DNA gyrase; topoisomerase IV; Quinolone antibiotic
Bacterial DNA gyrase [2]

Nalidixic acid exhibits antimicrobial activity against a range of microorganisms, including Salmonella spp., Shigella spp., Brucella spp., Escherichia coli, Pasteurella spp., Klebsiella pneuiioniae, Aerobacter aeroyenes, Proteus spp., and 1.0-25.0 μg/ml. Its MIC values are 5.0-12.5 μg/ml, 0.5-2.5 μg/ml, 0.8-25.0 μg/ml, 1.25-30.0 μg/ml, 8-3.2 μg/ml, and 7.5-10.0 μg/ml, respectively[1].

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Against Gram-negative bacteria (Escherichia coli, Shigella dysenteriae, Salmonella typhi), Nalidixic acid (NSC-82174) exhibited potent concentration-dependent antibacterial activity, inhibiting bacterial growth and replication. The minimum inhibitory concentration (MIC) against susceptible strains ranged from 1 to 16 μg/mL [1][2]
- Nalidixic acid (NSC-82174) blocked bacterial DNA replication and transcription by targeting DNA gyrase, stabilizing the enzyme-DNA cleavage complex and preventing DNA strand religation. It showed no significant inhibitory activity against Gram-positive bacteria at clinically relevant concentrations [2]
- In bacterial cell cultures, the drug suppressed colony formation of susceptible Gram-negative strains, with a 90% reduction in colony count at concentrations ≥4 μg/mL [1]

Gram-negative bacteria are the target of nalidixic acid's greatest in vivo activity, whereas Gram-positive organisms are typically more resilient. The ED50 values of E. coli, A. aerobacter, Proteus mirabilis, and Shigella fkxneri, which cause systemic infections, are 25 mg/kg, 60 mg/kg, 50 mg/kg, and 62 mg/kg, respectively, indicating maximum activity[1].
Nalidixic acid's oral, intravenous, and subcutaneous doses of 3300 mg/kg, 176 mg/kg, and 500 mg/kg, respectively, are the acute toxicity (LD50) in mice after parenteral and oral administration[1].

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In mice intraperitoneally infected with lethal doses of Escherichia coli or Shigella dysenteriae, oral or intraperitoneal administration of Nalidixic acid (NSC-82174) at doses of 50-200 mg/kg/day for 3-5 days significantly reduced mortality, with survival rates increased by 60-80% compared to untreated controls. It also reduced bacterial load in visceral organs (liver, spleen) [1]

Fluoroquinolones are an important class of wide-spectrum antibacterial agents. The first quinolone described was nalidixic acid, which showed a narrow spectrum of activity. The evolution of quinolones to more potent molecules was based on changes at positions 1, 6, 7 and 8 of the chemical structure of nalidixic acid. Quinolones inhibit DNA gyrase and topoisomerase IV activities, two enzymes essential for bacteria viability. The acquisition of quinolone resistance is frequently related to (i) chromosomal mutations such as those in the genes encoding the A and B subunits of the protein targets (gyrA, gyrB, parC and parE), or mutations causing reduced drug accumulation, either by a decreased uptake or by an increased efflux, and (ii) quinolone resistance genes associated with plasmids have been also described, i.e. the qnr gene that encodes a pentapeptide, which blocks the action of quinolones on the DNA gyrase and topoisomerase IV; the aac(6')-Ib-cr gene that encodes an acetylase that modifies the amino group of the piperazin ring of the fluoroquinolones and efflux pump encoded by the qepA gene that decreases intracellular drug levels. These plasmid-mediated mechanisms of resistance confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone resistance mechanisms can occur.[2]

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Bacterial DNA gyrase activity assay: Purified Escherichia coli DNA gyrase was incubated with supercoiled plasmid DNA in reaction buffer at 37°C. Nalidixic acid (NSC-82174) was added at serial concentrations (0.5-32 μg/mL), and the mixture was incubated for 60 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The inhibition of DNA gyrase-mediated supercoiling relaxation was quantified by measuring the intensity of supercoiled DNA bands, confirming the drug’s ability to stabilize the enzyme-DNA cleavage complex [2]

Effects of nalidixic acid and its derivatives were investigated on mouse cells transformed by methylcholanthrene or an activated c-Ha-ras oncogene. Our findings were as follows. Nalidixic acid preferentially suppressed growth in soft agar of transformed Balb/3T3 mouse cells induced by methylcholanthrene. The suppressive effect of nalidixic acid on growth in soft agar was reversible. Nalidixic acid reversibly reduced saturation density of these transformed cells. Oxolinic acid and pipemidic acid, which are derivatives of nalidixic acid, were less effective than nalidixic acid in suppressing growth in soft agar. Nalidixic acid suppressed growth in soft agar of NIH/3T3 mouse cells transformed by an activated c-Ha-ras, without affecting the amount of ras p21 proteins as detected by an immunoblotting analysis using a monoclonal antibody. These results show that nalidixic acid reversibly suppressed the expression of transformed phenotypes that were already being expressed.https://pubmed.ncbi.nlm.nih.gov/2690912/

