Antitumor effect of KT6124, a novel derivative of protein kinase inhibitor K-252a and its mechanism of action

Summary Novel derivatives of K-252a, (8R*,9S*,11S*)-(–)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo [a,g]-cycloocta[cde]trinden-1-one, an inhibitor of protein kinases and calmodulin-dependent phosphodiesterase, were synthesized and evaluated for their antitumor activity in vitro and in vivo. Of ten derivatives tested, four were active against the P388 murine leukemia i. p.-i. p. system, although K-252a was inactive. Among these derivatives, KT6124 was selected for further biological evaluation studies because its efficacy was the highest. KT6124 was also active against sarcoma 180 and B16 melanoma. It exerted a relatively broad spectrum of antiproliferative activity against 20 human tumor cell lines in vitro. To determine the mechanism(s) of action underlying the antitumor activity of KT6124, we tested the drug for inhibition of protein kinases, including Ca²⁺-and phospholipid-dependent protein kinase (PKC), in intact A431 human epidermoid carcinoma cells in comparison with the PKC-inhibitory activity of K-252a. KT6124 did not antagonize the action of phorbol 12-myristate 13-acetate (PMA) in A431 cells, whereas K-252a did, suggesting that KT6124 may not act on protein kinases in the cells. The interaction of KT6124 with DNA in living cells was examined by the alkaline elution method. KT6124 apparantly exhibited DNA scission both dose-and time-dependently in the target cells. The DNA breakage was dependent on proteinase K treatment, suggesting its possible interaction with DNA-related enzyme(s). These results indicate that KT6124 exerts antitumor activity by acting on DNA or on DNA-related enzyme(s) in tumor cells rather than via the inhibition of protein kinases.     

Classification of Diabetes Mellitus by Studies of C-Peptide Secretory Capacity

Residual pancreatic B-cell function was investigated in children with diabetes mellitus in whom classification of the type of disease was difficult at the first visit. Intravenous glucagon tests were performed at the first visit and subsequently, the C-peptide responses compared. Based on our data on a limited number of patients, we propose C-peptide concentrations of 3.0 to 3.5 ng/ml at the peak or at 6 min after injection of glucagon, as the critical level which distinguishes non-insulin dependent from insulin-dependent diabetes mellitus. However, the degree of obesity, clinical stage and other factors also need to be considered in the classification of diabetes mellitus.     

Hepatic conversion of 1-(tetrahydro-2-furanyl)-5-fluorouracil into 5-fluorouracil in patients with hepatocellular carcinoma

The pharmacokinetics of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) and its conversion into 5-fluorouracil (FUra) in liver tissue were studied in ten patients with hepatocellular carcinoma (HCC). The plasma concentration of FT after its intravenous injection (dosage: 800 mg) was computerfitted to a bi-exponential function (C = Ae-alpha t + Be-beta t), indicating a two-compartment disposition. The pharmacokinetic parameters did not significantly differ between the five patients with, and the five without cirrhosis of the liver. The plasma concentrations of FUra likewise showed no significant difference between the two groups. The rates of FT degradation in the liver tissue homogenate were similar for four of the patients with cirrhosis (0.10 +/- 0.05 mumol/g liver protein/30 min) and four of those without it (0.13 +/- 0.05). The rates of cytochrome P-450-dependent FUra formation in the microsomal fraction of liver tissue from two patients (1.1 and 1.3 nmol/mg microsomal protein/30 min) were dramatically reduced to less than half of those of two control subjects (2.4 and 2.7). The estimated rates of FUra formation in the soluble fraction (105,000 X g supernatant fraction) from the two patients (0.1 and 0.13 nmol/mg protein/30 min) were almost identical to those from the controls (0.12 and 0.14), suggesting that the rate in the soluble fraction from HCC patients may not be as strongly affected as the rate in the microsomal fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal fluorine-18-fluorodeoxyglucose uptake in benign esophageal leiomyoma

A benign esophageal leiomyoma with abnormally increased fluorine-18-fluorodeoxyglucose uptake on positron emission tomography (PET) was resected thoracoscopically. The tumor, of which the maximum standardized uptake value of the lesion was 4.7, was well defined and 38 mm in diameter. Neither mitotic activity nor degeneration was found histologically; and immunoreactivity for CD34, CD117, MIB-1, and glucose transporter-1 was negative immunohistochemically. A diagnosis of gastrointestinal stromal tumor was ruled out by an oncogenic kinase gene mutation study. This case cautions against PET-dependent evaluation for malignant potential of esophageal submucosal tumors.     

