Efficacy of SDZ RAD compared with CsA monotherapyand combined Rad/FTY720 treatment in a murine cardiac allotransplantation model.

OBJECTIVE: The macrolide immunosuppressant RAD and the immunomodulator FTY720 have distinct mechanisms ofaction. We investigated the efficacy of RAD (everolimus, certican) alone or in combination with FTY720 on graft survival (GS)and histology in comparison with CsA, using mouse strains with strong MHC disparity. METHODS: Heterotopic cardiac grafting was performed using the C57B1/6 to C3H strain combination. Osmotic mini-pumps filled with CsA or RAD were implanted subcutaneously. IFTY720 was administered as a single daily dose by gavage. Peripheral lymphocyte count (PLC) was determined at 1, 4 and 8 weeks or on the day of sacrifice. Body weight was recorded on the day of surgery and weekly. Grafts were histologically evaluated. MAIN FINDINGS: In placebo-treated mice the allografts were rejected after 7 days. Monotherapy with 10 and 30 mg/kg/day CsA achieved 10 and 22.5 days median survival time (MST), while 0.1, 0.3, 1 and 3 mg/kg/day RAD resulted in 10.5, 20, > 56 and > 56 days MST, respectively. FTY720 lowered the PLC significantly, while the lower CsA dose and RAD did not influence the PLC. Adding FTY720 to the 0.6 mg/kg/day dose of RAD extended GS modestly but reduced significantly the perivascular infiltration and endothelialitis in the grafts compared with RAD monotherapy. CONCLUSIONS: Underthe conditions of the present experiment RAD was more potent than CsA in extending the GS. Combining FTY720 and RADwas well tolerated with respect to weight gain and lack of clinically detectable infections in the mice. The 2-drug regimens suppressed the inflammatory allo-response better than RAD monotherapy.

Combined FTY720/cyclosporine treatment promotes graft survival and lowers the peripheral lymphocyte count in a murine cardiac allotransplantation model.

BACKGROUND: FTY720 lowers the peripheral lymphocyte count (PLC) by accelerating the migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of combined FTY720+cyclosporine (CsA) treatment versus monotherapy on prolonging graft survival and on lowering the PLC. METHODS: BALB/c hearts were heterotopically grafted in C3H mice. FTY720 was administered alone or in combination with CsA. PLC and body weight were determined on day 7, day 28, or the day of rejection. RESULTS: Combining FTY720 with CsA prolonged, dose-dependently and significantly, the allograft survival. FTY720, but not CsA, lowered the PLC dose-dependently. The granulocyte count was not reduced in any group. FTY720 concentrations were not influenced by the CsA co-administration. CONCLUSIONS: Combined FTY720 and CsA treatment was well tolerated, promoted graft survival, and suppressed the inflammatory allo-response. The PLC lowering correlated well with the antirejection effects in the two-drug regimens, suggesting that the PLC might guide FTY720 therapy at low doses.

Combined FTY720/cyclosporine A treatment promotes graft survival and lowers the peripheral lymphocyte count in DA to lewis heart and skin transplantation models.

OBJECTIVE: The immunomodulator, FTY720, lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of mono- vs. combined-FTY720/CsA therapy on graft survival (GS) and on lowering the PLC in a solid organ and a skin graft model, using strains with strong MHC disparity. METHODS: Heterotopic cardiac or tail skin grafting was performed using the DA (RT1a) to Lewis (RT1(1)) rat strain combination. FTY720 was administered as a single daily dose by gavage alone or in combination with subcutaneously delivered CsA. PLC, body weight and drug concentrations were determined on day 7, 28, or the day of rejection. MAIN FINDINGS: In placebo-treated animals the heart and skin allografts rejected after 6 and 8 days. FTY720 delayed rejection of both the solid organ and skin grafts. The maximal effect was achieved at 1 mg x kg(-l) x day(-1) FTY720, resulting in a median survival time (MST) of 14 days for both allotransplants comparable to the effect achieved by 1 mg x kg x day(-1) CsA in both models. In the cardiac graft experiment with CsA co-administration, doses of 0.3 and 1 mg/kg were used. Under these conditions very small doses of FTY720 were effective in maintaining grafts throughout the treatment period. Adding higher FTY720 doses to the 1 mg x kg(-1) x day(-1) CsA was needed to effectively extend the skin GS, e.g. 0.3 mg x kg(-l) x day(-1) FTY720 prolonged GS from 13 to 47.5 days MST, i.e. well beyond the 28 day-treatment period. CsA did not influence the PLC at clinically relevant doses. FTY720 lowered the PLC significantly and dose-dependently, at doses lower than those needed for the prolongation of both cardiac and skin GS with FTY720 monotherapy. In rats with skin grafts the PLC was markedly lowered up to 1 mg x kg(-1) x day(-1) FTY720, whereas, in the heart model, it was lowered up to 0.1 mg x kg(-1) x day(-1). Independently of the graft type, within the combination regimens 0.3 mg x kg(-1) x day(-1) FTY720 achieved a maximal PLC depletion. CONCLUSIONS: Combining FTY720 and CsA was very well tolerated with respect to weight gain and lack of any clinically detectable infections. In the strain combination used FTY720 monotherapy was less effective than previously reported in maintaining grafts. The two-drug regimens extended strikingly the GS for both models. However, the prolongation of the heart GS was smoothly dose-related with FTY720 doses ranging from 0.01 to 1 mg x kg(-1) x day(-1) , whereas, the skin graft prolongation was modest at doses up to 0.1 mg x kg(-1) x day(-1) and remarkably enhanced at 0.3 and 1 mg x kg(-1) x day(-1) FTY720.

