Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy

Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of iloprost at a dose of 10 μg, at a rate of 0.1 μg/kg/h, twice daily for two weeks.The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen . In the one patient tested, thermography revealed an increase in skin temperature by more than 2°C.Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy.     

Effects of prostacyclin and its stable analog, iloprost, upon insulin secretion in isolated pancreatic islets

We investigated the effects of prostacyclin in three concentrations of 2.7 nM, 53.8 nM and 267 nM on insulin release by isolated rat islets. The lowest concentration produced no significant effects. The middle concentration led to stimulation followed by inhibition of the reaction studied, while the highest concentration strongly depressed insulin release. These effects of prostacyclin appeared to be specific, because they were mimicked by its stable analog, iloprost, but not by its metabolite, 6-keto-PGF1 alpha. The results suggest that prostacyclin generated in islets might exert locally an influence upon insulin release.     

Effects of prostacyclin and its stable analog, iloprost, upon insulin secretion in isolated pancreatic islets

We investigated the effects of prostacylin in three concentrations of 2.7 nM, 53.8 nM and 267 nM on insulin release by isolated rat islets. The lowest concentration produced no significant effects. The middle concentration led to stimulation followed by inhibition of the reaction studied, while the highest concentration strongly depressed insulin release. These effects of prostacyclin appeared to be specified, because they were mimicked by its stable analog, iloprost, but not by its metabolite, 6-keto-PGF_1α. The results suggest that prostacyclin generated in islets might exert locally an influencing upon insulin release.     

Evaluation of a prostacyclin analog, iloprost, and a thromboxane A2 receptor antagonist, daltroban, in experimental intimal hyperplasia.

This study evaluated the efficacy of a prostacyclin analog, iloprost, and a thromboxane A2 receptor antagonist, daltroban, as inhibitors of experimental intimal hyperplasia. The vascular injury model used is based on an endothelial injury induced by a brief infusion of air into an isolated segment of the common carotid artery in the rat. Iloprost and daltroban were administered by continuous IV infusion for two weeks. The infusion rates were 0.1 micrograms/kg/min for iloprost and 0.1 mg/kg/hr for daltroban; these dosing rates are associated with significant alterations in eicosanoid-related pharmacologic effects. The animals were sacrificed at two weeks and the carotid arteries fixed in situ for light microscopy. The myointimal thickening was measured as the intima to media area (I/M) ratio. The control animals developed marked intimal thickening, with an I/M ratio of 0.76 +/- 0.12 (mean +/- SEM; N = 7). There was no inhibition of intimal hyperplasia (P greater than 0.05) after either iloprost (I/M ratio: 1.04 +/- 0.13; N = 8) or daltroban (I/M ratio: 0.70 +/- 0.04; N = 6). It is concluded that neither of these two modulators of eicosanoid activity, iloprost and daltroban, inhibit intimal hyperplasia following experimental endothelial injury.     

Protective effects of CG-4203, a novel stable prostacyclin analog, in traumatic shock

We studied the effects of CG-4203, a novel stable prostacyclin analog, in a severe model of traumatic shock in rats. Traumatic shock was produced by Noble Collip drum trauma and was characterized by marked hypotension, a 4- to 5-fold increase in plasma cathepsin D and myocardial depressant factor activities, and survival time of 95 +/- 15 minutes. Treatment with CG-4203 (100 ng/kg/min) significantly prolonged survival time to 194 +/- 20 min (p less than 0.002). Traumatized rats treated with CG-4203 exhibited significantly lower plasma activities of the lysosomal hydrolase cathepsin D (p less than 0.05). Furthermore, the plasma accumulation of myocardial depressant factor (MDF) activity was also significantly blunted in traumatized CG-4203 treated rats when compared with traumatized rats receiving only the vehicle (p less than 0.01). Our results suggest that a combination of membrane stabilizing and anti-proteolytic effects and inhibition of platelet aggregation may mediate the protective effects of CG-4203 in traumatic shock.     

