Epithelial delamination is protective during pharmaceutical-induced enteropathy.

Intestinal epithelial cell (IEC) shedding is a fundamental response to intestinal damage, yet underlying mechanisms and functions have been difficult to define. Here we model chronic intestinal damage in zebrafish larvae using the nonsteroidal antiinflammatory drug (NSAID) Glafenine. Glafenine induced the unfolded protein response (UPR) and inflammatory pathways in IECs, leading to delamination. Glafenine-induced inflammation was augmented by microbial colonization and associated with changes in intestinal and environmental microbiotas. IEC shedding was a UPR-dependent protective response to Glafenine that restricts inflammation and promotes animal survival. Other NSAIDs did not induce IEC delamination; however, Glafenine also displays off-target inhibition of multidrug resistance (MDR) efflux pumps. We found a subset of MDR inhibitors also induced IEC delamination, implicating MDR efflux pumps as cellular targets underlying Glafenine-induced enteropathy. These results implicate IEC delamination as a protective UPR-mediated response to chemical injury, and uncover an essential role for MDR efflux pumps in intestinal homeostasis.

Integrative, genome-wide association study identifies chemicals associated with common women's malignancies.

BACKGROUND: Breast cancer, cervical cancer, and ovarian cancer are three of the most commonly diagnosed malignancies in women, and more cancer prevention research is urgently needed. METHODS: Summary data of a large genome-wide association study of female cancers were derived from the UK biobank. We performed a transcriptome-wide association study and a gene set enrichment analysis to identify correlations between chemical exposure and aberrant expression, repression, or mutation of genes related to cancer using the Comparative Toxicogenomics Database. RESULTS: We identified five chemicals (NSC668394, glafenine, methylnitronitrosoguanidine, fenofibrate, and methylparaben) that were associated with the incidence of both breast cancer and cervical cancer. CONCLUSION: Using a transcriptome-wide association study and gene set enrichment analysis we identified environmental chemicals that are associated with an increased risk of breast cancer, cervical cancer, and ovarian cancer.

Differential Interaction of Dantrolene, Glafenine, Nalidixic Acid, and Prazosin with Human Organic Anion Transporters 1 and 3.

In renal proximal tubule cells, the organic anion transporters 1 and 3 (OAT1 and OAT3) in the basolateral membrane and the multidrug resistance-associated protein 4 (MRP4) in the apical membrane share substrates and co-operate in renal drug secretion. We hypothesized that recently identified MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin also interact with human OAT1 and/or OAT3 stably transfected in human embryonic kidney 293 cells. These four drugs were tested as possible inhibitors of p-[3H]aminohippurate (PAH) and [14C]glutarate uptake by OAT1, and of [3H]estrone-3-sulfate (ES) uptake by OAT3. In addition, we explored whether these drugs decrease the equilibrium distribution of radiolabeled PAH, glutarate, or ES, an approach intended to indirectly suggest drug/substrate exchange through OAT1 and OAT3. With OAT3, a dose-dependent inhibition of [3H]ES uptake and a downward shift in [3H]ES equilibrium were observed, indicating that all four drugs bind to OAT3 and may possibly be translocated. In contrast, the interaction with OAT1 was more complex. With [14C]glutarate as substrate, all four drugs inhibited uptake but only glafenine and nalidixic acid shifted glutarate equilibrium. Using [3H]PAH as a substrate of OAT1, nalidixic acid inhibited but dantrolene, glafenine, and prazosin stimulated uptake. Nalidixic acid decreased equilibrium content of [3H]PAH, suggesting that it may possibly be exchanged by OAT1. Taken together, OAT1 and OAT3 interact with the MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin, indicating overlapping specificities. At OAT1, more than one binding site must be assumed to explain substrate and drug-dependent stimulation and inhibition of transport activity.

Increased Cortical Cerebral Blood Flow in Asymptomatic Human Immunodeficiency Virus-Infected Subjects.

