Hydroxynalidixic acid

Similar metabolites were found produced by man (19)(20)(21)t monkeys( ), dogs (8), chickens(22)> calves(23) and microorganisms(4). The ratios of these metabolites, however, were found to vary with the individual. The overall conversion of nalidixic acid to hydroxynalidixic acid had been reported by McChesney(S) to be normally about 32% bicarbonate supplementation increased this to about 40%. Bicarbonate supplementation also increased the amount of total naphthyridine excreted in the biologically active form. 

Portmann and co-workers then studied the kinetic pathways in man for hydroxynalidixic acid, the active primary metabolite.(26) The rate constants for glucuronide formation, oxidation to the dicarboxylic acid and excretion of hydroxynalidixic acid were calculated. Essentially total absorption of hydroxynalidixic acid was found in every case. Good agreement between experimental and theoretical plasma levels, based on the first order rate approximations used for the model, was found. Again, the disappearance rate constant, kdoi was found to be very similar for each subject, although the individual excretion and metabolic rate constants varied widely. The disappearance rate constant, k was defined as the sum of the excretion rate constant, kg j and the metabolic rate constants to the glucuronide and dicarboxylic acid, kM-j and kgj, respectively. 

This data was then used in another study by Portmann and co-workers(24) of nalidixic acid metabolism in man in which a more elaborate model was developed and various rate constants were reported (Figure 10). This model was based on the oral administration of 1 g of nalidixic acid. Theoretical curves for plasma levels of nalidixic and hydroxynalidixic acid vs. time agreed with experimental values. 

The first spectrofluorimetric methods reported for the determination of nalidixic acid and its metabolites in biological fluids did not differentiate between nalidixic acid and hydroxynalidixic acid. The determination of free nalidixic acid and the hydroxy-metabolite in human urine plasma and feces was performed by extraction by toluene from acidified biological fluid and subsequent fluorimetric measurement at 325/375 nm of sample re-extracted into aqueous solution.(8) Conjugated nalidixic and hydroxynalidixic acids were determined by acid hydrolysis and then toluene extraction for fluorimetric measurement of the total drug. The conjugated nalidixic acid was then determined by difference. 

A refined method involving extraction of two aliquots of biological material, buffered at two different pH s, by toluene, and re-extraction into aqueous solution allowed the simultaneous determination of nalidixic and hydroxynalidixic acid(24)(26) by their differential extractabilities and fluorescent intensities. This method was extended to the differential determination of the conjugated forms of nalidixic and hydroxynalidixic acid. 

A thin-layer gas chromatographic system was devised by Pittman and Shekosky(39) for chicken tissue and feces. A TLC system of benzene methanol acetic acid (9 1 1) was used for prior separation the spots were then removed and esterified in 14% BF in methanol. A 4 ft. 3% OV-17 column at 240° was used. Retention times of about 7 minutes for nalidixic acid, 10 minutes for hydroxynalidixic acid and 17 minutes for the dicarboxylic acid were reported. 

A pulse polarographic system for detection of the dicarboxylic acid in the presence of nalidixic and hydroxynalidixic acids was devised by Koss and Warner.(45) The reduction potential in the system used was -.54V vs. SCE. 

Microbiological assay procedures for nalidixic acid have also been used for biological samples. Since nalidixic and hydroxynalidixic acids have the same order of antibacterial activity in-vitro, then cannot be determined separately. 

High Pressure Liquid Chromatography. In plasma or urine nalidixic acid, 7-hydroxynalidixic acid and 7-carboxynalidixic acid, sensitivity 500 ng/ml, UV detection—G. Cuisinaud et al., J. Chromat., 1980,181 Biomed. Appl., 7, 399-406. 

Disposition in the Body. Readily absorbed after oral administration. The major metabolite, 7-hydroxynalidixic acid, is active other metabolites include glucuronide conjugates of nalidixic acid and 7-hydroxynalidixic acid, and a 7-carboxy metabolite. 

Following a single oral dose of 1 g to 11 female subjects, a mean peak plasma concentration of nalidixic acid of 27.3 pg/ml was attained in 1.5 hours concentrations of 7-hydroxynalidixic acid reached a maximum of about 10 pg/ml within 1 hour. After repeated oral administration of 1 g twice daily to the same subjects, a peak plasma concentration of 33 pg/ml of nalidixic acid was reported on the 7th day. Urinary concentrations of nalidixic acid plus 7-hydroxynalidixic acid following single or multiple dose administration were in excess of about 200 xg/ml for 8 hours in all subjects, and in some cases greater than 50 pg/ml for 12 hours (N. Ferry etal., Clin. Pharmac. Ther., 1981, 29, 695-698). 

Protein Binding. In plasma, nalidixic acid about 93%, 7-hydroxynalidixic acid about 60%. 

Nalidixic acid is a highly protein bound oral quinolone (>90%), that undergoes major hepatic metabolism (80%) to active (hydroxynalidixic acid) and inactive metabolites [216]. The parent drug and its metabolites are rapidly excreted in the urine [217]. Most of the antibacterial effect is due to the biologically active hydroxynalidixic acid, which is 16 times more active than the parent compound. Nalidixic acid has a terminal half-life of about two hours. The drug does not accumulate in tissues even after prolonged administration the kidney is the only organ in which this may occur. Furthermore, nalidixic acid does not diffuse into prostatic fluid [214]. 

In this reaction C is the product of a Phase I reaction and is more active than A. An example would be the hydroxylation of the urinary anti-infective, nalidixic acid (A), to hydroxynalidixic acid (C), which is about 16 times more potent as an anti-infective compared to nalidixic acid. 

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