Effects of a prostacyclin analog iloprost on kidney function, renin-angiotensin and kallikrein-kinin systems, prostanoids and catecholamines in man

Iloprost (ZK 36 374), a stable analog of carbaprostacyclin, was infused for 72 h to nine patients with advanced obliterative arterial disease. Iloprost caused a marked vasodilation and a compensatory increase in cardiac output. The glomerular filtration rate increased by 45% and tubular reabsorption of sodium and water were reduced by 80% and 107%, respectively. The urine excretion rate increased by 122%. Tubular handling of potassium and calcium were not influenced by iloprost but magnesium reabsorption was stimulated. The renin-angiotensin system was not activated while serum angiotensin converting enzyme activity was decreased. Kallikrein excretion in urine was increased 4.4-fold but plasma kininogen, a substrate for kallikrein in producing vasoactive kinins, was unaffected by the drug. Plasma levels of 6-keto-PGF_1α and TxB₂ were decreased and their excretion in urine increased. Plasma catecholamines were not changed by iloprost. Several of the changes persisted for at least the first postinfusion day. The results indicate that iloprost increases urine excretion rate by increasing glomerular blood flow and by inhibiting sodium and water reabsorptions. The kinin-forming system, but not the renin-angiotensin system or plasma catecholamines, may be activated. The decrease in plasma level of prostanoids can be, at least partly, due to their increased excretions in urine.     

Effects of a prostacyclin analog iloprost on kidney function, renin-angiotensin and kallikrein-kinin systems, prostanoids and catecholamines in man

Iloprost (ZK 36 374), a stable analog of carbaprostacyclin, was infused for 72 h to nine patients with advanced obliterative arterial disease. Iloprost caused a marked vasodilation and a compensatory increase in cardiac output. The glomerular filtration rate increased by 45% and tubular reabsorption of sodium and water were reduced by 80% and 107%, respectively. The urine excretion rate increased by 122%. Tubular handling of potassium and calcium were not influenced by iloprost but magnesium reabsorption was stimulated. The renin-angiotensin system was not activated while serum angiotensin converting enzyme activity was decreased. Kallikrein excretion in urine was increased 4.4-fold but plasma kininogen, a substrate for kallikrein in producing vasoactive kinins, was unaffected by the drug. Plasma levels of 6-keto-PGF1 alpha and TxB2 were decreased and their excretion in urine increased. Plasma catecholamines were not changed by iloprost. Several of the changes persisted for at least the first postinfusion day. The results indicate that iloprost increases urine excretion rate by increasing glomerular blood flow and by inhibiting sodium and water reabsorptions. The kinin-forming system, but not the renin-angiotensin system or plasma catecholamines, may be activated. The decrease in plasma level of prostanoids can be, at least partly, due to their increased excretions in urine.     

Platelet sensitivity in vitro to the prostacyclin analogue iloprost in diabetic patients

Iloprost is a chemically stable analogue of prostacyclin, with similar vasodilator and anti-platelet actions. Platelet sensitivity to the inhibitory action of Iloprost has been tested in vitro. Platelet-rich plasma from six healthy subjects and from six patients with type 1 diabetes mellitus was incubated with different concentrations of Iloprost, and then stimulated with ADP (at threshold aggregating concentration) and collagen 4 micrograms/ml. The half-maximal inhibitory concentration (IC50) of Iloprost was calculated and no differences were found between patients and controls. The results of this study suggest that diabetic patients without complications do not differ from healthy subjects in their platelet sensitivity to Iloprost.     

Phosphofurylalanine, a stable analog of phosphohistidine.

Phosphorylated histidine residues occur in a number of signal-transduction pathways in bacteria as well as in eukaryotes. Phosphohistidine is hydrolytically labile and therefore difficult to study, this by contrast to stable phosphoserine, phosphothreonine or phosphotyrosine. Here we report the design and enantioselective synthesis of (4'-phospho-2'-furyl)alanine 1, a non-hydrolyzable analog of 1-phosphohistidine. This novel amino-acid should be useful to synthesize peptides incorporating a stable analog phosphohistidine.     