BACKGROUND: Human immunodeficiency virus (HIV)-infected individuals are at high risk for ischemic stroke. To investigate the physiological basis for this risk, we used magnetic resonance imaging (MRI) to measure oxygen extraction fraction (OEF) and cerebral blood flow (CBF) in treatment-naive asymptomatic HIV-infected subjects and controls. METHODS: In treatment-naive asymptomatic HIV-infected subjects and age-, gender-, and race-matched controls, OEF was measured by MRI asymmetric spin-echo echo-planar imaging sequences and CBF was measured by MRI pseudocontinuous arterial spin labeling. RESULTS: Twenty-six treatment-naive HIV-infected subjects and 27 age-, gender-, race-matched controls participated. Whole-brain, gray matter (GM), and white matter OEF were not different between the groups (all P > .70). Unexpectedly, HIV-infected subjects had significantly higher CBF in cortical GM (72.9 ± 16.2 mL/100 g/min versus 63.9 ± 9.9 mL/100 g/min; P = .01) but not in subcortical GM (P = .25). CONCLUSIONS: The observed increase in cortical GM CBF in treatment-naive HIV-infected subjects is unexpected, contrary to CBF decreases reported in HIV-infected subjects on treatment, and may represent an initial increase in metabolic activity due to an HIV-mediated inflammation.

The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway.

Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR > 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.

Bioactivation of glafenine by human liver microsomes and peroxidases: identification of electrophilic iminoquinone species and GSH conjugates.

Glafenine (Privadol; 2,3-dihydroxypropyl 2-[(7-chloro-4-quinolinyl) amino]benzoate) is a non-narcotic analgesic agent widely used for the treatment of pains of various origins. Severe liver toxicity and a high incidence of anaphylaxis were reported in patients treated with glafenine, eventually leading to its withdrawal from the market in most countries. It is proposed that bioactivation of glafenine and subsequent binding of reactive metabolite(s) to critical cellular proteins play a causative role. The study described herein aimed at characterizing pathways of glafenine bioactivation and the metabolic enzymes involved. Two GSH conjugates of glafenine were detected in human liver microsomal incubations using liquid chromatography tandem mass spectrometry. The structures of detected conjugates were determined as GSH adducts of 5-hydroxyglafenine (M3) and 5-hydroxy glafenic acid (M4), respectively. GSH conjugation took place with a strong preference at C6 of the benzene ring of glafenine, ortho to the carbonyl moiety. These findings are consistent with a bioactivation sequence involving initial cytochrome P450-catalyzed 5-hydroxylation of the benzene ring of glafenine, followed by two electron oxidations of M3 and M4 to form corresponding para-quinone imine intermediates that react with GSH to form GSH adducts M1 and M2, respectively. Formation of M1 and M2 was primarily catalyzed by heterologously expressed recombinant CYP3A4 and to a lesser extent, CYP2C19 and CYP2D6. We demonstrated that M3 can also be bioactivated by peroxidases, such as horseradish peroxidase and myeloperoxidase. In summary, these findings have significance in understanding the bioactivation pathways of glafenine and their potential link to mechanisms of toxicity of glafenine.

Correction of the Delta phe508 cystic fibrosis transmembrane conductance regulator trafficking defect by the bioavailable compound glafenine.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation Delta Phe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a Delta Phe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 microM). Glafenine increased the surface expression of Delta Phe508-CFTR in baby hamster kidney (BHK) cells to approximately 40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o(-) monolayers and intestines isolated from Delta Phe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (I(sc)) response to forskolin + genistein, and this effect was abolished by 10 microM CFTR(inh)172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects.

A Rapid Screenable Assay for Compounds That Protect Against Intestinal Injury in Zebrafish Larva.

