Development and validation of a sensitive and specific radioimmunoassay for the determination of cicaprost in biological samples.

Cicaprost is a potent, chemically and metabolically stable PGI2-mimetic. Pharmacodynamic effects were observed after oral administration of approximately 10 micrograms in man when plasma levels were in the low pg-range. The present report describes the development of a selective antiserum and a tracer with high specific activity and their use for the RIA determination of Cicaprost in biological samples. Cicaprost-[3H]-methylester with a specific activity of 819 GBq/mmol was used as a tracer. RIA-analyses were carried out with 0.05-0.5 ml plasma adjusted to pH 2 with 1 N HCl and extracted with 2.5 ml diethylether. Separation of antiserum bound and unbound Cicaprost was achieved by the charcoal method. Extraction recovery of Cicaprost was approximately 90% at pH approximately 2. The detection limit of the assay was 10-20 pg/ml plasma. Coefficients of variations were 6, 3 and 9% (within-day, n = 5) and 25, 12 and 10% (day-to-day, n = 11) at 50, 100 and 200 pg/ml. HPLC-chromatograms of plasma extracts did not reveal any peak apart from Cicaprost, demonstrating the specificity of the method. The present RIA for Cicaprost exhibits high specificity and sensitivity and will be used for further bioanalyses in pharmacokinetic study.

Development, validation and practical use of a sensitive and specific radioimmunoassay for the determination of iloprost.

Iloprost is a potent, clinically effective PGI2-mimetic. Therapeutic plasma levels are in the low pg-range and currently analyses of biological samples are performed by GC/MS after antiserum-column extraction. Although this method exhibits high sensitivity and specificity it permits only limited numbers of samples to be analyzed owing to time-consuming work-up. The present report describes the development of a novel highly selective antiserum and its use for the RIA determination of iloprost in biological samples. An antiserum was raised against "iloprost-9-pentynyl"-BSA in rabbits. Iloprost-[3H]-methylester with a specific activity of 66.9 Ci/mmol was used as a tracer. RIA-analyses were carried out with 0.05-0.5 ml plasma adjusted to pH2 with 1 N HCl and extracted with 2.5 ml diethylether. Separation of antiserum bound and unbound iloprost was achieved by the charcoal method. Extraction recovery of iloprost was approximately 90% at pH less than or equal to 4. The detection limit of the novel assay was 1-2 pg/tube corresponding to 5-10 pg/ml plasma (if 0.1-0.2 ml plasma was used). Coefficients of variations were 8% and 2% (within-day, n = 3) and 17% and 12% (day-to-day, n = 5) at 50 and 100 pg/ml. RIA- and GC/MS-levels of iloprost measured in human samples were similar (p less than 0.001). Cross-reactivity HPLC-chromatograms of plasma extracts did not reveal any peak apart from iloprost. The RIA-method exhibits both a similar specificity and detection limit to GC/MS and will be used for further analyses.

Pharmacokinetics and pharmacodynamics of cicaprost in healthy volunteers after oral administration of 5 to 20 micrograms.

In a Phase I study, the tolerability, pharmacodynamics and pharmacokinetics of cicaprost have been investigated in 6 male volunteers given 5, 10, 15 and 20 micrograms as tablets of the beta-cyclodextrin clathrate. Individual inhibition of platelet aggregation and changes in facial colour (measured by chromametry) were dose-dependent and reached a maximum 30 to 60 min post-dose. The maximum inhibition of platelet aggregation was about 40%. After 3 to 4 h pre-treatment values had returned. Blood pressure remained within the normal range. The peak plasma level of cicaprost was reached within 15 to 90 min after drug intake. Both Cmax- and AUC were individually dose-dependent. The terminal half-life in plasma of cicaprost was approx. 1 h, and its total clearance amounted to 4-7 ml.min-1.kg-1. The time courses of the plasma levels and of the pharmacodynamic actions were in agreement. Interindividual differences were observed in the occurrence of unwanted effects (e.g. headache). Thus, cicaprost is an orally available PGI2-mimetic, for which effects on platelet aggregation and vascular perfusion have been demonstrated in healthy volunteers after doses of 5 to 15 micrograms.

