First-in-human phase I study of SOR-C13, a TRPV6 calcium channel inhibitor, in patients with advanced solid tumors.

Introduction This was an open-label, dose escalation (3 + 3 design), Phase I study of SOR-C13 in patients with advanced tumors of epithelial origin. Primary objectives were to assess safety/tolerability and pharmacokinetics. Secondary goals were to assess pharmacodynamics and efficacy of SOR-C13. Methods SOR-C13 was administered IV QD on days 1-3 and 8-10 of a 21-day cycle. Doses were 2.75 and 5.5 mg/kg (20-min infusion) and 1.375, 2.75, 4.13 and 6.2 mg/kg (90-min infusion). Toxicity was assessed by National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Dose limiting toxicity (DLT) was assessed within the first treatment cycle. Tumors were evaluated, using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, after two cycles. Results Twenty-three patients were treated. No drug-related serious adverse events occurred. DLTs occurred in six patients: asymptomatic, drug-related, transient Grade 2 hypocalcemia (4 patients), and unrelated Grade 3 anemia and Grade 3 atrial fibrillation, 1 patient each. Calcium and vitamin D supplementation eliminated further Grade 2 hypocalcemia. One Grade 3 treatment emergent adverse event, urticaria, was definitely related to SOR-C13. Four possibly drug-related, Grade 3 events (alanine aminotransferase and aspartate aminotransferase elevation, headache, and hypokalemia) were observed. Of 22 evaluable patients, 54.5% showed stable disease ranging from 2.8 to 12.5 months. The best response was a 27% reduction in a pancreatic tumor with a 55% reduction in CA19-9 levels at 6.2 mg/kg. Conclusion SOR-C13 was safe and tolerated up to 6.2 mg/kg. The Maximal Tolerated Dose (MTD) was not established. Stable disease suggested antitumor activity.

Molybdate stabilized rat uterine progesterone receptors: evidence for two mechanisms.

Molybdate (Mo), EGTA, or protease inhibitors substantially increase detectable rat uterine progesterone (Pg) receptors. Rehomogenization experiments demonstrated that receptor levels decreased in the absence of Mo or protease inhibitors and were not regenerated. Thus Mo prevents an EGTA and protease-inhibitor-sensitive loss of uterine Pg receptors during homogenization. This effect was compared with receptor stabilization at elevated temperature. In contrast to the stability of receptors in the presence of Mo, receptors decreased rapidly to minimal levels by 30 min at 30 degrees C in TESHG (10 mM Tris, 1.5 mM EGTA, 12 mM thioglycerol, 10% glycerol) or TG buffers. The ability of EGTA to mimic receptor stabilization by Mo during homogenization, compared with its ineffectiveness at 30 degrees C, suggested fundamentally different mechanisms for these two phenomena. Similarly, 0.3 M KCl prevented Mo stabilization of the receptors at 30 degrees C, but did not change their recovery after homogenization. Results with protease inhibitors were also consistent: addition of 2-5 mM leupeptin and 500 microM PMSF to TG during homogenization resulted in substantially increased (P less than 0.01) receptor recovery, but leupeptin (+/- the temperature-labile PMSF) did not prevent the Pg receptor losses at 30 degrees C. The transformation state of the receptors may be important, since receptors were untransformed in the presence of either EGTA or Mo. Moreover, KCl transformed the receptors in parallel to their instability at 30 degrees C. In conclusion, Mo stabilizes Pg receptors during temperature elevation by a different mechanism from that involved during homogenization. Although the parallel effects by EGTA, molybdate, and the protease-inhibitors during homogenization is consistent with inhibition of Ca2+-dependent proteolysis, other possible mechanisms must be considered in future studies.

Rat uterine progesterone receptor analyzed by [3H]R5020 photoaffinity labeling: evidence that the A and B subunits are not equimolar.