This chapter describes a method to assay compounds modulating NSAID-induced intestinal injury in zebrafish larvae. The assay employs the NSAID glafenine, which causes intestinal epithelial cell damage and death by inducing organelle stress responses (endoplasmic reticulum and mitochondrial) and blocking the unfolded protein response pathway. This epithelial damage includes sloughing of intestinal cells into the lumen and out the cloaca of the zebrafish larvae. Exposing larvae to acridine orange highlights this injury when visualized under fluorescence microscope; injured fish develop intensely red-staining intestines, as well as a "tube" or cord of red color extending through the intestine and out the cloaca. Using this rapid visually screenable method, various candidate compounds were successfully tested for their ability to prevent glafenine-induced intestinal injury. Because this assay involves examination of larval zebrafish intestinal pathology, we have also included our protocol for preparation and analysis of zebrafish histology. The protocol includes numerous steps to generate high-quality zebrafish histology slides, as well as protocols to establish accurate anatomic localization of any given tissue cross-section-processes that are made technically difficult by the small size of zebrafish larvae.

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy-Causing Mutants of SLC4A11.

PURPOSE: Protein misfolding, causing retention of nascent protein in the endoplasmic reticulum (ER), is the most common molecular phenotype for disease alleles of membrane proteins. Strategies are needed to identify therapeutics able to correct such folding/trafficking defects. Mutations of SLC4A11, a plasma membrane transport protein of the human corneal endothelial cell layer, cause cases of congenital hereditary endothelial dystrophy, Harboyan syndrome, and Fuchs' endothelial corneal dystrophy. Most SLC4A11 mutations induce SLC4A11 misfolding and retention in the ER. METHODS: An assay amenable to high-throughput screening was developed to quantify SLC4A11 at the plasma membrane, enabling a search for potential traffic-correcting small molecules. The assay was validated by comparing cell surface abundance of SLC4A11 mutants measured in the assay to observations from confocal immunofluorescence and values from cell surface biotinylation. Functionality of mutant proteins was assessed, using a confocal microscopic green fluorescent protein (GFP) water flux assay where relative rates of cell swelling are compared. RESULTS: A small-scale screen revealed that the nonsteroidal anti-inflammatory drugs (NSAIDs), glafenine, ibuprofen, and acetylsalicylic acid dissolved in 0.2% dimethyl sulfoxide (DMSO), partially rescued the trafficking defect in some SLC4A11 mutants, expressed in HEK293 cells. These SLC4A11 mutants retained functional activity when rescued to the plasma membrane by glafenine treatment. Glafenine was effective with an EC50 of 1.5 ± 0.7 µM. CONCLUSIONS: These data suggest that glafenine, and perhaps other NSAIDs, hold potential as therapeutics for misfolded membrane proteins, like SLC4A11. The high throughput approach described here can be modified to identify correctors of other misfolded plasma membrane proteins that cause eye disease.

Effects of nonsteroidal anti-inflammatory drugs on prostaglandins and renal function.

In the past 15 years, there has been an explosion in the number of nonsteroidal anti-inflammatory drugs on the market. Along with this explosion have come increasing reports of the physiologic and pathologic changes seen in the kidneys. This report reviews the effects of prostaglandins on the kidney and the physiologic changes that result when prostaglandin synthesis is blocked. The world literature on renal complications of nonsteroidal anti-inflammatory drugs is reviewed and 274 cases of acute renal disease associated with their use are reported. The following cases are described: nephrotic syndrome (34); acute interstitial nephritis (51); acute tubular necrosis (29); papillary necrosis (53); poor perfusion with renal failure (40); acute glomerulitis or vasculitis (13); and unspecified renal failure (102). Fenoprofen appeared to be more nephrotoxic than other nonsteroidal anti-inflammatory drugs and resulted in multiple renal lesions in the same patient.

Tiapride versus glafenine: a double-blind comparative study in the management of acute rheumatic pain.