Ascending contraction and descending relaxation in the distal colon of mice lacking interstitial cells of Cajal.

Recently an essential role of interstitial cells of Cajal (ICC) within myenteric plexus (ICC-MY) was suggested in ascending contraction and descending relaxation in the mouse ileum. The role of ICC in these neural reflexes was examined in the distal colonic segments prepared from the wild type and c-kit mutant, W/W(V) mice, in the present study. Localized distension of the segments from the wild type mice by using a small balloon resulted in ascending contraction and descending relaxation. In the segments from the mutant mice, localized distension also induced these neural reflexes similar to those observed in the wild type mice. Immunohistochemical examination demonstrated that ICC-MY and ICC present in muscle layers (ICC-IM) were severely disrupted in the mutant mouse, but only ICC, present within submucosal plexus (ICC-SMP), remained unchanged. In the small strips with ICC-SMP absent prepared from the mutant mouse, electrical field stimulation induced contraction or relaxation in the absence or presence of atropine, respectively. It was suggested that ICC have no important role in the ascending and descending neural reflexes in the mouse distal colon, this is in direct contrast to the role of ICC-MY in the ileum.     

Cancer detection using a PET tracer, 11C-glycylsarcosine, targeted to H+/peptide transporter.

H+/peptide transporter, PEPT1, is functionally expressed in some human cancer cell lines and might be a candidate molecular target for detection of cancers in vivo using PET. The aim of the present study was to establish a novel tumor-imaging technology using a PET tracer targeted to H+/peptide transporter(s). We also compared the tracer with 18F-FDG, focusing on the specificity of their accumulation between tumor and inflammatory tissues. METHODS: A dipeptide PET tracer, 11C-glycylsarcosine (11C-Gly-Sar), was injected intravenously into athymic mice transplanted with human pancreatic, prostate, and gastric cancer cells. The distribution patterns of 11C-Gly-Sar and 18F-FDG in the tumor-bearing mice, and in mice with inflammatory tissue, were assessed by imaging with a positron planar imaging system (PPIS). Tissue distributions of tracer radioactivity were also measured. The expression levels of PEPT1 and PEPT2 (PEPTs) proteins in tumor xenografts and inflammatory tissue were examined by immunohistochemical analysis. The messenger RNA expression levels of PEPTs in 58 available cancer cell lines were quantified by means of real-time polymerase chain reaction. RESULTS: All 3 tumor xenografts were well visualized with the PPIS after injection of 11C-Gly-Sar. Expression of PEPTs in those xenografts was confirmed by immunohistochemical analysis. Tumor-to-blood concentration ratios of 11C-Gly-Sar increased in a time-dependent manner and were much higher than unity. Most of the radioactivity found in the tumor tissue was recovered as the intact tracer. These results indicated that 11C-Gly-Sar was taken up by the PEPTs in tumor xenografts. It is noteworthy that 11C-Gly-Sar was minimally present in inflammatory tissues that expressed no PEPT1 or PEPT2 protein, whereas 18F-FDG was highly accumulated, with the values of the selectivity index being >25.1 and 0.72 for 11C-Gly-Sar and 18F-FDG, respectively. The mRNAs of PEPT1 and PEPT2 were expressed in 27.6% and 93.1%, respectively, of the cancer cell lines examined in the present study. CONCLUSION: The present study indicates that 11C-Gly-Sar is a promising tumor-imaging agent and is superior to 18F-FDG for distinguishing between tumors and inflammatory tissue. Because PEPTs were ubiquitously expressed in various types of tumor cells examined, 11C-Gly-Sar could be useful for the detection of many types of cancers.