Pembrolizumab plus chemotherapy in Japanese patients with persistent, recurrent or metastatic cervical cancer: Results from KEYNOTE-826.

Pembrolizumab plus chemotherapy with or without bevacizumab demonstrated prolonged progression-free survival (PFS) and overall survival (OS) versus chemotherapy in patients with persistent, recurrent, or metastatic cervical cancer in the phase 3, randomized, double-blind, placebo-controlled KEYNOTE-826 study. We report outcomes in patients enrolled in Japan. Patients received pembrolizumab 200 mg or placebo Q3W for up to 35 cycles plus chemotherapy (paclitaxel 175 mg/m2 + cisplatin 50 mg/m2 or carboplatin AUC 5) with or without bevacizumab 15 mg/kg. Dual primary endpoints were PFS per RECIST v1.1 by investigator assessment and OS in the global population; these were evaluated in patients with tumors with PD-L1 combined positive score (CPS) ≥1, all-comers, and PD-L1 CPS ≥10. Fifty-seven patients from Japan were randomized (pembrolizumab plus chemotherapy, n = 35; placebo plus chemotherapy, n = 22). Pembrolizumab plus chemotherapy improved PFS versus placebo plus chemotherapy in patients with PD-L1 CPS ≥1 (n = 51; hazard ratio [HR; 95% CI], 0.36 [0.16-0.77]), all-comers (n = 57; 0.45 [0.22-0.90]), and patients with PD-L1 CPS ≥10 (n = 25; 0.36 [0.12-1.07]). HRs (95% CI) for OS were 0.38 (0.14-1.01), 0.41 (0.17-1.00), and 0.37 (0.10-1.30), respectively. Incidence of grade 3-5 AEs was 94% in the pembrolizumab group and 100% in the placebo group. Consistent with findings in the global KEYNOTE-826 study, pembrolizumab plus chemotherapy with or without bevacizumab may prolong survival versus placebo plus chemotherapy with or without bevacizumab and had a manageable safety profile in Japanese patients with persistent, recurrent, or metastatic cervical cancer.

Involvement of the ßγ subunits of G proteins in the cAMP response induced by stimulation of the histamine H1 receptor.

Stimulation of the histamine H1 receptor has been shown to enhance adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation in various cell types but, to date, the mechanism by which this occurs is still unclear. In the present study, we examined the possibility that the betagamma subunits of G proteins (G betagamma) are involved in this process in cultured Chinese hamster ovary cells transfected with the human histamine H1 receptor (CHO-H1). Histamine increased intracellular cAMP levels in a concentration-dependent manner in CHO-H1 cells, and this histamine action was abolished by pyrilamine (1 microM). Inhibition of histamine H1 receptor-G(q) protein coupling by stable expression of the C-terminal peptide of G alpha(q) protein significantly attenuated the cAMP accumulation induced by histamine. By comparison, neither BAPTA/AM (50 microM), an intracellular Ca2+ chelator, nor GF 109203X (1 microM), an inhibitor of protein kinase C, influenced the cAMP response. Histamine H1 receptor-mediated cAMP accumulation was significantly inhibited by transient transfection of CHO-H1 cells with the C-terminal peptide of beta-adrenoceptor kinase I (residues 542-685), a scavenger of G betagamma. Stable expression of the C-terminal peptide of the G alpha(s) protein, but not treatment with pertussis toxin (200 ng/ml for 24 h), attenuated the histamine H1 receptor-mediated cAMP accumulation. These results suggest that stimulation of histamine H1 receptors activates adenylyl cyclase through the release of G betagamma subunits from G proteins, thereby elevating intracellular cAMP levels.

Relaxant effect of YM976, a novel phosphodiesterase 4 inhibitor, on bovine tracheal smooth muscle.

Effects of 4-(3-chlorophenyl)-1,7-diethylpyrido[2,3-d]pyrimidin-2(1H)-one (YM976), a novel and selective phosphodiesterase type 4 inhibitor, on tension and adenosine 3',5'-cyclic monophosphate (cAMP) content of bovine tracheal smooth muscle were compared with those of rolipram and theophylline. YM976, rolipram and theophylline relaxed the tracheal preparations contracted with histamine in a concentration-dependent manner. The relaxant effects of YM976 and rolipram were more potent than those of theophylline. These phosphodiesterase inhibitors-induced relaxations were dramatically diminished when tracheal smooth muscle was contracted with methacholine instead of histamine. Pretreatment of the tracheal preparations with YM976 (10 microM) or rolipram (10 microM), but not with theophylline (1 mM), shifted the concentration-response curves for contractile responses to histamine; however, the same procedure failed to affect concentration-response relationships for methacholine-induced contractions. At 1 and 10 microM, both YM976 and rolipram increased the tissues cAMP content. These results suggest that YM976 relaxes tracheal smooth muscle, probably through the cAMP-dependent mechanism.

