Adjuvant immunotherapy with autologous dendritic cells for hepatocellular carcinoma, randomized phase II study.

Our previous phase I/IIA study showed that autologous dendritic cells (DCs) pulsed with tumor-associated antigens are well tolerated in patients with hepatocellular carcinoma (HCC). In this randomized, multicenter, open-label, phase II trial, we investigated the efficacy and safety of this DC-based adjuvant immunotherapy with 156 patients, who treated for HCC with no evidence of residual tumor after standard treatment modalities. Patients were randomly assigned to immunotherapy (n = 77; injection of 3 × 107 DC cells, six times over 14 weeks) or control (n = 79; no treatment). The primary end point was recurrence-free survival (RFS), and the secondary endpoints were immune response and safety. The RFS between the immunotherapy and control groups was not significantly different (hazard ratio [HR], 0.97; 95% confidence interval [CI], 0.60-1.56; p = 0.90). However, post-hoc subgroup analyses revealed that DC immunotherapy significantly reduced the risk of tumor recurrence of non-radiofrequency ablation (non-RFA) group patients (n = 83, HR, 0.49; 95% CI, 0.26-0.94; p = 0.03), whereas unexpectedly increased the risk of recurrence in RFA group (n = 61, p = 0.01). Tumor-specific immune responses were significantly enhanced (both p < 0.01) in the immunotherapy group. Baseline serum interleukin (IL)-15 was statistically correlated with RFS prolongation (HR, 0.16; 95% CI, 0.03-1.58; p = 0.001) within the immunotherapy groups. Overall adverse events were more frequent in the immunotherapy group (p < 0.001) but were mainly mild to moderate in severity. In conclusion, adjuvant immunotherapy with DC vaccine reduces the risk of tumor recurrence in HCC patients who underwent standard treatment modalities other than RFA. Baseline IL-15 might be a candidate biomarker for DC-based HCC immunotherapy.

Newly discovered orally active pure antiestrogens.

In order to develop orally active pure antiestrogens, we incorporated the carboxy-containing side chains into the 7alpha-position of the steroid scaffold and found that 17-keto derivative CH4893237 (12b) functioned as a pure antiestrogen with its oral activity much superior to clinically used pure antiestrogen, ICI182,780. Results from the pharmacokinetic evaluation indicated that the potent antiestrogen activity at oral dosing in mice attributed to both improved absorption from the intestinal wall and metabolic stability in liver.

Discovery of thiochroman derivatives bearing a carboxy-containing side chain as orally active pure antiestrogens.

In order to search for alternatives to the sulfoxide moiety in the long side chain of pure antiestrogens, several molecules that may interact with water in a fashion similar to ICI164,384 were designed and it was found that compounds with the carboxy, the sulfamide, or the sulfonamide instead of the sulfoxide moiety also functioned as pure antiestrogens. Interestingly, the compound possessing the carboxy moiety showed superior antiestrogen activity compared to ICI182,780 when dosed orally. Results of the pharmacokinetic evaluation indicated that the potent antiestrogen activity at oral dosing attributed to both the improved absorption from the intestinal wall and the metabolic stability of the compound in liver.

Discovery of thiochroman and chroman derivatives as pure antiestrogens and their structure-activity relationship.

In order to develop pure antiestrogens, a series of 7-hydroxy-3-(4-hydroxyphenyl)-3-methylchroman and 7-hydroxy-3-(4-hydroxyphenyl)-3-methylthiochroman derivatives with sulfoxide containing side chains at the 4-position were designed, synthesized, and evaluated. Among them, compounds 14b and 24b functioned as pure antiestrogens with the ability to downregulate ER, and their in vitro and in vivo antiestrogen activities were similar to those of ICI182,780. In addition, the structure-activity relationship indicated that the (3RS,4RS)-configuration between the 3- and 4-position, the methyl group at the 3-position, the 9-methylene chain between the scaffold and the sulfoxide moiety, and the terminal perfluoroalkyl moiety play an important role in increasing estrogen receptor binding and oral antiestrogen activities.

Role of aspartate 351 in transactivation and active conformation of estrogen receptor alpha.

