Hypolipidemic Activity of N,N-Dimethyl-n-Octadecylamine Borane in Rodents

Hypolipidemic Activity of N,ECDimethyl-mOctadecylamine Borane in Rodents IRISH. HALL", T. S. GRIFFIN*,

E.L. DOCKS*, R. J. BROTHERTON*,

AND

GEORGE FUTCH'

Received January 24, 1986, from the 'Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Accepted for publication April 14, 1986. Chapel Hi//, Chapel Hi//, NC 27574, and *US. Borax Research Corporation, Anaheim, CA 92807.

Abstract 0 N,NDimethyl-n-octadecylamine borane proved to be an effective hypolipidemic agent in rodents, lowering both serum cholesterol and triglyceride levels by >40% after 16 d of administration. The agent lowered serum lipid levels by inhibiting the enzymatic activity of rate-limiting enzymes of both de novo cholesterol and triglyceride pathways in the liver. The reduction led to lower levels of lipids in the liver and small intestine tissues, with a reduction of the cholesterol and triglyceride levels of the serum low density lipoprotein (LDL) fraction. Concurrently, the cholesterol level of high density lipoproteins (HDL) was significantly elevated in rats after 14 d of treatment. The drug caused an increased rate of cholesterol clearance from the body, essentially via the feces. The ability of the agent to modulate the cholesterol levels of LDL and HDL fractions suggests that the agent should be an effective agent for the treatment of hyperlipidemic states in humans.

Previously, series of trimethylamine cyanoboranes and trimethylamine carboxyboranes were shown to possess potent hypolipidemic activity at the intraperitoneal doses of 520 mgkg per day in CF1 mice.' These derivatives lowered serum cholesterol levels probably by suppressing 3-hydroxy3-methylglutaryl coenzyme A (HMG CoA) reductase activity. Reduction of serum triglyceride levels was correlated with the inhibition of fatty acid synthetase by the agents.' Subsecarboxyat0)-bisquently, tetrakis-p-(trimethylamine-borane (trimethylamine-carboxyborane)-dicopper(I1)(c21H6sB6C1.12N6O12) was observed to be a potent hypolipidemic agent at the low dose of 2.5 m g k g in mice.2The dicopper complex was observed to lower ATP-dependent citrate lyase, acetyl CoA synthetase, and phosphatidate phosphohydrolase in vivo and to accelerate cholesterol excretion from the body.2A series of long-chain trialkylamine boranes was synthesized and evaluated as hypolipidemic agents at 8 and 30 m g k g per day in CF1 mice. A number of these derivatives were active as hypolipidemic agents in both the CF1 mouse and SpragueDawley rat models. We have selected a representative compound, NJV-dimethyl-n-octadecylamineborane (21, which was one of the more potent analogues, for a detailed study of its effects on lipid metabolism in rodents. NJV-Dimethyl-noctadecylamine borane (2), in a preliminary screening, appeared to be equally potent as the previous agents; however, it was not as toxic. Nfl-Dimethyl-n-octadecylamine borane (2) was prepared by the transamination of triethylamine borane (1) with the corresponding amine.3 The reaction proceeds smoothly to completion because triethylamine, which has a lower boiling point, is removed at elevated temperatures.

700 /Journal of Pharmaceutical Sciences Vol. 75, No. 7, July 7986

Recrystallization of the residue from petroleum ether yielded 2 as a white solid which showed the characteristic E H band at 2300 cm-' in the infrared spectrum. Nfl-Dimethyl-noctadecylamine was prepared by methylation of n-octadecylamine with formaldehyde and formic acid according to the method of Clarke, et al.4 Triethylamine borane (1) was conveniently prepared from triethylamine hydrochloride and sodium borohydride in the presence of a small amount of water.6

