Iloprost, a prostacyclin analog, inhibits the invasion of ovarian cancer cells by downregulating matrix metallopeptidase-2 (MMP-2) through the IP-dependent pathway.

Recent studies have shown that a bioactive lipid prostacyclin (PGI2) plays a role in various cancers, including lung cancer. However, the specific function of PGI2 in ovarian cancer progression has not been determined. This study investigated the effects of PGI2 on cell growth, migration, and invasion in ovarian cancer cells using iloprost, a stable PGI2 analog. Iloprost significantly inhibited migration and invasion, but not cell growth, in a dose-dependent manner in human ovarian cancer cells (A2780 and SKOV3). Interestingly, the cell surface Gs protein-coupled PGI2 receptor IP was enhanced in human ovarian cancer cells. The inhibitory effect of iloprost on migration and invasion was entirely reversed by an IP antagonist (CAY10449) and IP siRNA, whereas the knockdown of peroxisome proliferator-activated receptor δ (PPARδ), a nuclear receptor of PGI2, did not rescue the effect of iloprost. Additionally, iloprost markedly decreased the expression of matrix metallopeptidase-2 and -9 (MMP-2 and MMP-9), which may be induced in the process of ovarian cancer metastasis. IP siRNA inhibited iloprost-reduced MMP-2 expression but not MMP-9 expression. Moreover, inhibition of protein kinase A (PKA) and overexpression of Akt and p38 rescued the inhibition of invasion and the reduction of MMP-2 expression by iloprost. Furthermore, iloprost-induced activation of PKA was associated with PKA-mediated Akt and p38 inactivation in ovarian cancer cells. Taken together, these results demonstrate that iloprost inhibits ovarian cancer cell invasion by downregulating MMP-2 expression via the IP-mediated PKA pathway. This study is the first to reveal a novel role for iloprost and to clarify its underlying mechanism in human ovarian cancer cells.

Relaxant effect of the prostacyclin analogue iloprost on isolated human radial artery: An approach for the reversal of graft spasm.

Radial artery graft spasm in the perioperative or postoperative period of coronary bypass surgery necessitates urgent treatment due to risk of graft failure and mortality. Herein, we evaluated the effect of iloprost, a prostacyclin (PGI2) analogue, against the contractions produced by noradrenaline and potassium chloride on isolated human radial artery. Following the determination of endothelial and vascular relaxing capacities of the arteries, iloprost (10-9M-10-6M) was cumulatively applied on rings precontracted submaximally with the spasmogens. In some rings, the response to iloprost was assessed following pretreatment with nitric oxide (NO) synthase inhibitor, l-NAME (3×10-4M,30min). Iloprost produced complete relaxations on radial artery rings precontracted with noradrenaline whereas, only moderate relaxations against the contractions induced by potassium chloride. Notably, the relaxation to iloprost was remarkably blunted in radial arteries with impaired endothelial function. Moreover, the relaxation to iloprost was unchanged in rings pretreated with l-NAME. Our results demonstrated that iloprost could be a potent relaxant agent in reversing radial artery spasm, particularly initiated by noradrenaline, possibly acting via an endothelium-mediated mechanism unrelated to NO.

Effect of prostaglandin I2 analogs on macrophage inflammatory protein 1α in human monocytes via I prostanoid receptor and cyclic adenosine monophosphate.

AIMS: Inflammation plays critical roles in atherosclerosis. Chemokines are responsible for leukocyte trafficking and involve in inflammatory diseases. Macrophage inflammatory protein 1α (MIP-1α) has been implicated in atherosclerotic lesion formation. Prostaglandin I2 (PGI2) analog, used in pulmonary hypertension, has been reported to have anti-inflammatory functions. However, little is known about its role in the MIP-1α production in human monocytes. METHODS: We investigated the effects of 3 conventional (iloprost, beraprost, and treprostinil) and 1 new (ONO-1301) PGI2 analogs, on the expression of MIP-1α expression in human monocytes. Human primary monocytes from control subjects and THP-1 cell line were treated with PGI2 analogs, with or without lipopolysaccharide (LPS) stimulation. Supernatants were harvested to measure MIP-1α levels by enzyme-linked immunosorbent assay. To explore which receptors involved the effects of PGI2 analogs on the expression of MIP-1α expression, I prostanoid (IP) and E prostanoid, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-r receptor antagonists were used to pretreat THP-1 cells. Forskolin, a cyclic adenosine monophosphate (cAMP) activator, was also used to further confirm the cAMP involvement on the effect of PGI2 analogs in MIP-1α production. RESULTS: Three PGI2 analogs could suppress LPS-induced MIP-1α production in THP-1 cells and human primary monocytes. ONO-1301 had a similar effect. CAY 10449, an IP receptor antagonist, could reverse the suppressive effects on MIP-1α production of iloprost. Forskolin, a cAMP activator, also suppressed MIP-1α production in THP-1 cells. CONCLUSIONS: Prostaglandin I2 analogs suppressed LPS-induced MIP-1α production in human monocytes via the IP receptor and cAMP pathway. The PGI2 analog may be potential in the treatment for atherosclerosis.

Development of a common fully stereocontrolled access to the medicinally important and promising prostacyclin analogues iloprost, 3-oxa-iloprost and cicaprost.