The peripheral lymphocyte count predicts graft survival in DA to Lewis heterotopic heart transplantation treated with FTY720 and SDZ RAD.

OBJECTIVE: The new immunomodulator 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride (FTY720) lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. This effect is dose-dependent at low (up to 0.1 mg/kg per day) doses in rats. We investigated the correlation between PLC and the later rejection, when FTY720 was combined with RAD. METHODS: Heterotopic cardiac grafting was performed using the DA-Lewis strain combination. FTY720 and RAD were administered as single daily doses by gavage alone and in combination starting 3 days before to 28 days after transplantation. Graft survival was monitored daily by palpation. PLC was determined at 1 and 4 weeks, body weight (BW) weekly. Histologic evaluation of grafted hearts was performed after rejection. MAIN FINDINGS: FTY720 at doses of 0.03, 0.1 and 0.3 mg/kg per day prolonged graft survival dose-dependently from 6 (placebo) to 7, 9.5 and 15 days median survival time (MST). RAD at doses of 0.3, 1 and 3 mg/kg per day delayed rejection to 8.5, 18 and 37.5 days MST. Very small FTY720 doses added to the lower RAD doses were effective in maintaining grafts throughout the treatment period and with normal weight gain, as opposed to regimens with 1 mg/kg or more per day RAD, which resulted in delayed weight gain. FTY720 lowered the PLC significantly and dose-dependently. The PLC correlated well with graft survival [Spearman rank correlation (n = 30, rs = -0.75)]. CONCLUSIONS: Fully effective FTY720 + RAD combination regimens caused no side effects with respect to the rats' general well-being or weight gain and were better tolerated than equiactive RAD monotherapy, suggesting a broader therapeutic window for the combinations. Under the experimental conditions, the PLC decrease showed an interesting correlation with the anti-rejection effects in these two-drug regimens. Thus, in rats the PLC is helpful for monitoring the biological activity of FTY720 at low doses (< 0.1 mg/kg per day), i.e. in the range of the steep part of its dose-response relationship.

Prevention of graft vessel disease by combined FTY720/cyclosporine. A treatment in a rat carotid artery transplantation model.

BACKGROUND: Graft vessel disease (GVD) is an important problem often responsible for late graft loss. The effects of FTY720, an immunomodulator with a novel mechanism of action were investigated in combination with cyclosporine A (CsA) in a carotid artery allograft model. METHODS: A segment of the carotid artery of Lewis rats was replaced by a DA allograft. Seven groups of eight rats were treated for 8 weeks with vehicle (P), CsA 0.3 (C0.3), 1 (C1) or 3 (C3) mg x kg(-1).day(-1) or a combination of CsA 1 with FTY 0.01 (C1F0.01), 0.03 (C1F0.03), and 0.1 (C1F0.1) mg x kg(-1).day(-1). Lumen area was estimated by magnetic resonance imaging, peripheral lymphocyte count and drug concentrations were determined at 1 and 8 weeks. Neointima, media, and lumen area were measured morphometrically. Intimal and adventitial infiltration of mononuclear cells, and medial smooth muscle cells number was assessed using a score. RESULTS: FTY720 did not influence CsA blood concentrations. FTY720 but not CsA decreased the PLC dose dependently. Magnetic resonance imaging revealed that treatment groups have larger lumen size than group P. Histological and morphometric evaluation showed that all aspects of GVD were dose dependently suppressed by treatment and lumen narrowing was prevented. CONCLUSIONS: CsA, at clinically relevant blood levels, suppressed GVD only partly. The addition of FTY720 was well tolerated and completely suppressed GVD development. In vivo lumen size did not correlate with the histologically estimated neointimal thickness.

