Design, synthesis, and biological evaluation of anti-HIV double-drugs. conjugates of HIV protease inhibitors with a reverse transcriptase inhibitor through spontaneously cleavable linkers.

Based on the prodrug concept as well as the combination of two different classes of anti-HIV agents, we designed and synthesized a series of anti-HIV double-drugs consisting of HIV protease inhibitors conjugated with a nucleoside reverse transcriptase inhibitor in an effort to enhance the antiviral activity. For the conjugation, a series of linkers that conjoins the two different classes of inhibitors has been investigated. Double-drugs using a succinyl amino acid linker were shown to release the parent drugs via spontaneous imide formation at a faster rate compared to compounds using a glutaryl amino acid linker, as expected from the energetically favorable cyclization to the five-membered ring. Among the double-drugs, KNI-1039 (3b) with a glutarylglycine linker exhibited extremely potent anti-HIV activity compared with that of the individual components. Double-drug 3b was relatively stable in culture medium, whereas it regenerated active species in cell homogenate. These results suggested that the synergistic enhancement of anti-HIV activities of 3b may be due to their ability to penetrate into the target cell and subsequent regeneration of two different classes of anti-HIV agents in the cytoplasm.

Gastric distension-induced pyloric relaxation: central nervous system regulation and effects of acute hyperglycaemia in the rat.

1. The pylorus plays an important role in the regulation of gastric emptying. In addition to the autonomic neuropathy associated with long-standing diabetes, acute hyperglycaemia per se has effects on gastric emptying. In this study, the role of the central nervous system in modulating the effects of hyperglycaemia on gastric distension-induced pyloric relaxation was investigated. 2. Gastric distension-induced pyloric relaxation was significantly reduced by subdiaphragmatic vagotomy, hexamethonium (20 mg kg(-1)) and N (G)-nitro-L-arginine methyl ester (L-NAME; 10 mg kg(-1)), a nitric oxide synthase (NOS) biosynthesis inhibitor, in anaesthetized rats. In contrast, neither splanchnectomy nor guanethidine (5 mg kg(-1)) had an effect. 3. An intravenous (I.V.) infusion of D-glucose (20 %) for 30 min, which increased blood glucose concentrations from 5.4 to 12.8 mM, significantly inhibited gastric distension-induced pyloric relaxation. 4. An intracerebroventricular (I.C.V.) injection of D-glucose (3 micromol) also significantly inhibited gastric distension-induced pyloric relaxation without affecting peripheral blood glucose concentrations. 5. I.V. infusion of D-glucose significantly elevated hypothalamic neuropeptide Y (NPY) concentrations. 6. Intracerebroventricular (I.C.V.) administration of NPY (0.03--3 nmol) and a Y1 receptor agonist, [leu(31), pro(34)] NPY (0.03--3 nmol), significantly inhibited gastric distension-induced pyloric relaxation in a dose-dependent manner. 7. I.C.V. administration of a Y1 receptor antagonist, BIBP 3226 (30 nmol), and of a NPY antibody (titre 1:24 000, 3 microl) abolished the inhibitory effects of hyperglycaemia on gastric distension-induced pyloric relaxation. 8. Taken together, these findings suggest that gastric distension-induced pyloric relaxation is mediated via a vago-vagal reflex and NO release. Acute hyperglycaemia stimulates hypothalamic NPY release, which, acting through the Y1 receptor, inhibits gastric distension-induced pyloric relaxation in rats exposed to acute elevations in blood glucose concentrations.

Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15.

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

Clinical and basic aspects of an anorexiant, mazindol, as an antiobesity agent in Japan.

The Japanese Mazindol study group investigated the action of an anorexiant, mazindol, and found that it reduced food intake by directly suppressing neurons in the lateral hypothalamus, inhibited gastric acid secretion, increased motor activity, decreased glucose absorption, and inhibited insulin secretion. It thus appears that the main effect of mazindol is to decrease food intake through suppressing feeding centers in the hypothalamus. A multicenter open study of mazindol in Japan revealed that loss of body weight and relative body weight in 14 wk were 4.6 kg and 9.2%, respectively, with suppression of appetite in the majority of obese patients. A multicenter double-blind study demonstrated that mazindol was superior to the placebo in the treatment of simple obesity. We also suggest that mazindol is effective in the maintenance of reduced body weight after obesity therapy and in the treatment of obesity-related diseases such as diabetes, hypertension, or hyperlipidemia.

