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.     

Surgical experience of subacute pulmonary thromboembolism with severe pulmonary hypertension.

Surgical treatment for subacute pulmonary arterial thromboembolism has previously been considered to be inappropriate. We undertook a pulmonary arterial thrombectomy and removal of a floating right heart thrombus in a patient who had been symptomatic for over a month. The pulmonary arterial pressure, which had been equal to the systemic pressure preoperatively, decreased gradually and almost normalized one month postoperatively. Pulmonary perfusion scintigraphy revealed a dramatic improvement and the patient returned to normal life activities.     

Use of Glycopeptidolipid Core Antigen for Serodiagnosis of Mycobacterium avium Complex Pulmonary Disease in Immunocompetent Patients

We report the development of a serodiagnostic method for Mycobacterium avium complex (MAC) disease with an enzyme immunoassay (EIA) with the MAC-specific glycopeptidolipid (GPL) core as the antigen. In this study, we confirmed by EIA that the GPL core antibody was in the sera of immunocompetent patients with MAC disease. The EIA for quantifying the GPL core antibody was evaluated as a clinical tool for serodiagnosis of pulmonary MAC disease. A significant increase in GPL core antibodies (immunoglobulins G, A, and M) was detected in sera of patients with MAC pulmonary diseases when they were compared to patients who were colonized with MAC, patients with Mycobacterium kansasii disease or tuberculosis, and healthy subjects. The sensitivities and specificities of the GPL core-based EIA for diagnosis of MAC pulmonary disease were 72.6% and 92.2%, respectively, for IgG, 92.5% and 95.1%, respectively, for IgA, and 78.3% and 91.0%, respectively, for IgM. The best sensitivity and specificity were obtained by measuring immunoglobulin A antibodies against GPL core antigen. The level of GPL core antibodies reflected disease activity, since it decreased in cured MAC patients who had responded to chemotherapy. Measurement of serum antibodies against GPL core is useful for both diagnosis and assessment of disease activity in MAC disease of the lung.     

Myocardial protective effect of lidocaine during experimental off-pump coronary artery bypass grafting.

Off-pump coronary artery bypass grafting (OPCABG) has recently gained popularity. During OPCABG, patients remain vulnerable to ischemic-reperfusion injury due to a temporary coronary occlusion without any active cardioprotection. Some strategies such as ischemic preconditioning (IP) and an intracoronary shunt have been applied with a view to minimizing the effects of ischemia, but the effects of these strategies remain controversial. This study was carried out to investigate the protective effect of lidocaine against myocardial ischemic-reperfusion injury. Twenty-one pigs were assigned to three groups, each consisting of seven pigs. In the control group, using a left internal thoracic artery (LITA) bypass circuit, the left anterior descending coronary artery (LAD) was occluded for 45 min followed by two hours of reperfusion. In the IP group, five min of occlusion followed by 15 min of reperfusion was performed. In the lidocaine group, 2 mg/kg of lidocaine was administered directly into the LAD just before the LAD occlusion. Infarct size expressed as a percentage of the area at risk was significantly smaller in the lidocaine group (2.7+/-4.2%) than in the control group (79.9+/-6.0%, p<0.001) or the IP group (57.0+/-25.9%, p<0.001). Lidocaine exhibited a potent myocardial protective effect in the present OPCABG model.     

Ketamine Suppresses Platelet Aggregation Possibly by Suppressed Inositol Triphosphate Formation and Subsequent Suppression of Cytosolic Calcium Increase

Background: Ketamine has been shown to suppress platelet aggregation, but its mechanisms of action have not been defined. The purpose of the current study is to clarify the effects of ketamine on human platelet aggregation and to elucidate the underlying mechanisms of its action.

Methods: Platelet aggregation was measured using an eight-channel aggregometer, and cytosolic free calcium concentration was measured in Fura-2/AM-loaded platelets using a fluorometer. Inositol 1,4,5-triphosphate (IP3) was measured with use of a commercially available IP3 assay kit. To estimate thromboxane A2 (TXA2) receptor binding affinity and expression, Scatchard analysis was performed using [3H]S145, a specific TXA2 receptor antagonist. TXA2 agonist binding assay was also performed. The membrane-bound guanosine 5'-triphosphatase activity was determined using [[gamma]-32P]guanosine triphosphate by liquid scintillation analyzer.