Activators of protein kinase A induce a glibenclamide-sensitive 86Rb+ efflux in rat isolated aorta.

The effect of activators of protein kinase A on membrane K+ permeability and the interaction of these compounds with cromakalim, an opener of ATP-sensitive K+ channels (K(ATP) channels), were investigated. Membrane K+ permeability was assessed by measuring 86Rb+ efflux from rings of rat aorta. Forskolin, an activator of adenylate cyclase, and isobutylmethylxanthine (IBMX), a nonselective phosphodiesterase inhibitor, induced small, concentration-dependent increases in tracer efflux up to 20-40% over the basal level. The effect of forskolin was abolished by the K+ channel blocker tedisamil (1 microM) and partially inhibited by glibenclamide (1 microM), a relatively selective blocker of K(ATP) channels. Further studies were conducted in the presence of 35 mM KCI in the bath in order to increase the size of the 86Rb+ efflux stimulated by forskolin and IBMX. At high concentrations, these compounds produced a biphasic effect with a peak increase being followed by a lower plateau value. Glibenclamide inhibited the 86Rb+ efflux response to forskolin and IBMX by 50-80%. The K+ channel blockers tedisamil (1 microM), Ba2+ (1 mM) and tetraethylammonium (10 mM) also reduced the peak response to forskolin by about 50% and abolished or greatly inhibited the plateau response. In addition to the small effect on basal 86Rb+ efflux, forskolin (0.3 microM) increased cromakalim-induced 86Rb+ efflux 3.4 times. At higher concentrations, however, a concentration-dependent inhibition was observed with an IC50 value of 7.6 +/- 0.4 microM. 1,9-dideoxyforskolin, which does not increase cAMP, increased neither basal nor cromakalim-induced 86Rb+ efflux; however, it inhibited cromakalim-stimulated tracer efflux with an IC50 value of 22 +/- 2 microM. It is concluded that forskolin and IBMX, probably by increasing intracellular cAMP levels, induce a 86Rb+ efflux from rat aorta, the major part of which is glibenclamide-sensitive and may pass through K(ATP) channels. In addition, low concentrations of forskolin greatly facilitate the K(ATP) channel opening effect of cromakalim whereas high concentrations block the channel; this blocking effect of forskolin is unrelated to the cAMP elevating action.

Ba2+ differentially inhibits the Rb+ efflux promoting and the vasorelaxant effects of levcromakalim and minoxidil sulfate in rat isolated aorta.

The K+ channel openers activate ATP-sensitive K+ channels (KATP) in vascular smooth muscle and induce relaxation. In this study, the relationship between these two effects was examined in rings of rat aorta using levcromakalim and minoxidil sulfate as the openers and Ba2+ as the K+ channel blocker; K+ channel opening was assessed by determining the rate constant of 86Rb+ efflux from the preparation. Ba2+ inhibited the 86Rb+ efflux stimulated by levcromakalim in a noncompetitive manner with an IC50 value of 29 microM and a Hill-coefficient of 1.2. At concentrations >300 microM, Ba2+ increased the tension of rat aortic rings concentration-dependently. Levcromakalim relaxed contractions to Ba2+ (0.5 and 1 mM) with potencies similar to those determined against KCl (25 mM) or noradrenaline as spasmogens (EC50 values 15-40 nM). The vasorelaxant effect against Ba2+ was inhibited by the KATP channel blockers, glibenclamide and tedisamil, and abolished in depolarizing medium (55 mM KCl). At 3 mM Ba2+, levcromakalim was still able to transiently induce complete relaxation; however, within 1 h oscillations in tension developed, leading to a stable level of only 15% relaxation. A similar level of relaxation was achieved against 10 mM Ba2+ whereas the combination of 0.5 mM Ba2+ and 3 microM tedisamil blocked the relaxant effect of levcromakalim completely. With minoxidil sulfate as the KATP channel opener the results of the 86Rb+ efflux and tension experiments were similar to those obtained with levcromakalim. It is concluded that Ba2+ is more potent in inhibiting the K+ channel opening than the vasorelaxant effects of the openers. On the basis of the 86Rb+ efflux experiments it is estimated that at least 97% of the channels opened by the activators can be blocked without major effects on vasorelaxation suggesting a dissociation between the two effects. However, if the block is pushed to extremes (> or = 99.95%) the vasorelaxant effect of the openers is also abolished suggesting a link between both effects. This paradoxon remains to be solved.

Binding of the K+ channel opener [3H]P1075 in rat isolated aorta: relationship to functional effects of openers and blockers.

