Determination of nevirapine, an HIV-1 non-nucleoside reverse transcriptase inhibitor, in human plasma by reversed-phase high-performance liquid chromatography.

A sensitive and rapid high-performance liquid chromatography method has been developed to measure the levels of the HIV-1 non-nucleoside reverse transcriptase inhibitor nevirapine in human plasma. The sample pre-treatment consists of a protein precipitation with perchloric acid. A Hypersil ODS column is used at ambient temperature and a wavelength of 280 nm is used for ultraviolet detection. The mobile phase contains acetonitrile and a 60 mM phosphate buffer pH 4.5 (30:70, v/v). The detection limit of the method is 0.05 mg/l using 150 microl of plasma. The lower and upper limit of quantitation are 0.1 mg/l and 10 mg/l, respectively. The average recovery of nevirapine is 101.8% with a variation of 4.6%. The average inter-assay precision is 2.4%, the average intra-assay precision 2.9% and the average accuracy 97%.

Long-term stability of morphine and bupivacaine mixture for spinal use.

From clinical studies it has been proven that morphine in combination with bupivacaine is applicable in cancer pain. The availability of a ready to use parental dosage form of morphine and bupivacaine is comfortable for health care workers. Stability of a morphine or a bupivacaine preparation or a combination of both in a PVC cassette or polypropylene syringe for spinal use is examined in several studies. Apart from one study no data on long-term stability of morphine-bupivacaine mixture is available. A forced degradation study and a shelf-life study at room temperature (20-25 degrees C) were started on morphine hydrochloride 0.2 mg/ml and bupivacaine hydrochloride 7.5 mg/ml in 50 ml sterilized glass bottles type II. The results of the stability study showed that this mixture was stable up to 18 months at room temperature, whereafter morphine showed a slight degradation (5%) and bupivacaine remained stable.

Simultaneous determination of the HIV-protease inhibitors indinavir, nelfinavir, saquinavir and ritonavir in human plasma by reversed-phase high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the four licensed HIV-protease inhibitors indinavir, nelfinavir, saquinavir and ritonavir. An aliquot of 500 microl plasma, spiked with internal standard, was extracted with 0.5 ml 0.1 M NH4OH and 5 ml methyl tert.-butyl ether. After evaporating, the residue was dissolved in eluent consisting of acetonitrile-50 mM phosphate buffer, pH 5.63 (40:60, v/v). Subsequently, the eluent was washed with hexane. Chromatography was performed using a C18 reversed-phase column and gradient elution with a linear increase of acetonitrile from 36 to 66%. Ultraviolet detection at 215 nm was used. Linearity of the method was obtained in the concentration range of 45-30 000 ng/ml for all four analytes. The method was validated extensively and stability tests under various conditions were performed. The assay is now in use to analyse plasma samples from patients treated with (combinations of) HIV-protease inhibitors.

Safety of lidocaine-prilocaine cream application four times a day in premature neonates: a pilot study.

UNLABELLED: Although safety is established for lidocaine-prilocaine cream application to the heel once a day in neonates, it is often necessary to repeat heel lances several times a day in the clinical situation. A pilot safety study applying 0.5 g lidocaine-prilocaine cream to the heel covering an area of 5 cm2 with an occlusive dressing during 30 min four times a day was carried out. Twelve neonates (5 male, 7 female) with a gestational age of 30.1-36.3 weeks (mean 31.6 weeks) and a birth weight of 1100-2910 g (mean 1665 g) were enclosed. To establish safety, methaemoglobin levels and plasma concentrations of lidocaine, prilocaine and o-toluidine were measured until 24 h after the final application. Methaemoglobin levels were no different from baseline measurements, ranging from 0.2-1.1% and 0.1-0.7% respectively. Plasma concentrations of lidocaine and prilocaine were very low, maxima at 0.230 and 0.223 mg/l respectively. Plasma o-toluidine concentrations remained below the detection limit (0.025 mg/l). CONCLUSION: Application of 0.5 g lidocaine-prilocaine cream to the heel under occlusion four times a day during 30 min is safe in preterm neonates. Establishing safety by measuring the methaemoglobin level by daily application is recommended.

Stability of dithranol in creams.

The stability of the anthrachinone derivative dithranol in creams was studied during storage at temperatures of 4 degrees C and 20 degrees C. Aluminum-coated tubes with 0.1, 0.3 and 0.5% dithranol were stored and samples were analysed immediately and after 3, 6 and 12 months of storage. The 0.3% dithranol cream was also stored in polypropylene tubes. Drug concentration was analysed by high-performance liquid chromatography. All concentrations tested were stable for 12 months of storage at 4 degrees C in aluminum-coated tubes. This means that these low concentrations are sufficiently stable to be prepared in advance for at least 12 months if prepared as described and kept refrigerated. Polypropylene tubes should not be used.