Lidocaine attenuates muscarinic receptor-mediated inhibition of adenylyl cyclase in airway smooth muscle.

We examined how lidocaine affects muscarinic receptor-mediated inhibition of adenylyl cyclase in bovine tracheal smooth muscles. Lidocaine (100 microM) augmented the relaxant responses to forskolin in the bovine tracheal smooth muscle contracted with methacholine (0.3 microM). On the other hand, lidocaine failed to affect the relaxant effects of forskolin on the histamine (100 microM)- and KCl (40 mM)-contracted preparations. Lidocaine (100 microM) enhanced both basal and forskolin-stimulated cAMP accumulation in the presence of methacholine (0.3 microM). However, in the absence of methacholine, neither basal nor forskolin-stimulated cAMP accumulation was affected by lidocaine. Similar phenomenon was observed when the bovine tracheal smooth muscles were treated with methoctramine (0.03 microM). In radioligand binding experiments, lidocaine inhibited [3H]N-methyl scopolamine binding to cloned human muscarinic receptors (M(1)-M(5)) expressed in Chinese hamster ovary cells. These results suggest that lidocaine prevents muscarinic receptor-mediated signaling pathway and thereby reverses inhibition of adenylyl cyclase by methacholine in bovine tracheal smooth muscle.

Augmentation of rat urinary bladder relaxation mediated by beta1-adrenoceptors in experimental diabetes.

We examined how diabetes affects the beta-adrenoceptor subtypes mediating relaxation of rat urinary bladder smooth muscle contracted with carbachol. The relaxant responses to isoproterenol were larger in muscles from rats 8 to 10 weeks after induction of diabetes with streptozotocin (80 mg/kg, i.p.) as compared to the control muscles. In contrast, forskolin-induced relaxations did not differ significantly in the control and diabetes groups. Propranolol (1 microM) abolished the diabetes-induced augmentation of relaxant responses to isoproterenol. The relaxant responses to T-0509 ((-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)-amino]ethanol hydrochloride), a beta(1)-adrenoceptor agonist, were small but significantly augmented by diabetes. On the other hand, diabetes did not change the relaxations produced by clenbuterol, a beta(2)-adrenoceptor agonist, and BRL37344 ((+/-)-(R*,R*)-(4-[2-([2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]phenoxy)acetic acid), a beta(3)-adrenoceptor agonist. These results suggest that diabetes selectively augments the beta(1)-adrenoceptor-mediated relaxation of the rat urinary bladder smooth muscle.

Protease-activated receptor-2-mediated contraction in the rat urinary bladder: the role of urinary bladder mucosa.

The role of protease-activated receptor-2 (PAR-2) in the regulation of the rat urinary bladder contractility was investigated. Both trypsin and PAR-2 activating peptide (SLIGRL-NH(2)) produced a concentration-dependent contractile response in the urinary bladder preparations. These contractions were abolished by removal of the urinary bladder mucosa and were significantly reduced by indomethacin (10 microM). These results suggest that activation of PAR-2 stimulates release of prostaglandins from mucosal layer and thereby contracts rat urinary bladder smooth muscles.

Possible involvement of Ca2+-independent phospholipase A2 in protease-activated receptor-2-mediated contraction of rat urinary bladder.

Possible involvement of Ca(2+)-independent phospholipase A2 (iPLA2) was examined in protease-activated receptor-2 (PAR-2)-mediated contraction of the rat urinary bladder. Both PAR-2 activating peptide (PAR-2 AP; SLIGRL-NH2) and trypsin produced a concentration-dependent contractile response in the urinary bladder preparations. These contractions were significantly (p<0.01) attenuated by indomethacin (10 microM), an inhibitor of cyclooxygenase, or bromoenol lactone (BEL; 10 micro M), an inhibitor of iPLA2. On the other hand, the contractile responses to bradykinin were not significantly affected by BEL, although they were reduced by indomethacin. Arachidonyltrifluoromethyl ketone (AACOCF3; 30 microM), an inhibitor of cytosolic Ca(2+)-dependent phospholipase A2, did not affect the trypsin- and bradykinin-induced contractions. Both indomethacin and BEL had no inhibitory effect on the prostaglandin E2-induced contractions. These results suggest that PAR-2 activators and bradykinin stimulate the release of prostaglandins and thereby contract the rat urinary bladder smooth muscles. The release of prostaglandins by PAR-2 activators seems to be partly mediated by the iPLA2.

