Hypolipidemic, anti-obesity, anti-inflammatory, anti-osteoporotic, and anti-neoplastic properties of amine carboxyboranes.

The amine-carboxyborane derivatives were shown to be effective antineoplastic/cytotoxic agents with selective activity against single-cell and solid tumors derived from murine and human leukemias, lymphomas, sarcomas, and carcinomas. The agents inhibited DNA and RNA synthesis in preference to protein synthesis in L1210 lymphoid leukemia cells. Inosine-monophosphate dehydrogenase apparently is a target site of the compounds; similar effects on phosphoribosyl-pyrophosphate amido transferase, orotidine-monophosphate decarboxylase, and both nucleoside and nucleotide kinases were observed. Deoxyribonucleotide pool levels were reduced in the cells; DNA strand scission was observed with the agents. In rodents, the amine carboxyboranes were potent hypolipidemic agents, lowering both serum cholesterol and triglyceride concentrations, in addition to lowering cholesterol content of very low-density lipoprotein and low-density lipoprotein (LDL) and elevating high-density lipoprotein (HDL) cholesterol concentrations. De novo regulatory enzymes involved in lipid synthesis were also inhibited (e.g., hypocholesterolemic 3-hydroxy-3-methyl-Coenzyme A reductase, acyl-Coenzyme A cholesterol acyltransferase, and sn-glycerol-3-phosphate acyltransferase). Concurrently, the agents modulated LDL and HDL receptor binding, internalization, and degradation, so that less cholesterol was delivered to the plaques and more broken down from esters and conducted to the liver for biliary excretion. Tissue lipids in the aorta wall of the rat were reduced and fewer atherosclerotic morphologic lesions were present in quail aortas after treatment with the agents. Cholesterol resorption from the rat intestine was reduced in the presence of drug. Genetic hyperlipidemic mice demonstrated the same types of reduction after treatment with the agents. The agents would effectively lower lipids in tissue based on the inhibition of regulatory enzymes in pigs. These findings should help improve domestic meat supplies from fowl and pigs. The amine-carboxyboranes were effective anti-inflammatory agents against septic shock, induced edema, pleurisy, and chronic arthritis at 2.5 to 8 mg/kg. Lysosomal and proteolytic enzyme activities were also inhibited. More significantly, the agents were dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities. These compounds also affected cytokine release and white cell migration. Subsequent studies showed that the amine-carboxyboranes were potent anti-osteoporotic agents reducing calcium resorption as well as increasing calcium and proline incorporation into mouse pup calvaria and rat UMR-106 collagen.

Initially, the amine-carboxyborane derivatives were shown to be nontoxic in CF, male mice when administered at 20, 50, or 100 mg/kg / day, intraperitoneally (ip), for 7 days (17). Variables measured were survival, total body weight, daily food consumption, individual body weight, clinical chemistry values, hematopoietic values, and histologic evaluations of major organs. LD50 values (dose required for 50% deaths) of these compounds were between 1 and 2 g/kg, ip, in mice.
Then we examined these compounds for antineoplastic activity (9). Because these amine carboxyboranes were similar in structure and size to endogenous intermediary substrates, they could be functioning as antimetabolites (e.g., of amino acids or betaine). Once they are taken up into cells, a second mechanism of action is neutron capture. An identified organ or foci of tissues can be irradiated with neutrons, whereupon the boron atom will capture a neutron and decay to emit an alpha particle. This process causes local tissue damage and cell death. In vivo these boron derivatives have demonstrated strong activity against Ehrlich ascites carcinoma growth, Lewis lung carcinoma, and B,6 melanoma.
Marginal activity against P388 lymphocytic and L1210 lymphoid leukemia cells was also demonstrated. Selected compounds ( Table  1) were tested for cytotoxicity against murine and human tissue culture cells. These compounds were active against the growth of single tumor cells (e.g., L1210, Tmolt3, and HeLa-S 3; Table 1 in a concentration dependent manner (Figures 1, 2) and protein synthesis was sometimes marginally inhibited. The de novo nucleic acid synthesis of purines was reduced by the agents specifically at phosphoribosyl pyrophosphate (PRPP)-amido transferase and inosine monophosphate (IMP) dehydrogenase enzyme sites, the regulatory sites in the pathway ( Figure 3). Apparently, the latter enzyme is a major target of the amine-cyanoborane derivatives.