Development and application of a radioimmunoassay of the new progestagen gestodene.

A radioimmunoassay for the determination of gestodene (17-ethinyl-13-ethyl-17 beta-hydroxy-4,15-gonadien-3-one) in human plasma is described with regard to procedure, specificity, accuracy and reproducibility. Antiserum was raised against gestodene-3-O-(carboxymethyl)oxime-BSA in rabbits and [9,11-3H]-gestodene tracer was used with a specific radioactivity of 2.16 TBq/mmol. The final antiserum dilution was 1: 200,000. RIA was performed according to routine methods using diethylether plasma extracts and the charcoal separation technique. Cross-reactivity of antiserum with cortisol, 17 beta-estradiol, progesterone, testosterone and ethinylestradiol was less than 0.03%; levonorgestrel exhibited a 5% cross-reactivity. No cross-reactivity with metabolites of gestodene or ethinylestradiol was found. Accuracy and precision of the assay were tested using human plasma samples spiked with 1, 5 and 10 ng/ml gestodene. Accuracy was within 94 to 104% of the nominal values. Within-assay and between-assay coefficients of variation were in the range of 4.7-6.5% and 10.3-13.1%, resp. This RIA was used to follow plasma gestodene levels after single oral administration of 75 micrograms of gestodene combined with 30 micrograms ethinylestradiol as tablet and coated tablet in a cross-over design in 6 female test subjects. Plasma gestodene levels were equivalent after both treatments.

Radioimmunological determination of levonorgestrel and ethinylestradiol.

A sensitive and specific radioimmunoassay for ethinylestradiol (EE2) and levonorgestrel (LN) has been developed. Antisera were raised in rabbits after injection of EE2-6-carboxymethyloxime-aminocaproic acid and LN-3-carboxy-methyloxime linked to bovine serum albumin. The sensitivity of immunoassay was 15 pg/ml for EE2 and 10 pg/ml for LN. The cross-reactions showed a high specificity of the assay, and the accuracy and precision of this method satisfied the requirements for the investigation of pharmacokinetics. With this assay, although the recovery of the assay was not very high (the coefficient of variation 14.02% for EE2 and 10.27% for LN) when used to analyse diethylether extracts of biological fluids, a microquantitative metabolite of norethisterone (NET) and norethisterone-3-oxime (NETO), EE2 analogue in incubation solution with rat hepatocytes, was satisfactorily detected. The result showed that in vitro the amount of NETO transferred to EE2 analogue was three times as much as that of NET. It is necessary to further establish the identity and activity of the analogue, but these data will be found advantageous to the research of steroid-oxime.

Systemic availability and abortion-inducing effects of nocloprost after intravenous, subcutaneous and intragastric administration to pregnant guinea pigs.

Nocloprost was administered to 3 groups of 4 pregnant guinea pigs intravenously and subcutaneously in a dose of 30 micrograms/kg and intragastrically in a dose of 100 micrograms/kg. Plasma nocloprost levels were measured at defined times up to 24 h p. adm. with a specific radioimmunoassay and induction of abortion monitored simultaneously. In one animal per group uterus pressure was recorded continuously up to 8 hours p.adm. Animals were sacrificed 7 days p.adm. and the number and state of fetuses in utero evaluated. Systemic availability of unchanged drug was 100% after intravenous (AUCi.v. = 8.6 +/- 2.0 ng h/ml) and subcutaneous (AUCs.c. = 11.5 +/- 1.2 ng h/ml) administration and approximately 30% after intragastric administration (AUCi.g. = 8.9 +/- 2.0 ng h/ml). The incidence of abortion after intragastric administration corresponded to that after subcutaneous administration. After intravenous injection the abortion rate was somewhat less, indicating that equal AUC-values do not necessarily indicate identical pharmacological effects.

Pharmacokinetics of lisuride after subcutaneous infusion.

