C.P.W.G.M. Verwey-van Wissen

Isolation, identification and determination of sulfamethoxazole and its known metabolites in human plasma and urine by high-performance liquid chromatography

From human urine the following metabolites of sulfamethoxazole (S) were isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N4-acetyl-5-methylhydroxysulfamethoxazole (N4SOH) and sulfamethoxazole-N1-glucuronide (Sgluc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by beta-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N4-acetylsulfamethoxazole (N4), SOH, N4-hydroxysulfamethoxazole (N4OH), N4SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N4 could be detected in high concentrations, while the other metabolites were present in only minute concentrations. In urine, S and the metabolites and conjugates were present. The quantitation limit of the compounds in plasma are respectively: S and N4 0.10 micrograms/ml; N4SOH 0.13 micrograms/ml; N4OH 0.18 micrograms/ml; SOH 0.20 micrograms/ml; and Sgluc 0.39 microgram/ml. In urine the quantitation limits are: N4 and N4OH 1.4 micrograms/ml; S 1.5 micrograms/ml; N4SOH 1.9 micrograms/ml; SOH 3.5 micrograms/ml; and Sgluc 4.1 micrograms/ml. The method was applied to studies with healthy subjects and HIV positive patients.

Direct determination of codeine, norcodeine, morphine and normorphine with their corresponding O-glucuronide conjugates by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method has been developed for the detection, separation and measurement of codeine and its metabolites norcodeine, morphine and normorphine, with their glucuronide conjugates. The glucuronidase Escherichia coli type VIIA hydrolyses codeine-6-glucuronide completely and is used for the construction of the calibration curves of codeine-6-glucuronide. Enzymic hydrolysis of codeine-6-glucuronide depends on the specific activity of the glucuronidase applied. Examples are shown of a volunteer who is able to form morphine from codeine and one who is unable to do so.

Determination of naproxen and its metabolite O-desmethylnaproxen with their acyl glucuronides in human plasma and urine by means of direct gradient high-performance liquid chromatography

Naproxen is metabolized in humans by O-demethylation, and by acyl glucuronidation to the 1-O-glucuronide. Naproxen, its metabolite and the conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic deglucuronidation. The glucuronide conjugates were isolated by preparative chromatography from human urine samples. Mild acidic hydrolysis of one urinary conjugate resulted in naproxen. This conjugate was also formed by alkaline isomerization of isolated naproxen acyl glucuronide, indicating that the structure of this urinary conjugate must have been naproxen isoglucuronide (4-O-glucuronide). Mild acidic hydrolysis of another urinary conjugate resulted in O-desmethylnaproxen. This conjugate was also formed by alkaline isomerisation of isolated O-desmethylnaproxen acyl glucuronide, indicating that the structure of this urinary conjugate must have been O-desmethylnaproxen isoglucuronide (4-O-glucuronide). Calibriation curves were constructed by enzymic deconjugation of samples containing different concentrations of isolated naproxen acyl glucuronide, O-desmethylnaproxen acyl glucuronide, and the isoglucuronides of naproxen and O-desmethylnaproxen by mild acidic hydrolysis. The limit of quantitation of naproxen in plasma is 1.5 microgram/ml. The limits of quantitation in urine are: naproxen, O-desmethylnaproxen, naproxen acyl glucuronide and O-desmethylnaproxen acyl glucuronide, 1 microgram/ml; the isoglucuronide of naproxen and O-desmethylnaproxen, 1.5 microgram/ml. A pharmacokinetic profile of naproxen is shown, and some preliminary pharmacokinetic parameters of naproxen obtained from two human volunteers are given.

