Oral iloprost in the treatment of thromboangiitis obliterans (Buerger's disease): a double-blind, randomised, placebo-controlled trial. The European TAO Study Group.

OBJECTIVES: To assess efficacy and tolerability of two dosages of the oral prostacyclin analogue iloprost versus placebo in thromboangiitis obliterans (TAO). DESIGN: Placebo-controlled, double-blind, study; TAO patients randomised to iloprost 100, 200 micrograms, or placebo bid for 8 weeks, with 6 months' follow-up. METHODS: Three-hundred and nineteen TAO patients with rest pain, trophic lesions (or both) from 23 clinics in six European countries. Primary endpoint: total healing of most important lesion. Secondary endpoint: relief of rest pain without need of analgesics. Combined endpoint: alive without major amputation, no lesions, no rest pain, no use of analgesics. RESULTS: Total healing of lesions was not significantly different between treatment groups at any time point. For relief of rest pain without need of analgesics, low dose (LD) iloprost was significantly more effective than placebo at end of follow-up (placebo 49%; LD iloprost 63%; p = 0.020). This also applied to the combined endpoint (placebo 35%; LD iloprost 50%; p = 0.016). High dose iloprost (HD) failed to show significant treatment effects over placebo. CONCLUSIONS: Iloprost LD was significantly more effective than placebo for relief of rest pain without need of analgesics and for a combined endpoint at 6 months of follow-up, whilst both iloprost doses showed no significant effects vs. placebo on total healing of lesions.

Internalization and down-regulation of the prostacyclin receptor in human platelets.

The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 degrees C, addition of unlabelled ligand at either 37 degrees C or 4 degrees C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 degrees C or 4 degrees C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 degrees C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 degrees C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 degrees C, two classes of binding sites were present, whereas after preincubation at 37 degrees C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.

Pharmacokinetics and pharmacodynamics of intra-graft iloprost in femorodistal bypass surgery.

Intra-graft injection of the prostacyclin analogue, iloprost, was performed at the end of femorodistal bypass procedures in 12 patients with severe peripheral arterial occlusive disease. Iloprost plasma levels were measured and compared with changes in haemodynamics. There was a high initial iloprost plasma level (mean 625 pg/ml) which dropped to a mean of 50 pg/ml after 15 min. This correlated with an immediate reduction in systolic blood pressure which had returned to pretreatment levels after 15 min. In contrast, the vascular resistance distal to the graft showed a reduction after 5 min which was maintained for at least 20 min after iloprost injection and the mean blood flow through the graft increased steadily throughout the same period of measurement. The study showed an effect of iloprost on blood pressure which correlated with plasma levels, but the time course of the changes in distal vascular resistance and graft blood flow demonstrated an effect more prolonged than the half-life of iloprost.

Pharmacokinetics and tolerability of oral iloprost in thromboangiitis obliterans patients.

OBJECTIVE: Iloprost is a potent PGI2 mimetic, which has been shown to be therapeutically effective in several vascular disorders. Due to its rapid clearance from the central compartment, iloprost is administered mainly by i.v. infusion, which limits its use to hospitalized patients. In order to improve pharmacotherapeutic use of this PGI2 mimetic, an oral extended-release (ER) dosage form has been developed, which should mimic plasma level profiles as observed after i.v. infusion and serve as a therapeutic equivalent. METHODS: This trial was performed to investigate the tolerability and pharmacokinetics of iloprost administered perorally, compared with i.v. infusion, in 12 patients suffering from thromboangiitis obliterans (TAO). A dose titration was carried out for 1 week with i.v. iloprost, followed by a p.o. titration and treatment phase of 3 weeks' duration. Pharmacokinetics was investigated at the individually tolerated dose levels; i.e., on days 5-7 (i.v. infusion at 2, 2.5 and 3 ng.kg-1.min-1), and twice during p.o. treatment after b.i.d. administration of 50, 100, 150, 200 or 300 micrograms. RESULTS: Individual tolerability of iloprost varied: 7 patients out of 12 tolerated the maximum i.v. dose of 3 ng.kg-1.min-1; six tolerated the maximum oral dose of 600 micrograms. No patients withdrew from the study due to adverse events. Flush and headache were the most common adverse events and seemed to be related to the study drug. After i.v. infusion of iloprost, dose-normalized (3 ng.kg-1.min-1), steady-state plasma levels were 260 pg.ml-1. Terminal half-life was 0.57 h. Total clearance ranged from 8 to 17 ml.min-1.kg-1. Peroral administration of the ER formulation resulted in dose-dependent Cmax and AUC values. AUC values of the first and second daily dose interval, i.e., 0-5 h and 5-11 h after first dosing, were almost identical. Absolute bioavailability was 24%, with the exception of two patients who tolerated only 50 micrograms b.i.d. and exhibited a bioavailability of approx. 60%. The AUC values observed in weeks 2 and 4 were identical, demonstrating low day-to-day variability of iloprost plasma level profiles in TAO patients. CONCLUSION: Based upon pharmacokinetic data, the ER formulation provides an equivalent to the i.v. infusion of iloprost and broadens the range of therapy to nonhospitalized patients. The availability of capsules with 50 and 100 micrograms iloprost enables individual dose titration and pharmacotherapy. Beneficial effects, as observed with i.v. iloprost in TAO patients, should therefore be achievable by peroral pharmacotherapy using the new ER formulation.

