Soluble vascular cell adhesion molecule (VCAM) is associated with treatment effects of interferon beta-1b in patients with secondary progressive multiple sclerosis.

BACKGROUND: Subcutaneous IFNbeta-1b (Betaferon) is an established immunomodulatory treatment for relapsing remitting MS and active secondary progressive multiple sclerosis (SPMS). It modulates cytokine and adhesion molecule expression but long term in vivo effects of IFNbeta-1b on the immune system are not known in multiple sclerosis. OBJECTIVE: To address the effects of IFNbeta-1b on serum levels for soluble adhesion molecules and cytokine receptors from MS patients. METHODS: Serial blood samples were obtained from 40 patients of the frequent MRI subgroup (20 patients each from the placebo and the IFNbeta-1b treatment group), participating in the European multi-center clinical trial with IFNbeta-1b for secondary progressive MS, at regular intervals for up to 36 months. Soluble adhesion molecules (sVCAM, sICAM-1, sL-Selectin) as well as TNF-receptor I and II were analysed in the serum of patients by enzyme linked immunosorbent assays (ELISAs). Monthly brain MRI was performed in 34 of these patients (16 patients from the placebo and 18 from the IFNbeta-1b group) during months 1-6 and 19-24 to monitor disease activity as assessed by newly occurring gadolinium (Gd) enhancing lesions. RESULTS: An early and significant increase in sVCAM and sTNF-RII serum levels was detected in 16 out of 20 patients (80 %) treated with subcutaneous IFNbeta-1b already at month 1 but was absent in all but one patient during placebo treatment (p < 0.01). Raised sVCAM and TNF RII serum levels during months 1-6 inversely correlated with less MRI activity in the 19-24 months treatment interval in the IFNa-1b treatment group ( p = 0.0093 for TNF-RII; p = 0.047 for VCAM). CONCLUSIONS: sVCAM and sTNF RII levels in the serum of SPMS patients are increased during IFNbeta-1b therapy and may at least in part explain some of the treatment effects, like reduced immune cell transmigration.

Expression profiling identifies responder and non-responder phenotypes to interferon-beta in multiple sclerosis.

Autoimmune diseases such as multiple sclerosis are characterized by complex genetic traits and pathomechanisms that translate into clinical heterogeneity. This wide heterogeneity of multiple sclerosis as well as different biological responses to immunomodulatory drugs can be expected to contribute to differential treatment responses. Strategies that dissect the relationship between the treatment response and the biological characteristics in individual patients are valuable not only as a clinical tool, but also in leading to a better understanding of the disease. Here we address the in vitro and ex vivo RNA expression profile under one approved therapy of multiple sclerosis, interferon-beta (IFN-beta, Betaseron), by cDNA microarrays and demonstrate that non-responder and responder phenotypes to IFN-beta as assessed by longitudinal gadolinium-enhanced MRI scans and clinical disease activity differ in their ex vivo gene expression profile. These findings will help to better elucidate the mechanism of action of IFN-beta in relation to different disease patterns and eventually lead to optimized therapy.

Interferon-beta treatment of experimental autoimmune encephalomyelitis leads to rapid nonapoptotic termination of T cell infiltration.

