The Probenecid-story - A success in the fight against doping through out-of-competition testing.

The effectiveness of doping control in sport has been improved continuously during the past 50 years. One of the major steps forward was the introduction of unannounced and targeted out-of-competition testing in order to control the misuse of anabolic-androgenic steroids (AAS), mainly during the end of the 1980s. It also led to the misuse of masking agents in case a surprise control was performed. Athletes tried to be "prepared", when the doping control officer showed up. The disclosure of the masking agent probenecid in 1987 is a perfect example of a memorable finding, of a suspected and purported case of performance manipulation. Probenecid and its metabolites were identified in five urine samples collected from Norwegian athletes in an out-of-competition test, while they were staying and training in the USA. Probenecid is a drug that reduces the urinary excretion of AAS from the body. It was the first time that it had showed up in a doping control sample. The athletes were sanctioned for hampering the analysis of their urine sample, although probenecid was not yet specified on the Prohibited List. Its detection was the result of a successful collaboration of laboratories and investigative diligence and enthusiasm following up suspicious observations in the actual samples. Immediately afterwards probenecid was added to the Prohibited List for 1988 as well as including the manipulation of doping control samples.

Competitive inhibition of renal tubular secretion of gemifloxacin by probenecid.

Probenecid interacts with transport processes of drugs at several sites in the body. For most quinolones, renal clearance is reduced by concomitant administration of probenecid. The interaction between gemifloxacin and probenecid has not yet been studied. We studied the extent, time course, site(s), and mechanism of this interaction. Seventeen healthy volunteers participated in a randomized, two-way crossover study. Subjects received 320 mg gemifloxacin as an oral tablet without and with 4.5 g probenecid divided in eight oral doses. Drug concentrations in plasma and urine were analyzed by liquid chromatography-tandem mass spectrometry. WinNonlin was used for noncompartmental analysis, compartmental modeling, and statistics, and NONMEM was used for visual predictive checks. Concomitant administration of probenecid increased plasma gemifloxacin concentrations and amounts excreted in urine compared to baseline amounts. Data are average estimates (percent coefficients of variation). Modeling showed a competitive inhibition of the renal tubular secretion of gemifloxacin by probenecid as the most likely mechanism of the interaction. The estimated K(m) and Vmax for the saturable part of renal elimination were 9.16 mg/liter (20%) and 113 mg/h (21%), respectively. Based on the molar ratio, the affinity for the renal transporter was 10-fold higher for gemifloxacin than for probenecid. Since probenecid reached an approximately 200-times-higher area under the molar concentration-time curve from 0 to 24 h than gemifloxacin, probenecid inhibited the active tubular secretion of gemifloxacin. Probenecid also reduced the nonrenal clearance of gemifloxacin from 25.2 (26%) to 21.0 (23%) liters/h. Probenecid inhibited the renal tubular secretion of gemifloxacin, most likely by a competitive mechanism, and slightly decreased nonrenal clearance of gemifloxacin.

The effect of the interaction of pyrazinamide and probenecid on urinary uric acid excretion in man.

Complex interactions occur between pyrazinamide (PZA) and probenecid in man involving both the metabolism and distribution of the drugs, and their effects on renal tubules. Pretreatment with PZA prolonged the half-life (T 1/2) of probenecid without changing its plasma-binding. As the rate of probenecid metabolism is decreased, its uricosuric action tends to be prolonged and the effect of PZA lessened. The PZA-suppressible urate level is increased to values well above control after the administration of probenecid; it is less after alkalinization of urine, although still larger than the value for PZA-suppressible urate after the administration of PZA alone. Urinary probenecid excretion is much greater when urine is alkalinized. These observed drug interactions, plus the known effect of probenecid to block secretion of PZA, have to be considered in evaluating the effect of the two drugs given together, compared to the effect of each drug given separately.

Urinary excretion of probenecid and its metabolites in humans as a function of dose.

