrolytic activity was then measured and compared to a control without inhibitor. The irreversible nature of inhibition was tested by dialyzing the enzyme-inhibitor mixture for 24 hours and then reassaying activity. [2]
For kinetic studies, a purified type III β-lactamase was used. The initial velocity of substrate (ampicillin or cephaloridine) hydrolysis was measured at various substrate concentrations, in the presence and absence of clavulanic acid (0.25-0.5 μg/ml), without preincubation. Data were analyzed using Lineweaver-Burk plots to determine Km and Ki values. [2]

Minimal Inhibitory Concentration (MIC) Determination: MICs were determined by agar or broth dilution in Mueller-Hinton medium with an inoculum of 10⁵ colony-forming units (CFU), incubated at 35°C for 18 hours. [2]
Synergy Studies: Synergy was tested using a checkerboard broth dilution method. Twofold dilutions of ampicillin (or other β-lactam) and clavulanic acid were combined in Mueller-Hinton broth. Tubes were inoculated with 10⁵ CFU of the test organism and incubated overnight at 35°C. The minimal bactericidal concentration (MBC) was determined by plating 0.01 ml from clear tubes onto agar. Synergy was defined as a fourfold reduction in the MIC or MBC of both agents. [2]
Killing Curve Studies: An overnight culture of E. coli (type III β-lactamase producer) was diluted in fresh Mueller-Hinton broth to approximately 6 × 10⁴ CFU. Clavulanic acid (0.8 or 1.6 μg/ml), ampicillin (6.3 μg/ml), or a combination of both was added. Samples were taken at various time points (0, 2, 4, 6 hours), and viable counts were determined by plating on agar. [2]

Pharmacokinetic Study:** Immunocompetent C57BL/6 mice received a single intraperitoneal dose of clavulanic acid (13 mg/kg). Blood was collected from the periorbital plexus of three anesthetized mice per time point at 5, 10, 15, 30, 60, 90, 120, and 240 minutes post-dose. Serum drug concentrations were determined by a bioassay using *Klebsiella pneumoniae* ATCC 29665 as the indicator organism. Pharmacokinetic parameters (Cmax, AUC, t₁/₂, T>MIC) were calculated. [3]
* **Efficacy Study (Pneumonia Model):** C57BL/6 mice were anesthetized and inoculated intratracheally with 50 μL of a bacterial suspension (approximately 8.6 log₁₀ CFU/mL of A. baumannii strain Ab11 or Ab51) mixed 1:1 with 10% porcine mucin. Treatments began 4 hours after inoculation. Mice were randomly allocated to three groups (n=15 per strain per group): untreated control, clavulanic acid (13 mg/kg i.p.), or imipenem (30 mg/kg i.m.). The dosing interval for clavulanic acid was 2.5 hours for Ab11 and 2 hours for Ab51 to maintain T>MIC at 40% of the dosing interval. Animals were observed for 24 hours. Immediately after death or at the end of the experiment, blood and lung samples were collected for quantitative culture. Results were expressed as log₁₀ CFU/g of lung and frequency of sterile blood cultures. [3]
* **Toxicity Control:** A group of 10 uninfected mice were treated with the same dose regimen of clavulanic acid to evaluate its toxicity. No toxicity was observed. [3]

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Pharmacokinetic Study: Immunocompetent C57BL/6 mice received a single intraperitoneal dose of clavulanic acid (13 mg/kg). Blood was collected from the periorbital plexus of three anesthetized mice per time point at 5, 10, 15, 30, 60, 90, 120, and 240 minutes post-dose. Serum drug concentrations were determined by a bioassay using Klebsiella pneumoniae ATCC 29665 as the indicator organism. Pharmacokinetic parameters (Cmax, AUC, t₁/₂, T>MIC) were calculated. [3]
Efficacy Study (Pneumonia Model): C57BL/6 mice were anesthetized and inoculated intratracheally with 50 μL of a bacterial suspension (approximately 8.6 log₁₀ CFU/mL of A. baumannii strain Ab11 or Ab51) mixed 1:1 with 10% porcine mucin. Treatments began 4 hours after inoculation. Mice were randomly allocated to three groups (n=15 per strain per group): untreated control, clavulanic acid (13 mg/kg i.p.), or imipenem (30 mg/kg i.m.). The dosing interval for clavulanic acid was 2.5 hours for Ab11 and 2 hours for Ab51 to maintain T>MIC at 40% of the dosing interval. Animals were observed for 24 hours. Immediately after death or at the end of the experiment, blood and lung samples were collected for quantitative culture. Results were expressed as log₁₀ CFU/g of lung and frequency of sterile blood cultures. [3]
Toxicity Control: A group of 10 uninfected mice were treated with the same dose regimen of clavulanic acid to evaluate its toxicity. No toxicity was observed. [3]