Experimental Section Nfl-Dimethyl-n-octadecylamine-n-Octadecylamine (35.73 g, 0.13 mol), 37% formalin (64.78 g, 0.80 moll, and 98% formic acid (136.25 g, 2.90 mol) were combined and stirred at reflux for -22.5 h. (Caution: initial vigorous gas evolution causes foaming). After cooling the mixture to room temperature, 25 mL of 6 M HCl was added followed by 225 mL of 15%NaOH. The slurry containing the free amine was vacuum-filtered through no. 41 filter paper. The solution was extracted with ether (3 x 100 mL), dried over Na2S04, and the ether was removed under reduced pressure to give 14.98 g (39%yield) of an amber oil. This material was purified by distillation (bp 205-208 "C, 16 mmHg), giving two fractions for a combined yield of 10.12 g (26%).The two fractions had identical infrared spectra; IR (neat) showed no N-H band; 'H NMR (CDC13): 6 0.9-1.2 (37, m, CH,S, CH2CH3) and 2.2 ppm (6, s, N(CH3)2). Aml.-(from fraction 2) Calc. for C20H43N:C, 80.70; H, 14.59; N, 4.71. Found: C, 81.18; H, 14.46; N, 4.56. N,A'-Dimethyl-n-Octadecylamine Borane (2)-To a one-necked, round-bottomed flask (with thermometer well), equipped with a magnetic stirrer, a distillation head, and a condenser, were added 3.87 g (0.0336 mol) of triethylamine borane and 10.0 g (0.0336 mol) of NJ-dimethyl-n-octadecylamine. The flask was heated slowly, with stirring, to remove the triethylamine by distillation (maximum reaction temperature, 240 "C). After removing 3.2 mL of triethylamine by distillation, heating was interrupted and 10 mL of toluene was added to the cooled flask. Distillation was continued until 9.0 mL was removed (last few drops removed under 15-20 mm reduced pressure. The solid in the flask was recrystallized from petroleum ether (bp 30-60 "C) to give 7.58 g (72%yield) of white crystals, mp 56.0-57.5 "C (lit.3mp 50-52 "C). Hypolipidemic Screenings in Normal Rodents-The test compound was suspended in an aqueous 1% carboxymethylcellulose solution, homogenized, and administered intraperitoneally to CF1 male mice (-25 g) for 16 d, or orally, by a n intubation needle, to Sprague-Dawley male rats (-350 g) for 14 d. On days 9 and 14 or 16, blood was obtained by tail-vein bleeding and the serum was separated by centrifugation for 3 min. The serum cholesterol levels were determined by a modification of the Liebermann-Burchard reaction.6 Serum was also collected on day 14 or 16 and the triglyceride content was determined by a commercial kit (Hycel Triglyceride Test Kit, Fisher). Testing in Hyperlipidemic Mice-CF, male mice (-25 g) were placed on a commercial diet (US.Biochemical Corporation, Basal Atherogenic Test Diet) which produced a "hyperlipidemic" state.7 After the serum cholesterol and triglyceride levels were observed to be elevated in mice, the test drug (8mg/kg per day) was administered intraperitoneally for an additional 12-d period. Serum cholesterol and triglyceride levels were measured at that time. 0022-3549/86/0700-0706$0 7 .OO/O 0 7986, American Pharmaceutical Association