Pyrimidine de novo synthesis was also inhibited by compounds 1  4). Orotidine monophosphate (OMP) decarboxylase was the major site at which the agents functioned as inhibitors. Nucleoside and nucleotide kinase activities were also inhibited ( Figure 5). Ribonucleoside reductase, dihydrofolate reductase, DNA polymerase ax and topoisomerase II activities were inhibited by specific boron agents, but not by all of the agents. Inhibition was dependent on the substituted moieties of the basic structure of the amine-carboxyboranes. This class of boron derivatives reduced the deoxyribonucleotide [d(NTP)] pools after incubation for 60 min at 100 pM ( Figure 6). L1210 deoxyriboguanosine triphosphate, deoxyribocytosine triphosphate, and deoxyribothymidine triphosphate pools were reduced, whereas  Crossover in pharmacologic activity between antineoplastic, antibacterial, antiviral, immunomodulatory, and antiinflammatory activities is well documented for a number of chemically unrelated agents. Currently, evidence is accumulating to support the contention of a crossover between antineoplastic and hypolipidemic activity. For example, compactin, a hypocholesterolemic 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase) inhibitor, inhibited DNA synthesis in L929 tumor cells (18). Similar types of crossover in activity between the 2,3-dihydrophthalazine-1,4-dione (19), sesquiter-    (Figure 12), but not in the small intestine (data not shown). On the other hand, lipids, particularly cholesterol and triglycerides, were increased in the feces ( Figure 13) and bile ( Figure 14). Further studies in rats showed that the serum lipoprotein lipid content was altered by drug treatment (Figures  15-18  The ideal clinical hypolipidemic agent should lower VLDL cholesterol content, since this is the means by which cholesterol is delivered to peripheral tissues, including the aorta plaques. In patients with atherosclerosis, the LDL cholesterol concentration is high and the HDL cholesterol oncentration is low. An effective hypolipidemic agent must reverse this ratio. Reversing the LDL / HDL cholesterol ratio has been shown to protect man from myocardial infarctions. The HDL fraction is responsible for the uptake of free cholesterol from peripheral cells (including aorta plaques) and conducting cholesterol to the liver for metabolism to cholic acid followed by excretion in the bile. Because modulations of the serum lipoprotein fractions were favorable for treatment of atherosclerosis, we initiated~~~~~~~~~~~~~~~~~~~~~~~~~~~~.  studies to understand further the agents' effects on cellular lipid regulation (23). LDL receptors are located on peripheral cells which bind with high affinity to ape-B lipoproteins. LDL cholesterol complexes are taken up by the cell and merge with lysosomal vesicles from which hydrolytic enzymes are released. These enzymes digest the complex and release free cholesterol. These LDL receptors regulate the activities of HMG-CoA reductase, acyl-CoA cholesterol acyl transferase (ACAT), and cholesterol 7at hydrolase. We found the amine  carboxyboranes caused a decrease in LDL receptor binding of '25I-LDL and its internalization ( Figure 19). Further, the degradation of the LDL-cholesterol complexes was reduced, whether the complex entered the cell through the high-affinity transport mechanism or a nonreceptor transport process ( Figure 20). The net effect was that less free cholesterol was released in the cell. Macrophages, as scavenger mechanisms,   aorta wall and more lipid excretion in the bile, suggesting that the drugs accelerated the HDL cholesterol reverse transport process. The trimethylamine-carboxyborane derivative 5 accelerated cholesterol, triglyceride and neutral lipid excretion into the bile ( Figure 14). Furthermore, the agent increased the bile flow by 48%. Individual bile acids demonstrated different concentrations after drug treatment; however, there was no evidence that the agent induced lithogenic effects like many hypolipidemic agents. In situ rat intestinal loop studies showed that agent 5 interfered with cholesterol absorption/reabsorption from the gut ( Figure 25); however, it had no effect on cholic acid reabsorption (data not shown). This property of the agent would also account for the observed reduction of serum cholesterol over time.