Six parkinsonian patients received a constant subcutaneous infusion of 60 micrograms lisuride per hour in the abdominal region for 2 hours. Plasma levels of the unchanged drug were measured by radio-immunoassay. During infusion, a steady state plasma level of 0.78 +/- 0.19 ng/ml was achieved. After discontinuation of the infusion, concentrations declined with a half-life of 1.4 +/- 0.4 hour. The total clearance of lisuride was 20 +/- 6 ml/min/kg. Due to the low interpatient variability of plasma levels, a good control of clinical effects is to be expected.

Development of antibody-mediated extraction followed by GC/MS (antibody/GC/MS) and its application to iloprost determination in plasma.

A new analytical principle has been developed combining the features of both radioimmunoassay and GC/MS. Its application in eicosanoid analysis was tested with the prostacyclin analogue, iloprost. The iloprost antibody, generally employed in RIA measurements, was coupled to Sepharose 4B and used as stationary phase for extraction of the drug. Variations in recovery were corrected by using deuterated iloprost as an internal standard. The samples were derivatized and quantitated by negative-ion chemical ionization-mass spectrometry. Reproducibility was 2.3 % at the 50 pg/ml level and the limit of detection was 5 pg for 1 ml samples. With plasma volumes of up to 20 ml, 0.25 pg/ml could be determined. Antibody/GC/MS proved superior to radioimmunoassay due to its higher specificity and sensitivity and superior to GC/MS with conventional clean-up procedures because of a higher sample capacity.

Development of a radioimmunoassay and its application to the pharmacokinetics of the stable prostacyclin analogue, nileprost, in the rat.

The development of a sensitive radioimmunoassay for measuring concentrations of nileprost , a stable prostacyclin analogue, is described. The RIA method, furthermore, is applied to evaluate the pharmacokinetics of nileprost in the rat. Antiserum against nileprost was raised in rabbits by immunizing with the drug coupled to bovine serum albumin via the carboxylic group. Nileprost is extracted from plasma samples using a reversed-phase Sep- PakR cartridge. The limit of detection is less than 10 pg/ml. At the 1.25 ng/ml level intra- and inter-assay variations of 6 and 16% were calculated. Following intravenous injection of 200 micrograms/kg to female Wistar rats, a three-phasic decline in plasma levels was observed with half-lives of 10 min, 30 min and 7.4 h. Oral doses of 200 and 2,000 micrograms/kg were very rapidly absorbed exhibiting maximum concentrations in the plasma of 20 and 30 ng/ml as early as 5 min postadministration . Bioavailability of nileprost was calculated to be 21 and 13% of the dose. The disposition half-life was 5-7 h in both cases.

The pharmacokinetics of fluocortolone and prednisolone after intravenous and oral administration.

The pharmacokinetics of fluocortolone and of prednisolone were examined following a single intravenous injection of 5 mg and oral administrations of 10 and 20 mg to five healthy male volunteers. After intravenous injection the plasma levels of fluocortolone decreased biexponentially with half-lives of 9 +/- 5 min and 1.3 +/- 0.3 h. Total plasma clearance of fluocortolone was calculated to be 7.0 +/- 1.5 ml/min/kg. Oral administrations of fluocortolone revealed maximum plasma levels of 86 +/- 12 ng/ml (10-mg dose) and 174 +/- 34 ng/ml (20-mg dose) after 1.4 +/- 0.2 h. Intravenously administered prednisolone was rapidly distributed (t 1/2 alpha = 5 +/- 3 min) but more slowly eliminated from plasma than fluocortolone (t1/2 beta = 3.1 +/- 0.8 h). Correspondingly a total plasma clearance of 2.2 +/- 0.2 ml/min/kg was calculated. Oral administrations revealed maximum prednisolone plasma levels of 172 +/- 25 ng/ml (10-mg dose) and 278 ng/ml (20-mg dose) after 1.6 +/- 0.7 h. The absolute bioavailability of both corticosteroids was higher than 80% and independent of both dose levels investigated.

Pharmacokinetics and pharmacodynamics of the ergot derivative, transdihydrolisuride, in man.

