Akiko Hof

Stereoselectivity at the calcium channel: opposite action of the enantiomers of a 1,4-dihydropyridine.

The stereoisomers of the new dihydropyri-dine derivative 202–791 [isopropyl 4-(2,1,3-benzoxadi-azol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecar-boxylate] were synthesized separately and tested on isolated rabbit aortic rings for effects on depolarization-induced contraction and depolarization-stimulated uptake of 45Ca2+. The racemic mixture enhanced contraction of rabbit aortic rings at low levels of depolarization but inhibited contraction and 45Ca2+ uptake at high levels of depolarization. The IC50 values were 2.0 × 10−7 and 1.7 × 107 M, respectively. The R enantiomer inhibited contraction and 45Ca2+ uptake with IC50 values of 3.2 × 10−8 and 4.3 × 10−8 M, respectively. This compound showed no stimulant activity. By contrast, the S enantiomer of 202–791 shifted the concentration-response curve for depolarization-induced contraction in an almost parallel fashion to the left, thus enhancing contraction. The EC50 value for this effect at a KCl concentration of 16 mM was 1.8 × 10−7 M. This compound enhanced 45Ca2+ uptake concentration dependently at all levels of depolarization tested. Thus, the stereoisomers of a dihydropyridine derivative may behave as a calcium entry blocker or a calcium entry enhancer on vascular smooth muscle, depending only on the stereochemistry. If asymmetric compounds elicit effects suggesting a dualistic action at the calcium channel, then the stereoisomers should be prepared.

Effect of PY 108-068, a new calcium antagonist, on general hemodynamics and regional blood flow in anesthetized cats: a comparison with nifedipine.

The hemodynamic effects of PY 108–068 (PY), a new benzoxadiazolyle dihydropyridine derivative with calcium antagonistic effects on vascular smooth muscle, were investigated in chloralose-urethane-anes-thetized open-chest cats. Regional blood flow was measured with tracer microspheres. PY lowered blood pressure and increased cardiac output similarly to nifedipine (N). Unlike the latter drug, PY decreased heart rate without showing evidence of cardiodepression. Right atrial pressure increased slightly less than with N. All these effects were dose dependent in the dose range from 1 to 43 μg/kg i.v.. PY and N increased myocardial blood flow to the same extent initially, and redistributed it in favor of the outer layer of the left ventricle. The activity of sublingually administered PY was comparable to that of a similar intravenous dose. An approximately three times larger intraduodenal dose was needed for similar effects. Our results provide preliminary in vivo evidence that it is possible to separate the negative chronotropic from the negative inotropic effects of calcium antagonists.

Stereoselectivity at the calcium channel: different profiles of hemodynamic activity of the enantiomers of the dihydropyridine derivative PN 200-110.

Summary: The enantiomers of PN 200-110 (PN), a highly potent calcium antagonist, were synthesized by a stereoselective synthesis. Cross-contamination was <0.2% for the (S)-enantiomer and 0.5% for the (R)-enantiomer. Both isomers inhibited depolarization-induced contraction of rabbit aorta, the (S)-enantiomer being much more active: The pD′2 values were 9.1 and 6.9 for the (S)- and the (R)-enantiomer, respectively. This activity of (R)-PN on potential-operated channels can be attributed to the contamination with 0.5% of (S)-PN. In anesthetized cats, the (S)-enantiomer lowered blood pressure and heart rate dose-dependently (0.3–10 μg/kg i.v.). The (R)-enantiomer had almost no effect on heart rate (HR) and blood pressure (BP) at doses up to 300 μg/kg. However, both enantiomers increased cardiac output and blood flow to the heart and the brain. The effects on regional blood flow were tissue-dependent, the (R)-enantiomer being surprisingly potent in the subendocardium of the left ventricle. The qualitative and quantitative differences between the activities of the two enantiomers suggest that the results obtained cannot merely be explained by traces of the more active enantiomer contaminating the less active enantiomer. These results, together with those found with the enantiomers of a nitro-substituted dihydropyridine, suggest that calcium channels appear to be able not only to discriminate between enantiomers but also to respond differently to them.

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.

Studies on the mechanism of action of the vasoconstrictive dihydropyridine, CGP 28392.