Animal Weights and Food Intake-Periodic animal weights were obtained during the experiments and expressed as a percentage of the animal’s weight on day zero. After dosing for 14 d with the test drug, selected organs were excised, trimmed of fat, and weighed. Food consumption was determined daily. Toxicity Studies-The acute toxicity (LD6,, va1ues)a was determined in CF1 male mice (-25 g) by intraperitoneal administration of test drug (100 mg/kg to 2 gkg) as a single dose. The number of deaths was recorded over a 7-d period for each group. Enzymatic Studies--In vitro enzymatic studies were performed using 10% homogenates of CF1 male mouse liver and 50-200 pM of test drug. In vivo enzymatic studies were performed using 10% liver homogenates (prepared in 0.25 M sucrose + 0.001M (EDTA, pH 7.2) from CF1 male mice obtained after intraperitoneal administration of the agents for 16 d at a dose ranging from 4 to 16 mgkg per day. The enzyme activities were determined by following literature procedures:7 acetyl coenzyme A ~ y n t h e t a s e ;adenosine ~ triphosphatedependent citrate 1yase;’O mitochondrial citrate exchange;11.12 cholesterol 7a-hydroxylase;13 3-hydroxy-3-methylglutarylcoenzyme A;14J5 acetyl coenzyme A carboxylase activity;l6 fatty acid synthetase activity;I7 sn-glycerol-3-phosphate acyl transferase activity;18 phosphatidate phosphohydrolase activity;lg cholesterol acyl transferase;Z0 and heparin activated hepatic lipoprotein lipase.21Protein was determined for all enzyme assays by the method of Lowry et a1.22 Liver, Small Intestine, and Fecal Lipid Extraction-In CFI male mice that had been administered NJV-dimethyl-n-octadecylamine borane for 16 d, the liver, small intestine, and fecal materials (24-h collection) were removed, extracted,23.24 and analyzed for cholesterol levels,6 triglyceride levels (Bio-Dynamics, bmc Triglyceride Kit), neutral lipid content,26 and phospholipid content.ls [3H]Cholesterol Distribution in Rats-N,N-dimethyl-n-octadecylamine borane was orally administered for 14 d to Sprague-Dawley rats (-300 g). On day 13,lOpCi of [3Hlcholesterol was administered orally, by intubation needle, to male rats according to the procedures described previ~usly.~ Some tissue samples were combusted in a tissue oxidizer (Packard) or plated on filter paper, dried, and digested for 24 h in Hyamine Hydroxide (1M (2-[2-@-1,1,3,3-tetramethylbutylcresoxy)ethoxylethyl)dimethylbenzylammoniumhydroxide) (New England Nuclear) at 40°C and counted (Fisher Scintiverse in a Packard scintillation counter). Results are expressed as disintegrationlmin (dpm) per total organ. Bile Cannulation Study-Sprague-Dawley male rats (-300 g) were orally treated with test drugs at 20 mgkg per day for 14 d. After anesthetization the bile duct was cannulated as previously de~cribed.~ 1, 2-[3HlCholesterol (40.7mCi/mmol; 10 pCi) was administered orally 18 h prior to commencing the surgery. Bile was collected over the next 6 h and the volume (mL) was measured. Aliquots were counted (Fisher Scintiverse in a Packard scintillation counter) as well as analyzed for [3Hlcholesterol content.6

Plasma Lipoprotein Fractions-Sprague-Dawley male rats (-300 g) were orally administered test drugs a t a dose of 20 mgkg per day. Blood was collected from the abdominal vein and lipoprotein fractions were obtained by the methods of Hatch and Lees27 and Have1 et aLZaEach of the fractions was analyzed for cholestero1,B triglyceride (Bio-Dynamicshmc Triglyceride Kit), neutral lipids,26 and protein levels.22

Results In CF1 male mice, the most effective intraperitoneal dose of N,N-dimethyl-n-octadecylamineborane was 8 mgkg per day which reduced serum cholesterol levels by 41% and serum triglyceride levels by 59%. In Sprague-Dawley rats, after oral administration of 20 mgkg per day, serum cholesterol levels were reduced by 54% and serum triglyceride levels were lowered by 35% (Table I). In hyperlipidemic-induced mice, where the serum cholesterol levels had been elevated to 375 mg% compared with the control value of 128 mg%, 12 d of administering the drug lowered the level to 180 mg%; a 52% reduction. Serum triglyceride levels in the hyperlipidemic mice were elevated to 367 mgldL compared with the normal value of 137 mg/dL. Drug treatment for 12 d lowered the triglyceride levels to 187 mg%, a 49% reduction. N,N-dimethyl-n-octadecylamine borane effectively reduces the liver mitochondrial citrate exchange, ATP dependent citrate lyase, and acetyl CoA synthetase activities by >50% at all concentrations tested in vitro (Table 11).Cholesterol synthesis (HMG CoA reductase) was reduced in a concentrationdependent manner, resulting in a 38% reduction with the administration of 200 p M of the drug. Cholesterol 7a-hydrolase activity was unaffected by the presence of drug; however, acyl CoA cholesterol acyl transferase activity was reduced by 41 and 55% with 100 and 200 pM of drug, respectively. Acetyl CoA carboxylase, fatty acid synthetase, and sn-glycer01-3-phosphate acyl transferase activities were unaffected by the drug in vitro. The activity of the regulator enzyme, phosphatidate phosphohydrolase, was reduced by 67% at 200 pM of drug. Hepatic lipoprotein lipase activity, which was induced by heparin, was reduced by 23% at 100 and 200 pM concentrations of drug. In vivo N,N-dimethyl-n-octadecylamine borane reduced acetyl CoA synthetase by 32% at 16 mgl kg per day (Table 111). ATP-dependent citrate lyase activity was reduced by 62% a t 16 mgkg per day; HMG CoA reductase activity was maximally reduced by 34% a t 4 mgkg