Plasma levels and urinary excretion of the dopamine agonist, transdihydrolisuride (TDHL), were measured by radioimmunoassay in healthy male volunteers given TDHL 50 micrograms i.v. and oral doses of 200, 400 and 800 micrograms. Plasma prolactin was also measured by radioimmunoassay. Following i.v. injection, the concentration of TDHL declined with a half-life of 37 +/- 19 min. The total clearance was 38 +/- 27 ml/min/kg and the apparent volume of distribution was 1.3 +/- 0.41/kg. The bioavailability of oral TDHL was proportional to the dose; after 200, 400 and 800 micrograms the bioavailability was 20 +/- 25%, 31 +/- 24% and 48 +/- 26%. TDHL was almost totally metabolized and less than 0.5% of the dose was excreted unchanged in urine in 24 h. Plasma prolactin levels were depressed by 66 +/- 15%, 75 +/- 11% and 80 +/- 7% after TDHL 200 micrograms, 400 micrograms and 800 micrograms. The effect lasted for more than 12 h after the lowest dose and for more than 24 h after 400 and 800 micrograms. Side effects, mainly nausea and headache, only occurred at the two highest dose levels.

Pharmacokinetics and biotransformation of the prostacyclin analogue, ZK 36 374. III. Development of a radioimmunoassay and its application to the pharmacokinetics of ZK 36 374 in the rat.

The development of a radioimmunoassay for ZK 36 374, a chemically stable prostacyclin analogue, and its application to the pharmacokinetics of the drug in the rat is described. Antiserum against ZK 36 374 was raised in rabbits by immunization with ZK 36 374 coupled to bovine serum albumin via the carboxylic group. ZK 36 374 is extracted from plasma samples and purified from matrix constituents by means of octadecyl silyl cartridges. At a plasma concentration of 2.5 ng/ml intra- and interassay variations are 6 and 7%, respectively. The limit of detection is 0.5 ng/ml. After intravenous administration of 200 micrograms/kg to female Wistar rats a biphasic decline of the drug concentration in the plasma was observed with half-lives of 5 min and 3 h. Oral doses of 200 and 2000 micrograms/kg were very rapidly absorbed reaching maximum plasma levels of 3 and 32 ng/ml as early as 10-15 min p.admin.. Bioavailability at the two dose levels was calculated to be 8 and 13% of dose. Disposition was biphasic, as well, with a somewhat prolonged beta-phase after the highest dose.

A highly specific radioimmunological determination of sulprostone (1) using antisera against two different haptens.

A sensitive and specific radioimmunoassay for sulprostone in serum has been developed. The use of antisera raised against two different haptens allows the determination of sulprostone alone or together with its main metabolites. Radioactive tracers with high specific radioactivity were synthesized by coupling the haptens to [125I]-iodohistamine.

Radioimmunoassay of plasma lisuride in man following intravenous and oral administration of lisuride hydrogen maleate: effect on plasma prolactin level.

The development of a sensitive radioimmunoassay for the determination of lisuride in plasma is described. The antiserum against lisuride-4-hemisuccinate-BSA was raised in rabbits. Using this method the plasma levels of lisuride were monitored following one intravenous (25 microgram) and two oral (100 microgram and 300 microgram) doses of lisuride hydrogen maleate in three female and three male volunteers (intra-individual comparison). The plasma prolactin was also determined by radioimmunoassay. Following i. v. injection, the concentration of lisuride declined in three phases, with half-lives of 5 min, 25 min and 2 h. The total plasma clearance of 800 +/- 250 ml X min-1 was in the range of "plasma flow" through the liver. In agreement with the high rate of biotransformation, the bioavailability of lisuride administered orally was 10% +/- 7% of the 100-microgram dose, and 22% +/- 7% of the 300-microgram dose. The plasma prolactin was lowered to 3%-18% of its pretreatment value depending on the route of administration and the dose. The reduction appeared to be short-lived and to be directly dependent on the plasma concentration of lisuride. Following intravenous injection, the prolactin level declined after a so far unexplained lag-time of 0.5 h.

Effect of cyproterone acetate and its metabolites on the adrenal function in man, rhesus monkey and rat.