The effects of the vasoconstrictive dihydropyridine, CGP 29392, upon tension development and 45Ca2+ uptake by vascular smooth muscle were studied using isolated rabbit aorta. CGP 28392 did not elicit contractile responses when administered alone but lowered the threshold for the contractile response to elevated extracellular K+. CGP 28392 also increased the magnitude of the contractions elicited by submaximal concentrations of K+. This effect was competitively antagonized by PY 108-068, a Ca antagonist of the dihydropyridine class. Similarly, the relaxing effects of PY 108-068 upon KCl-constricted rabbit aorta were competitively antagonized by CGP 28392. The calcium content of unstimulated tissues was only minimally increased by CGP 28392 but the 45Ca2+ uptake stimulated by depolarizing levels of K+ was markedly augmented by this agent. Further analysis of the data indicated that CGP 28392 did not alter the relationship between tension development and 45Ca2+ uptake. In contrast, CGP 28392 was much less effective in augmenting the contractile response and 45Ca2+ uptake elicited by noradrenaline. These results thus support the hypothesis that CGP 28392 predominantly facilitates Ca2+ entry through potential-operated Ca2+ channels.

Differential effect of cyclosporine A and SDZ RAD on neointima formation of carotid allografts in apolipoprotein E-deficient mice

Background. Apolipoprotein E-deficient (apoE-/-) mice spontaneously develop hypercholesterolemia and atherosclerotic lesions. This may be an appropriate background for the development of graft vessel disease. The authors therefore investigated the effects of cyclosporine A (CsA) and SDZ RAD (RAD, everolimus, Certican) on neointima formation of carotid allografts in apoE-/- mice. To ascertain that equipotent immunosuppressive doses were used, the authors also investigated their effects on cardiac allografts using the same wild-type strain combination. Methods. Heterotopic heart allotransplantation was performed in the BALB/c to C57BL/6 strain combination. Graft survival was monitored daily. Orthotopic carotid artery allotransplantation was performed from BALB/c to apoE-/- (C57BL/6) mice. Groups of mice were treated for 8 weeks with placebo; CsA 10, 20, and 30 mg/kg/d; or RAD 0.3, 1.0, and 3.0 mg/kg/d using ALZET minipumps. Body weight, CsA blood levels, serum lipids, and histology were examined 8 weeks after transplantation or on the day of rejection. Results. Compared with the placebo group, CsA or RAD did not affect body weight. Both CsA and RAD prolonged the survival of cardiac grafts in a dose-dependent manner. CsA blood levels were not different between wild-type and apoE-/- recipients. CsA increased total cholesterol and low-density lipoprotein, but not high-density lipoprotein dose-dependently and significantly, but RAD did not affect the lipids. RAD but not CsA significantly attenuated neointima formation. Conclusions. These results suggest that RAD at a dose preventing organ rejection may also prevent transplant vasculopathy even in the presence of hyperlipidemia.

Vasoconstrictor and vasodilator effects in normal and atherosclerotic conscious rabbits.

1 Rabbits were fed a cholesterol‐rich diet for 5 two‐week intervals. Polyvinyl catheters were then implanted into the femoral artery and vein. Dose‐response curves to acetylcholine (ACh), noradrenaline (NA), phenylephrine (Phen) and angiotensin II (AII), were obtained in 6 cholesterol‐fed and 6 control rabbits before and after isradipine (code name PN200–110) 100μgkg−1. After these experiments the animals were killed and aortic rings were suspended in an organ bath. ACh but not nitroprusside‐induced relaxation was impaired in atherosclerotic but not in control preparations. 2 ACh decreased blood pressure dose‐dependently in both groups of rabbits even though ACh did not relax the aortae of the same rabbits in vitro. 3 Blood pressure effects reflect mostly changes in resistance vessels. The pressor effects of NA, Phen and AII were enhanced in atherosclerotic compared with normal rabbits. 4 After a dose of 100μgkg−1 isradipine the dose‐response curves of all agents were shifted to the right. The differences between atherosclerotic and control rabbits disappeared, except for the AII‐induced pressor response, which remained enhanced in atherosclerotic animals. The calcium antagonist thus only partly corrected the atherosclerosis‐associated hyperresponsiveness to vasoconstrictor agents.

Mechanism of the vasodilator effects of the cardiotonic agent DPI 201-106

Summary: DPI 201-106 (DPI) is a new cardiotonic agent. In experiments on cats anaesthetized with chloralose/urethane, it lowered blood pressure and caused peripheral vasodilatation at doses between 0.3 and 3 mg/kg, infused i.v. Furthermore, DPI lowered heart rate and increased coronary blood flow (measured with tracer microspheres). The mechanism of this vasodilator action was investigated in experiments in vitro using rabbit aorta. DPI inhibited depolarization-induced contraction. The effect was strongly dependent on the contact time. The pIC50 (−log IC50) values for 15 min, 1, and 2 h pretreatment were 4, 5.2, and 5.7, respectively. 45Ca2+ uptake into rabbit aorta was inhibited by DPI with pIC50 values of 4.3, 5.1, and 5.7, respectively, for the pretreatment periods indicated above. The excellent agreement between effects on tension development and calcium uptake suggests that the mechanism of vasodilatation observed in vivo is related to calcium antagonism.

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.