β-lactam
Penicillin binding protein 2 (PBP2) [2]

The minimal inhibitory concentration (MIC) of Amdinocillin against the Escherichia coli strain (WE 6933) was determined to be 0.5 μg/mL in nutrient broth and Mueller-Hinton agar.[1]

Treatment of catheter-associated infections can be achieved with high dosages (400 mg per kg of body weight/d) of mecillinam (Amdinocillin, 400 mg per kg)[1].
In a rabbit model of catheter-associated bacteriuria, intravenous Amdinocillin at 400 mg/kg/day (divided into four doses over 72 h) rapidly eliminated E. coli from catheter urine: at 48 h, four of five rabbits had sterile urine; at 72 h, all treated rabbits had sterile catheter urine, and bladder wall, bladder urine, catheter tip, and urethral catheter samples were all sterile (no detectable bacteria).[1]
At 200 mg/kg/day, four of five rabbits did not clear E. coli after 72 h, but counts were low; bladder tissue was sterile in all rabbits and bladder urine sterile in four of six rabbits; bacterial growth was absent from four of six catheter tips and two of six urethral catheters.[1]
At 100 mg/kg/day, E. coli was not eliminated from catheter urine after 72 h, but counts were significantly decreased; bladder urine and bladder wall showed growth in only two of four and two of five rabbits, respectively; catheter tip and urethral catheter remained infected in three of five rabbits; all samples had significantly fewer bacteria than controls.[1]
At 50 mg/kg/day, no significant reduction in catheter urine cfu count except at 72 h; catheter tip, urethral segment, bladder urine, and bladder wall remained heavily colonized in most rabbits, though counts were significantly lower than controls; bacteria were eliminated from bladder urine and bladder wall in two of six rabbits.[1]
Scanning and transmission electron microscopy showed gradual deterioration of bacterial biofilm and reduction in bacterial numbers with increasing antibiotic dosages. At 400 mg/kg, no biofilm or bacteria were visible; at lower doses, biofilm appeared “spongy” with pores, cracks, and crevices.[1]

Minimal inhibitory concentration (MIC) of Amdinocillin (Mecillinam; FL 1060) was determined using Etest® strips. Bacteria were grown overnight, diluted 500-fold in phosphate-buffered saline (PBS), spread evenly on Mueller Hinton (MH) agar plates, and an Etest® strip was placed on the agar. Results were analyzed after ~24 hours. When appropriate, MH agar plates were supplemented with 0.3 mM or 0.75 mM cysteine. [2]
To isolate Amdinocillin (Mecillinam; FL 1060) resistant mutants, independent overnight cultures of E. coli MG1655 were diluted in PBS, and approximately 10⁷ CFUs were spread on MH agar plates containing 4, 8, 16, or 32 mg/L mecillinam (corresponding to 32×, 64×, 128×, and 256× the wild-type MIC). Colonies were picked after 1 and 2 overnight incubations and restreaked on plates with the same mecillinam concentration. Mutation frequencies were calculated as the median number of colonies per plate divided by the total CFUs. [2]
Fitness (relative growth rate) of Amdinocillin (Mecillinam; FL 1060) resistant mutants was measured in MH medium and in sterile filtered human urine. Overnight cultures were diluted to ~1-3 × 10⁶ CFU/mL, and 300 μL were added to a 100-well honeycomb plate in quadruplicate. Growth was monitored at 37°C with shaking for 16 hours using a Bioscreen C Analyzer, with OD₆₀₀ measurements every 4 minutes. Relative growth rates were calculated by dividing the generation time of the parental strain (MG1655) by that of the mutants. [2]
To determine the effect of cysteine on resistance, strains were grown for 48 hours on M9 glucose (0.4%) agar plates with or without 0.3 mM cysteine to screen for cysteine auxotrophy. [2]

Animal Model: High dosages (400 mg per kg of body weight/d) of mecillinam (Amdinocillin, 400 mg per kg) are effective in treating catheter-associated infections[1].
Dosage: 50, 100, 200, and 400 mg per kg.
Administration: Intravenously each day in four divided doses over a 72-h period.
Result: High dose of 400 mg/kg exhibited significant activity.

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Animal model: Male New Zealand white rabbits were catheterized with size 8 Fr. Foley catheters with preconnected drainage bags under sterile conditions. An intravenous line was placed in the jugular vein, and a 5% dextrose-electrolyte infusion maintained urine output >500 mL/day. The catheter drainage tube was inoculated with 0.1 mL of 10⁹ cfu/mL of auxotrophically marked E. coli (streptomycin-resistant, lactose-negative) just distal to the catheter, and clamped for 15 min. Antibiotic therapy began 72 h after inoculation when catheter urine contained ≥10⁵ cfu/mL. Amdinocillin was administered intravenously at 50, 100, 200, or 400 mg per kg body weight per day in four divided doses over 72 h. Control rabbits received no antibiotic. Urine samples were obtained from the catheter at 0, 6, 12, 18, 24, 48, and 72 h, treated with penicillinase (1.6×10⁴ IU/mL), serially diluted, and plated on Mueller-Hinton and MacConkey agar with streptomycin (100 μg/mL). At 72 h, rabbits were euthanized, residual urine collected, and bladder wall (1 cm²), catheter tip (1 cm), and urethral catheter segment (1 cm) were sonicated in PBS and plated. Catheter and bladder samples were processed for scanning electron microscopy (Hitachi S-450, 20 kV) and transmission electron microscopy (Hitachi 600, 50 kV) with ruthenium red staining.[1]

Absorption, Distribution and Excretion
Oral absorption is poor. Biological Half-Life The biological half-life in patients with normal renal function is approximately 1 hour. In patients without kidneys, the biological half-life is prolonged to 3 to 6 hours.

