The Hypolipidemic Activity of Boronated Nucleosides in Male Mice and Rats

The boronated nucleosides with varying bases and sugar moieties were shown to be potent hypolipidemic agents in rodents. The 3′– aminocynaoborane dideoxythymidine derivative caused reductions in serum cholesterol and triglyceride levels, tissue lipids, VLDL and LDL cholesterol levels while elevating HDL cholesterol levels in rodents. The agents suppressed rat hepatic acetyl CoA synthetase, HMG-CoA reductase, acyl-CoA cholesterol acyl transferase, phosphatidylate phosphohydrolase and lipoprotein lipase activities while elevating cholesterol-7α-hydroxylase activity from 25 to 100 μM.

Introduction" Previously 2'-deoxyribonucleoside cyanoboranes and phosphate-boronated nucleotides were shown to be hypolipidemic agents in rodents at 8 mg/kg/day ip and orally [i]. In rats the cholesterol levels of VLDL and LDL fractions were reduced and the HDL-cholesterol content was significantly increased with reductions of both serum cholesterol and triglyceride levels after 14 days at 8 mg/kg/day of these boronated deoxyribonucleosides. These boronated nucleosides and nucleotides were shown to be safe for therapeutic use [2].
We have now expanded the types of nucleosides with boron substitutions at different nitrogen moieties. The current investigation is an effort to establish their hypolipidemic activity in mice and rats at 8 mg/kg/day.

Methods:
Source of materials: All of the compounds have previously been synthesized and the chemical and physical characteristics reported [3][4][5]. All isotopes were purchased from New England Nuclear.
Substrates and co-factors were obtained from Sigma Chemical Co. Sprague Dawley male rats were obtained from Charles River Laboratory.
CF 1 mice were obtained from Jackson Laboratory. Weekly blood samples were obtained by tail vein bleeding.
Animal weight, organ weight and food consumption effects: Control and treated normalipidemic Sprague Dawley male rat(~230g) weights were obtained and expressed as a percentage of the initial body weight (week zero). Food consumption (gm/day/rat) was noted for two weeks for control and treated rats [l].
Tissue lipid levels: Normalipidemic Sprague Dawley male rats (.230 g) which were treated orally for two weeks with compound 6 at 8 mg/kg/day, were sacrificed and tissue samples of the liver, small intestinal mucosa and aorta were removed.
A 24 hr fecal sample was also obtained. A 10% homogenate in 0.25 M sucrose + 0.001M EDTA, pH 7.2, was prepared for each tissue. An aliquot (2 ml) of the homogenate was extracted [7][8]  Normalipidemic Sprague Dawley male rats(-230g) treated for two weeks with compounds 6 at 8 mg/kg/day, orally were anesthetized with ether and blood (~I0 ml) was collected from the abdominal vein. Serum was separated from whole blood by centrifugation at 3500 rpm.
Aliquots of the serum were separated into chylomicrons, VLDL, HDL and LDL by ultracentrifugation as modified for normal rats [12,13].
Each of the fractions was analyzed for cholesterol, triglyceride, neutral lipids, phospholipid and protein levels.
Enzymatic studies: Compounds 3, 6, 13 and 14 were examined for their ability to inhibit i_n vitro activities of hepatic enzymes involved in lipid synthesis and metabolism.
Data denoted in Tables 1-5 represent the means + standard deviations of each group expressed as a percentage of the control value.
The Student's "t" test was applied between control groups and the individual drug treatment groups using the raw data.