In quail, treatment with amine-carboxyborane derivatives for 8 weeks reduced serum cholesterol concentrations and numbers of atherosclerotic lesions in the aorta ( Figure 26). Some of the boron derivatives of this chemical class should be effective in reducing tissue lipids. These agents were effective in the treatment of genetic hyperlipidemic, normalogenic and hypolipidemic diseased mice, lowering lipid concentrations in an analogous manner, as indicated in the CF1 mouse and rat studies. Further studies have indicated that the activities of the same lipid regulatory enzymes in miniature pig liver was inhibited by compound 5, which suggests that the compounds may be useful in agriculture in improving the quality of the meat products from domestic animals (24).
Another area where these compounds have demonstrated good pharmacologic activity is as antiinflammatory agents.
These agents were particularly useful in the inhibition of induced edema, reduction of local pain associated with inflammation, and inhibition of centrally induced pain (25) (Figure 27). But more important, the agents protected against septic shock from lipopolysaccharides (LPS) better than any tested commercial agent. Selected agents were also demonstrated to be effective against chronic induced arthritis in rats at 2.5 mg/kg/day and active against pleurisy in rats. Subcutaneous implantation of sponges containing LPS (26) in mice showed that agents blocked PMN myeloperoxidase (MPO) activity's ( Figure  28) and macrophage/monocyte N-acetylglucosaminidase (NAG) activity's ( Figure  29) migration to the inflammation sites. These derivatives inhibited the activities of lysosomal enzymes from a number of tissues [e.g., PMNs, hepatocytes, and leukocytes, with the concentration necessary for 50% inhibition of enzyme activity (IC50 value) in the range of 10-6M (Figures 30,  31)]. Although another characteristic of these agents is their potent inhibition of trypsin, elastase, and neutral cathepsin activities, in our hands they were less potent inhibitors of collagenase type I and II activities with IC50 values from 10to 10-5M (data not shown ). The agents proved to be dual inhibitors of prostaglandin cyclo-oxygenase and 5'lipoxygenase (27,28), with IC50 values in the range of 10-6M. IC-21 macrophages incubated with the agents secreted less interleukin-1 (Il-1) and tumor necrosis factor-a (TNFa) cytokines (Figures 32,33). However, after treatment with the agents at 8 mg /kg in mice in vivo 11-2 concentrations were low in the blood and TN-a was high ( Figure 34). This relationship between high amounts of TNF -a in bacterial or malarial infections and septic shock is not unique. The deleterious effects nor-mally attributed to TNF--a (e.g., cachexia and endotoxic shock) may be incorrect. Rather, deleterious effects may be the result of other concurrently released cytokines, like 11-6 or 11-8, and not TNF-a.
Examination of the derivatives for their ability to block calcium resorption as antiosteoporotic agents was our next priority. We first demonstrated that inorganic calcium, phosphorus, and hydroxyproline were low in the urine but high in the blood after 21-day treatment at 8 mg/kg/day in mice. Four-day-old rat pup calvaria bone exchanged less calcium to the medium in the presence of drug from 10 -to 10-4M for 48 hr (29) (Figure 35). In rat UMR-106 cultured osteosarcoma cells, calcium resorption was blocked by the agent (30). These derivatives were more active than calcitonin and the bis-phosphate standard in blocking calcium resorption. Concurrent incorporation of calcium into the cell collagen was increased in rat UMR-106 cells, IC-21 macrophages, and Be Sal human osteoporosis cells in the presence of the agents (Figure 36). In addition, an increase in labeled collagen incorporation into cellular collagen was also observed in these cells as well as in the pup calvaria cultures ( Figure 37) and UMR-106 cells (Figure 38). The exchange of proline to the medium over the next 48 hr was reduced significantly in the presence of drugs (Figure 39). In a lactating rat model in which rats were dosed orally for 14 days at 8 mg/kg/day, the amine boranes increased bone volume, weight, density, and ash weight, while elevating bone and serum calcium levels. In conclusion, the amine-carboxyborane derivatives demonstrated promise as therapeutic agents for a number of disease states.