A double-blind study was carried out in 42 patients suffering from acute rheumatic pain to compare the analgesic effectiveness and tolerance of tiapride with that of glafenine, a widely used analgesic in Europe. Patients were allocated at random to receive either 100 mg tiapride or 200 mg glafenine 3-times daily over a period of 14 days. Pain intensity was rated daily by the patients using a visual analogue scale and an overall assessment of response to treatment was made by both patients and physician at the end of the study. The results showed that, whilst both treatments resulted in a marked reduction in mean pain scores, pain disappeared completely in 16 (76%) of the 21 patients treated with tiapride compared with 9 (43%) of the 21 receiving glafenine. There was also a significant difference in favour of tiapride in the physician's overall assessment of response which was considered as excellent in 71% of the patients on tiapride compared with 31% receiving glafenine. Both treatments were well tolerated and few side-effects were reported. Drowsiness occurred in 6 patients on tiapride but this was only mild in 5 and moderate in the other patient.

Comparative analgesic efficacy and tolerability of ketorolac tromethamine and glafenine in patients with post-operative pain.

In a randomized, single-dose, double-blind, parallel comparative trial of analgesic efficacy, 96 adult patients received either 10 mg ketorolac tromethamine or 400 mg glafenine orally the morning after surgery if they requested pain relief medication. Each patient provided a baseline pain assessment and then received the assigned medication. Patients assessed pain intensity and pain relief and reported any adverse events in interviews held 30 minutes after drug administration and then hourly for 6 hours. The demographic characteristics, baseline pain intensity, and surgical categories of the 47 patients who received ketorolac tromethamine and the 49 who received glafenine were similar. Both drugs provided prompt, sustained pain relief throughout the 6-hour observation period, and there were no statistically significant differences between the two groups in any of the efficacy measures analyzed. The global assessment recorded by patients suggested a slight clinical advantage for ketorolac tromethamine (32.6% of 'excellent' responses) as compared to glafenine (12.5% 'excellent'). The differences in overall response were statistically significant (p = 0.017). Fourteen (30%) patients who received ketorolac tromethamine and 17 (35%) who received glafenine reported adverse experiences that began or seemed to worsen after administration of the study drugs. The most prominent were drowsiness and sleeping, both of which are common in post-surgical patients.

Influence of agents affecting voltage-dependent calcium channels and dantrolene on the anticonvulsant action of the AMPA/kainate receptor antagonist LY 300164 in mice.

It was previously documented that calcium (Ca(2+)) channel inhibitors intensified the protective effects of conventional antiepileptics against electroconvulsions in mice. The aim of this study was to evaluate the effects of Ca(2+) channel inhibitors (nifedipine, nicardipine and flunarizine) on the anticonvulsant action of the new AMPA/kainate receptor antagonist, 7-acetyl-3-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxazolo[4,5-h][2,3]-benzodiazepine (LY 300164), against maximal electroshock (MES)-induced seizures in mice. Dantrolene (an inhibitor of Ca(2+)release from intracellular stores) was also included. Nifedipine (30 mg/kg) and flunarizine (15 mg/kg) raised the threshold for electroconvulsions, being ineffective at lower doses. Nicardipine (up to 30 mg/kg) and dantrolene (up to 20 mg/kg) did not affect this parameter. Flunarizine (10 mg/kg), nicardipine (20 mg/kg) and dantrolene (20 mg/kg) potentiated the efficacy of LY 300164 against MES. However, nicardipine (at 20 mg/kg) raised the free plasma concentration of LY 300164. Nifedipine (30 mg/kg), given even in a dose raising the electroconvulsive threshold, did not significantly alter the protective effect of LY 300164 against MES. Furthermore, the Ca(2+) channel agonist-BAY k-8644 (at 5 mg/kg) did not influence the protection offered by LY 300164 against MES. Finally, this Ca(2+) channel activator did not affect the enhanced efficacy of LY 300164 by Ca(2+) channel modulators. The only exception was the combination of LY 300164 with flunarizine. Combined treatment with LY 300164 and dantrolene (20 mg/kg), compared to LY 300164 alone, resulted in an impairment of motor performance in mice. Ca(2+) channel inhibitors were without effect upon this parameter evaluated in the chimney test. As shown in the passive avoidance task, LY 300164 alone (at its ED(50)) or combined with agents affecting neuronal Ca(2+) concentration did not disturb long-term memory. The present results suggest that agents preventing influx of Ca(2+) ions into neurons may enhance the protective action of LY 300164.