Inhibitory mechanism of BRL37344 on muscarinic receptor-mediated contractions of the rat urinary bladder smooth muscle.

We examined the inhibitory mechanism of BRL37344, a beta-adrenoceptor agonist that is considered to be specific to beta(3)-subtype, on muscarinic receptor-mediated contraction of the rat urinary bladder smooth muscle. BRL37344 produced apparently biphasic concentration-relaxation curves in the urinary bladder smooth muscle contracted with carbachol (0.6 microM). The first and second phases had estimated p D(2) (-logEC(50)) values of 7.80+/-0.34 and 4.62+/-0.18, respectively ( n=6). The first component of the BRL37344 concentration-response curve was not affected by propranolol (1 microM), whereas it was inhibited by higher concentrations of the drug (10 microM or 30 microM). The second component was completely resistant to propranolol. On the other hand, BRL37344 produced monophasic concentration-relaxation of 30 mM KCl-precontracted urinary bladder smooth muscle with a p D(2) value of 8.34+/-0.18 ( n=6). Pretreatment of the urinary bladder smooth muscles with BRL37344 (30, 100 and 300 microM) significantly ( P<0.05) shifted the concentration-response curves for carbachol-induced contractions. In radioligand binding experiments, BRL37344 concentration-dependently displaced the specific binding of [(3)H] N-methyl scopolamine to muscarinic receptors on rat urinary bladder smooth muscle membranes. Additionally, BRL37344 inhibited [(3)H] N-methyl scopolamine binding to cloned human muscarinic receptors (M(1)-M(5)) expressed in Chinese hamster ovary cells. These results suggest that BRL37344 attenuates muscarinic receptor-mediated contractions through prevention of the agonists binding to their receptors, in addition to stimulation of beta(3)-adrenoceptors, in rat urinary bladder.

MaxiK channel-triggered negative feedback system is preserved in the urinary bladder smooth muscle from streptozotocin-induced diabetic rats.

MaxiK channel, the large-conductance Ca2+-sensitive K+ channel, facilitates a negative feedback mechanism to oppose excitation and contraction in various types of smooth muscles including urinary bladder smooth muscle (UBSM). In this study, we investigated how the contribution of MaxiK channel to the regulation of basal UBSM mechanical activity is altered in streptozotocin-induced diabetic rats. Although the urinary bladder preparations from both control and diabetic rats were almost quiescent in their basal mechanical activities, they generated spontaneous rhythmic contractions in response to a MaxiK channel blocker, iberiotoxin (IbTx). The effect of IbTx on the mechanical activity was significantly greater in diabetic rat than in control animal. Similarly, the basal mechanical activity was increased with apamin, an inhibitor for some types of small conductance Ca2+-sensitive K+ channels, and this effect was more pronounced for diabetic rat. However, in both control and diabetic animals, IbTx action was stronger than that of apamin. Diabetes also enhanced the responses to BayK 8644, an L-type Ca2+ channel agonist. The extent of this enhancement in diabetic bladder vs. control was, however, almost the same as that attained with IbTx. Expression levels for MaxiK channel as well as apamin-sensitive K+ channels and L-type Ca2+ channel were not altered by diabetes, when determined as their corresponding mRNA levels. These results indicate that diabetes can potentially increase the basal UBSM mechanical activity. However, in diabetic UBSM, the main negative-feedback system triggered by MaxiK channel is still preserved enough to counteract the possible enhancement of this smooth muscle mechanical activity.

Influence of receptor number on the cAMP response to forskolin in Chinese hamster ovary cells transfected with human beta2-adrenoceptor.

We examined the basal adenosine 3',5'-cyclic monophosphate (cAMP) levels and forskolin-induced cAMP accumulation in cultured Chinese hamster ovary cells (CHO) expressing different levels of human beta(2)-adrenoceptors. Both the basal and forskolin-induced cAMP accumulation in the cells that express higher density of beta(2)-adrenoceptors (CHO-beta(2)/H; 560 fmol/mg protein) were larger than those in the cells that express lower density of beta(2)-adrenoceptors (CHO-beta(2)/L; 270 fmol/mg protein). In addition, isoproterenol-induced cAMP accumulation was also augmented as the number of beta(2)-adrenoceptors was increased. ICI 118,551, a selective beta(2)-adrenoceptor antagonist with inverse agonist properties, decreased all the levels of cAMP observed in both cell lines. These results suggest that the agonist-independent (constitutive) activity of beta(2)-adrenoceptors plays a key role in the control of forskolin-induced cAMP accumulation.