Table &Effects of NHDlmethyl-mOctadecylamlneBorane on Serum Cholesterol and Trlglycerlde Levels In Rodents’

CF, Mice Compound Control (1Yo carboxymethylcellulose) Treated, mg/kg/d

Serum Cholesterol

Serum Triglycerides

Serum Triglycerides

Day 16

Day 9

Day 16

Day 9

Day 14

Day 9

Day 14

1 0 0 2 Sb

1 0 0 2 6’

1 0 0 2 7‘

10026’

100 k 6‘

1OOk 6g

100 2 7h

100k8’

-

45 2 5’ 49 5’ 41 2 3’ 56 2 7’ 63 t 8‘ 77 k 7’ 93 k 6 113 2 4

82 k 8

46 2 5’

82 2 7

98

96

7’ 5’ 4) 5’ 6’ 7’ 8’ 6

61 59 59 61 61 79 79 82

150

88’7

87 2 5

20

-

Clofibrate, mg/kg/d

Serum Cholesterol

Day 9

71 t 6’ 69 2 5’ 79 k 7’ 62 f 6’ 60 f 5’ 82 k 7 83 2 6 81 5 6

2 4 8 12 16 20 40 60

Sprague-Dawley Rats

2 2 2 2 2 2 2 2

-

“Expressed as percent of control (mean ? SD);n mg%. l p 5 0.001.

=

80 2 9 87 k 7 58 2 6’ 73 -t 8’

-

6. b118 mg%. ‘122

’

75 2 5’

-

2

12

* 15

-

65

5

6’

93 2 6

mg%. ‘137 mg%. ‘141 mg%. ‘73 mg%. g78 mg%. h i l o mg%. ‘112

Journal of Pharmaceutical Sciences/ 707 Vol. 75, No. 7, July 1986

Table l H n Vltro E M S of N,HDlm~yi-rrOctadecylamlneBorane Derlvatlves on CF, Mouse Liver Enzyme Actlvltles ~

Control

Enzyme Parameter

~~

Liver Enzyme Activity, % of ControF ~

50 pM

100 pM

200 pM

~

Mitochondria1 citrate exchange ATP-dependent citrate lyase Acetyl CoA synthetase HMG CoA reductase Cholesterol 7a-hydroxylase Cholesterol acyl transferase Acetyl CoA carboxylase Fatty acid synthetase Phosphatidate phosphohydrolase srrGlycerol-3-phosphateacyl transferase Hepatic lipoprotein lipase

100 t 100 l o o f gc l o o ? 7d 1002 98 100 f 5' 1 0 0 f 88 100 t 6h l o o t 8' 100 f 71 loof 8 k 1 0 0 2 6'

17 2 3" 16 t 2"' 42 5 3" 82 2 7 10429 68 5 7" 101 2 7 87 2 7 8329 99 2 8 8446

2 3 2 5m 21 f 7" 3 6 2 4'" 722 112 f 10 5 9 % 6" 100 f 10 7 6 f 4" 7 4 t 6'" 103f 5 7 7 2 am

32 f 4" 5 * 1" 32 5 6" 62 f 6'" 119 f 6 45 f 4" 98 f 7 72 f 5" 33 f 5" 9057 77 f 8*

nExpressedas mean f SD; n = 6. b30.8% exchange of mitochondria1 citrate. C30.5-mgcitrate hydrolyzedig wet tissue/20 min. d28.5mg acetyl CoA formed19 wet tissue/20 min. '384,900 dpm cholesterol formed19 wet tissue/60 min. '224,000 dprnlpg microsomal protein. O4,808 dpm/mg microsomal protein/20min. h32,010dpm/g wet tissue/30 rnin. '37,656 dpm/g wet tissue/20 min. '16.7 pPJg wet tissuell5 min. k537,800dpm/g wet tissue/20 min. '278,583 dpm/g wet tissue/h. "p 5 0.001. Table l l H n Vlvo Effect8 of N,KDlm~hyi-rrOctadecylamlneBorane on CF, Mouse Liver Enzyme Activities'