P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine], an analogue of the K+ channel opener pinacidil, was shown to be a K+ channel opener in rat aorta, based on (i) its ability to stimulate 86Rb+ efflux, (ii) its ability to relax contractions in response to noradrenaline under normal conditions (5 mM KCl) but not under depolarized conditions (55 mM KCl), and (iii) the sensitivity of these effects to inhibition by the sulfonylurea glibenclamide. In these assays, P1075 was approximately 20 times more potent than cromakalim. Using a tritiated derivative, [3H] P1075, specific binding could not be detected in microsomal preparations from various tissues. However, in rat aortic strips specific binding of [3H]P1075 has been observed and was reduced by lowering the temperature or by decreasing intracellular ATP levels via metabolic inhibition. Specific [3H]P1075 binding was influenced neither by depolarization (55 mM KCl) nor by lowering the pH from 7.4 to 6.0. Binding was inhibited by representatives from all major families of K+ channel openers, with potencies that correlated well with the potencies obtained in 86Rb+ efflux and relaxation studies. However, stimulation of 86Rb+ efflux occurred at 40 times higher concentrations than did binding (and vasorelaxation). Of the various inhibitors of the K+ channel openers tested, only the sulfonylureas inhibited [3H] P1075 binding with the same rank order of potencies as that required for inhibition of P1075-induced 86Rb+ efflux, although at higher concentrations. The results show that binding of [3H] P1075 is independent of membrane potential but decreases concomitantly with the intracellular ATP level. The excellent correlation between the potencies of the openers and sulfonylurea blockers in binding assays and functional studies suggests that the 'drug receptor' labeled by [3H]P1075 in rat isolated aorta is of functional relevance. However, the fact that binding of the openers occurred at concentrations considerably lower than those required for K+ channel opening and that binding of the sulfonylureas was only reflected at concentrations higher than those needed to block the channel requires complex models to link binding and effect, possibly involving two agonist binding sites coupled by negative cooperativity.

Identification of a specific binding site for K+ channel openers in rat aorta.

The K+ channel openers (KCOs) form a structurally heterogeneous group of compounds which relax smooth muscle by opening K+ channels in the plasmalemma. At present it is not known whether these drugs open the same K+ channel in smooth muscle and, if so, whether they bind to the same site of this channel. To address these questions we present the first binding study with KCOs in a smooth muscle preparation. In intact rat aortic strips, the novel tritiated KCO, 3H-P1075 (N-cyano-N'-(1,1-dimethyl[2,2,3,3(3)H]propyl)-N"-3-pyridylguanidine++ +), a potent pinacidil analogue, showed saturable specific binding of high affinity (KD = 6 +/- 1 nM, Bmax = 23 +/- 3 fmol/mg tissue wet weight). Specific binding of 3H-P1075 was inhibited stereospecifically by representatives from all major families of K+ channel openers, with a rank order of potency that correlated well with the potencies for vasorelaxation in rat aorta. The sulfonylurea glibenclamide, a relatively specific blocker of ATP-sensitive K+ channels and an inhibitor of the effects of KCOs, also inhibited 3H-P1075 binding and increased the rate of dissociation of 3H-P1075 from the tissue in a concentration-dependent manner. Lowering temperature (from 37 degrees C to 2 degrees C) and decreasing intracellular ATP levels by metabolic poisoning, diminished specific 3H-P1075 binding by reducing Bmax. However, depolarization (KCl = 55 mM) or lowering pH from 7.4 to 6.0 did not influence binding. The data demonstrate the existence of a specific binding site for the KCO 3H-P10756 in rat isolated aorta, which seems to be of functional relevance.(ABSTRACT TRUNCATED AT 250 WORDS)

Cromakalim inhibits contractions of the rat isolated mesenteric bed induced by noradrenaline but not caffeine in Ca(2+)-free medium: evidence for interference with receptor-mediated Ca2+ mobilization.