Skin irritation by dithranol cream. A blind study to assess the role of the cream formulation.

In connection with a national cost-effective evaluation study of short contact dithranol therapy for psoriasis, the question arose whether dithranol cream irritation is influenced by constituents of the vehicle. To establish the role of the different components of the vehicle in the mechanism of dithranol irritation, the dithranol 3% cream used in the evaluation study and its vehicle with nine different combinations of its components were tested in a blind study. The creams were applied for 15, 30 and 45 min on the backs of 12 healthy volunteers. Irritation was scored as erythema by visual and colorimeter scoring. The dithranol creams with salicylic acid among their stabilizers showed 42% more irritation than the dithranol creams with only sorbic acid or no stabilizers at all. Stability tests showed no significant degradation of dithranol in the two less irritating creams when kept at 4 degrees C for 11 months. Salicylic acid in the cream aggravates dithranol-induced erythema.

Determination of indinavir, an HIV-protease inhibitor, in human plasma by reversed-phase high-performance liquid chromatography.

A sensitive high-perforrmance liquid chromatographic assay has been developed to determine the concentrations of the HIV-protease inhibitor indinavir in human plasma. The sample pretreatment involved a protein precipitation procedure using 100 microl of human plasma and 400 microl of acetonitrile. Chromatography was carried out on an Octadecyl column using a mobile phase of acetonitrile-water (40:60, v/v). The water phase contained 50 mM phosphate buffer pH 6 and 4 g/l tetramethylammoniumchloride. Ultraviolet detection at 210 nm was used. The method has been validated with regard to specificity, detection limit, lower and upper limit of quantitation, recovery, accuracy, and inter- and intra-assay precision. Stability tests under various conditions were performed. The bioanalytical assay is now in use for the determination of indinavir in several clinical pharmacokinetic studies in HIV-infected patients.

Chiral cognisance: a road to safer and more effective medicinal products.

A quarter of all synthetic medicinal drugs contain a mixture of equal proportions of two molecules that have the same chemical constitution but differ in the spatial arrangement of their constituent atoms such that each is a mirror-image of the other, like the right and left hand. Biologically, receptors which are stereospecific react with only one of the two components of the mixture to produce the desired therapeutic effect, while the other is inactive or may interact with different receptors to cause undesirable, even toxic, effects. Development of syntheses that produce a preponderance of the required form, and efficient separation of mixtures, will result in safer and more effective medicinal products.

Interindividual variation in the capacity-limited renal glucuronidation of probenecid by humans.

A dose of 1,000 mg probenecid was administered orally to 14 human volunteers in order to quantify the maximal rate of formation and excretion of probenecid acyl glucuronide in the urine. Probenecid showed dose-dependent pharmacokinetics. Plasma protein binding of probenecid was high, being somewhat higher in males (90.7 +/- 1.4%) than in females (87.9 +/- 1.4%; p = 0.0019). It was shown that probenecid is metabolized by cytochrome P-450 into at least two phase I metabolites. Each of the metabolites accounted for less than 12% of the dose administered; the main metabolite probenecid acyl glucuronide, representing 42.9 +/- 13.2% of the dose, was only present in urine and not in plasma. The renal excretion rate-time profile of probenecid acyl glucuronide showed a plateau value in the presence of an acidic urine pH. This plateau value was maintained for about 10 h at the dose of 1,000 mg. The height of the plateau value depended on the individual and varied between 250 and 800 micrograms/min (15-50 mg/h). It was inferred that probenecid acyl glucuronide is formed in the kidney during blood-to-lumen passage through the tubular cells. We conclude that the plateau value in the renal excretion rate of probenecid glucuronide reflects its Vmax of formation.

Capacity-limited renal glucuronidation of probenecid by humans. A pilot Vmax-finding study.

Probenecid shows dose-dependent pharmacokinetics. When in one volunteer the dose is increased from 250 to 1,500 mg orally, the t1/2 increased from 3 to 6 h. The Cmax was 14 micrograms/ml with a dosage of 250 mg, 31 micrograms/ml with 500 mg, 70 micrograms/ml with 1,000 mg and 120 micrograms/ml with 1,500 mg. The tmax remained 1 h for all four dosages. The AUC/dose ratio increased with the dose, indicating nonlinear elimination. The total body clearance declined from 64.5 ml/min for 250 mg to 26.0 ml/min for 1,500 mg. The renal clearance of probenecid remained constant, 0.6-0.8 ml/min. Protein binding of probenecid is high (91%) and independent of the dose. The phase I metabolites show lower protein binding values (34-59%). The protein binding of probenecid glucuronide in vitro (spiked plasma) is 75%. Probenecid is metabolized by cytochrome P-450 to three phase I metabolites. Each of the metabolites accounts for less than 10% of the dose administered; the percentage recovered in the urine is independent of the dose. The main metabolite probenecid glucuronide is only present in urine and not in plasma. The renal excretion rate--time profile of probenecid glucuronide shows a plateau value of approximately 700 micrograms/min (46 mg/h) with acidic urine pH. The duration of this plateau value depends on the dose: 2 h at 500 mg, 10 h at 1,000 mg and 20 h at 1,500 mg. It is demonstrated that probenecid glucuronide must be formed in the kidney during its passage of the tubule. The plateau value in the renal excretion rate of probenecid value reflects its Vmax of formation.