Compound

Acetyl CoA Synthesis

ATP-Dependent Citrate Lyase

HMG CoA Reductase

Cholesterol 7aHydroxylase

Cholesterol Acyl Transferase

Control (1% carboxymethylcellulose) Treated, mglkgld

loot8

look7

100?8

100 f 8

100 t 7

4

76 2 7b

87 f 8

8

100f8 80 f 70 68 f 56

86f7 80 f 6b 38 f 46

12 16

Control (1y0 carboxymethylcellulose) Treated, mg/kg/d 4 8 12 16

66 5 7' 78 f 6b 79 f 6b 81 f 7

88 f 7 79 f 8 73 t 7 b 65 f 8

96 t 6 48?5b 73 2 76 108 f 8

Acetyl CoA Carboxylase

Fatty Acid Synthetase

sn-Glycerol3-Phosphate Acyl Transferase

Phosphatidate Phosphohydrolase

100 f 6

10029

10026

100 f 7

9826 97 f 7 98 f 7 104 f 7

92 f 6

89 2 86 2 74 2 67 2

93 f 7 97 f 8 96 f 7

#Activitieswere determined after 16 d of dosing and are expressed as % control (mean rc_ SD); n per day. Cholesterol 7u-hydroxylase activity was reduced by 35%at 16 mgkg per day, acyl CoA cholesterol acyl transferase activity was reduced by 35% at 16 mgkg per day, and acyl CoA cholesterol acyl transferase activity was lowered by 52% at 8 mgkg per day. sn-Glycerol-3-phosphateacyl transferase and phosphatidate phosphohydrolase activities were only moderately reduced at 16 mgkg per day by 33 and 40%, respectively. The lipid content of livers from mice treated with NJVdimethyl-n-octadecylamineborane a t 8 mgkg per day showed that cholesterol and triglyceride levels were reduced after 16 d of dosing (Table IV). Phospholipid content was elevated by 67-92% a t 8-16 mgkg per day. In SpragueDawley rats treated for 14 d, cholesterol content was reduced by 12%, triglyceride and neutral lipid contents were reduced by 88,and phospholipid was elevated 14% (Table V). In the small intestine of rats, a similar pattern of reduction was observed with a slightly higher level of reduction of triglycerides and neutral lipids (2440%). Lipid fecal excretion in the rats after 14 d of dosing demonstrated a 55% increase with high levels of cholesterol (40%) and neutral lipids (37%) being eliminated. The serum lipoprotein fractions from rats demonstrated that the low density lipoprotein (LDL)-cholesterol content was reduced by 34% and the high density lipoprotein (HDL)-cholesterol content was elevated by 62% 708 / Journal of Pharmaceutical Sciences Vol. 75, No. 7, July 1986

6 5 76 6'

= 6; see Table II

96 t 8 90 f 7 76 t 7' 60 f 5b

for control values. p < 0.001.

(Table V). Triglyceride content was elevated in the very low density protein (VLDL) and HDL fractions. The [3H]cholesterol distribution study in rats showed a large accumulation of [3H]cholesterol in the stomach (194%), large intestine (5981,and the chyme (144%) compared with the control values (Table VI). Fecal excretion of cholesterol was significantly elevated at this time (29%). The [3H]cholesterol content in the major organs demonstrated only an 11% increase in the lung, but all other organs were equal to or less than the control values. Organ weights aRer two weeks of administration of the drug were not significantly different from the control weights. The adrenal weight showed no difference after drug treatment indicating no hypertrophy of the cortex because of induced steroidogenesis. Daily food consumption was reduced by 14% after 7 d of administration of the drug. The [3Hlcholesterol absorption from the intestine after oral administration of drug showed a 20% reduction in plasma levels after 24 h. Whereas this is not conclusive, it does suggest a reduction in the absorption of the drug from the intestine.

Discussion NJV-Dimethyl-n-octadecylamineborane proved to be an effective hyperlipidemic agent at 8 mgkg per day in mice and

Table IV-Efhts

of N,HDimethyl-rrO~d~ylamine Borane on CF, Mouse Liver Lipld Content'

Compound

Lipid, mg

Cholesterol

Triglyceride

Neutral Lipids

Phospholipid

Protein

Control (1% carboxymethylcellulose) Treated, mglkgld

10026

100 f 7c

100 f 5d

100 2 6'

100 f 7'

100 f 6g

10027 10028 84 f 6 91 f 5

89 f 8 86 f 5 82 f 6' 78 f 56

92 f 6 88f6 89 f 5 108 f 7

94 t 5 97 '' 4 10026 99 2 7

101 f 7 192 f 8' 184 2 9b 167 2 7'

84 f 7 98 2 5 97 2 6 82 ? 6

4 8 12 16

'Effects were determined after 16 d of intraperitoneal administration; results are expressed as % control (mean f SD); n = 6. 'p 5 0.001. 12.24 mg cholesteroWg tissue. d4.77 mg of triglyceridelgof tissue. '28.35 mg of neutral lipid/gtissue. '4.39 mg phospholipid(P#g of tissue. O4.5 mg of protein/g tissue.