A high performance liquid radiochromatographic assay for the simultaneous analysis of iloprost and misoprostol.

A high-performance liquid chromatographic (HPLC) method utilizing ultraviolet absorbance coupled with radioisotove detection was developed for the precise and simultaneous determination of iloprost and misoprostol. This assay allows complete resolution of iloprost diastereoisomers and has a total run time of approximately twenty minutes. Samples were prepared for chromatographic analysis by extracting a mixture of tritiated drugs from rat plasma with acetonitrile. The resulting solutions were chromatographed on a reversed phase Zorbax Rx-C8 column using 0.02M potassium phosphate (pH 3.0), acetonitrile, and methanol (46:30:24, v/v) at a flow rate of 1.7 mL/min. 2-Naphthoic acid was employed as an internal standard. The correlation coefficient for varying concentrations of tritiated iloprost (12.7 Ci/mmol specific activity) from 2.18 ng/mL to 21.8 ng/mL was 0.995, and the correlation coefficient for concentrations of tritiated misoprostol (50 Ci/mmol specific activity) from 0.617 ng/mL to 6.17 ng/mL was 0.993. The high selectivity and sensitivity of this assay make it useful for the simultaneous quantitation of iloprost and misoprostol.

Platelet PGI2 receptor affinity is reduced in pre-eclampsia.

Prostacyclin (PGI2) receptors were studied in platelet membrane preparations from women with normal pregnancy, pregnancy-induced hypertension (PIH) or pre-eclampsia. Patient groups showed no differences in gestational week at delivery. A markedly lower birth weight, however, was found in pre-eclampsia. No differences between groups could be detected in platelet PGI2 receptor number. In contrast, the binding affinity to the PGI2 mimetic iloprost was considerably reduced in pre-eclampsia, whereas receptor affinity between PIH and normal pregnancy did not differ significantly.

Inter-species extrapolation of pharmacokinetic data of three prostacyclin-mimetics.

Cica-, eptalo- and iloprost are chemically and metabolically stabilized derivatives of prostacyclin which maintain the pharmacodynamic profile of the endogenous precursor. While iloprost is still subject to beta-oxidative degradation of the upper side chain, cicaprost is highly metabolically stable. Eptaloprost was synthesized to realize the pro-drug concept in PGI2-mimetics and was designed to be activated to cicaprost by single beta-oxidation. All three prostacyclin-mimetics were studied in various animal species (mouse, rat, rabbit, monkey, dog and pig) and in man to determine their pharmacokinetic profiles. Based upon this data, it was of interest whether an inter-species extrapolation of pharmacokinetic parameters can be performed to show the predictive value of animal experimentation. Allometric inter-species extrapolation is performed by modelling pharmacokinetic data (Y) as exponential functions (x) of species characteristics (e.g. body weight, W) as: Y = .aWx. For total clearance and volumes of distribution at steady state, a clear-cut correlation with x-values of 0.6-0.8 and 1.0-1.1 could be shown for all three compounds. For cicaprost, which was excreted unchanged in several species, renal and non-renal clearance was also mathematically scalable. Due to the use of different compartment models to describe plasma disposition, different sets of half-life data were obtained and could not be extrapolated reasonably. However, mean residence time showed a dependency on body weight with 0.25 as power function. In case of cicaprost, only the dog, which extensively metabolizes the compound, could not be enrolled in inter-species extrapolation. Excretion half-lives or residence times did not show a significant correlation to body weight or maximum life time potential. The present inter-species extrapolation showed a dependency from species body weight for model-independent pharmacokinetic data, e.g. clearance, volume of distribution at steady state and correspondingly mean residence time. The disposition profile of these compounds can therefore be predicted. Preliminary information on bio-degradation is an additional prerequisite for extrapolation. These data demonstrate that basic physiologically determined processes, which show some evolutionary allometric dependency, also influence the disposition of prostacyclin-mimetics. An extrapolation of data from animal to man could easily be realized giving additional justification for animal studies in pharmacology, toxicology and pharmacokinetics.