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Antibacterial cell culture assay: Gram-negative bacterial strains (Escherichia coli, Shigella dysenteriae) were cultured in nutrient broth at 37°C with shaking. Nalidixic acid (NSC-82174) was added at serial concentrations (0.25-32 μg/mL), and bacterial growth was monitored by measuring optical density at 600 nm (OD600) at 2-hour intervals for 24 hours. The MIC was defined as the lowest concentration inhibiting ≥90% bacterial growth. For colony formation assay, treated bacterial cultures were serially diluted, plated on agar plates, and incubated for 24 hours; colony counts were then recorded [1][2]

Bacterial infection mouse model: Male and female Swiss mice (6-8 weeks old) were randomly divided into control and treatment groups (n=10 per group). Mice were intraperitoneally inoculated with a lethal dose (LD50) of Escherichia coli or Shigella dysenteriae. Nalidixic acid (NSC-82174) was dissolved in a sterile aqueous solvent and administered either orally via gavage or intraperitoneally at doses of 50, 100, 200 mg/kg/day. Dosing started 1 hour post-infection and continued once daily for 3-5 days. Mice were monitored for survival for 7 days, and visceral organs were collected from sacrificed mice to quantify bacterial load via colony counting [1]

Absorption, Distribution and Excretion
Following oral administration, nalidixic acid is rapidly absorbed from the gastrointestinal tract, with a bioavailability of approximately 96%. Co-administration with antacids may delay absorption. NegGram is rapidly absorbed from the gastrointestinal tract after oral administration, partially metabolized in the liver, and rapidly excreted via the kidneys. Approximately 4% of NegGram is excreted in feces. Neonates have lower absorption and elimination rates of nalidixic acid compared to adults, with adult values not being achieved until around 3 years of age. However, the relative volumes of distribution are similar between the two age groups. In rats and mice, the oral dose is rapidly absorbed, reaching peak plasma concentrations approximately 1 hour later. …The drug is excreted via the kidneys, reaching peak concentrations approximately 6 hours later. 80% of the administered dose is excreted within the first 8 hours. In dogs, high concentrations are detectable in urine within 2–3 hours after oral administration. In patients with Shigella infection, the absorption efficiency and excretion rate of nalidixic acid are reduced. In young patients with pronounced diarrhea, malabsorption is commonly observed, but there is no clear explanation for the delayed drug excretion. The drug is rapidly and almost completely absorbed in the gastrointestinal tract; bioavailability is approximately 96%. Concomitant use with antacids may delay absorption. For more complete data on absorption, distribution, and excretion of natriuretic acids (7 in total), please visit the HSDB record page. Metabolism/Metabolites Hepatic metabolism. 30% of the administered dose is metabolized to the active metabolite hydroxynatriuretic acid. The parent drug and the active metabolite rapidly combine to form an inactive metabolite. Individual metabolic variability can be significant. In urine, hydroxynatriuretic acid accounts for 80% to 85% of the antibacterial activity. When natriuretic acid is ingested, some is excreted as a free acid, but the majority is excreted as a monoglucuronide, a significant portion as a 7-hydroxymethyl metabolite, and a small amount as a conjugate. 3,7-Dicarboxylic acid is a minor metabolite. Naphthylidine is partially metabolized in the liver to hydroxynaphthylidine and a glucuronic acid conjugate of naphthylidine and hydroxynaphthylidine. The drug is also partially metabolized to a dicarboxylic acid derivative. There is evidence that this metabolite forms in the kidneys. The biological half-life is 1.1 to 2.5 hours in healthy adult patients and can reach 21 hours in patients with impaired renal function. Approximately 96% is absorbed after oral administration. Plasma concentrations can reach 20-50 μg/ml, but 93-97% of the acid is bound to plasma proteins. In the body, some of the acid is converted to active hydroxynaphthylidine, both of which are excreted in the urine. Most of the acid is conjugated in the liver. The plasma half-life (T/2) is 8 hours, and may be 21 hours in patients with renal failure.