Absorption, Distribution and Excretion
Clavulanic acid is well absorbed in the gastrointestinal tract after oral administration. In a study of four subjects, the minimum absorption rate was 73%, and the mean absolute bioavailability was 64%. In a pharmacokinetic study of eight healthy volunteers, the mean Cmax was 2.098 ± 0.441 μg/mL. The mean Tmax reported in this study was 1.042 ± 0.80 hours. Another pharmacokinetic study reported Tmax ranging from 40 to 120 minutes. Approximately 40% to 65% of clavulanic acid is excreted unchanged in the urine within 6 hours after administration. Metabolites of clavulanic acid are primarily excreted via urine, feces, and carbon dioxide in exhaled air. Clavulanic acid can be eliminated via renal and non-renal routes. Approximately 17% of the dose of radiolabeled clavulanic acid is excreted in exhaled air, and 8% is excreted in feces. A study of four healthy volunteers showed a volume of distribution of 12 liters after administration of radiolabeled clavulanic acid. Clavulanic acid is distributed in various tissues and interstitial fluids. Clinically significant concentrations have been detected in the gallbladder, abdomen, skin, fat, and muscle tissues. Therapeutic concentrations of clavulanic acid have also been detected in bile, pus, synovial fluid, and peritoneal fluid. Animal studies have shown that clavulanic acid can cross the placenta. A pharmacokinetic study in four healthy volunteers showed a clearance of 0.21 L/min after administration of radiolabeled clavulanic acid. Another study showed a mean clearance of 12.20 L/h/70 kg. Dosage adjustments may be necessary for patients with renal failure.

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Metabolism/Metabolites
Clavulanic acid is primarily metabolized to two metabolites: 2,5-dihydro-4-(2-hydroxyethyl)-5-oxo-1H-pyrrole-3-carboxylic acid and 1-amino-4-hydroxy-but-2-one. One pharmacokinetic study found that the first metabolite accounted for 15.6% of the dose, and the second metabolite accounted for 8.8%.

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Biological Half-Life
According to reports, clavulanic acid has a half-life similar to that of amoxicillin, lasting 45-90 minutes. A study of radiolabeled clavulanic acid in four healthy volunteers determined its half-life to be 0.8 hours.

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In C57BL/6 mice, after a single intraperitoneal dose of 13 mg/kg of clavulanic acid, the following pharmacokinetic parameters were determined: Cmax = 13.38 mg/L; AUC = 8.17 mg·h/L; t₁/₂ = 0.24 h. [3]
The T>MIC (time above MIC) for clavulanic acid was 0.93 h for strain Ab11 (MIC = 2 mg/L) and 0.79 h for strain Ab51 (MIC = 4 mg/L). [3]
The Cmax/MIC ratio was 6.69 for strain Ab11 and 3.35 for strain Ab51. The AUC/MIC ratio was 4.0 h for Ab11 and 2.0 h for Ab51. [3]
In humans, after a 200 mg dose of clavulanic acid, the reported Cmax is 11.4 mg/L and the t₁/₂ is 0.8-1 hour. Levels in bronchial mucosa reach 118% of serum levels. [3]

Protein Binding
Amoxicillin has a plasma protein binding rate of approximately 25%.

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[1]. Clavulanic acid: a review. Biotechnol Adv. Jul-Aug 2008;26(4):335-51

[2]. Clavulanic acid, a novel inhibitor of beta-lactamases. Antimicrob Agents Chemother. 1978 Nov;14(5):650-5.

[3]. In vitro activity and in vivo efficacy of clavulanic acid against Acinetobacter baumannii. Antimicrob Agents Chemother. 2009 Oct;53(10):4298-304.

[4]. Tannic acid exhibits anti-inflammatory effects on formalin-induced paw edema model of inflammation in rats. Hum Exp Toxicol. 2019 Nov;38(11):1296-1301.

Clavulanic acid is an antibiotic isolated from Streptomyces clavuligerus. It is a suicidal β-lactamase inhibitor that inhibits the activity of bacterial β-lactamases. Clavulanic acid has multiple effects, including antibacterial, anti-anxiety, and EC 3.5.2.6 (β-lactamase) inhibition. It is a conjugate of clavulanic acid. Clavulanic acid is a β-lactamase inhibitor, often used in combination with amoxicillin or ticarcillin. By preventing β-lactamases from degrading the antibiotic, it broadens the antibacterial spectrum against susceptible bacterial infections, thus combating antibiotic resistance. Clavulanic acid is derived from Streptomyces clavuligerus. When used in combination with amoxicillin, clavulanic acid is often called Augmentin, Co-Amoxiclav, or Clavulin. Clavulanic acid is a β-lactamase inhibitor. Clavulanic acid's mechanism of action is as a β-lactamase inhibitor. It has been reported to be present in Streptomyces cattleya and Streptomyces clavuligerus, with supporting data. Clavulanic acid is a semi-synthetic β-lactamase inhibitor isolated from Streptomyces. It contains a β-lactam ring that binds firmly to the active site of β-lactamase or its vicinity, thereby inhibiting enzyme activity. This protects other β-lactam antibiotics from β-lactamase catalysis, thus enhancing their antibacterial activity. This drug is often used in combination with antibiotics sensitive to β-lactamases (such as penicillin and cephalosporins) to treat infections caused by β-lactamase-producing microorganisms. Streptomyces clavuligerus is a β-lactam antibiotic produced by actinomycetes. It is a suicide inhibitor of bacterial β-lactamases. When used alone, it has weak antibacterial activity against most microorganisms, but when used in combination with other β-lactam antibiotics, it can prevent microbial β-lactamases from inactivating the antibiotics.