Results
The boronated nucleosides demonstrated potent hypolipidemic activity in mice at 8 mg/kg/day ip. Compounds I, 3, 4, 5, 6, II, 12, 13, 14, and 15 decreased serum cholesterol levels at least 40% by day 16 [ Table i]. Serum triglyceride levels were reduced greater than 30% on day 16 by compounds 3, 7, 12, 13, 14, and 15. All of the derivatives afforded better hypolipidemic activity than the standards clofibrate at 150 mg/kg/day or lovastatin at 8 mg/kg/day in mice. Compound 6 was selected for further studies as being representative of this class of derivatives. This compound reduced serum cholesterol levels in rat 36% and serum triglyceride levels were reduced 32% on day 14 after oral administration [ Table 2]. Rat tissue lipids after 14 days of drug administration were not elevated. Aorta wall cholesterol was reduced 24% after administration of compound 6 [ Table 3]. Fecal triglycerides and phospholipids were elevated significantly after 14 days, i.e. 104% and 50%, respectively while cholesterol levels were reduced 24%.
Small intestinal mucosa and aorta wall triglyceride levels were reduced as was aorta wall phospholipids and protein content.      Table  4]. Triglyceride content was increased in the chylomicron but lowered in the VLDL and HDL fractions. Phospholipids were lower in the chylomicron and VLDL fractions but was elevated in the LDL fraction.
Protein content was reduced in the chylomicron and VLDL fractions but was elevated in the LDL and HDL fractions.
In vitro hepatic enzyme activities showed similar patterns of effects for the four compounds tested over 60 min [ Table 5]. Cytoplasmic acetyl CoA synthetase activity was suppressed significantly by all of the agents following a concentration dependent response. ATP-dependent citrate lyase activity was also reduced by the compounds except by compound 14. HMG-CoA reductase activity was suppressed moderately by 19% by compound 3 at I00 tM but compounds 13 and 14 caused greater than 50% reduction.
Acetyl-CoA carboxylase activity was elevated by compounds 3 and 13 at all concentrations whereas compounds 6 and 14 caused only 15% reduction at I00 tM. sn-Glycero l-3-phosphate acyl transferase activity was essentially unaffected by the compounds.
Phosphatidylate phosphohydrolase activity was markedly reduced by compounds 3, 13 and 14 from 72% to 85% whereas compound 6 caused only 18% reduction. Hepatic lipoprotein lipase activity was reduced 47-48% by compounds 3 and 13 and 24% by compound 6 but the activity was elevated 127% by compound 14 at I00 M.

Discussion
The boronated nucleosides were potent hypolipidemic agents in rodents at 8 mg/kg/day both by po and ip administration. The N boronated guanosine derivative 3 as well as 3'-aminocyanoborane dideoxythymidine 6, the  Enzymatic studies demonstrated that four of these derivatives inhibited rat hepatic cytoplasmic acetyl-CoA synthetase markedly and ATP-dependent citrate lyase activity moderately which would reduce acetyl-CoA necessary for both fatty acid and cholesterol syntheses. The reduction of HMG-CoA reductase activity, the regulatory enzyme for cholesterol synthesis, by some of the agents should add to the overall reduction of tissue, lipoprotein and serum cholesterol levels.
The agents, 13 and 14, which demonstrated good inhibition of the activity of this enzyme were more effective in lowering cholesterol levels than the compounds, 3 and 6, which only lowered acetyl CoA synthetase activity. All of the agents lowered hepatic acyl-CoA cholesterol acyl transferase activity, the enzyme responsible for cholesterol ester synthesis.
Assuming that the same inhibition occurred in the aorta wall, of this enzyme activity by the agents then plaque growth should be reduced since their size depends directly on the deposition of cholesterol esters.
Cholesterol-7--hydroxylase activity was elevated by the agents. This is the regulatory enzyme for converting cholesterol to bile acids for excretion into the bile.
The lowering of triglyceride levels by the agents is probably due to their ability to reduce the activity of phosphatidylate phosphohydrolase, the enzyme responsible for conversion of phospholipids to triglycerides.
The reduction of hepatic lipoprotein lipase by three of the agents would lower the removal of triglycerides from lipoproteins for delivery to the liver or other tissues. In vivo studies with the 3'-aminocyanoborane-dideoxythymidine showed that rat serum cholesterol and triglyceride levels were reduced at 8 mg/kg/day and was comparable in hypolipidemic effects to lovastatin at 8 mg/kg/day and clofibrate at 150 mg/kg/day in rats. This agent, compound 6, achieved lowering of VLDL and LDL cholesterol content while elevating HDL cholesterol levels. Modifying this ratio is important in choosing a hypolipidemic therapeutic agent in that VLDL and LDL lipoprotein conduct cholesterol to peripheral tissues including the aorta walls leading to deposition and tissue accumulation whereas HDL conducts free cholesterol from peripheral tissue to the liver for excretion in the bile. Hyperlipidemic patients have high VLDL and LDL cholesterol content and low HDL-cholesterol content; thus, if a therapeutic agent reverses this ratio this is useful clinically [24]. This compound effectively lowered aorta wall cholesterol levels after 14 days administration but there was no observable elevation in cholesterol excretion in the feces on day 14, but triglyceride excretion and phospholipid excretion was elevated at this time. The pharmacological properties of these boronated nucleosides as hypolipidemic agents are similar to those of boronated derivatives reported previously [I].
These studies demonstrate that the compound have potential as hypolipidemic agents but further structure activity relationship studies are need before an agent can be selected for clinical development.