Infusion of fish oil emulsion: effects on platelet aggregation and fatty acid composition in phospholipids of plasma, platelets, and red blood cell membranes in rabbits.

An emulsion of fish oil was manufactured to contain 10 g of fish oil/100 mL. Of this, 3 g were eicosapentaenoic acid (EPA) and 1 g was docosahexaenoic acid (DHA). We administered 100 mL of the emulsion into six rabbits intravenously on days 1, 4, 7, 10, and 13. Blood samples were taken on days 0 and 16. The EPA content in phospholipids of plasma, platelets, and red blood cell (RBC) membranes increased 16, 4, and 5 times, respectively. The DHA content in phospholipids of plasma and RBC membranes increased two times whereas that in platelet phospholipids did not increase significantly. Platelet aggregation induced by collagen (10 micrograms/mL) and ADP (5 microM) was depressed significantly after infusion of the fish oil emulsion. In control experiments with soybean oil emulsion, there were almost no significant changes. Therefore, fish oil emulsion may be beneficial to patients who cannot take n-3 fatty acids orally but need them.

Antiatherogenic action of eicosapentaenoic acid (EPA) in multiple oral doses.

The possible antiatherogenic action of eicosapentaenoic acid (EPA) was pharmacologically investigated using purified and ethylesterified fish oil containing 75% EPA (EPA-E) in multiple oral doses in rats and rabbits. EPA-E showed dose-dependent prevention of thrombus formation in a vascular shunt or sudden death caused by arachidonic acid injection in rats. EPA-E in daily doses ranging from 3 to 30 mg/kg slightly altered platelet aggregability and prostacyclin-like activity generated from arterial ring preparations of rats, but these alterations were not statistically significant. Further, EPA-E showed no effect on blood viscosity of rats. In cholesterol-fed rabbits, EPA-E in daily doses of 10 and 30 mg/kg moderately lowered the levels of plasma cholesterol, beta-lipoprotein, triglyceride and phospholipid, but these changes showed neither dose-dependency nor time-dependency. In this experiment, EPA-E moderately altered atherogenic plaque formation and platelet aggregability, but these alterations were not statistically significant. EPA-E showed no effect on prostacyclin-like activity generated from arterial ring preparations and blood viscosity of cholesterol-fed rabbits. It is, therefore, proposed that the antithrombotic action of EPA-E may be partially related to its effects on platelet aggregability and prostacyclin generation, but the major mechanism remains unclear.

Infusion of emulsified trieicosapentaenoyl-glycerol into rabbits--the effects on platelet aggregation, polymorphonuclear leukocyte adhesion, and fatty acid composition in plasma and platelet phospholipids.

Using 90%-pure free eicosapentaenoic acid, we synthesized 1,2,3-trieicosapentaenoyl-glycerol (EPA-TG) and manufactured an emulsion of EPA-TG with purified phosphatidylcholine from krill as an emulsifier. After two intravenous injections of the EPA-TG emulsion into rabbits, the EPA content in plasma and platelet phospholipids increased markedly. ADP- and collagen-induced platelet aggregation and polymorphonuclear leukocyte adhesion to glass beads were depressed significantly. No significant changes were observed in serum lipids and liver function. In control experiments which were performed in exactly the same manner except that soybean oil emulsion was used instead of the EPA-TG emulsion, there were almost no significant changes. Our results suggest that an EPA-TG emulsion is applicable to those patients who need both intravenous alimentation and preventive care of thrombosis, such as postoperative patients.

Acute and chronic complications of laser angioplasty: vascular wall damage and formation of aneurysms in the atherosclerotic rabbit.