The hormone-binding components of the rat uterine progesterone receptor were investigated by the methods of [3H]R5020 photoaffinity labeling and sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis. Two specifically labeled peaks were observed at mol wt of 85,600 +/- 1,200 and 109,600 +/- 1,200 (n = 31), resembling the A and B progesterone receptor components previously described in other systems. However, in contrast to the equimolar ratio reported in other systems, the level of subunit A observed was consistently greater than that of B (A/B ratio = 3.2 +/- 0.3; n = 31). The unusual A/B ratio prompted a complete validation of the photolabeling procedure in this system. Although the levels of specific binding increased, there was no change in the A/B ratio with varying [3H]R5020 concentrations (5-80 nM) or with time of UV exposure (0.5 min to 3 h). Although adsorption to hydroxylapatite indicated that specific [3H]R5020 binding was reduced by 72.0 +/- 6.4% within 5 min of UV exposure, relabeling the irradiated preparations with [3H]R5020 resulted in little change in specific [3H]R5020 binding. TLC analysis of [3H]R5020 (Rf = 0.48 +/- 0.01; n = 4) after irradiation demonstrated rapid photolysis resulting in a 94.3 +/- 2.5% (n = 3) loss of authentic [3H]R5020 within 5 min. After photolysis, at least two new tritiated products were recovered with Rf values of 0.20 +/- 0.03 and 0.72 +/- 0.02. Analysis by adsorption to hydroxylapatite indicated that the photolysis products competed for specific [3H]R5020-binding sites in cytosol with only 10-fold lower relative binding activity than authentic R5020. Thus, these compounds probably account for the increase in specific photolabeling of the A and B peaks achieved when UV exposure is prolonged from 5 to 30 min. Further study indicated that the A/B subunit ratio in this system was not changed under a variety of in vitro conditions, including the absence or presence of molybdate, sulfhydryl protective reagents (dithiothreitol and thioglycerol), protease inhibitors (phenylmethylsulfonylfluoride and leupeptin), glycerol (0%, 10%, and 30%, vol/vol), or 1.5 mM EGTA, or after precipitation with 40% ammonium sulfate. This consistency of the A/B ratio under a wide variety of adverse in vitro conditions suggests that in vitro artefacts may not account for the ratio's deviation from unity. Estrogen withdrawal (48 h) enhanced by progesterone treatment (0.5 mg for 24 h) resulted in only a modest reduction in the A/B ratio to 1.9 +/- 0.1.(ABSTRACT TRUNCATED AT 400 WORDS)

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+ uptake by cultured adult rat ventricular cardiac muscle cells.

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and its previously described cardiac receptors play roles in regulating intracellular calcium homeostasis in cardiac muscle cells. This question was addressed by assessing whether 1,25-(OH)2D3 influences 45Ca2+ uptake by homogeneous cultures of adult rat ventricular cardiac muscle cells. Twenty-four h prior to the measurement of 45Ca2+ uptake, the cells were transferred to serum-free medium ([Ca2+], 1.0 mM) containing 1.0 nM 1,25(OH)2D3 or vehicle. The cells were then incubated with 45Ca2+ for periods up to 60 min at room temperature, followed by removal of excess external 45Ca2+ by washing repeatedly with La3+. Pretreating the cells with 1,25-(OH)2D3 caused 3-fold stimulation (p less than 0.005) of 45Ca2+ uptake. Stimulation of 45Ca2+ uptake required a prolonged (8-12 h) exposure to 1,25-(OH)2D3, suggesting a receptor-mediated phenomenon. Concentrations of 0.01-10 nM 1,25-(OH)2D3 yielded a dose-response curve which peaked at 1.0 nM and decreased at higher concentrations. Steroid specificity was established by the failure of 1.0 nM levels of 25-hydroxyvitamin D3, estradiol-17 beta, and progesterone to change 45Ca2+ uptake. Sucrose gradient analysis confirmed the presence of a specific 3-4 S 3H-1,25-(OH)2D3 binding component both in freshly isolated and in cultured ventricular cardiac muscle cells. The stimulatory effect of 1,25-(OH)2D3 on 45Ca2+ uptake was abolished by the concomitant incubation of the cells with cycloheximide or actinomycin D, demonstrating a requirement for protein and nucleic acid synthesis. In conclusion, these data demonstrate that 1,25-(OH)2D3 stimulates 45Ca2+ uptake in adult ventricular cardiac muscle cells by a mechanism resembling a receptor-mediated phenomenon.

Juvenile hormone increases ouabain-binding capacity of microsomal preparations from vitellogenic follicle cells.

A comparison has been made of the effects of juvenile hormone (JH) on the binding characteristics for ouabain of microsomes prepared from brain and from cells of the follicular epithelium surrounding previtellogenic or vitellogenic oocytes in Rhodnius. JH has no effect on the binding of ouabain to brain microsomes and decreases the Kd, but does not alter the Bmax for previtellogenic follicle cells. For vitellogenic follicle cells. Scatchard analysis reveals a curvilinear relationship, which is interpreted as indicating that a new population of JH-sensitive ouabain-binding sites develops as the follicle cell enters vitellogenesis. These results are related to earlier data obtained on the effect of JH on ATPase activity, volume changes in isolated follicle cells, and the development of spaces between the cells of the follicular epithelium.