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Drug Indications
Clavulanic acid, when used in combination with other antibiotics, can prevent the emergence of drug-resistant strains and enhance their antibacterial therapeutic effects.
The following diseases, when the pathogens produce β-lactamases, can be treated with amoxicillin/clavulanic acid or ticarcillin/clavulanic acid in combination: acute otitis media caused by Haemophilus influenzae and Moraxella catarrhalis; sinusitis caused by Haemophilus influenzae and Moraxella catarrhalis; lower respiratory tract infections caused by Haemophilus influenzae, Staphylococcus aureus, Klebsiella spp., and Moraxella catarrhalis; skin and soft tissue infections caused by Staphylococcus aureus, Escherichia coli, and Klebsiella spp.; urinary tract infections caused by Escherichia coli, Klebsiella spp., Enterobacter spp., Serratia marcescens, or Staphylococcus aureus; and gynecological infections caused by various bacteria, including Pseudomonas melanogenans, Enterobacter spp., Escherichia coli, and Klebsiella spp. Septicemia caused by Staphylococcus aureus and Staphylococcus epidermidis; septicemia caused by various bacteria (including Klebsiella spp., Escherichia coli spp., Staphylococcus aureus, or Pseudomonas spp.); bone and joint infections caused by Staphylococcus aureus; intra-abdominal infections caused by Escherichia coli, Klebsiella pneumoniae, or Bacteroides fragilis. Regarding drug susceptibility: It is important to note that this product is only indicated for infections confirmed or highly suspected to be caused by susceptible bacteria. Bacterial culture and drug susceptibility testing should be performed whenever possible, and this should be used as the basis for determining whether to use this antibiotic. Clavulanic acid should not be used if β-lactamase production is not detected in microbiological testing. When these tests are not possible, the local infection pattern and drug susceptibility test results can be used to determine whether clavulanic acid is appropriate. Ticarcillin combined with clavulanate potassium has shown particular efficacy in mixed infections and can be used as empirical treatment before determining the drug susceptibility of the causative bacteria. The ticarcillin-clavulanate potassium combination may be an effective monotherapy for treating infections that usually require multiple drug combinations.

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Mechanism of Action
The structure of potassium clavulanate contains a β-lactam ring, which can irreversibly bind to β-lactamases, thereby preventing β-lactamases from inactivating certain β-lactam antibiotics and effectively treating infections caused by Gram-positive and Gram-negative bacteria that are sensitive to β-lactam antibiotics.

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Clavulanic acid is a naturally occurring β-lactamase inhibitor produced by Streptomyces clavuligerus. Its chemical name is Z-(2R,5R)-3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylic acid. It has weak intrinsic antibacterial activity but is a potent inhibitor of many bacterial β-lactamases. [2]
The primary mechanism of action of clavulanic acid is the irreversible inhibition of β-lactamases. It acts as a "suicide inhibitor," binding to the enzyme's active site and preventing the hydrolysis of co-administered β-lactam antibiotics like ampicillin or amoxicillin. This restores the antibacterial activity of these antibiotics against β-lactamase-producing organisms. [2]
The combination of clavulanic acid with ampicillin or amoxicillin is synergistic against many clinically important β-lactamase-producing bacteria, including S. aureus, E. coli, Klebsiella, Salmonella, and Shigella. However, it is not effective against organisms that produce Richmond type I β-lactamases, such as P. aeruginosa and some strains of Serratia and Citrobacter, as these enzymes are not significantly inhibited. [2]

199.048

58001-44-8

Potassium clavulanate cellulose;Clavulanate lithium;61177-44-4;Clavulanate potassium;61177-45-5

5280980

Off-white to light yellow solid powder

1.7±0.1 g/cm3

545.8±50.0 °C at 760 mmHg

117.5-118
117.5 - 118 °C

283.9±30.1 °C

0.0±3.3 mmHg at 25°C

1.644

-1.98

2

5

2

14

324

2

C(=C/1\[C@H](C(=O)O)N2C(=O)C[C@H]2O1)/CO

HZZVJAQRINQKSD-PBFISZAISA-N

InChI=1S/C8H9NO5/c10-2-1-4-7(8(12)13)9-5(11)3-6(9)14-4/h1,6-7,10H,2-3H2,(H,12,13)/b4-1-/t6-,7-/m1/s1

(2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Clavulanate; Acide clavulanique; Acido clavulanico; Clavulansaeure; Antibiotic MM 14151; acidum clavulanicum;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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 : ~13.89 mg/mL (~69.74 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.55 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 25.0 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: ≥ 2.5 mg/mL (12.55 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 25.0 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 3: 10% DMSO+90% (20% SBE-β-CD in Saline): ≥ 2.5 mg/mL (12.55 mM)

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Solubility in Formulation 4: 12.5 mg/mL (62.76 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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