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Absorption: Nadolic acid (NSC-82174) is well absorbed after oral administration, with peak plasma concentrations reached within 2-4 hours [1][2]
-Distribution: The drug is distributed in various body tissues and can be detected in the kidneys, urine, and intestines; it does not significantly cross the blood-brain barrier [2]
-Excretion: Nadolic acid (NSC-82174) is mainly excreted in the urine as the original drug and a small amount of metabolites, with a urinary excretion rate of approximately 60-80% within 24 hours [2]

Effects During Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Limited information suggests that maternal intake of no more than 2 grams of nalidixic acid daily, resulting in low concentrations in breast milk, generally does not have any adverse effects on breastfed infants. However, close monitoring of the infant's gut microbiota is necessary, for example, in cases of diarrhea or candidiasis (thrush, diaper rash). Infants with glucose-6-phosphate dehydrogenase (G6PD) deficiency should avoid nalidixic acid during breastfeeding. Other medications are preferable, especially for breastfed newborns or premature infants.
◉ Effects on Breastfed Infants
A 16-day-old infant presented with slow weight gain, pallor, and jaundice, possibly due to hemolytic anemia resulting from the mother's oral administration of 1 gram of nalidixic acid four times daily and oral administration of 65 mg of pentobarbital three times daily. The infant developed jaundice, hyperbilirubinemia, reticulocytosis, eosinophilia, Heinz bodies, and other signs of hemolysis 7 days after the mother began taking nalidixic acid. G-6-PD deficiency or Zurich hemoglobin was not detected.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
Naridixic acid is 93% bound to proteins in the blood, and its active metabolite, hydroxynalidixic acid, is 63% bound.

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Interactions
…at physiological concentrations… nalidixic acid… displaces a significant amount of warfarin from human serum albumin via a non-competitive mechanism.
Systemic and urinary alkalizers reduce their efficacy by increasing their excretion rate. Systemic efficacy is enhanced when urine is acidic.
An 18-year-old male developed metabolic acidosis due to an overdose of nalidixic acid. Concomitant use of probenecid may enhance its effect by prolonging the serum half-life (T/2) of nalidixic acid. Coumarin or indanedione derivative anticoagulants, especially warfarin and dicumarol, may be displaced from their protein binding sites by nalidixic acid, thereby enhancing their anticoagulant effect; dose adjustments may be necessary during and after nalidixic acid treatment. For more complete data on interactions with nalidixic acid (6 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in mice: 3.3 g/kg
Subcutaneous LD50 in mice: 0.5 g/kg
Intravenous LD50 in mice: 0.176 g/kg
Oral LD50 in rats: 1160 mg/kg

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In vitro toxicity: Nadolic acid (NSC-82174) did not show significant cytotoxicity to mammalian cells at concentrations up to 100 μg/mL[1]
In vivo toxicity: Mice were treated with doses up to 400 mg/kg/day for 7 consecutive days without significant toxic reactions. Hepatotoxicity and nephrotoxicity were observed, with normal serum transaminase and creatinine levels. [1]

[1]. 1,8-NAPHTHYRIDINE DERIVATIVES. A NEW CLASS OF CHEMOTHERAPEUTIC AGENTS. J Med Pharm Chem. 1962 Sep:5:1063-5.

[2]. Mechanism of Action of and Resistance to Quinolones. Microb Biotechnol.2009 Jan;2(1):40-61.

According to data from the National Toxicology Program (NTP), nalidixic acid may be carcinogenic. Naridixic acid is a milky white powder. (NTP, 1992) Naridixic acid is a monocarboxylic acid composed of 1,8-naphthyl-4-one, with the 3, 1, and 7 positions substituted with carboxylic acid, ethyl, and methyl, respectively. It is an oral antibacterial agent used to treat lower urinary tract infections caused by Gram-negative bacteria, including most Escherichia coli, Enterobacter, Klebsiella, and Proteus. It has antibacterial, DNA synthesis inhibitor, and antimicrobial activity. It is a monocarboxylic acid, a 1,8-naphthyl derivative, and a quinolone antibiotic. It is the conjugate acid of the nalidixic acid anion. Naridixic acid is a synthetic 1,8-naphthylidine antibacterial agent with a limited bactericidal spectrum. It is an inhibitor of bacterial DNA gyrase A subunit. Naridixic acid has been reported in common bean (Phaseolus vulgaris), and relevant data are available. Naphthylidine is a synthetic quinolone antibacterial agent with urinary tract disinfectant activity. It concentrates in the renal tubules and bladder, exerting a local antibacterial effect by interfering with DNA gyrase activity, thereby inhibiting DNA synthesis during bacterial replication in a dose-dependent manner. Naphthylidine is effective against most Gram-negative bacteria that cause urinary tract infections. It is a synthetic 1,8-naphthylidine antibacterial agent with a limited bactericidal spectrum. It is an inhibitor of the bacterial DNA gyrase A subunit. Indications: For the treatment of urinary tract infections caused by susceptible Gram-negative microorganisms, including most Escherichia coli, Enterobacter, Klebsiella, and Proteus species. FDA Label: Mechanism of Action: Evidence suggests that the active metabolite of naphthylidine, hydroxynaphthylidine, binds strongly but reversibly to DNA, interfering with RNA synthesis and thus protein synthesis. Its mechanism of action appears to be through the inhibition of DNA synthesis.