Determination of indomethacin, its metabolites and their glucuronides in human plasma and urine by means of direct gradient high-performance liquid chromatographic analysis: Preliminary pharmacokinetics and effect of probenecid

Indomethacin is metabolized in humans by O-demethylation, and by acyl glucuronidation to the 1-O-glucuronide. Indomethacin, its metabolite O-desmethylindomethacin (DMI) and their conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic deglucuronidation. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. In plasma only indomethacin was present. No isoglucuronides were present in acidic urine of the volunteer. The possible metabolite deschlorobenzoylindomethacin (DBI) was not detectable in urine. Calibration curves were constructed by enzymic deconjugation of samples containing different concentrations of isolated indomethacin acyl glucuronide, DMI acyl glucuronide and DMI ether glucuronide. The limit of quantitation of indomethacin in plasma is 0.060 microgram/ml. The limits of quantitation in urine are: indomethacin 0.053 microgram/ml, DMI 0.065 microgram/ml, DMI acyl glucuronide 0.065 microgram/ml and DMI ether glucuronide 0.254 microgram/ml. A pharmacokinetic profile of indomethacin is shown, and some preliminary pharmacokinetic parameters of indomethacin obtained from one human volunteer are given. Probenecid inhibits the formation of both the ether and the acyl glucuronide of DMI.

Absolute bioavailability of chlorthalidone in man: A cross-over study after intravenous and oral administration

SummarySeven normal human volunteers each received a constant-rate infusion of chlorthalidone for 2 h, and the same (commonly 50 mg) single oral dose on separate occasions. The concentration of unchanged chlorthalidone was analyzed over a 100 to 220 h period in plasma, red blood cells, urine and faeces after both dosage forms. A three compartment model was required to describe the intravenous plasma concentrations in five of the subjects. A two compartment model sufficed to account for the decay of the oral plasma concentrations in all seven subjects. The mean plasma t1/2 after i.v. dosing was 36.5 h (±10.5 SD), and the mean plasma t1/2 after oral doses was 44.1 h (±9.6 SD). The mean red blood cell concentration t1/2 after i.v. doses was 46.4 h (±9.9 SD), and the mean red blood cell t1/2 after the oral doses was 52.7 h (±9.0 SD). The shorter i.v. half-live was not equally manifest in all subjects, being mainly apparent in three of them. In all cases the urinary excretion rate plots were parallel to the plasma concentration curves. As the faster decay after i.v. administration was not accompanied by increased renal clearance, the difference must have been due to non-renal mechanism. The mean total of 65.4 (±8.6 SD) % of the intravenous dose was excreted in urine over infinite time, whereas the mean total excretion after the oral dose was 43.8 (±8.5 SD) %. Faecal excretion ranged from 1.3–8.5% of dose in the i.v. study to 17.5–31.2% of dose in the oral study. The sum of the amounts present in urine plus faeces pointed strongly to an important metabolic route of elimination of chlorthalidone. Bioavailability estimates (F) from three sets of data were — a mean F of 0.61 from plasma concentrations, 0.67 from urinary excretion measurements and 0.72 from the erythrocyte concentrations. Simulations with a non-linear model indicated lesser validity of the estimate from erythrocyte concentrations. It was concluded that the average of plasma and urine data, F=0.64, yielded the best estimate of the oral availability of chlorthalidone 50 mg in man.

Age-dependent Pharmacokinetics of Lamivudine in HIV-infected Children

The recommended dose of lamivudine in children is higher when compared with adults: 4 mg/kg vs ~2 mg/kg (150 mg) and administered twice a day. Limited data are available to demonstrate that this increased dose results in adequate exposure to lamivudine in children with human immunodeficiency virus (HIV) infection. Data were selected from children who were using lamivudine for at least 2 weeks before a full pharmacokinetic (PK) study was conducted. Lamivudine PK parameters were significantly related to age. The age of 6 years appeared to be a cutoff for a change in PK parameters of lamivudine, with children <6 years of age (n=17) having a median area under the curve 43% lower and a median peak plasma concentration 47% lower (both P<0.001) than older children (n=34). In conclusion, further investigation of the relationship between decreased lamivudine exposure and treatment outcome and long‐term resistance development in younger children with HIV infection is warranted.

Isolation, identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and Rhesus monkey by high-performance liquid chromatography

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by beta-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N4-acetylsulfadiazine (N4), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N4 could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 microgram/ml (S 0.31, N4 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 microgram/ml). In urine it varies between 0.6 and 1.1 micrograms/ml (S 0.75, N4 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 micrograms/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.