We describe new fully stereocontrolled syntheses of the prostacyclin analogues iloprost (2), the most active component of the drugs Ilomedin and Ventavis, and 3-oxa-iloprost (3), a derivative that is expected to have a significantly higher metabolic stability than 2 perhaps allowing an oral application. The syntheses are based on the same strategy and chiral bicyclic building block as used in the synthesis of cicaprost (4), the third most potent analogue that exhibits, besides prostacyclin-like activities, antimetastatic activities. Reaction of the enantiopure C6-C13 bicyclic aldehyde 17 with Cl(3)CCOOH/Cl(3)CCOONa afforded trichlorocarbinol 24 which was converted via mesylate 25 to the C6-C14 bicyclic alkyne 9. The palladium-catalysed hydrostannylation of alkyne 9 gave with high regio- and stereoselectivity the alkenylstannane 26, Sn/Li exchange of which afforded the E-configured alkenyllithium derivative 8. Coupling of the C6-C14 building block 8 with the enantiopure C15-C20 building block, the N-methoxyamide 7, gave the C6-C20 bicyclic ketone 6 in high yield without epimerisation at C16. The configuration at C15 of iloprost (2) and 3-oxa-iloprost (3) was established through a highly diastereoselective reduction of ketone 6 with catecholborane and the chiral oxazaborolidine 28 which furnished alcohol (15S)-29. The highly stereoselective conversions of alcohol (15S)-29 to iloprost (2) and 3-oxa-iloprost (3), which include as key stereoselective steps an olefination with a chiral phosphonoacetate and a copper-mediated allylic alkylation, have already been described.

Tratamiento crónico combinado con iloprost y sildenafil en un paciente con hipertensión pulmonar primaria / Aerosolized iloprost combined with a phosphodiesterase inhibitor in the treatment of primary pulmonary hypertension

Iloprost, a prostacyclin analogue administered by inhalation, improves hemodynamic and functional class variables in patients with primary pulmonary hypertension. However, repetitive inhalations are required due to its short term effects. One potential approach to prolong and increase the effects of aerosolized iloprost might be to combine its use with phosphodiesterase inhibitors. We report a 36 years old female patient with primary pulmonary hypertension treated with this combination. After 18 months of therapy the patient had an improvement in functional class and in the 6 min walk distance despite persistence of high pulmonary pressures. Our case is in agreement with the reported beneficial effect of the association of sildenafil and iloprost. We postulate that functional improvement in primary pulmonary hypertension is not always related to a decrease in pulmonary artery pressure (Rev MÚd Chile 2004; 132: 353-6).

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.

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.

Effects of prostacyclin analogues in in vivo tumor models.

Much attention has recently focused on the role of tumor cell-platelet interaction in the metastatic cascade. Prostacyclin and stable prostacyclin analogues have been shown to inhibit specifically the formation of metastases in experimental tumor models. This action is based on their ability to reduce the attachment of tumor cells to platelets and to inhibit adhesion of tumor cells-platelet aggregates to the endothelial lining. To investigate the antimetastatic potential of two prostacyclin analogues (Iloprost and Eptaloprost, Schering AG), we have tested these compounds in the spontaneously metastasizing R 3327 MAT Lu prostate carcinoma of the Cop rat in two types of experiments. Treatment was performed for 33 days, starting one day before s.c. implantation of the tumor. The primary s.c.-implanted tumor remained in situ throughout the experiment. In the first test, Iloprost (0.3 micrograms/kg/min) and Eptaloprost (0.1 micrograms/kg/min) were administered via Alzet mini pumps s.c.. There was a considerable reduction of the number of visible lung metastases by Eptaloprost. In the second test, Eptaloprost was administered p.o. in doses of 0.1 and 0.5 mg/kg daily. The number of lung metastases was significantly reduced. Both compounds had no effect on the growth of the primary tumor in the first as well as in the second test. These data show that the prostacyclin analogue Eptaloprost has a significant antimetastatic activity in a spontaneously metastasizing tumor model and thus merits further investigation.

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.

Prostaglandin F2 alpha isopropyl ester versus iloprost phenacyl ester in rabbit and beagle eyes.

One and 2 micrograms of prostaglandin F2 alpha isopropyl ester (PGF2 alpha -IE) applied topically to the rabbit eye caused a biphasic response. The hypotensive phase was dose-dependent with a maximum reduction in intraocular pressure (IOP) of 9.4 +/- 1.7 mmHg at a dose of 2 micrograms. In beagles, 0.4 to 2 micrograms topical PGF2 alpha-IE resulted in a sustained IOP reduction; 2 micrograms produced the maximum reduction of 7-9 mmHg. No initial hypertensive response was observed. Iloprost phenacyl ester (Iloprost-PE) caused a greater decrease in IOP when dissolved in 0.5% hydroxypropyl methylcellulose (AT) than in saline. In rabbits, doses of 0.1 to 1 microgram in AT caused a biphasic response with a sustained IOP decrease fluctuating between 7 and 8 mmHg. In beagles 1 and 2 micrograms Iloprost-PE resulted in a mean IOP reduction of 5.8 +/- 0.4 mmHg and 5.8 +/- 0.5 mmHg (P less than 0.005), respectively; the decrease persisted for 5 hrs. No initial hypertensive response was observed. In beagles PGF2 alpha-IE induced a strong miosis lasting more than 6 hours; Iloprost-PE had no effect on pupil size. Both PG-esters induced a slight hyperemia in rabbit and beagle eyes. In rabbits Iloprost-PE affects the blood-aqueous barrier more than PGF2 alpha-IE, since higher protein concentrations are seen in the aqueous humor after application of Iloprost-PE. Neither PG-ester had a noticeable effect on aqueous protein in beagles. In rabbits, both PG-esters led to slightly increased aqueous humor cyclic-AMP concentrations. In beagles aqueous humor cyclic-AMP was elevated only after Iloprost-PE.(ABSTRACT TRUNCATED AT 250 WORDS)