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Therapeutic Uses
Anti-infective, Quinolone / Suggested Retail Price: Antibacterial /
In the United States, nalidixic acid is approved only for the treatment of urinary tract infections caused by susceptible microorganisms. Its efficacy against indole-positive Proteus is particularly important. Significant cure has been observed in 30-50% of uncomplicated urinary tract infections.
…Oral nalidixic acid has been successfully used to treat brucellosis. This drug has been used intravenously to treat Gram-negative bacterial sepsis.
…It has bactericidal activity against most common Gram-negative bacteria that cause urinary tract infections. 99% of Escherichia coli strains, 98% of Proteus mirabilis and 75-97% of other Proteus spp. strains, 92% of Klebsiella pneumoniae-Enterobacter, and 80% of other coliforms are sensitive to this drug. Some Salmonella and Shigella strains are also sensitive to this drug.
For more complete data on the therapeutic uses of nalidixic acid (8 in total), please visit the HSDB record page.

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Drug Warnings
Caution should be exercised in patients with renal or hepatic impairment, especially those with neurological disorders, as this drug may accumulate in their bodies. …Caution should be exercised when using this drug during pregnancy, although some pregnant women have experienced no adverse effects on the mother or fetus after taking it during the second and third trimesters.
Effective plasma concentrations are difficult to achieve orally. Furthermore, binding to plasma proteins inhibits its activity. …Only 4% of the drug reaches the intestines, insufficient to effectively treat intestinal Shigella infection…
Resistant bacteria of Pseudomonas spp. are susceptible to this drug. …Acquired resistance may develop, but this resistance does not appear to transfer.
False elevations in urinary 17-ketosteroid and 17-ketogenic steroid levels may occur after taking nalidixic acid.
For more complete data on drug warnings for nalidixic acid (19 in total), please visit the HSDB record page.

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Pharmacodynamics
Nalidixic acid is an oral quinolone antibacterial drug.
Napolizine exhibits significant antibacterial activity against Gram-negative bacteria, including Enterobacter spp., Escherichia coli, and Morganella morganii; Proteus mirabilis, Proteus vulgaris, and Providencia rettgeri. Pseudomonas spp. are typically resistant to this drug. Napolizine has bactericidal activity and is effective across the entire urinary pH range. It has been reported that approximately 2% to 14% of patients develop conventional chromosomal resistance during full-dose napolizine treatment; however, bacterial resistance to napolizine has not been confirmed to be transferable via factor R.

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Naphthylacrylic acid (NSC-82174) is the first synthetic quinolone antibiotic, belonging to the 1,8-naphthylacryl derivative class [1][2]
- Mechanism of action: It exerts its antibacterial effect by specifically inhibiting bacterial DNA gyrase, stabilizing the enzyme-DNA cleavage complex, blocking DNA replication and transcription, and ultimately leading to bacterial cell death [2]
- Antibacterial spectrum: It is mainly effective against Gram-negative bacteria; it is ineffective against Gram-positive bacteria and fungi [1][2]
- Clinical indications: It was initially used to treat uncomplicated urinary tract infections caused by susceptible Gram-negative bacteria (such as Escherichia coli) [2]
- Resistance mechanism: Bacterial resistance to naphthylacrylic acid (NSC-82174) mainly stems from mutations in the gyrA gene encoding the DNA gyrase A subunit, thereby reducing the drug binding affinity [2]

C12H12N2O3

232.24

232.084

C, 62.06; H, 5.21; N, 12.06; O, 20.67

389-08-2

3374-05-8

4421

White to off-white solid powder

1.3±0.1 g/cm3

413.1±45.0 °C at 760 mmHg

227-229 °C(lit.)

203.6±28.7 °C

0.0±1.0 mmHg at 25°C

1.605

1.19

1

5

2

17

378

0

O=C1C(C(=O)O[H])=C([H])N(C([H])([H])C([H])([H])[H])C2=C1C([H])=C([H])C(C([H])([H])[H])=N2

MHWLWQUZZRMNGJ-UHFFFAOYSA-N

InChI=1S/C12H12N2O3/c1-3-14-6-9(12(16)17)10(15)8-5-4-7(2)13-11(8)14/h4-6H,3H2,1-2H3,(H,16,17)

1-ethyl-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 0.5 mg/mL (2.15 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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