Absorption of the stable prostacyclin analogue iloprost through the ulcer base in chronic venous insufficiency.

Iloprost, a stable prostacyclin analogue, is known to have beneficial effects on the disturbed microcirculation. To develop a topical application for venous leg ulcers it is necessary to know the extent to which iloprost might be absorbed through the ulcer base. The aim of this study was to measure the absorption kinetics of iloprost solutions in increasing concentrations and doses (0.004% [15 micrograms]-0.006% [30 micrograms]) in 23 patients. There was considerable variation amongst the patients in the amount of iloprost absorbed. In 40% of patients no iloprost could be detected in the plasma, whereas in others up to 82% of the iloprost applied was absorbed through the ulcer base. High iloprost plasma levels provoked flushing in two patients. The highest plasma levels were always reached during the first hour after application. There was no direct relation between the ulcer size and the amount of iloprost absorbed. Iloprost concentrations up to the highest concentration applied (0.006%) were well tolerated locally.

Bioactivation of eptaloprost in animals and man.

Eptaloprost is a novel concept PGI2-mimetic, which is designed to be activated to the pharmacologically potent cicaprost via beta-oxidation. By pro-drug formation advantages in terms of sustained delivery of prostacyclin-mimetic activity were envisaged. The active metabolite is known to be metabolically stable and highly pharmacologically potent. In the present set of experiments the pharmacokinetics of eptaloprost was studied in rat, monkey and man by i.v. and ig administration of tritiated compound. Eptaloprost was completely and rapidly absorbed in all three species. Peak plasma levels of the parent compound were observed within 30 min postdose. Total clearance of the pro-drug accounted for 170, 62 and 66 ml/min/kg in rat, monkey and man. Disposition of eptaloprost exhibited half-lives of 0.1 to 0.5 h and mean residence times accounted for 0.15, 0.4 and 0.6 h in the three species. The active metabolite cicaprost was present in the central compartment with a slight delay as compared to eptaloprost. Its peak plasma levels were found within 0.25 to 0.5 h postdose. Disposition of radiolabel in plasma and 3H-excretion with the urine and feces was determined by the pharmacokinetic behaviour of cicaprost. In rats excretion was mainly biliary while monkeys and man excreted almost unchanged cicaprost in equal portions with urine and feces. Half-lives of renal excretion were in the range of terminal half-lives in the central compartment. Neither in animals nor in man eptaloprost administration resulted in an advantageous systemic profile of cicaprost. On the contrary the bioavailable dose fraction of cicaprost was lower as compared to cicaprost administration. A delay or an extension of cicaprost plasma levels was not observed. The present pharmacokinetic data of eptaloprost studied in three species demonstrated that a pro-drug concept based on simple beta-oxidative bioactivation could be successfully realized for a special PGI2-mimetic. An advantage resulting from oral pro-drug administration as compared to direct treatment with the active metabolite could not be shown. For long-lasting plasma levels of cicaprost a chemically determined retardation might require a more sophisticated pro-drug concept or alternatively pharmaceutical technology is required.

Comparison of bioanalytical determinations of Iloprost, a chemically stable PGI2 mimetic, by conventional radioimmunoassay (RIA) and scintillation proximity assay (SPA).

The scintillation proximity assay is a novel variant of classical radioimmunoassay. It can be performed as a single tube measurement because the separation of bound and unbound tracer fraction is avoided. In principle, microbeads are coated with anti-species antibodies that can couple with the respective antiserum used for RIA. By means of special cores, light emission takes place if labelled, antiserum-bound tracer is coupled to the anti-species antibody on the fluomicrosphere surface. In the present report, the novel assay was compared to a validated RIA for the bioanalysis of the PGI2 mimetic, Iloprost. Extraction recovery of Iloprost was approximately 90% at pH less than or equal to 4. The detection limit of the novel assay was 2-4 pg/sample, corresponding to 10-20 pg/ml plasma (if 0.2 ml plasma was used). Coefficients of variations were 9, 7 and 6% (within-day, n = 5) and 30, 11 and 10% (day-to-day, n = 10) at 50, 100 and 200 pg/ml. RIA and SPA levels of Iloprost measured in human plasma samples (n = 428) were similar. The SPA method exhibits both a similar specificity and detection limit to RIA and will be used for further analyses.

