The Hypolipidemic and Anti-Inflammatory Activity of Boronated Aromatic Amino Acids in CF1 Male Mice

The boronated aromatic amino acids were shown to be potent hypolipidemic agents in mice lowering both serum cholesterol and triglycerides after 16 days. Selective compounds were as effective as the clinical standards. Furthermore, the compounds were effective anti-inflammatory agents reducing local and central pain as well as suppressing LPS induced endotoxic shock in mice. These agents inhibited lysosomal and proteolytic enzymes of the liver and macrophages as a part of their mechanism of action.

Vol. 6, No. 6,1999 The Hypolipidemic and Anti-Inflammatory Activity of Boronated Aromatic Amino Acids in CF1    The Hypolipidemic and Anti-Inflammatory Activity of Boronated Aromatic Amino Acids in CF 1 Male Mice Anti-inflammatory Activity.
CF male mice (-25 g) were administered drugs at 8 mg/kg in 1% CMC I.P, at 3 h and again 30 min prior to the injection in 0.2 ml of carrageenan in 0.9% saline into the plantar surface of the high hind foot. Saline was injected into the left hind foot which served as the standard base line. Atier 3 h both feet were excised at the tibiolarsal (ankle) joint according to the modified method of Winter et al. [21], resulting in 84 mg increase in the paw weight of the control mice.
Male CF1 male mice (-25g) were administered test drugs at 8 mg/kg I.P. 20 min prior to the administration of 0.5 ml of 0.6% acetic acid, I.P. [22]. Aler 5 min the number of stretches, characterized by repeated contractions of the abdominal musculature accompanied by extension of the hind limbs for the next 10 min were counted. The control mice afforded 61 stretch reflexes in 10 min.
Hot Plate Tail Flick Activity -Central Analgesic Activity.
C FI male mice (-28g) were administered drugs at 8 mg/kg, I.P., 15 rain prior to placement on a hot plate ma.intained at 100 C. The time elapse for the tail to be raised from the surface of the hot plate was determined using a digital read-out connected to the hot plate [23]. The   were maintained in Dulbecco's modified medium (DMEM) + 15% fetal calf serum + P/S were homogenized.
All of the methods for the enzyme studies have been described previously [16]. Acid phosphatase, elastase, trypsin and cathepsin activities were determined atter 30 min incubation of drugs at 10 M prepared in 1% CMC at 37C. Acid phosphatase activity was determined using 0.1 M [3-glycerolphosphate in 0.1 M acetate buffer, pH 5.0. The reaction was stopped with 10% TCA and centrifuged at 3000 x g x 6 min. The supematant inorganic phosphate was determined by the spectrophotometric method. The net inorganic phosphate released from 30 min was corrected by subtracting the blank. Cathepsin activity was determined using 2% azocasein as the substrate in 0.1 M acetate buffer, pH 5.0 and the hydrolyzed acid-soluble peptide fragment was analyzed at 366 nm and corrected for the blank. Trypsin proteolytic activity was determined by the method of Schleuning and Fritz [25] using 6 mM N-benzoly-L-arginine ethyl ester (BAEE) in 0.1 M Tris buffer, pH 8.0. The hydrolyzed product was determined at 253 nm and the blank subtracted. Elastase activity was determined by the method of Kleinerman et al. [26] with 2 units of porcine pancreatic elastase (Sigma, Type III), N-succinyl-L-alanyl-L-alanine-p-nitroanilide (Sigma, 100 mg in 5 ml methyl-2-pyrrolidone)in 0.2 M Tris buffer, pH 8.0. The cleaved product p-nitroanilide was determined at 410 nm and the blank substracted. The control values for these assays are located in the Methods section.

RESULTS
The boronated amino acids and their metal complexes demonstrated significant hypolipidemic effects in mice at 8 mg/kg I.P. [ Table 1]. Compounds 6 and 14 caused greater than 40% reduction of total serum cholesterol levels after 16 days. Compounds 2, 4, 5, 7, 8, 10, 11, 13 and 15-19 afforded greater than 30% reduction of serum cholesterol after 16 days which was significantly better than the standards clofibrate and lovastatin at their therapeutic doses. Serum triglycerides were reduced 45% by compound 10. Compounds 2, 6, 8, 11 and VoI. 6, No. 6, 1999 The Hypolipidemic and Anti-Inflammatory Activity of Boronated Aromatic Amino Acids in CF1 Male Mice 1'7 lowered serum triglyceride levels greater than 30% which was greater than the standards' effects. L-Phenylalanine itself was not active in either assay. The addition of the methyl ester did result in an active compound indicating that the boron atom was not necessarily important for hypolipidemic activity in mice.
The boronated aromatic amino acids in vitro demonstrated the ability to suppress the activities of proteolyic and lysosomal hydrolytic enzymes in CF mouse liver homogenates and cultured mouse J774A macrophages [ Table 3]. In mouse liver compounds 3-5, ,7, 12-1'7 and 19 afforded greater than 40% inhibition of elastase activity. Liver cathepsin activity was reduced greater than 30% by compounds 4, 5, ,7, 9 13, 1"7 and 19. Liver trypsin proteolytic activity was suppressed 94% by compound ,7, but compounds 11-1,7 caused greater than 50% reduction of activity. In the cultured mouse macrophages elastase activity was reduced at least 30% by compounds 3, 5, 8 10, 12, 15 and 1'7. Macrophage cathepsin activity was inhibited greater than 35% by compounds 5, 11, 12, 14, 16, 1"7, and 19. Trypsin activity was reduced at least 30% by compounds 10 and 16. Macrophage acid phosphatase activity was reduced 40% by compound 5 and 30% by compound 13.  14 appear to be the best compounds in this respect and they were markedly more potent at 8 mg/kg than the standards clofibrate at 150 mg/kg and lovastatin at 8 mg/kg. There did not appear to significant differences between the phenylalanine, tyrosine, proline, histidine and tryptophan derivatives and their effects on lowering cholesterol levels.
(N-methylmethanamide)dihydro[[[ 1-(4-hydroxyphenylmethyl)-2-methoxy-2-oxoethyl ]amino]carbonyl]boron 11 was the only compound that was as active as morphine in reducing central pain although compound 13 was significantly effective. The phenylalanine derivatives did not demonstrate any ability to block central pain. The boronated aromatic acids did protect the mouse from LPS induced shock and many of the tested derivatives were more potent than the standards dexamethasone and pentoxifylline at their therapeutic doses. Those compounds without a boron atom, compounds 1 and 2 offered no protection against LPS induced shock. Previously amine-carboxyboranes were shown to inhibit proteolytic and lysosomal hydrolytic enzyme activities [16]. The present study would suggest that similar mechanisms of action are caused by the boronated aromatic amino acids for their anti-inflammatory activity. The aminecarboxyboranes also suppressed prostaglandin synthetase and cytokine, e.g. II-1 and TNF release as well as the migration of white cells to the inflammation site [16]. Whereas further investigation of this group of compounds is required, they may offer some therapeutic use in the future because of the low dose required for their in vivo activity as hypolipidemic and anti-inflammatory agents.