Ciprostene, a stable prostacyclin analog, produces peripheral vasodilation, platelet inhibition and increased clot dissolution in the cat

The effect of the stable prostacyclin analog ciprostene on hemodynamic parameters, platelet aggregation and clot dissolution was examined in the sodium pentobarbital anesthetized cat. Hemodynamic and platelet aggregation effects were measured in 5 cats following infusion of 5, 10, 20, 40 and 80 μg/kg/min of ciprostene. Drug was dissolved in Tyrode's buffer (pH 7.4) and all doses were infused for 20 minute intervals in ascending order. The hemodynamic data were consistent with peripheral vasodilation. The total peripheral resistance and mean aortic pressure decreased with incresing dose. No change in heart rate, cardiac index, or left ventricle dP/dt (contractility) was observed. All doses infused produced inhibition of ADP induced platelet aggregation. In vivo fibrinolytic activity was assessed with an aortic thrombus positioned at the bifurcation of the aorta. Five cats were infused with vehicle and 5 cats each were infused with 8 and 20 μg/kg/min ciprostene respectively. All infusions were via a 4F catheter positioned in the aorta proximal to the thrombus. Infusion time was 3 hours. Infusion of 8 μg/kg/min did not enhance dissolution of the aortic thrombus. However, the 20 μg/kg/min infusion significantly reduced the thrombus weight (mean = 13.2 mg) compared to vehicle (mean = 38.7 mg) (p < 0.03). The results suggest that ciprostene is a potent vasodilator and platelet inhibitor with clot dissolution properties.     

Ciprostene, a stable prostacyclin analog, produces peripheral vasodilation, platelet inhibition and increased clot dissolution in the cat

The effect of the stable prostacyclin analog ciprostene on hemodynamic parameters, platelet aggregation and clot dissolution was examined in the sodium pentobarbital anesthetized cat. Hemodynamic and platelet aggregation effects were measured in 5 cats following infusion of 5, 10, 20, 40 and 80 micrograms/kg/min of ciprostene. Drug was dissolved in Tyrode's buffer (pH 7.4) and all doses were infused for 20 minute intervals in ascending order. The hemodynamic data were consistent with peripheral vasodilation. The total peripheral resistance and mean aortic pressure decreased with increasing dose. No change in heart rate, cardiac index, or left ventricle dP/dt (contractility) was observed. All doses infused produced inhibition of ADP induced platelet aggregation. In vivo fibrinolytic activity was assessed with an aortic thrombus positioned at the bifurcation of the aorta. Five cats were infused with vehicle and 5 cats each were infused with 8 and 20 micrograms/kg/min ciprostene respectively. All infusions were via a 4F catheter positioned in the aorta proximal to the thrombus. Infusion time was 3 hours. Infusion of 8 micrograms/kg/min did not enhance dissolution of the aortic thrombus. However, the 20 micrograms/kg/min infusion significantly reduced the thrombus weight (mean = 13.2 mg) compared to vehicle (mean = 38.7 mg) (p less than 0.03). The results suggest that ciprostene is a potent vasodilator and platelet inhibitor with clot dissolution properties.     

Stabilization mechanism of prostacyclin by human serum: an approach by binding kinetics using a stable prostaglandin I2 analogue, iloprost

We used a gel filtration method and a stable prostaglandin I2 (prostacyclin) analogue, iloprost, to study the kinetics of prostaglandin I2 binding by human serum proteins. Binding equilibrium experiments conducted at physiological prostaglandin I2 concentration (nM) yielded a KD of 10(-9) and a capacity of approx. 50 nM for the serum binding protein(s). Kinetic measurements gave a dissociation rate constant of 10(-3) s-1. When binding equilibrium was established at various ligand concentrations ranging from nM to microM, a result indicating an unsaturable binding was obtained utilizing this method. On the other hand, saturation was achieved with a ligand concentration as high as 50-100 microM by another binding method. A KD of 7 X 10(-5) and a capacity of approx. 600 microM was obtained. This apparent discrepancy was resolved by performing parallel experiments using purified human serum albumin samples and serum. It is concluded that the large quantity of serum albumin, approx. 600 microM, in serum may compensate for its low KD (approx. 10(-5] for prostaglandin I2, thus simulating a binding protein with a KD of 10(-9) and a limited capacity. These data offer direct information regarding how prostaglandin I2 is stabilized by serum and is transported to the platelet prostaglandin I2 receptors. There is a strong implication that serum albumin is the major if not the only protein responsible for binding of prostaglandin I2.     

Effect of the prostacyclin analogue iloprost in sodium-depleted rats pretreated with captopril.