Pharmacokinetics of glafenine and glafenic acid in patients with cirrhosis, compared to healthy volunteers.

Pharmacokinetic parameters were evaluated in 12 patients with alcoholic cirrhosis and 12 healthy volunteers after a single 400 mg oral dose of glafenine. Glafenine (G) and its major active metabolite glafenic acid (GA) were measured at regular intervals using a specific high performance liquid chromatographic method. Glafenine absorption was significantly delayed in cirrhotic patients (CP) (Tmax = 2.8 +/- 1.3 hvs 1.5 +/- 0.4 h, p less than 0.01) and was dramatically reduced in 3 patients. The large hepatic 'first pass' effect observed in healthy volunteers was markedly reduced in CP (ratio Cmax GA/Cmax G = 3.6 +/- 2.9 vs 18.9 +/- 9.8, p less than 0.001; ratio areas under the curves AUC GA/AUC G = 2.3 +/- 2.3 vs 18.2 +/- 11.2, p less than 0.001). The elimination half-life of G was prolonged in the CP (13.0 +/- 13.1 h vs 1.5 +/- 0.5 h, p less than 0.01). In CP, GA elimination half-life was increased (12.0 +/- 13.4 h vs 4.3 +/- 1.3 h, NS) but the difference did not reach statistical significance because of large variability. The significant rise of G plasma concentrations (Cmax = 2.2 +/- 2.1 mg/L vs 0.7 +/- 0.2 mg/L, p less than 0.05) and its longer half-life would lead to an accumulation if the usual dosage regimen was prescribed for CP and could result in nephrotoxicity. On the other hand, lower dosage would be ineffective because only GA is active and nephrotoxic. Hence, G should be given with great caution to CP.

Ion suppression effects in liquid chromatography-electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries.

Ion suppression effects during electrospray-ionsation mass spectrometry (ESI-MS) caused by different sample preparation procedures for serum were investigated. This topic is of importance for systematic toxicological analysis for which LC-ESI-MS has been developed with transport-region collision-induced dissociation (ECI-CID) and mass spectra library searching. With continuous postcolumn infusion of two test compounds-codeine and glafenine-the ion suppression effects of extracted biological matrix obtained after a standard liquid-liquid extraction, a mixed-mode solid-phase extraction (SPE) method, a protein precipitation method and a combination of precipitation with polymer-based mixed-mode SPE have been investigated. Extracted ion chromatograms of codeine ([M+H](+), m/z 300) and glafenine ([M-H](-), m/z 371) were used for monitoring ion suppression. Severe ion suppression effects for codeine and glafenine were detected in positive and in negative ionisation modes, respectively, in the LC-front peak after serum clean-up with SPE (acid/neutral fraction) and protein precipitation as well as with protein precipitation combined with SPE. Less ion suppression of codeine in positive mode was found with liquid-liquid extraction of serum samples. No ion suppression was detected with the second fraction of the mixed-mode SPE (using RP-C(8) and cation-exchange phase) in both ionisation modes. All suppression effects were caused by polar and unretained matrix components, which were present after extraction and/or protein precipitation. However, no specific ion suppression was seen after elution of the polar LC-front throughout the whole gradient. It could be demonstrated, that ion suppression is not generally present at any retention time when using reversed-phase HPLC with rather long gradient programs, but may play an important role in case of high-throughput LC-MS analysis, when the analyte is not separated from the LC-front, or in flow injection analysis without chromatographic separation.

Fluorescence properties of glafenine.

The fluorescence properties of glafenine are partly caused by the anthranilic acid nucleus of the molecule. Correlations are made between fluorescence capacities and UV absorbance. Analytical determinations can be carried out in different solvents, e.g., ether, benzene, and ethanol. Linearity of the emission intensity with the concentration, limiting detectable sample concentrations, and Stokes shifts are reported.

