Chlorazanil

Formation of the diuretic chlorazanil from the antimalarial drug proguanil--implications for sports drug testing

Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries. A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil. In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil. While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance.

Enhancement of urine prostaglandin excretion by chlorazanil in dogs

In conscious dogs chlorazanil (2.5 mg/kg intravenously) markedly enhanced (5--10 fold) urinary excretion of prostaglandin E2 and F2alpha. The effect came to a peak at 15--30 min. following the administration. Chlorazanil did not modify renal blood flow or inulin clearance in non-pretreated or indomethacin pretreated dogs and the plasma renin activity remained unchanged. A marginal natriuretic and antikaliuretic activity by chlorazanil was similarly observed in non-pretreated dogs and dogs pretreated with indomethacin. The possible effects of the prostaglandins released by chlorazanil remained obscure. In vitro chlorazanil (10(-4) M) exhibited a moderate inhibition of 15-OH-prostaglandin dehydrogenase but an enhanced excretion of 15-keto-13, 14-dihydro PGE2alpha in vivo suggested that chlorazanil increased renal prostaglandin activity by increased prostaglandin synthesis, probably due to increased precursor availability. This was presumably mediated by some as yet unknown factor since chlorazanil ( 4 x 10(-5)M) failed to affect the release of precursor and prostaglandins from isolated Krebs-Henseleit perfused rabbit kidneys.

The effects of cortisol and chlorazanil on the mitotic rate in mouse liver and skin

Enhancement of urinary prostaglandin excretion by chlorazanil in rats. Effects of indomethacin

In conscious unloaded Sprague-Dawley female rats chlorazanil (10 mg/kg orally) markedly increased urinary prostaglandin E2-excretion from 51 +/- 9 to 813 +/- 112 ng/kg/6 hrs. Urinary flow rate increased from 12.8 +/- 0.6 to 42.0 +/- 1.4 ml/kg/6 hrs and urinary sodium excretion from 0.96 +/- 0.12 to 3.86 +/- 0.33 mmol/kg/6 hrs Urinary potassium excretion was unchanged. Indomethacin pretreatment (5 mg/kg orally) greatly induced the effect of chlorazanil on urinary prostaglandin E2-excretion. In addition indomethacin modified the excretory effects of chlorazanil, thus the enhancement of urinary flow rate was attenuated, the potassium excretion decreased, while sodium excretion tended to be potentiated. In non-pretreated condition chlorazanil variably affected urinary kallikrein excretion (TAMe-esterase activity) from 108 +/- 6 to 92 +/- 33 mEU/kg/6 hrs. After indomethacin pretreatment chlorazanil invariably reduced urine enzyme excretion from 111 +/- 6 to 32 +/- 4 mEU/kg/6 hrs.

Dissociation between renal medullary PGE2-synthesis and urine PGE2-excretion. Antagonism by bumetanide of chlorazanil induced urine PGE2-excretion in rats

Normal conscious female Sprague-Dawley rats were treated with chlorazanil (3 mg/kg i.p.), and urine was collected for 3 hours. Urine prostaglandin E2-excretion increased from 25 +/- 3 to 271 +/- 32 ng/kg/3 h. The enhancement of urine PGE2-excretion was inhibited by pretreatment with bumetanide (75 mg/kg p.o.). In separate experiments the papillary quantity of PGE2 was determined in freshly homogenized tissue. The basal level (14 +/- 2 ng PGE2/papilla) was increased by chlorazanil to 51 +/- 11 ng PGE2/papilla and 24 +/- 7 ng PGE2/papilla at one and two hours respectively after drug administration. The capacity of chlorazanil to increase medullary PGE2 accumulation was unaffected by bumetanide pretreatment. The results show that bumetanide dissociated the medullary PGE2 level from the excretion of PGE2 in urine, when the former was elevated by chlorazanil.