Previous studies have shown that administration of captopril to sodium-depleted rats decreases the glomerular filtration rate (GFR) and blunts the increase in glomerular prostacyclin synthesis normally occurring in response to sodium depletion. To clarify the relationship between these two responses, iloprost, a stable analogue of prostacyclin, was administered to Na-depleted, captopril-treated (LNC) rats. At a dosage not affecting systemic blood pressure (12.5 ng/kg/min), iloprost increased GFR in LNC rats by 25% (from 0.26 +/- 0.03 to 0.35 +/- 0.03 ml/min/100 g body wt, P less than 0.01), without significant effects on renal plasma flow. No effect was observed in control rats. The results suggest that altered prostacyclin synthesis could contribute to the decrease of GFR in this model.     

Comparison in anesthetized dogs of the anti-aggregatory and hemodynamic effects of prostacyclin and a chemically stable prostacyclin analog, 6a-carba-PGI2 (carbacyclin)

The intravascular anti-aggregatory and systemic and hemodynamic effects of prostacyclin and carbacyclin were compared by intravenous infusion in pentabarbital anesthetized dogs. Ten times as much carbacyclin was needed to produce comparable inhibition of platelet aggregation in the lumen of partially obstructed circumflex coronary arteries. These doses of carbacyclin caused similar decreases in total peripheral resistance as equi-effective anti-aggregatory doses of prostacyclin. There was a trend for the decrease in blood pressure with carbacyclin to be less than that produced by equi-effective anti-aggregatory doses of prostacyclin because carbacyclin caused somewhat greater increases in cardiac output. Changes in heart rate were similar with both substances. During carbacyclin and prostacyclin infusion resistance in normal (unobstructed) coronary arteries decreased. Both substances had comparable effects on pulmonary vascular resistance, right atrial pressure and left ventricular dp/dt at equivalent anti-aggregatory doses both before and after atropine (1 mg/kg) and hexamethonium (5 mg/kg). During 5 to 6 hour infusions of carbacyclin there was no evidence of desensitization of dog platelets to the anti-aggregatory activity. These results show that carbacyclin has a similar spectrum of activity as prostacyclin and is about one-tenth as potent.     

The effects of 15AU81, a chemically stable prostacyclin analog, on the cardiovascular and renin-angiotensis systems of anesthetized dogs

15AU81 is a chemically stable tricyclic benzindene analog of prostacyclin. Prostaglandins, PGI2 and PGE2, and their analogs have demonstrated acute hemodynamic benefit in congestive heart failure. The purpose of this study was to evaluate the cardiovascular effects of 15AU81 in anesthetized dogs in order to assess this agent's potential for clinical study in the treatment of congestive heart failure. Four hour intravenous infusions of 15AU81 at 0.1, 0.3, 1.0, or 3.0 micrograms/kg/min produced dose-dependent decreases in mean arterial blood pressure of 10%, 43%, 55% and 68% respectively with associated decreases in total peripheral resistance of 20%, 32%, 56% and 73%, respectively. 15AU81 dilated the pulmonary circulation; however, the effects of 15AU81 on the pulmonary vascular bed were not dose dependent and not stable over time. Maximal decreases in mean pulmonary artery blood pressure achieved were 9%, 23%, 22%, and 18% with an associated decrease in pulmonary vascular resistance of 9%, 33%, 30% and 23%, at doses of 15AU81 of 0.1, 0.3, 1.0 and 3.0 micrograms/kg/min respectively. The onset of vasodilator effects were rapid, reaching a maximum within 5-10 min with an equally rapid recovery when the infusion was discontinued. Cardiac and hormonal effects associated with 15AU81 treatment included decreases in inotropy, and lusitropy and increases in heart rate and plasma angiotensin II concentration. The increase in plasma angiotensin II concentrated correlated significantly with the decrease mean arterial blood pressure. These effects are interpreted to be generally secondary to the potent and profound vasodilator activity of 15AU81 and not likely due to direct cardiac or neurohumoral effects. The hemodynamic profile of 15AU81 suggests that, through its rapid onset and ability to reduce loading conditions of the right and left ventricles and its chemical stability over natural prostacyclin, this prostacyclin analog may have utility in the management of chronic congestive heart failure.     