Recurrent acute renal failure due to nonaccidental poisoning with glafenin in a child.

A 7-year-old boy experienced recurrent attacks of acute renal failure over an 18 month period. Each attack was accompanied by a fluorescent yellow discoloration of his urine. Laboratory data and the kidney biopsy were consistent with acute tubulo-interstitial nephritis. Repeated contacts with the boy in a play setting and thorough toxicologic examination of his urine finally led to the conclusion that the mother systematically poisoned her child with glafenin. The clinical and experimental literature on glafenin-related disease is reviewed. This is the first report of glafenin intoxication in a child. This is also the first case of kidney disease as an expression of child abuse.

Analgesic and non-steroidal anti-inflammatory drug-associated acute renal failure: a prospective collaborative study.

During a one-year period analgesic and non-steroidal anti-inflammatory drug-(NSAID) associated acute renal failure (ARF) was recorded in 147 of 398 patients registered in 58 nephrology units. This figure represented 36.9% of drug-associated ARF, and 6.8% of total patients with ARF hospitalized during the same period. Drugs involved were primarily glafenin (79), NSAID (62), paracetamol (5) and phenacetin (1 case). Hypersensitivity reactions were documented in 32 patients. Acute tubular necrosis was found in 20, and interstitial nephritis (AIN) in 9 of 34 biopsied patients. All patients in the glafenin group and 71.4% in the NSAID group recovered fully or regained previous renal function (p less than 0.01). Permanent renal damage (9.5% of total cases) was more frequent in patients with AIN than in those with other types of ARF (p less than 0.001). Preventive measures should be especially directed to older patients receiving NSAID, by avoiding the combined use of drugs potentiating their action and by correcting any predisposing factor to ARF.

Determination of glafenine in dosage forms and serum by thin layer densitometry and high performance liquid chromatography.

New thin layer densitometry and high performance liquid chromatography (HPLC) methods are described for quantitative determination of glafenine in dosage forms in the presence of its photo-degradation products and in serum in the presence of its metabolites. Mobile phases consisting of toluene-isopropyl alcohol-dimethylformamide-water (18:3:1:0.5) and methanol-water-phosphoric acid (80:120:0.5) are found to be efficient for reasonable separation and adequate resolution of glafenine from associated substances by thin layer chromatography (TLC) and HPLC techniques, respectively. The methods are used for the study of glafenine purity, stability, bioavailability, bioequivalence and tablet dissolution rate. The results obtained by TLC and HPLC techniques are in good agreement and offer the advantages of reproducibility and accuracy.

Spectrophotometric determination of two N-(4-quinolyl) anthranilic acid derivative (glafenine and floctafenine).

Spectrophotometric methods were developed for the determination of glafenine and floctafenine. The first method depends upon the determination of glafenine in raw material and tablets as well as in the presence of its main degradation product glafenic acid (up to 40%). Differential first derivative spectral response at 245 nm in 0.1 N hydrochloric acid, where the corresponding degradation product exhibits no contribution in 0.1 N sodium hydroxide. The method allows the determination of 2.5-30 microg ml(-1). The second method depends upon the reaction of floctafenine with 2,3-dichloro 5,6-dicyano-p-benzoquinone (DDQ) in acetonitrile to give highly colored complex that could be measured quantitatively at (about) lambda(max) 538 nm. The method permits the determination of 40-180 microg ml(-1) or by measuring the first derivative spectral response of the color at 610 nm. The method permits the determination of floctafenine in presence of thiocolchicoside. The methods mentioned both simplicity and sensitivity, having excellent precision and accuracy (100.31 +/- 0.63, 100.78 +/- 0.77 and 99.90 +/- 0.56 for glafenine and floctafenine, respectively). The results were of comparable accuracy and reproducibility with the reported methods.