Characterization of oral sustained release preparations of iloprost in a pig model by plasma level monitoring.

Iloprost (5-[(E)-1S,5S,6R,7R)-7-hydroxy-6-[(E)-3S,4RS)-3-hydroxy-4- methyl-octen-6-inyl]-bicyclo[3.3.0]-oct-3-ylidene)-pentanoic acid) is a chemically stable PGI2-mimetic with high pharmacological potency. Therapeutic efficacy in various disease stages (e.g. peripheral arterial occlusive disease, M. Raynaud and thromboangiitis obliterans) was shown after repeated once-a-day infusion treatment over several weeks. In order to facilitate drug therapy an oral dosage form is desirable. As a first step, a suitable animal model was needed to screen several formulation variants prior to characterization of promising candidates in man. After intravenous infusion treatment, the pig exhibited - similar to man - strictly dose-dependent steady state plasma levels and a total iloprost clearance of approximately 26 ml/min/kg (man approximately 20 ml/min/kg). Partial similarity of physiology and anatomy of the GI-tract and the possibility to administer intact capsule dosage forms led to a series of screen experiments with several sustained release preparations (pellets and matrix tablets) of iloprost exhibiting different in-vitro drug release profiles. A good correlation of in-vitro dissolution and in vivo plasma level data was obtained for all preparations containing the pellet neutral polymer. For the other formulations slight differences between duration of liberation and plasma level or time of maximum dissolution rate and tmax of plasma levels was observed. In the case of ionized polymers or matrix tablets, in vitro dissolution profiles were slightly different from in vivo data. This might be due to different dissolution behaviour in the gastro-intestinal tract. The pig seems to be a model that is suitable for verifying drug liberation profile in-vivo. Based upon plasma levels obtained in animals, selection of a formulation for characterization in man can be made.

Pharmacokinetics of iloprost in patients with hepatic dysfunction.

In the present experiment the pharmacokinetics of iloprost was studied in eight hospitalized patients suffering from liver cirrhosis (mean age: 56 years, 3 females, 5 males, child-classification: A [n = 1], B [n = 5], C [n = 2]/mean 14C-aminopyrine breath test: 3.9% dose/2 h). Iloprost was administered as a 1 h-i.v. infusion with 1 ng/kg/min to all the test subjects. Steady state plasma levels of 93 +/- 31 pg/ml were observed at the end of infusion. The terminal half-life of iloprost was 28 +/- 24 min. From AUC values of 126 +/- 60 pg.h/ml a total clearance of 10 +/- 5 ml/min/kg was calculated. The study demonstrated that iloprost clearance was reduced by a factor of 2 in patients suffering from hepatic dysfunction compared with healthy subjects. Individual dose titration is the recommended dose regimen for iloprost therapy in all patients. Therefore, apart from a reduction of the starting dose (of approximately 50%) for titration, special recommendations are not necessary for patients with impaired liver function.

Pharmacokinetics of iloprost in patients with severe peripheral arterial occlusive disease.

The pharmacokinetics of iloprost were studied in 12 hospitalized patients suffering from severe peripheral arterial occlusive disease (PAOD) stages III or IV according to Fontaine. The patients were 8 males and 4 females aged from 49 to 83 years. Apart from PAOD, several other concomitant diseases were present, e.g. myocardial and/or renal insufficiency, diabetes and hypertension. Patients were treated daily with i.v. infusions of iloprost at dosages of 1.0-3.3 ng/kg/min over a period of 4 to 6 h. Dose-normalized steady-state plasma levels ranged from 39 to 100 pg/ml (65 +/- 20 pg/ml). The total clearance accounted for 16 +/- 5 ml/min/kg. Post-infusion disposition in the plasma was biphasic with half-lives of 4 +/- 2 min and 37 +/- 8 min. The plasma level profiles obtained on days 4 and 14 of treatment in 3 patients were similar. Sex specific kinetic differences were not observed. In comparison to healthy volunteers, studied in an earlier trial, total clearance was slightly lower and consequently steady state levels were increased (p less than 0.05) in PAOD patients. Half-lives in the plasma were not significantly different.

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.