A GLC assay was used to study the excretion of probenecid and its metabolites in the urine of human subjects following oral doses of 0.5, 1, and 2 g. From 75 to 88% of the dose was found in the urine. The major metabolite, probenecid acyl glucuronide, accounted for 34-47% of the dose. Approximately equal amounts (10-15%) of the mono-N-propyl, secondary alcohol, and carboxylic acid metabolites were excreted in the unconjugated from with only traces in the conjugated form. The primary alcohol metabolite was not found in measurable amounts. The terminal half-lives for excretion of all metabolites were in the range of 4-6 hr, were independent of dose, and were limited by their rates of formation. A prolonged time course of excretion of the metabolites, particularly at higher doses, suggests that probenecid, being poorly soluble in water, precipitates from solution in the GI tract, forming a depot of drug from which absorption is dissolution rate limited. The urinary excretion of unchanged probenecid, which accounts for 4-13% of the dose, is dependent on both the pH and flow rate of urine.

Negligible excretion of unchanged ketoprofen, naproxen, and probenecid in urine.

On the average, 0.6% of a dose of ketoprofen or naproxen or 1.2% of a dose of probenecid was found in the urine of normal male volunteers assayed immediately after its collection. Between approximately 60 and 85% of the dose of these drugs can be excreted in the urine as conjugates, which rapidly hydrolyze at body temperature, at room temperature, and even during frozen storage, thereby regenerating the parent drug. Since urine collections involved sample retention in the bladder at 37 degrees for collection intervals as long as 2--3 hr, the given percentages excreted unchanged probably are overestimates. It is possible that no unchanged ketoprofen, naproxen, or probenecid is excreted in urine. This study contrasts with previous reports of up to 50% of a dose of ketoprofen and 15--17% of doses of naproxen and probenecid being excreted in urine as the parent compound. Those reports probably reflect primarily the duration of frozen sample storage between collection and assay along with the urine collection schedules employed the speed of the clinical procedures, and the analytical procedures used. Attention should be given to potential conjugate hydrolysis whenever the pharmacokinetics of carboxylic acids are studied.

Probenecid disposition by parallel Michaelis-Menten and dose-dependent pseudo-first-order processes.

Re-evaluation of published data on the urinary excretion of probenecid [4-(dipropylamino)sulfonylbenzoic acid, 1] and its metabolites as a function of orally administered dose has revealed that the elimination process is comprised of five parallel pathways. Excretion of the major metabolite, the acyl glucuronide 2 (35-45% of dose), follows Michaelis-Menten kinetics. The three oxidized metabolites, the mono-N-propyl, carboxylic acid, and secondary alcohol derivatives 3, 4, and 5, respectively, (totaling 30% of dose), each adhere to pseudo-first-order kinetics in which the elimination rate constant, as a result of product inhibition, is a function of the administered dose. Four to 13% of the dose is eliminated unchanged in apparent first-order fashion. A mathematical relationship between elimination half-life and dose, under conditions of product inhibition, is derived. Computer simulation of the elimination process confirms the experimental observation that the fraction of dose excreted in the form of each metabolite remains relatively constant over the dose range of 0.5 to 2 g. This study is believed to represent the first application of parallel Michaelis-Menten and dose-dependent pseudo-first-order processes to drug disposition. It demonstrates that the observation of constant proportions of excreted metabolites over a relatively wide range of doses does not provide evidence that the elimination process remains first-order.

Rapid high-performance liquid chromatographic assay for the simultaneous determination of probenecid and its glucuronide in urine. Irreversible binding of probenecid to serum albumin.

A reversed-phase high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of probenecid and its glucuronide in urine has been developed. The genuine glucuronide conjugate was isolated from urine by the use of solid-phase extraction on Amberlite XAD-2 and finally purified by the use of preparative HPLC on a Sepharon Hema 1000 RP-18 column. The purity of the product obtained was 88.9%. The isolated glucuronide was used as a standard sample. Of a p.o. dose of 500 mg to two volunteers, 26 and 29% were excreted as the ester glucuronide, while 1.0 and 2.7% were excreted unmetabolized. The stability of the ester glucuronide was investigated in aqueous buffers, buffered urine and human serum albumin solutions. The glucuronide was unstable in neutral and mildly alkaline solutions, and special precautions have to be taken during sampling and sample treatment in order to preserve the genuine glucuronide. The presence of human serum albumin in the solution stabilized the glucuronide against isomerization/rearrangements but catalysed the hydrolysis of the glucuronide. When incubating human serum albumin with the ester glucuronide, probenecid was shown to be covalently bound to the protein probably via a transacylation reaction.

[Solid-phase extraction and RP-HPLC screening procedure for diuretics, probenecid, caffeine and pemoline in urine].