Acute and chronic vascular responses to laser exposure in atherosclerotic rabbits were studied. In 7 rabbits fed an atherogenic diet for 3 to 5 months before the study to induce aortic atherosclerosis, a flexible quartz fiber, 400 micron core diameter, attached to an argon ion laser was passed anterogradely or retrogradely to the atherosclerotic ascending aorta. The laser was turned on using power intensities of 1 to 2 W for 3 seconds. After laser treatment, the aortas were studied acutely in 3 rabbits and chronically in 4 rabbits after recovery for 1 to 14 days. In 2 rabbits studied acutely, the argon laser produced a vaporized crater within the atherosclerotic plaque at the endothelial surface; however, in 1 there was also vascular damage extending deep into the medial layer. In addition, aortic aneurysm with muscular wall damage occurred in 2 of the 4 animals studied chronically. Thus, vascular complications may arise when catheter laser angioplasty is randomly applied without visualizing specific plaque targets or without using safe dose increments of power intensities and durations of exposure. This study suggests caution in the clinical use of intensive phototherapy to cardiovascular lesions and stresses the need for further understanding of laser vascular consequences before application of laser angioplasty in patients.

Studies on the mechanism of antiaggregatory effect of Moutan Cortex.

The effects of Moutan Cortex and one of its major components, paeonol, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets were studied. One week oral administration of water extract of Moutan Cortex [Moutan Cortex (w), 3 g/day] significantly reduced platelet aggregation and thromboxane B2 (TXB2) formation induced by collagen, epinephrine and ADP. Paeonol dose-dependently inhibited ADP and collagen induced platelet aggregation in vitro. Moutan Cortex (w) and paeonol dose-dependently inhibited the conversion of exogenous [14C]AA to [14C]heptadecatetraenoic acid [( 14C]HHT) and [14C]TXB2 by washed human platelets, while both of them increased its conversion to [14C]12-hydroxy eicosatetraenoic acid [( 14C]12-HETE). High dose of Moutan Cortex (w) inhibited the release of [14C]AA from prelabeled platelets in vitro, while paeonol did not. These results suggest that a reduction in platelet aggregation by the oral administration of Moutan Cortex might be ascribed to a decrease in thromboxane synthesis and that paeonol might play an important role in the antiaggregatory effect of Moutan Cortex because of its potent inhibitory effect on platelet aggregation and thromboxane formation.

Serum HDL-cholesterol-increasing and fatty liver-improving actions of Panax ginseng in high cholesterol diet-fed rats with clinical effect on hyperlipidemia in man.

Effects of Panax ginseng on plasma and hepatic lipids were investigated in the high cholesterol diet-fed rats and in patients with hyperlipidemia. Oral administration of red ginseng powder reduced plasma total cholesterol, triglyceride and NEFA, while plasma HDL-cholesterol was elevated. Platelet adhesiveness was also reduced by ginseng administration. The plasma lipid-improving actions were also observed in patients with hyperlipidemia. Hepatic cholesterol and triglyceride contents were decreased and phospholipid increased by ginseng administration in the high cholesterol diet-fed rats, corresponding to improvement of the fatty liver.

Plasma lipid-lowering action of ginseng saponins and mechanism of the action.

Elevation of plasma levels of cholesterol and triglyceride was reduced by the intramuscular injection of ginseng principles fraction 4 (saponin content, ca. 1/2). The elimination of intraperitoneally injected 4-[14C]-cholesterol from plasma was accelerated by fraction 4 administration. Fecal excretion [14C]bile acids and [14C]sterols after intraperitoneal injection of 4-[14C]-cholesterol was significantly increased by fraction 4 administration.

Plasma lipid-lowering and lipogenesis-stimulating actions of ginseng saponins in tumor-bearing rats.

Ascited hepatoma (AH41C or AH130) was transplanted to male rats Donryu strain. Plasma cholesterol, triglyceride (TG) and non-esterified fatty acid levels were reduced with oral administration of ginseng principle fraction 3 (saponin content, ca. 1/5). Incorporation of 1-[14C]-acetate into total lipids and fatty acids in adipose tissue was increased by fraction 3 administration in both normal and tumor-bearing rats. The incorporation increased in the earlier stage of tumor growth and decreased in the later one. Incorporation of 1-[14C]-acetate into total lipid, free and esterified cholesterol, TG and phospholipid in the liver was also enhanced by fraction 3 administration in both normal and tumor-bearing animals. In vitro addition of ginseng principle fraction 4 (saponin content, ca. 1/2) increased incorporation of 1-[14C]-acetate into lipid fractions in adipose tissue and liver. Incorporation of 1-[14C]-acetate into lipid fractions in ascites hepatoma cells remained unchanged with both oral administration of fraction 3 and in vitro addition of fraction 4. DNA and protein synthesis in the tumor cells was not changed with in vitro addition of fraction 4.