Zonisamide: Antihyperalgesic efficacy,the role of serotonergic receptors on efficacy in a rat model for painful diabetic neuropathy

Aims

The purpose of this study was to compare the changes of antihyperalgesic effectiveness of zonisamide (25 and 50 mg/kg), an antiepileptic drug, on the early and late phases of neuropathy and to investigate the role of serotonergic descending inhibitory pain pathways in antihyperalgesic effectiveness of zonisamide in the streptozotocin-induced rat model for painful diabetic neuropathy.

Main methods

The hot-plate and tail-immersion, to determine thermal thresholds, and paw pressure withdrawal tests, to determine mechanical thresholds, were performed as hyperalgesia tests. To investigate the role of serotonergic pathway, 1 mg/kg ketanserin (5-HT_2A/2C antagonist) and ondansetron (serotonin 5-HT₃ receptor antagonist) were used.

Key findings

Zonisamide enhanced pain thresholds significantly in the 3rd, 6th and 8th weeks as the reference drugs morphine (5 mg/kg) and carbamazepine (32 mg/kg, tested only in the 3rd week). There were no observed differences on the potency of antihyperalgesic effect between weeks and between doses. Each antagonist reversed the effect of zonisamide in the hot-plate and tail-immersion tests significantly, but, relatively in the paw pressure withdrawal tests.

Significance

These results support the role for zonisamide in the management of diabetic neuropathic pain in all phases. Serotonin 5-HT_2A/2C and 5-HT₃ receptors are involved in the antihyperalgesic effect of zonisamide by enhancement of thermal threshold, and partially by mechanical threshold, so they may not mediate mechanical hyperalgesia in diabetic neuropathy.     

TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Triply-convergent synthesis of a homochiral 3,3-dimethyl, 15-cyclohexyl prostacyclin analog

Allylic bromocuprate reagent undergoes synfacial SN2′ addition to homochiral allyl ammonium salt to provide vinyl sulfone as a single stereoisomer. Addition of homochiral acetylenic anion to vinyl sulfone smoothly provides the bicyclic sulfone which is further transformed to prostacyclin analog . Analog was only a weak inhibitor of platelet aggregation having an IC₅₀ of 0.48 μM.     

Effects of new chemically and metabolically stable prostacyclin analogues (iloprost and ZK 96480) on early consequences of a transient cerebral oligemia, in the rat

Transient incomplete cerebral ischemia (oligemia) has been obtained in rats by associating mild systemic hypotension with bilateral carotid artery occlusion for 60 min. Continuous i.v drug administration for 3 days, performed with Alzet osmotic minipumps so that to deliver 167 ng.kg-1.min-1 Iloprost, 5 ng.kg-1.min-1 ZK 96480 or their respective vehicle, started 1 hour post-oligemia. Both compounds which are stable prostacyclin analogues reduced the edematous reaction and the post-oligemic accumulation of calcium in the brain tissue but above all they improved, mainly ZK 96480, the learning capacity of injured animals. These results indicate that regarding its therapeutic effect toward early consequences of a transient cerebral oligemia, ZK 96480 has the same profile as Iloprost at a dose 20-fold lower. Thus, these data encourage further clinical studies, in ischemic stroke, in which PGI2 and Iloprost have been shown promising.     

A prostacyclin analogue, iloprost, augments the ovulatory response of the LH-stimulated in vitro perfused rat ovary.

Ovaries from immature rats, primed with pregnant mare's serum gonadotropin (PMSG; 20 IU, on day 28), were perfused in vitro in a recirculating system for 21 h from the morning of day 30 of age. Stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) in vitro at 0 h of perfusion resulted in 2.4 +/- 0.75 (mean +/- SEM) ovulations per treated ovary, whereas no ovulations occurred in the unstimulated group. When the addition of LH was supplemented hourly for 10 h with a stable prostacyclin analogue, Iloprost, at concentrations of 0.01 microM or 0.1 microM, the ovulation rate increased significantly (p less than 0.05) to 6.6 +/- 1.3 and 10.2 +/- 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 microM) did not cause any follicular ruptures when added by itself at every hour up to 10 h. The addition of Iloprost did not affect the release of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone or estradiol from unstimulated or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positive modulatory role of prostacyclin in the LH-induced ovulatory process for the rat.