We investigated the possible mechanisms how interferon (IFN)-beta may control T cell infiltration in the CNS in experimental autoimmune encephalomyelitis (EAE). Adoptive transfer (AT) EAE was induced in groups of six female Lewis rats. Animals were treated with 3 x 10(5) units of recombinant rat IFN-beta s.c. once at 18 hr, or with 10 mg/kg methylprednisolone (MP) i.v. twice at 18 and 6 hr prior to dissection, or with a combination of both. T cell apoptosis was detected by immunohistochemistry on paraffin sections of spinal cord, using morphological criteria and TUNEL staining. Double labeling of immune cells was done for tumor necrosis factor (TNF)-alpha and metalloproteinase (MMP) 2. Disruption of the blood-brain barrier (BBB) was visualized by staining for albumin. In severe EAE, an increase of T cell apoptosis was seen after IFN-beta alone (all data presented as mean +/- SD: 24.5% +/- 2.2%, P < 0.05, vs. 19.4% +/- 3.1% in controls), and in combination with MP (29.4% +/- 7.3%, P < 0.05 vs. controls). Only the combination therapy decreased T cell infiltration (53.9 +/- 17.7 cells/mm(2), P < 0.05, vs. 99.5 +/- 35.2 cells/mm2 in controls). In moderate EAE, the rate of T cell apoptosis was slightly increased after IFN-beta (21.2% +/- 5.2% vs. 17.4% +/- 5.0% in controls), whereas MP alone (25.5% +/- 3.5%, P < 0.01 vs. controls) and the combination therapy (22.4% +/- 4.8%, P < 0.05 vs. controls) had a clear augmenting effect. IFN-beta tended to decrease T cell infiltration (46.1 +/- 12.7 cells/mm2) compared to controls (59.2 +/- 18.5 cells/mm2). The rate of TNF-alpha-expressing T cells was significantly decreased by IFN-beta and in combination with MP. Also, TNF-alpha expression in macrophages was significantly reduced by IFN-beta and by the combination therapy. The rate of MMP2-expressing macrophages was lower after IFN-beta but clearly decreased only in combination with MP. BBB disruption was ameliorated after IFN-beta but significantly only in combination with MP. Our study indicates that IFN-beta affects the immunopathological process in EAE in several ways, but apoptosis appears as a minor component. In view of treatment of MS relapses, the synergistic effects in this study corroborate the use of a combination therapy with high-dose MP and IFN-beta.

Pharmacodynamic comparison of single doses of IFN-beta1a and IFN-beta1b in healthy volunteers.

In a study in 75 volunteers, preparations of interferon-beta1b (IFN-beta1b) and IFN-beta1a were compared in terms of the resulting serum concentrations of three biologic markers, neopterin, human Mx protein, and 2',5' oligoadenylate synthetase. Each preparation was tested at five dose levels, the middle dose being that recommended for use in patients with multiple sclerosis on the basis of large clinical trials. Five randomly chosen volunteers each received a single subcutaneous dose of one of the IFN or of IFN-beta1a given intramuscularly. The amounts of each marker induced were dose related. There were no major differences between the results with the two IFN or in the duration of the changes in the markers after the two routes of injection. The data indicated that 8 million international units (MIU) of IFN-beta1b and 6 MIU of IFN-beta1a had very similar effects. Even after the highest single dose tested, the increase in the biologic markers were not sustained for a full week.

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.

The PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban synergistically inhibit TXA2-dependent platelet activation.

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban (BM 13177) were investigated for possible synergistic effects on platelet aggregation in human platelet rich plasma in vitro. Iloprost and sulotroban synergistically inhibited U 46619, collagen, and the second wave of ADP-induced platelet aggregation. Iloprost and sulotroban at concentrations showing little or no inhibition alone resulted, in combination, in marked or complete inhibition of U 46619 or collagen induced aggregation. Combination of iloprost 10(-10) M, which had no effect on the concentration-response curve (CRC) to U 46619, with sulotroban 5 x 10(-6) M, which shifted the CRC to U 46619 by a factor of 3 to the right, resulted in a rightward shift of the U 46619 CRC by a factor of 4.5. To attain a 4.5-fold shift with either compound alone, a concentration of 5 x 10(-10) M iloprost or 10(-5) M sulotroban was required. A similar mutual enhancement of inhibitory effects was seen for combinations of the PGI2-analogue cicaprost (ZK 96.480) with sulotroban or the TXA2-receptor antagonist SQ 29548 with iloprost. When the TXA2-dependent part of collagen-induced aggregation was fully inhibited by sulotroban, the concentrations of iloprost necessary for 90% inhibition were reduced by a factor of 2.5 - 3. In the presence of acetylsalicylic acid, the synergistic action of sulotroban and iloprost was reduced and merely additive effects against U 46619-induced platelet aggregation were found, suggesting that the release of endogenous TXA2 plays an important role for the synergistic effect of the two compounds. The combination of a PGI2-analogue and a TXA2-antagonist may lead to a safer and more effective control of platelet activation than with either compound alone.

Synergistic antiplatelet and antithrombotic effects of a prostacyclin analogue (iloprost) combined with a thromboxane antagonist (sulotroban) in guinea pigs and rats.