Direct high pressure liquid chromatographic analysis and preliminary pharmacokinetics of enantiomers of oxazepam and temazepam with their corresponding glucuronide conjugates.

Three high pressure liquid chromatographic systems for the separation of oxazepam, temazepam and their glucuronides (system A), the separation of their R,S glucuronide diastereomers (system B) and the chiral separation of the parent drugs (system C) are described. Preliminary pharmacokinetics of R,S-oxazepam and R,S-temazepam in a human volunteer reveal that the protein binding of the glucuronides is lower than that of the parent drugs, but that there is no difference in protein binding between the R-oxazepam/temazepam and S-oxazepam/temazepam and their corresponding glucuronides. The S-glucuronide is the main metabolite formed and excreted by man. The plasma ratio R/S-glucuronide is 1:1 for both oxazepam and temazepam. The renal clearance of R-temazepam, and S-temazepam are similar, and those of R-oxazepam and S-oxazepam tend to be different.

Pharmacokinetics of baclofen in spastic patients receiving multiple oral doses.

The pharmacokinetics of racemic baclofen as determined from plasma and urine data in six spastic patients treated with individualized oral doses, 30-80 mg daily, are presented. Peak plasma concentrations were achieved 1.9 h (+/- 0.7) after a dose. The fluctuation in the plasma concentration was great, ranging from 188 to 439%. The total body clearance averaged 175 ml.min-1 (+/- 44), plasma protein binding 35% (+/- 6). Baclofen was for the greater part excreted unchanged by the kidney, 65% (+/- 16). Its apparent renal equalled the creatinine clearance. The contribution of the renal clearance to the total body clearance can explain the previously described toxicity when renal impairment is present. The results agree with earlier reports on single doses in healthy subjects.

Pharmacokinetics of intravenously administered dantrolene and its 5-hydroxy metabolite in dogs.

Dantrolene, a direct acting muscle relaxant used orally for spasticity, has appeared to be effective in the prevention and treatment of malignant hyperthermia in man and animals when administered intravenously. Its pharmacokinetics following intravenous administration have been studied in dogs. Concentrations of dantrolene and its metabolites in plasma, urine, and bile were determined by high-performance liquid chromatography. Recovery of unchanged drug and reduced metabolites was negligible; of the hydroxy metabolite 2% was found in the urine and about 25% in the bile. The half-life of 5-hydroxydantrolene was shorter than that of the parent drug as demonstrated by administration of the metabolite. The apparent renal clearance of 5-hydroxydantrolene was independent of creatinine clearance, urine flow and pH, and appeared to be reduced in the presence of probenecid. Bile to plasma ratios of the hydroxy metabolite were high with biliary concentrations far exceeding the maximum solubility in water. The results of this pilot study indicate that hydroxylation is primarily responsible for the excretion of the dantrolene molecule from the body.

HPLC analysis and pharmacokinetics of the enantiomers of R,S-oxazepam and R,S-temazepam with their corresponding glucuronide conjugates in urine and plasma of man.

R,S-Oxazepam and R,S-temazepam can be separated in their enantiomers by means of a chiral AGP column. The corresponding R- and S-glucuronide conjugates can be separated on a normal reversed-phase C18 column. Man conjugates the S-enantiomer of oxazepam and temazepam both better than the R-enantiomer. The urinary recovery of the glucuronides following either R,S,-oxazepam or R,S-temazepam almost amounts to 100% of the dose administered.

Plasma and urinary excretion kinetics of oral baclofen in healthy subjects.

Baclofen, a centrally acting muscle relaxant, is used in the treatment of spasticity. Its pharmacokinetics has been derived from plasma and urine data in four healthy subjects, whose renal function was simultaneously measured. After oral administration of a single 40 mg dose, baclofen was mainly excreted unchanged by the kidney, 69 (14)%. The half-life, calculated from extended least squares modelling (ELSMOS) both of plasma and urine data was 6.80 (0.68) h, which is longer than reported in most studies based solely on plasma data. The renal excretion rate constant had the high mean value of 0.35 (0.24) h-1, and the apparent renal clearance of baclofen equalled the creatinine clearance. Passive tubular reabsorption is relatively unimportant, since no dependence was observed on variables urine flow or pH. Although active tubular secretion may contribute to its renal clearance, as shown by the effect of co-administration of probenecid, glomerular filtration appears to be the dominant transport mechanism.