Direct gradient reversed-phase high-performance liquid chromatographic determination of salicylic acid, with the corresponding glycine and glucuronide conjugates in human plasma and urine

A gradient reversed-phase HPLC analysis for the direct measurement of salicylic acid (SA) with the corresponding glycine and glucuronide conjugates in plasma and urine of humans was developed. The glucuronides were isolated by preparative HPLC from human urine samples. The concentration of the glucuronides in the isolated fraction were determined after enzymatic hydrolysis. Salicylic acid acyl glucuronide (SAAG) was not present in plasma. No isoglucuronides were present in acidic and alkaline urine of the volunteer. The limits of quantitation in plasma are: SA 0.2 microgram/ml, salicyluric acid (SU) 0.1 microgram/ml, salicylic acid phenolic glucuronide (SAPG) 0.4 microgram/ml and salicyluric acid phenolic glucuronide (SUPG) 0.2 microgram/ml. The limit of quantitation in urine is for all compounds 5 micrograms/ml. Salicylic acid acyl glucuronide is stable in phosphate buffer pH 4.9 during 8 h at 37 degrees C; thereafter it declines to 80% after 24 h. The subjects urine was therefore acidified by the oral intake of 4 x 1.2 g of ammonium chloride/day. With acidic urine, hardly any salicylic acid is excreted unchanged (0.6%). It is predominantly excreted as salicyluric acid (68.7%).

Determination of furosemide with its acyl glucuronide in human plasma and urine by means of direct gradient high-performance liquid chromatographic analysis with fluorescence detection Preliminary pharmacokinetics and effect of probenecid

Furosemide is metabolized in humans by acyl glucuronidation to the 1-O-glucuronide (Fgluc). Furosemide (F) and the conjugate can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic deglucuronidation. The glucuronide conjugate was isolated by preparative HPLC from human urine samples. Furosemide and its acyl glucuronide were present in plasma. No isoglucuronides were present in acidic urine of a volunteer. Calibration curves were constructed by enzymic deconjugation of samples containing different concentrations of isolated F-acyl glucuronide. The limit of quantitation of F in plasma is 0.007 microgram/ml, Fgluc 0.010 microgram/ml. The limits of quantitation in urine are respectively: F 0.10 microgram/ml, Fgluc 0.15 microgram/ml. A pharmacokinetic profile of furosemide is shown, and some preliminary pharmacokinetic parameters of furosemide obtained from one human volunteer are given. Probenecid does not inhibit the formation of the acyl glucuronide of F, but inhibits the renal clearance of both compounds.

Drug-drug interactions between raltegravir and pravastatin in healthy volunteers.

Background:To evaluate the potential drug-drug interaction between raltegravir and pravastatin. Methods:This was an open-label, randomized, 3-period, cross-over, single-centre trial in 24 healthy volunteers. Subjects received the following treatments: pravastatin 40 mg every day for 4 days, raltegravir 400 mg twice a day for 4 days, and pravastatin 40 mg every day + raltegravir 400 mg twice a day for 4 days. The treatments were separated by washout periods of 10 days. On day 4 of each treatment period, blood samples for pharmacokinetics were collected throughout a 24-hour period. Results:Geometric mean ratios (90% confidence interval) for pravastatin + raltegravir versus pravastatin alone were 0.96 (0.83 to 1.11) for AUC0-24 and 1.04 (0.85 to 1.26) for Cmax. The mean low-density lipoprotein cholesterol decrease after 4 days of pravastatin was 0.42 mmol/L both in the presence and the absence of raltegravir. The geometric mean ratio (90% confidence interval) AUC0-12, Cmax, and C12 for raltegravir + pravastatin versus raltegravir alone were 1.13 (0.77 to 1.65), 1.31 (0.81 to 2.13), and 0.59 (0.39 to 0.88), respectively. Conclusions:Raltegravir did not influence the pharmacokinetics or the short-term lipid-lowering effects of pravastatin, whereas pravastatin increased the Cmax but decreased the C12 of raltegravir. The effects of pravastatin on raltegravir pharmacokinetics are not likely to be clinically relevant.