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Urine trough concentrations of Amdinocillin measured 24 h after initiation of therapy were: 40 ± 15 μg/mL for 50 mg/kg/day, 135 ± 39 μg/mL for 100 mg/kg/day, 390 ± 90 μg/mL for 200 mg/kg/day, and 890 ± 310 μg/mL for 400 mg/kg/day.[1]

No specific toxicity data reported. All rabbits remained clinically normal throughout the experiment. Five rabbits were excluded due to catheter occlusion (not drug-related). At the highest dose (400 mg/kg/day), no adverse effects were noted.[1]

[1]Amdinocillin Treatment of Catheter-Associated Bacteriuria in Rabbits. J Infect Dis. 1989 Jun;159(6):1065-72.;

[2] Amdinocillin (Mecillinam) resistance mutations in clinical isolates and laboratory-selected mutants of Escherichia coli. Antimicrob Agents Chemother. 2015 Mar;59(3):1718-27.

Mecillinam is a penicillin with a 6β-substituent of [(azacycloheptan-1-yl)methylene]amino; it is a broad-spectrum penicillin antibiotic that specifically binds to penicillin-binding protein 2 (PBP2) and is effective only against Gram-negative bacteria. It can be used as both an antibacterial and anti-infective agent. It is an amidinopenicillic acid derivative with broad-spectrum antibacterial activity. It is poorly absorbed orally and is used to treat urinary tract infections and typhus. Ampicillin is not marketed in the United States. Ampicillin is a semi-synthetic broad-spectrum β-lactam penicillin with antibacterial activity. Ampicillin specifically binds to and inactivates penicillin-binding protein 2 (PBP2) located on the inner membrane of bacterial cell walls. Inactivation of penicillin-binding protein (PBP) interferes with the cross-linking of peptidoglycan chains, which is crucial for the strength and rigidity of bacterial cell walls. This interrupts bacterial cell wall synthesis, leading to weakened cell walls and ultimately cell lysis. Given that most β-lactam penicillins bind almost exclusively to PBP1 and PBP3, ampicillin can synergize with other penicillins to treat bacterial infections. It is an amidinopenicillic acid derivative with broad-spectrum antibacterial activity. See also: ampicillin ester (in salt form). Indications: For the treatment of urinary tract infections caused by certain strains of Escherichia coli, Klebsiella, and Enterobacter. Primarily used against Gram-negative bacteria. Mechanism of Action: Ampicillin is a potent and specific antagonist of penicillin-binding protein-2 (PBP2). It is effective against Gram-negative bacteria by inhibiting the activity of PBP2, thereby preventing cell wall synthesis. PBP2 is a peptidoglycan elongation initiation enzyme. Peptidoglycan is a polymer composed of sugars and amino acids and is a major component of bacterial cell walls.

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Pharmacodynamics
Ampicillin is a novel semi-synthetic penicillin effective against a variety of Gram-negative bacteria. Its antibacterial activity stems from its specific and high-affinity binding to penicillin-binding protein-2 (PBP2). Ampicillin is effective against a wide range of Gram-negative bacteria when used alone. However, Pseudomonas and non-fermenting Gram-negative bacteria often develop resistance. Ampicillin exhibits significant synergistic effects against a variety of Enterobacteriaceae when used in combination with various β-lactam antibiotics. However, this synergistic effect has not been observed against Gram-positive bacteria or Pseudomonas spp. Ampicillin is not resistant to β-lactamases. Bacteria that produce high levels of plasma-mediated β-lactamases are resistant to this drug. Concomitant use with probenecid leads to a significant increase in ampicillin plasma concentrations and delays its excretion.

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Amdinocillin (mecillinam) is a β-lactam antibiotic with low protein binding, high diffusibility, high urinary concentrations, and broad activity against gram-negative bacteria. In this study, it eradicated bacterial biofilms on Foley catheters at high doses (400 mg/kg/day). At lower doses (50–200 mg/kg/day), it first cleared bacteria from the bladder wall, then bladder urine, but the catheter biofilm was more resistant. The drug rapidly eliminated planktonic bacteria and caused dose-dependent biofilm deterioration visible by electron microscopy.[1]

439.214

C, 55.36; H, 7.12; N, 12.91; O, 14.75; S, 9.85

32887-01-7

Mecillinam-d12

36273

Solid powder

1.3±0.1 g/cm3

581.0±60.0 °C at 760 mmHg

156°C

305.2±32.9 °C

0.0±1.6 mmHg at 25°C

1.604

2.97

1

5

3

22

500

3

CC1([C@@H](N2[C@H](S1)[C@@H](C2=O)N=CN3CCCCCC3)C(=O)O)C

BWWVAEOLVKTZFQ-CXUHLZMHSA-N

InChI=1S/C15H23N3O3S/c1-15(2)11(14(20)21)18-12(19)10(13(18)22-15)16-9-17-7-5-3-4-6-8-17/h9-11,13H,3-8H2,1-2H3,(H,20,21)/b16-9+

(2S,5R,6R)-6-(azepan-1-ylmethylideneamino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Mecillinam FL 1060 AMDINOCILLIN FL-1060Coactin Penicillin HX Mecillinamum Hexacillin FL1060

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 : 65~250 mg/mL (199.73 ~768.21 mM)
H2O : ~100 mg/mL (~307.29 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (19.21 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 62.5 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: ≥ 6.25 mg/mL (19.21 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 62.5 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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Solubility in Formulation 3: ≥ 6.25 mg/mL (19.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (307.29 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.)