Effects of tocopherol deficiency on lipid metabolism in the arterial wall of rats on normal and high cholesterol diets.

The effects of dietary tocopherol deficiency on arterial wall enzymes involved in lipid synthesis and hydrolysis were studied in rats receiving normal diets and diets supplemented with 1% cholesterol. Arterial wall lipase and cholesterol esterase were associated with both the lysosome and microsome fractions, whereas acyl CoA synthetase, triglyceride synthesizing activity, cholesteryl ester synthesizing activity and cytidine diphosphatecholine-1,2-diacyl glycerol choline phosphotransferase (CPT) were found mainly in the microsomal fraction. When tocopherol was depleted from either the normal or high cholesterol diets, the following changes occurred in the arterial wall: (1) increase in thiobarbituric acid reactive substances; (2) decrease in lysosomal acid lipase and acid cholesteryl esterase; (3) decrease in the microsomal enzymes, acyl CoA synthetase, triglyceride synthesizing activity, cholesteryl ester synthesizing activity, neutral lipase and neutral cholesteryl esterase; and (4) increase in microsomal CPT. The results of these studies suggest that dietary tocopherol plays an important role in both lipid synthesis and degradation in the arterial wall, and the results may account for the accumulation of lipids and lipoperoxides in atherosclerotic lesions.

Effect of hypophysectomy and growth hormone administration on somatostatin content in the rat hypothalamus.

The effect of hypophysectomy and bovine GH administration on somatostatin (SRIF) content in the rat hypothalamus was investigated. SRIF content was determined by a specific radioimmunoassay method described elsewhere. The total SRIF content of the rat hypothalamus as well as its content per milligram wet weight had decreased 4 weeks after hypophysectomy but was restored significantly in those rats which were subjected to bovine GH administration for 7 days 3 weeks after hypophysectomy. Furthermore, in nonoperated rats, increase of hypothalamic SRIF content was observed after 7 days GH administration. These results indicate that growth hormone may influence the SRIF content of hypothalamus and it seems likely that a feedback mechanism between pituitary GH and hypothalamic SRIF exists.

Changes in pancreatic somatostatin content in spontaneously diabetic mice, as determined by radioimmunoassay and immunohistochemical methods.

A specific RIA for somatostatin (SRIF) was used to determine the SRIF content of the pancreas and hypothalamus in spontaneously diabetic C57BL/KsJ dbdb and C57BL/6J obob mice. In addition, SRIF- and glucagon-containing cells were examined in the pancreatic islets with an immunohistochemical technique. In dbdb mice, immunoassayable pancreatic SRIF content was increased, as was the number of SRIF- or glucagon-containing cells. In obob mice, immunoassayable pancreatic SRIF content was also increased, but no increase was noted in the number of SRIF- or glucagon-containing cells. The hypothalamic SRIF content of either strain was not different from that of controls. These results regarding pancreatic SRIF content were in accord with some but not all previous reports. These differences may be related to the age of the mice or to the conditions in which they were bred. The pancreatic SRIF increase in both dbdb and obob mice may be attributable to hyperglucagonemia, hyperglycemia, or a decrease in insulin action. Further work is necessary to clearly delineate the mechanism.

Stimulatory effect of ginsenosides on DNA, protein and lipid synthesis in rat bone marrow and participation of cyclic nucleotides.

Effects of several kinds of ginsenosides, saponins from Panax ginseng on DNA, RNA, protein and lipid synthesis in bone marrow were investigated. Single i.p. injection of 0.5--1 mg/100 g body weight of ginsenosides Rb2, Rc, Re and Rg1 4 h prior to the sacrifice increased DNA synthesis in bone marrow cells. RNA, protein and lipid synthesis were also increased. The direct addition of ginsenosides Rb1, Rb2 and Rc mixture (GNS) enhanced DNA synthesis. Cyclic AMP levels in bone marrow cells were decreased 20 min after i.p. injection of ginsenosides Rb2, Rc and Rg1 and the direct addition of ginsenosides Rb2, Rc and Rg1 also decreased cyclic AMP levels. While cyclic GMP levels were increased by administration of ginsenosides Rb2, Re and Rg1. Relationship between chemical structure and actions of ginsenosides and the role of cyclic nucleotides in the stimulatory action of ginsenosides on DNA synthesis in bone marrow cells were discussed.