Table V-Lipid Extraction of Sprague-Dawiey Ral Tissue and Rat Blood Llpoproteln After led Oral Admlnistratlon of N,Hdlmethyl-m Octadecyiamlne Borane' Tissues Rat Liver Control Treated Rat Small Intestine Control Treated Rat Fecal Material Control Treated

Lipid, mg

Cholesterol Content

Triglyceride Content

Neutral Lipid Content

Phospholipid Content

10027 100 f 9

100 f 7' 88 2 6

1002 92k 5

100 f 6d 92 f 7

100 f 8' 114 f 9

100 t 6' 104 ? 5

100 f 6 8324

100 f 7Q 91 f 6

1005 5 h 76 2 4"

100 f 5' 70 f 4''

100 f 71 110 f 8

100 f 7" 89 t 8

10027 155 & 5"

100 f 6' 140 f 5"

1002 6"' 1052 8

100 f am 137 f 9

100 f 7O 76 f 5

100 f 5 p 96 2 6

100 f 7q 98 f 8

1002 7' 84% 5

100 f 8' 98 f 9

100 f 7' 82 f 5ii

100 2 6" 93 2 7

100 f 6" 111 f 5

100% 6" 210 2 10"

100 f 7" 98 f 8

100 f 8 Y 90 f 9

100 f 72 93 f 8

100 f 6" 66 f 5"

1002 7 8 0 2 6"

100 f 7bb 90 f 8

100 f 85 f 9

100 t 8" 95 2 8

100 f 162 f 6"

100 2 6" 1182 5"

100 f 7QQ 93 f 8

100 f 6hh 94 f 7

100 ? 6" 101 t 7

Rat Blood Lipoprotein Chyiomicron Control Treated

QLDL

Control Treated LDL Control Treated HDL Control Treated

~

Protein Content

~~

'Expressed as percent of control (mean f SD); n = 6.'44.1 1 mg neutral lipid/gtissue. '6.37 mg triglyceride/gtissue. d7.19 mg phospholipid(P)/g tissue. '4.5 mg proteinlg wet tissue. '7.82 mglg. O6.98 mg/g. 1.12 mglg. '2.06 glg. /42 mg/g. '28.47 mg/g. '33.94 mglg. 1.86 mglg. " 1.239 kglg. '6.99 mglg. p337 pg1mL. q67 M m L . '420 M m L . '149 pglmL. '184 pglmL. "190 &mL. "98 pg1mL. w22pg1mL. "26 pglmL. y50 pg1mL. 2210 pg1mL. "10 pg/mL. &45 pglmL. OC41pg/mL. *122 pg1mL. "544 pglmL. "620 pg/mL. w27 pg/mL. hh153pg1mL. "657 pglmL. up < 0.001.

20 mgkg per day in rats. This agent was effective alter both intraperitoneal and oral administration. In hyperlipidemicinduced mice, the drug lowered the elevated levels of serum cholesterol and triglycerides. However, a longer time of Percent administration of the drug may be necessary to reduce the Control elevated blood lipids to within normal levels. The LDS0of the drug was 500 mgkg; thus, using the agent a t 8 mgkg per day 76.6 for its hypolipidemic effects is within a safe therapeutic 77.8 range. Reduction of the activities of enzymes leading to the 111.9 synthesis of cytoplasmic acetyl CoA would theoretically 98.5 reduce the synthesis of cholesterol fatty acids and triglycer80.0 99.9 ides in the hepatmyte. Apparently, the drug is able to 293.9 markedly lower mitochondria citrate exchange, ATP-depen104.4 dent citrate lyase, and acetyl CoA synthetase activities. 158.61 Inhibition of any of these would lead to less acetyl CoA in the 243.65 cytoplasm. The ability of the drug to specifically lower 128.90 cholesterol, as well as cholesterol ester synthesis, should result in less cholesterol stored in various tissues. The tissue lipid analysis studies, as well as the cholesterol distribution