Results: Ketamine (500 [mu]m) suppressed aggregation induced by adenosine diphosphate (0.5 [mu]m), epinephrine (1 [mu]m), (+)-9,11-epithia-11,12-methano-TXA2 (STA2) (0.5 [mu]m), and thrombin (0.02 U/ml) to 39.1 +/- 30.9, 46.3 +/- 4.3, -2.0 +/- 16.8, and 86.6 +/- 1.4% of zero-control, respectively. Ketamine (250 [mu]m-1 mm) also suppressed thrombin- and STA2-induced cytosolic free calcium concentration increase dose dependently. Although ketamine (2 mm) had no effect on TXA2 receptor expression and its binding affinity, it (1 mm) suppressed intracellular peak IP3 concentrations induced by thrombin and STA2 from 6.60 +/- 1.82 and 4.39 +/- 2.41 to 2.41 +/- 0.98 and 1.90 +/- 0.86 pmol/109 platelets, respectively, and it suppressed guanosine triphosphate hydrolysis induced by thrombin (0.02 units/ml) and STA2 (0.5 [mu]m) to 50.3 +/- 3.2 and 67.5 +/- 5.5%versus zero-control, respectively.     

A high percentage yield of tyrosine hydroxylase-positive cells from rat E14 mesencephalic cell culture.

In the ventral mesencephalon of the E14 rat fetus, 90% of the dopaminergic, tyrosine hydroxylase positive (TH+) cells are localized in 1.0 mm3 of tissue. This same ventral mesencephalic region also contains 90% of the dopamine content of the E14 ventral brainstem (2.2 +/- 0.3 nmol/mg protein). When cells were prepared for culturing from this localized area, and plated at a density of 2.5 x 10(5) cells/cm2, 17-21% of the cells were TH+, at 4 and 12 h, and at 1, 5, 7 and 10 days after plating. The percentage of TH+ cells was also 17-21% when examined at 4 h, 12 h or 5 days after plating at densities ranging from 7.8 x 10(3) to 2.5 x 10(5) cells/cm2. However, cell survival at a density of less than 6.2 x 10(4) cells/cm2 was poor after 5 days in culture. Based on the degree of neurite elongation and complexity, cell maturation appeared to be complete at 5 days in culture (DIV5), and appeared to be maintained at this level up to DIV10. By DIV14, neurite retraction was evident, and the cells were more rounded. These signs may indicate the inception of senescence in the cultures. A benztropine-sensitive, concentration-dependent dopamine uptake mechanism was demonstrated in the cultures at DIV7, and DA could be released from preloaded cells using 50 mM K+. Five morphological subtypes of TH+ cells were identified in the cultures. This primary culture of the ventral mesencephalic, dopaminergic area, with a high percentage of TH+ cells, is suitable for use in acute biochemical and cellular studies, between DIV 5 and DIV10.     

HISTOPATHOLOGIGAL STUDY OF HYPERTROPHIC CARDIOMYOPATHY WITH PROGRESSION TO LEFT VENTRICULAR DILATATION

The heart of seven cases of fatal congestive heart failure with dilated left ventricle, developing in 5 patients with symptomatic hypertrophic cardiomyopathy (HCM) and 2 patients with histologically widespread disarray of both ventricles, was morphologically investigated. These 7 cases showed myocardial widespread disarray and massive fibrosis, the mean percent area of fibrosis was 40.6% and 59.4% at upper and lower levels of left ventricles, respectively. Fibrosis was most extentsive in the lateral wall, and followed by anterior, posterior and interventricular walls. The severity of cell infiltration in left ventricle was completely matched to that of fibrosis and was most extensive in subepicardial area followed by middle and subendocardial areas of left ventricle. The intima and medial thickness of intramural small arteries in the fibrotic areas was significantly larger (p<0.05) than that of nonfibrotic areas, which suggested that the effect of intramural small artery was not essential for pathogenesis of massive fibrosis. ACTA PATHOL. JPN. 37: 1041 -1052, 1987.     

Discrepancy of xenon concentrations between end-tidal and blood collection methods in xenon-enhanced computed tomographic measurement of cerebral blood flow

Summary Using xenon-enhanced computed tomography for the study of cerebral blood flow, simultaneous measurements of end-tidal and arterial blood xenon concentrations using the blood collection method were performed to investigate the validity of substituting the end-tidal for the arterial blood xenon concentration. Simultaneous measurement by both methods was performed 68 times in 27 patients. There was no statistical correlation between the arterial blood accumulation rate constant obtained by arterial blood and end-tidal samples, nor between the arterial blood saturation value obtained by the two methods, even when correction was made for age. In brain tissue, all parameters calculated using the end-tidal concentration were lower than those using arterial blood. We therefore suggest that cerebral blood flow values calculated using end-tidal xenon concentration are useful only for qualitative cerebral blood flow mapping, and not applicable to absolute values of cerebral blood flow.