The effect of oral administration of 17 alpha-acetoxy-6-chloro-1,2 alpha-methylene-4,6]pregnadiene-3,20-dione (cyproterone acetate, Androcur) on the adrenal function in male hypersexual subjects and in female rhesus monkey was investigated on the basis of cortisol and ACTH levels in serum or plasma and excretion of cortisol and 17-ketosteroids. In addition, cyproterone acetate and its main metabolite alcohol of the main metabolite were characterized for treatment of male hypersexual subjects and female rhesus monkeys did not reveal any signs of adrenal suppression. Cyproterone acetate and its metabolites gave on indication of any appreciable anti-inflammatory effect in the adjuvant edema test in rats. However, there was a general increase in the level of blood glucose and liver glycogen as well as a reduction in body weight and organ weight (spleen, thymus and adrenal) in rats, in which 15 beta-hydroxy cyproterone was slightly more active with the exception of adrenal weight tests. It can be concluded that adult man and rhesus monkey are much less sensitive, is so at all, to some corticosteroid-like activities of cyproterone acetate and its main metabolites than the rat.

Kinetics and biotransformation of lormetazepam. II. Radioimmunologic determinations in plasma and urine of young and elderly subjects: first-pass effect.

A specific and sensitive radioimmunoassay has been developed for a new benzodiazepine, lormetazepam. After intravenous injection, lormetazepam levels in plasma fell in three (alpha, beta, gamma) dispositional phases, two of them (alpha, beta) mainly reflecting different distribution processes. The terminal (gamma) phase correlated well with the rate of renal elimination of glucuronides. Oral doses were completely absorbed with widely varying absorption half-lifes (t1/2s) amounting to an average of 0.67 +/- 0.53 hr. Dose-dependent maximum plasma levels were reached in about 2 hr. Lormetazepam undergoes first-pass metabolism of about 20% of an oral dose. Total clearance was in the range of 200 ml/min. There was a trend toward slower terminal disposition phase in elderly subjects. In younger subjects, the terminal phase t1/2 was about 10 hr. Lormetazepam glucuronide peak plasma levels were reached by about 6 hr. Thereafter, the level fell in one (elimination) phase, with a t1/2 of 12 hr in young subjects and with a significantly (p < 0.05) different t1/2 of about 20 hr in the elderly. Renal clearance was calculated as about 30 to 40 ml and did not show an age-dependent difference. Recovery of lormetazepam glucuronide with urine amounted to 70% to 80% of the dose during 72 hr after intravenous injection in both age groups.

Investigations of pharmacokinetics of ethinyloestradiol to specific consideration of a possible first-pass effect in women.

Ethinyloestradiol-3H was given intravenously and orally to four and three women, respectively, in a dose of 60 micrograms and 3 mg. To another three female volunteers, 100 micrograms of ethinyloestradiol was administered by both routes in succession. Drug concentration in plasma and total radioactivity in plasma, urine and faeces were measured for different periods of time. Intraindividual comparison of the area under the drug level vs. time curve after intravenous and oral administration of 100 micrograms showed that ethinyloestradiol is subject to an about 60% first-pass effect in women. The time course of ethinyloestradiol concentration in plasma can be described by a 3-compartment model after intravenous injection and by a 2-compartment model after oral administration, because an early disposition phase with a half-life of about 15 minutes only becomes visible after i.v. injection. On an average, the terminal half-life of unchanged ethinyloestradiol level and total radioactivity was calculated to be about 1 day. However, a high variability was found with this parameter as well as with the rate and degree of elimination in urine.

PIP: Investigations of pharmacokinetics of ethinyl estradiol (EE) to specific consideration of a possible 1st-pass effect in women are reported. Tritiated EE was given intravenously and orally to 4 and 3 women, respectively, in a dose of 60 mcg and 3 mg. 3 female volunteers received 100 mcg of EE by both routes in succession. EE concentration in plasma and total radioactivity in plasma, urine, and feces were measured for different periods of time. Intraindividual comparison of the area under the drug level vs time curve following iv and oral administration of 100 mcg showed that EE is subject to about 60% 1st-pass effect in women. The time course of EE concentration in plasma can be described by a 3-compartment model after iv injection and by a 2-compartment model after oral administration, because an early disposition phase with a 1/2-life of about 15 only became visible after iv injection. The terminal 1/2-life of unchanged EE level and total radioactivity was calculated to be about 1 day.