A solid-phase extraction and reversed phase high performance liquid chromatographic method (RP-HPLC) was developed for the rapid determination of 13 diuretics (belonging to five different pharmacological groups), probenecid, caffeine and pemoline in urine. Two ml urine sample was first adsorbed on a XAD-2 column, then eluted with ether-ethyl acetate (1:1). The eluate was evaporated to dryness and reconstituted in methanol. The methanolic solution was injected into a HP LiChrosorb RP-18 column, using phosphate buffer (pH 3) and acetonitrile as the mobile phase and monitored at 216 nm, 230 nm, and 275 nm on a diode array ultraviolet detector. The extraction recoveries of 16 drugs were above 75%. The limits of detection ranged from 0.3-3.0 micrograms/ml of urine. All drugs were separately administered to healthy volunteers, positive urine samples were collected, and urinary excretion-time curves of some drugs were reported.

Column-switching techniques for screening of diuretics and probenecid in urine samples.

A method based on high-performance liquid chromatography using column-switching is described for the screening of diuretics and probenecid in urine samples. The system uses a 20- x 2.1-mm i.d. precolumn, packed with a Hypersil ODS-C18, 30-microns stationary phase, for the on-line sample cleanup and enrichment. Untreated urine samples are directly injected, and the precolumn is flushed for 1 min with water to eliminate polar matrix components. The retained analytes are then back-flushed by means of a six-port switching valve onto a Hypersil ODS-C18 analytical column (5 microns, 250- x 4-mm i.d.), where they are separated using an acetonitrile/phosphate buffer (pH = 3) gradient elution. Under these conditions, the separation and identification of diuretics and probenecid can be achieved with satisfactory selectivity and sensitivity. The described procedure is very simple and rapid since no off-line manipulation of the sample is required, the total analysis time being ca. 15 min.

Effects of urinary pH on renal interactions between probenecid and cefsulodin in rabbits.

The effect of urinary pH on renal interaction of cefsulodin and probenecid was tested in rabbits. Probenecid was reabsorbed in acidic urine (fractional excretion [FE] = 8 +/- 4%) and secreted in alkaline urine (FE = 492 +/- 258%). Renal excretion of cefsulodin alone was not affected by the urinary pH (FE = ca. 100%). In acidic urine, probenecid significantly reduced tubular secretion of cefsulodin (FE = 74 +/- 8%). An inverse pattern was observed in alkaline urine (FE = 122 +/- 18%).

Direct measurement of probenecid and its glucuronide conjugate by means of high pressure liquid chromatography in plasma and urine of humans.

Probenecid with its phase-I metabolites, and phase-II glucuronide conjugate can be analysed by a gradient high pressure liquid chromatographic method. Probenecid glucuronide in plasma with pH 7.4 is not stable and declines to 10% of the original value within 6 h (t1/2 approximately 1 h). Probenecid glucuronide is stable in urine with pH 5.0, moderately unstable at pH 6.0 (t1/2 approximately 10 h), and unstable at pH 8.0 (t1/2 approximately 0.5 h). Probenecid glucuronide is stable in water and 0.01 mol/l phosphoric acid in the autosampler of the high pressure liquid chromatograph. The decrease in concentration in water is 5.5% during 9 h and 0% in diluted acid. Probenecid glucuronide and the phase-I metabolites were not detectable in plasma. The main compound in fresh urine is the phase-II conjugate probenecid glucuronide (62% of a 500 mg dose); the phase-I metabolites are present and only a trace of probenecid is present. The percentage of the dose of the phase-I metabolites varies between 5 and 10, while hardly any probenecid is excreted unchanged (0.33%).

Improved detection limits for screening of diuretics by coupled liquid chromatography and ultraviolet-visible spectrophotometry.

Experimental conditions have been studied in order to improve the sensitivity for the analysis of diuretics and probenecid in urine samples by high-performance liquid chromatography with ultraviolet detection. Sample clean-up and chromatographic parameters have been optimized to obtain a suitable sensitivity for the detection or quantification of each diuretic using an HP-Hypersil ODS-C18 column (5 microns, 250 mm x 4 mm I.D.), taking into account the pharmacological properties of each compound. The reliability of this method was tested by analysing urine samples after a minimum single-dose administration of chlorthalidone and probenecid.