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.

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.

[Severe immunoallergic hemolytic anemia caused by a glafenine metabolite]. / Anémie hémolytique immuno-allergique grave provoquée par un métabolite de la glafénine.

Three weeks after a surgical operation, a 74-year old woman was admitted to hospital for severe haemolytic anaemia. A strongly positive IgG type direct Coombs test pointed to a iatrogenic origin, but a search for anti-molecule antibodies directed against all medicines taken by the patient after surgery was negative. We extended our immunological investigations and were able to demonstrate the presence of IgG type antibodies directed against an ex vivo metabolite of glafenine.

[Urinary calculi caused by glaphenine. Role of radiology in diagnosis. Apropos of a case]. / Les lithiases urinaires dues à la glafénine. Place de la radiologie dans le diagnostic. A propos d'un cas.

The authors report a case of urinary calculi secondary to glafenine which posed a difficult problem in the radiological diagnosis. They review the literature of iatrogenic urinary calculi and discuss the place of radiology (Excretory Urography, Ultrasound and Computed Tomography) in the diagnosis.

[Renal pelvis stones made of glafenic acid in two patients under prolonged treatment with analgesics (author's transl)]. / Calculs pyéliques d'acide glafénique chez deux patientes traitées de façon prolongée par des analgésiques.

Two women aged 62 and 69 years who had been taking glafenine at normal dosage over a period of 4 years developed a renal calculus. In the first case, 6 small slightly radioopaque stones were extracted by pyelotomy, presenting a crystalline surface and yellow, soft, and amorphous section. They consisted of 50% calcium oxalate, 33% glafenic acid, and 10% proteins. In the second case, pyelography showed a sizable round and radiotransparent defect in the renal pelvis. At pyelotomy, a large, soft, and greenish stone was extracted, presenting a yellow and amorphous section, without calcium, but consisting of 75% glafenic acid, and 25% proteins. Through IR spectrography, glafenine metabolites found in the stones represented 33% in our first case and 75% in our second case. Through other methods, such as UV spectrophotometry and chromatography, 26% and 61% are respectively found. The metabolites are glafenic acid and hydroglafenic acid, in an identical proportion of 9 to 1 in both cases.

Biotransformations of glafenine in the rat and in man.

The biotransformations of a therapeutic dose of the non-narcotic analgesic, glafenine, have been studied in the rat and in man. In the rat, the ester bond is extensively hydrolysed to give glafenic acid which is the major metabolite excreted in bile and in urine. Two minor pathways have been identified one leading by hydroxylation of the benzene ring of glafenine or glafenic acid in para of the amino-substituent to the corresponding phenols, the other, by oxidation of the quinoline nitrogen of glafenic acid, to its N-oxide. In vivo this N-oxide is partly reduced into the parent compound. Hydroxyglafenic acid is the product of both direct oxidation of glafenic acid and hydrolysis of hydroxyglafenine. The glyceric esters are conjugated as glucuro-ethers and/or sulfo-esters and the carboxylic metabolites as acyl glucuronides. The conjugation rate, high for glafenine, its phenol homologue and glafenic acid, is low for hydroxyglafenic acid and the N-oxide. The analogous urinary excretion patterns in man and in the rat suggest a similarity in the biotransformation of glafenine in these two species.

Comparison of the biological effects in rat of high doses of two 4-amino-7-chloroquinoline derivatives: chloroquine and glafenine.

When given orally in elevated but nonlethal doses (150 to 450 mg/kg, on 2 consecutive days), glafenine induces in rats (body weight 100 g) a transient nephritis with an increase in blood urea, hypertrophy of adrenals, and some changes in the serum proteinogram. These effects do not appear to be due to the 4-amino-7-chloroquinoline structure from which glafenine is derived, as they are not observed with the structural analogue chloroquine given at equimolar doses under the same conditions. Further, they do not appear to be due to glycerol, the by-product of metabolic glafenine hydrolysis. The responsible molecule appears to be either glafenine itself or its acid metabolite 4-(0-carboxyphenylamino) 7-chloroquinoline.

[The use of analgesic and antiphlogistic agents in small animals (author's transl)]. / De toepassing van analgetica en antiflogistica bij de kleine huisdieren

The metabolism of many analgesic and antiphlogistic agents in dogs and cats differs markedly from that in man or other animals. When the known data on the subject are borne in mind, potentially toxic agents such as acetosal, phenylbutazone, ibuprofen and indomethacin may be used without much risk in various indications. It is pointed out that double-blind studies on the clincal effectiveness in patients unfortunately have not yet become available.