Estrogen-dependent transcriptional activation by estrogen receptor alpha (ERalpha) depends on the conformation of helices 3 and 12 in the ligand-binding domain. To better understand the function of helix 3 in ERalpha, we examined the role of charged residues, which are conserved in most steroid receptors in helix 3, in estrogen-dependent transactivation. The replacement of Asp-351 with lysine (D351K) or leucine (D351 L) completely abolished estrogen-dependent transactivation without affecting estrogen-binding, DNA-binding and homodimerization activities in ERalpha. The mutations dramatically reduced the ligand-dependent activation function 2 activity and impaired the ability of ERalpha to bind p160 coactivators. In addition, the D351K mutant effectively inhibited the transcriptional activation activity of wild-type ERalpha. Furthermore Asp-351 was required not only for the estrogen-dependent conformational change of wild-type ERalpha but also for the constitutive transcriptional activity and ligand-independent active conformation of ERalpha mutant Y537N. Similarly, in the orphan nuclear receptor called estrogen-related receptor 3 (ERR3), the replacement of Asp-273 (the corresponding amino acid to Asp-351 in ERalpha) with lysine abolished constitutive transcriptional activity of ERR3 without affecting DNA-binding activity and impaired the ability of the receptor to interact with p160 coactivators. These data suggest a role of Asp-351 in inducing and stabilizing the active conformation of ERalpha, and our results experimentally confirm the concept that Asp-351 in helix 3 interacts with the amide hydrogen of L540 in helix 12 to form a transcriptionally competent surface for binding p160 coactivators.

Evaluation of KCB-328, a new IKr blocking antiarrhythmic agent in pacing induced canine atrial fibrillation.

HYPOTHESIS: KCB-328 is a new potassium channel blocker, which prolongs action potential duration with exhibition of minimal reverse use dependence. We tested the efficacy and proarrhythmic potential of KCB-328, dofetilide and propafenone in the pacing induced canine model of atrial fibrillation (AF). METHODS: Mongrel dogs in complete heart block were paced for 1-6 weeks to produce AF, and given KCB-328 or dofetilide. A subset then received propafenone 14+/-3 days after testing the first drug. RESULTS: KCB-328 prolonged right and left atrial (RA and LA) activation times and AF cycle length (CL), terminating AF in 3 of 6 dogs. RA effective refractory period (ERP) and ventricular ERP and QT interval were prolonged. Dofetilide terminated AF in 1/6 dogs, and increased AF CL and ventricular ERP and QT interval. Dofetilide's reverse use dependency on the QT interval was greater than KCB-328. Propafenone prolonged RA and LA activation times and AF CL and terminated AF in 8 of 9 dogs. One death occurred with dofetilide, none with KCB-328 or propafenone. CONCLUSION: The spectrum of effect of the three drugs differed significantly: propafenone showed the greatest success in AF termination, and both propafenone and KCB-328 appeared less proarrhythmic than dofetilide in this model.

A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].

Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.

Differential recovery of action potential duration and HERG currents from the effects of two methanesulfonamide class III antiarrhythmic agents, KCB-328 and dofetilide.

A novel pure class III antiarrhythmic agent, 1-(2-amino-4-methanesulfonamidophenoxy)-2-[N-(3,4-dimethoxyphen-ethyl)-N-methylamino]ethane hydrochloride (KCB-328) prolongs action potential duration (APD) by blocking the rapid component delayed rectifier K+ current (IKr). However, KCB-328 manifests little of the reverse frequency dependence (RFD) that is a general characteristic of class III antiarrhythmic agents. We have studied the onset and recovery kinetics of KCB-328 and dofetilide on APD in guinea-pig papillary muscle and on human ether-a-go-go-related gene (HERG) channel, which encodes IKr, expressed in Xenopus oocytes. KCB-328 (1 microM) and dofetilide (0.1 microM) progressively increased the duration of post-rest AP at 1-Hz stimulation, with onset time constants of 6.4 +/- 0.6 seconds and 20.7 +/- 1.8 seconds, respectively. With a 100-second resting period, the effect of KCB-328 recovered by 70% with a time constant of 13.2 +/- 4.2 seconds, whereas that of dofetilide recovered only by 25%. Both drugs blocked activated HERG channels in a biexponential decay fashion, with faster time constants for KCB-328 (3 microM) than for dofetilide (0.3 microM). After a 300-second resting period, HERG current inhibited by KCB-328 was recovered more at depolarized membrane potentials than at hyperpolarized ones, with a time constant of 179.9 seconds during the rest at -60 mV. In contrast, recovery after dofetilide was negligible at all voltages tested. These results suggest that KCB-328 binds to IKr at a preferentially open state in a use-dependent manner, but that KCB-328 unbinds from the resting state more readily than dofetilide. The less striking RFD of KCB-328 than of dofetilide might be related to the faster recovery from its effect on IKr.