Table VCSprague-Dawiey Rat ['H]Choleaterol Dirtrlbution After 14 Day8 of Oral Admlnlstratlon of N,Hdlmethyl-rrOctsdecylamlne Borane Organ' Brain Heart Lung Liver Spleen Kidney Stomach Small intestine Large intestine Chyme Feces Adrenal

Organ Weight, g Control Treated 1.701 1.037 1.402 9.001 0.620 1.735 1.537 6.107 3.130 2.866 5.341 0.067

1.766

0.800 1.433 8.567 0.577 1.734 1.765 6.235 3.342 4.264 3.258 0.066

aAt 20 mglkg per day; n = 6.

DPG/g Total Organ Control Treated 19,655 15,049 55,637 43,265 279,924 313,096 2,260,398 2,227,049 279,993 224,049 137,238 137,101 87,747 257,946 3,003,579 3,134,369 244,838 388,332 706,210 1,720,704 713,502 919,720

-

-

Journal of Pharmaceutical Sciences / 709 Vol. 75, No. 7. July 1986

studies, support the reduction of cholesterol levels in tissues; furthermore, the cholesterol is being eliminated faster in feces. Reduction of the activity of the liver regulatory enzymes, sn-glycerol-3 phosphate acyl transferase and phosphatidylate phosphohydrolase, has been correlated with the lowering of serum triglyceride levels.1s The reduction of hepatic lipoprotein lipase activity by the drug should reduce the availability of triglycerides or fatty acids for tissue uptake from the blood. The effect of the drugs on lipoprotein content of cholesterol was encouraging. The cholesterol content of the LDL fraction was reduced; whereas the cholesterol content of the HDL fraction was elevated. Supposedly, the LDL fraction carries the cholesterol to tissues and the atherogenic plaque. High cholesterol content of the LDL fraction is associated with the growth in size of the plaque. The HDL fraction, on the other hand, conducts cholesterol out of the tissue to the liver for the purpose of excretion via the biliary route. The drug appears to be affecting this parameter since the HDL cholesterol and the fecal cholesterol levels are high after drug treatment. High cholesterol content in the HDL fraction has been reported to protect against cardiovascular infarctions in

References and Notes 1. Hall, I. H.; Das, M. K.; Harchelrood, F., Jr.; Wisian-Neilson, P.; McPhail, A. T., Spielvogel, B. F. J. Pharrn. Sci. 1981,70, 339341. 2. Hall, I. H.;Williams, W. L., Jr.; Gilbert, C. J.; McPhail, A. T.; S ilvogel, B. F. J.Pharm. Sct. 1984,73,973-977. 3. 8 w f o r d , R. F. Canadian Patent 738 333 1966. 4. Clarke, H.T.; Gillespie, H. B.; Weisshaus, S. Z. J. A m . Chem. Soc. 1933,55,4571. 5. Brown, M. P.; Older, D. G. US. Patent 3 227 762 1966. The latter also describes a one-vessel synthesis of 2 from triethylamine hydrochloride and NJV-dimethyl-n-octadecylamine, without isolation of the intermediate borane 1.

710 / Journal of Pharmaceutical Sciences Vol. 75, No. 7, July 1986

6. Ness, A. T.; Pastewka, J. V.; Peacock, A. C. Clin Chim Acta 1964,10,229-237. 7. Hall. I. H.: Voorstad. P. J.: ChaDman. J. M.. Jr.: Cocolas. G. H. J. Pharm. Sci. 1983,72,845-851: 8. Litchfield, J. T.,Jr.; Wilcoson, F. J.P h a r m o l . Exp. Ther. 1949, 96,99-113. 9. Goodridge, A. G. J. Biol. Chem. 1973,4318-4326. 10. Hoffman, M.; Weiss, L.; Wieland, 0. H. Anal. Biochem. 1978,84, 441-448. -~~ ~~11. Robinson, B. H.; Williams, G. R. Biochim. Biophys. A c t ~1970, 216,63-70. 12. Robinson, B. H.; Williams, G. R.; Halperin, M. L.; Leznoff, C. C. Eur. J. Biochem. 1970,15,263-272. 13. Shefer, S.; Hauser, S.; Mosbach, E. H. J.Lipid Res. 1978,9,328I

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