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban were investigated for possible cooperative effects on platelet function and experimental thrombus formation in guinea pigs and rats. Iloprost and sulotroban inhibit intravascular platelet aggregation in guinea pigs and rats induced by the stable endoperoxide U 46.619 and collagen, with iloprost being the more potent and (for collagen) more efficacious drug. Combinations of both compounds show synergistic or additive effects on in vivo platelet function. Thrombus formation in rats induced by vascular damage is strongly reduced by combining doses of iloprost and sulotroban (BM 13.177) which given alone are ineffective. These results suggest a cooperative enhancement of antiplatelet and antithrombotic effects for combinations of iloprost and sulotroban. In view of disadvantages of currently used platelet inhibitors this cooperativity may offer a new approach in antiplatelet therapy.

Potential therapeutic mechanisms of stable prostacyclin (PGI2)-mimetics in severe peripheral vascular disease.

In 2 randomized, double-blind studies, 109 diabetic patients with trophic lesions and 101 non-diabetics suffering from peripheral vascular disease (PVD) stage IV (Fontaine) received daily 6-hour i.v. infusions of iloprost (less than or equal to 2ng/kg/min) or of placebo over 28 days. Iloprost treatment was superior to placebo, showing ulcer healing in more than 60% of patients compared to less than 25% in the control group. The beneficial effects were sustained during a 1 year follow-up period. Platelet activation, adhesion, aggregation and release reaction on atherosclerotic lesions, impaired microvascular perfusion, loss of microvascular barrier function, increased white blood cell - vessel wall interaction and hemorheological disturbances are all believed to play a role in PVD. Stable PGI2-mimetics inhibit platelet activation by all endogenous mediators as well as platelet release of mitogenic factors (PDGF).

Pharmacological profile of a novel carbacyclin derivative with high metabolic stability and oral activity in the rat.

A novel carbacyclin derivative (16S)-13,14-dehydro-16,20-dimethyl-3-oxa-18,18,19,19-tetradehydro- 6a- carbaprostaglandin-I2 (3-oxa-analogue) has been synthesized in order to find chemically and metabolically stable prostacyclin-mimetics with a potency equal or even superior to PGI2. The 3-oxa-analogue was found to be stabilized against beta-oxidation, a main metabolic degradation step also for chemically stable PGI2-analogues. The compound is orally available and displays a long duration of 4.5-48 h of antiaggregatory and hypotensive action. The 3-oxa-analogue inhibits ADP-induced platelet aggregation with an IC50 of 3.0 nM. Following intravenous application the 3-oxa-analogue lowers diastolic blood pressure in a dose dependent manner, the ED20 being 0.1-0.2 micrograms/kg after injection and less than or equal to 0.05 micrograms/kg/min after infusion respectively. In vivo platelet aggregation is inhibited after i.v. infusion of the 3-oxa-analogue with an IC50 of 0.037 micrograms/kg/min. As compared to Iloprost, the 3-oxa-analogue is 5-12 fold more potent with respect to in vivo hypotensive and anti-aggregatory effects. The results of the present studies indicate that the 3-oxa-analogue has a pharmacological profile comparable to prostacyclin (PGI2) and Iloprost. Due to the fact that the 3-oxa-analogue is chemically and metabolically stable, long term oral treatment can be achieved in clinical conditions in which PGI2 and Iloprost have already been shown to be therapeutically useful principles.

Antifibrillatory action of the stable orally active prostacyclin analogues iloprost and ZK 96 480 in rats after coronary artery ligation.

Iloprost (ILO) and ZK 96 480 (96 480) are stable prostacyclin (PGI2) analogues with platelet aggregation-inhibiting and hypotensive activities equal or superior to PGI2 which in contrast to PGI2 show longlasting pharmacological effects also after oral application. PGI2 as well as ILO and 96 480 with i.v. infusion at equihypotensive doses in rats after coronary artery ligation reduce ventricular ectopic beats, markedly reduce or abolish the periods of ventricular tachycardia and entirely prevent ventricular fibrilloflutter. Even nonhypotensive doses of the prostanoids attenuate postligation arrhythmias. Catecholamine depletion by reserpine pretreatment also markedly reduced the incidence of arrhythmias. As PGI2 and ILO have previously been shown by others to preserve noradrenaline content of sympathetic nerve terminals in ischemic myocardium, prevention of excessive catecholamine loss from hypoxically compromised sympathetic nerve terminals might be involved in the antiarrhythmic action of PGI2, ILO and 96 480.