Combination Therapy of Pulmonary Arterial Hypertension with Vardenafil and Macitentan Assessed in a Human Ex Vivo Model.

PURPOSE: Treatment of pulmonary arterial hypertension (PAH) by vasodilator drug monotherapy is often limited in its effectiveness. Combination therapy may help to improve treatment and to reduce drug toxicity. This study assessed the combination of the endothelin receptor antagonist macitentan and the phosphodiesterase-5 inhibitor vardenafil in a human ex vivo model. METHODS: Study patients did not suffer from PAH. Human pulmonary arteries (PA) and veins (PV) were harvested from resected pulmonary lobes. Contractile forces of blood vessel segments in the presence and absence of the vasodilator drugs macitentan, its main metabolite ACT-132577, and vardenafil were determined isometrically in an organ bath. RESULTS: Macitentan 1E-7 M was sufficient to significantly abate endothelin-1-induced vasoconstriction in PA. A concentration of 1E-6 M was required for significant effects of macitentan on PV and of ACT-132577 on both vessel types. Combination of 1E-7 M macitentan and 1E-6 M vardenafil inhibited sequential constriction with endothelin-1 and norepinephrine of PA significantly more than either compound alone. Effects of 3E-7 M and 1E-6 M macitentan and effects of all doses of ACT-132577 were not further enhanced by 1E-6 M vardenafil. CONCLUSIONS: These data suggest that vasodilator effects of macitentan and vardenafil combined may surpass monotherapy in vivo if drug doses are adjusted properly. Vasodilation by the longer-acting metabolite ACT-132577 was not further enhanced by vardenafil.

Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo.

The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.