The effects of the K+ channel opener cromakalim on phasic contractions induced by noradrenaline and caffeine were studied in the rat isolated mesenteric bed. In the presence of 1.4 mM Ca2+, 1-s pulses of noradrenaline increased the perfusion pressure of the preparation concentration dependently (midpoint at 92 +/- 10 microM noradrenaline). Cromakalim (0.3 and 1 microM) inhibited these contractions in a non-competitive manner. Contractions elicited by 1-s pulses of noradrenaline (100 microM) were inhibited by the dihydropyridine Ca2+ antagonist isradipine by maximally 24 +/- 1%, indicating that only a minor component of this contraction depended on Ca2+ entry via dihydropyridine-sensitive Ca2+ channels. Cromakalim was a much more effective inhibitor of these contractions (maximum inhibition by 80%, midpoint of the inhibition curve at 171 +/- 15 nM). The effect of cromakalim was stereoselective, inhibited by the sulphonylurea glibenclamide, and abolished in partially depolarizing media (KCl = 35 and 50 mM). In Ca(2+)-free medium, cromakalim inhibited the contraction induced by noradrenaline (100 microM) by maximally 69 +/- 4%, with a midpoint at 58 +/- 14 nM. The effect of cromakalim was again stereoselective, inhibited by glibenclamide, and abolished in the presence of 50 mM KCl. Contractions induced by caffeine (10 and 100 microM) were not affected by cromakalim (1 microM). The results indicate that, in rat mesenteric resistance vessels, cromakalim interferes with the ability of noradrenaline, but not caffeine, to mobilize Ca2+ from intracellular stores. The antivasoconstrictor effect of cromakalim against noradrenaline is inhibited by glibenclamide and appears to be linked to the ability of cromakalim to hyperpolarize the cell membrane.

Comparison of the effluxes of 42K+ and 86Rb+ elicited by cromakalim (BRL 34915) in tonic and phasic vascular tissue.

The cromakalim-induced effluxes of 42K+ and 86Rb+ were compared in rat aortic segments and in guinea-pig portal vein. In both vessels, low concentrations of cromakalim (0.1 microM) increased the permeability to 86Rb+ 3-4 times less than that to 42K+; at 10 microM the difference was about a factor of 1.3-2. In rat aorta, the threshold concentration of cromakalim for 42K+ efflux was greater than or equal to 0.03 microM; with 86Rb+ as the tracer ion it was 0.1 microM. At similar concentrations, cromakalim relaxed the tension of aortic segments precontracted with 23 mM KCl (IC50 = 0.06 +/- 0.01 microM). However, no concomitant increase in 42K+ or 86Rb+ efflux could be detected from this stimulated preparation at these concentrations. In guinea-pig portal vein, 42K+ efflux measurements were performed in the presence and absence of the dihydropyridine Ca2+ entry blocker PN 200-110 (isradipine) yielding comparable results. In the presence of PN 200-110, where spontaneous activity and the K+ efflux associated with it were abolished, the threshold concentration of cromakalim for 42K+ efflux was 0.02 microM as compared to 0.06 microM for 86Rb+ efflux. In the absence of PN 200-110, spontaneous activity of the portal vein was inhibited by 70% and 90% at these concentrations. In double isotope experiments, the K+ channel inhibitor tetraethylammonium did not discriminate between the effluxes of 42K+ and 86Rb+ stimulated by cromakalim. It is concluded that in the two vascular tissues examined, cromakalim increased the permeability to 42K+ more than to 86Rb+, the difference being more marked at low cromakalim concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Similarities in the mechanism of action of two new vasodilator drugs: pinacidil and BRL 34915.

The present study compares the in vitro and in vivo activities of pinacidil with another new vasodilator drug, BRL 34915, claimed to act via the opening of K+ channels in vascular smooth muscle. In the rabbit aorta, BRL 34915 and pinacidil caused rightward shifts of the KCl concentration-response curve and noncompetitively antagonized angiotensin II contractions, yielding an IC50 of 5 and 10 microM, respectively. In 86Rb-loaded guinea pig portal veins, both BRL 34915 and pinacidil stimulated 86Rb+ efflux over the concentration range 0.3-30 microM. At saturating concentrations, the maximum efflux elicited by pinacidil was only one-third that of BRL 34915. Spontaneous activity measured simultaneously from the same vessels was inhibited by BRL 34915 and pinacidil with an IC50 of 12 and 32 nM, respectively. Effects on mechanical activity were thus observed at drug concentrations 100-fold lower than those required to stimulate 86Rb+ efflux. In anesthetized rats, both compounds rapidly lowered blood pressure, with BRL 34915 being threefold more potent than pinacidil in this respect. Tachycardia was more pronounced after BRL 34915 than after pinacidil. Angiotensin II pressor responses were poorly antagonized by these two vasodilators in rats, in marked contrast to the potent effects of Ca2+ antagonists on these responses at equieffective hypotensive doses. We conclude the pinacidil, like BRL 34915, is a potent antihypertensive that is able to enhance the K+ permeability of vascular smooth muscle. The similarities between these two drugs suggest they have a common mechanism of action. However, the discrepancy between the concentrations of these drugs necessary to stimulate 86Rb efflux, and those at which mechanical effects are seen in the portal vein, do not rule out the possibility that other actions may contribute to their vasodilator activities.