The Effects of Amine-Carboxyborane Related Derivatives on UMR-106 Bone Metabolism

The amine-carboxyboranes and related derivatives have been shown to be potent anti-inflammatory and anti-osteoporosis agents. Their action in part appears to be mediated by the modulation of cytokines, e.g. TNFα or IL-1. Previous studies have demonstrated that LPS induced macrophages release of TNFα maximally at 60 to 90 min. and IL-1 from 5 to 8 hr. The amine-carboxyboranes reduced significantly the release of these cytokines but also blocked TNFα high affinity binding to UMR-106 receptor at 90 min. at 10 μM, and IL-1 high affinity binding at 5 hr. at 12.5 μM. In addition, the agents suppressed IL-8 binding to CHO K1 high affinity receptor at 24 hr. at 50 μM and IL-2 binding to HuT-8 receptors at 25 μM at 90 min. and 5 hr. Correlation of metabolic events associated with osteoporosis showed that at 90 min., when TNFα receptor binding was reduced by the agents, calcium uptake into UMR-106 cells was reduced at 10 μM as well as the acid and alkaline phosphatases, and the prostaglandin cyclo-oxygenase activities and adhesion of leukocytes and macrophages to UMR-106 cell monolayers. At 5hr. when the agents reduced IL-1 binding to UMR-106 receptors, calcitonin and 1,25-dihydrovitamin D3 binding was reduced by the agents as was acid and alkaline phosphatase, and 5′-lipoxygenase activities and white blood cell adhesion. At this time calcium uptake and proline incorporation was increased significantly by the agents. At later times e.g. 18-48 hr. calcium uptake was still increased, and NAG activity was inhibited in the presence of the agents. These effects may be related more to the inhibition of other cytokine receptor binding, e.g. IL-8. Thus, many of the observed metabolic effects of amine-carboxyboranes as antiosteoporosis agents can be correlated with their inhibition of cytokine high affinity binding to target cell receptors.

macrophages release of TNF maximally at 60 to 90 min. and IL-I from 5 to 8 hr.
The amine-carboxyboranes reduced significantly the release of these cytokines but also blocked TNF high affinity binding to  receptor at 90 min. at i0 M, and IL-I high affinity binding at 5 hr. at 12.5 M. In addition, the agents suppressed IL-8 binding to CHO K1 high affinity receptor at 24 hr. at 50 M and IL-2 binding to HUT-8 receptors at 25 M at 90 min. and 5 hr.
Correlation of metabolic events associated with osteoporosis showed that at 90 min., when TNF receptor binding was reduced by the agents, calcium uptake into UMR-106 cells was reduced at i0 M as well as the acid and alkaline phosphatases, and the prostaglandin cyclo-oxygenase activities and adhesion of leukocytes and macrophages to UMR-106 cell monolayers. At 5hr. when the agents reduced IL-I binding to UMR-106 receptors, calcitonin and 1,25-dihydrovitamin D 3 binding was reduced by the agents as was acid and alkaline phosphatase, and 5'-lipoxygenase activities and white blood cell adhesion.
At this time calcium uptake and proline incorporation was increased significantly by the agents. At later times e.g. 18-48 hr. calcium uptake was still increased, and NAG activity was inhibited in the presence of the agents. These effects may be related more to the inhibition of other cytokine receptor binding, e.g.  Thus, many of Introduction Selected amine-carboxyborane derivatives at 8 mg/kg/day have been shown to prevent osteoporosis and loss of bone mass in rodents [l].
In vitro studies with CF 1 mouse pups calvaria and rat UMR-106 osteosarcoma cells have shown that these compounds over 48 hr. reduce the loss of calcium from the cells better than calcitonin and simple boron salts [l]. Indications from the previous in vivo and in vitro studies were that the Vol. 3, No. 1, 1996 The Effects of Amine-Carboxyborane Related Derivatives amine-carboxyboranes effectively lowered TNF and IL-I release from macrophages [l,2] which is asociated with a lowering of hydrolytic and proteolytic enzyme activities and secondary chemical effects from prostaglandins and leukotirenes released at the sites of inflammation [3,4] and of bone osteoporosis [l] The previous studies did not attempt to correlate the incubation time of the drugs with the maximum released of the cytokines into the medium. The present study has been performed in rat UMR-106 bone cells at times, i.e. 90 min. and 5 hr., which have been shown to be the time periods of maximum release of cytokines, TNF and IL-I, from macrophages. These time points were selected so that we can determine the direct effect of those cytokines on specific biochemical events of bone metabolism.
The selected compounds at 8 mg/kg/day orally have been shown in vivo in lactating ovariectomized rats to significantly elevate femur and humerus volume, weight, density and ash weight after 14 days[l]. Also, serum calcium and femur calcium levels were elevated whereas hydroxyproline levels were reduced in rodents by the amine-carboxyboranes.

Methods and Materials
The amine-carboxyborane derivatives were previously synthesized and the  [12] were used to determine prostaglandin formation from 3H(N)-arachidonic acid (I00 Ci/mmole) and UMR-106 cells (106). After 1 hr, the reaction was terminated with 2N HCI and the mixture was extracted with ether and evaporated.
The residue was dissolved in ethyl acetate and applied to silica gel TLC plates which were eluted with chloroform, methanol, water and acetic acid (90:8:1:0.8) The plates were developed with iodine vapor and the area appropriate to the prostaglandin standards was scraped and counted [12,13].
The dpm in each area was calculated as percent of the total dpm applied to the plate. 5'-Lipoxygenase Assay UMR-106 cells were incubated for 30 min with phosphate buffer (pH 7.2) containing 0.6 mM CaCI 2, and 1.0 mM MgCI 2 I0 mg Calcium Ionophore A23187 and 1 mCi 14C-arachidonic acid (i00 i/mmol). The reaction was terminated with 2 volumes of EtOAc'CH2CI2 containing 12 mg cold arachidonic acid.
The organic phase was evaporated to a residue which was applied to silica gel plates.

IL-I under similar
conditions was released from P388DI with the peak elevation being at 5 hr.
The IL-I levels remainder elevated through 8 hr. and then fell slowly back to normal levels. Thus, the following biochemical parameters were measured in the presence of the amine-carboxyboranes at these peak times of the two cytokines.
Incubation of 125I-TNF with UMR-106 cells showed peak high affinity binding at 90 min. [Table la and b].
Compound 1 at I0 M at 60 min caused at 34% reduction in the high affinity binding of TNF whereas Compounds 2 and 3 caused 18% and 33% reduction in binding at 90 min. When higher concentration of the drugs were employed at 90 min incubation, elevated high affinity binding of TNF to UMR-106 receptors was observed [ Table Ib].
00+/-5 a c 742 CPM/mg 231 CPM/mg 125I-Calcitonin high affinity binding to UMR-106 cell receptors was the highest from 60 to 120 min. and then declined.
The agents at i0 M caused a significant increase in proline incorporation at 5 and 8 hr. but not at 24 and 48 hr. Proline incorporation into non-collagen protein of UMR-106 cells was elevated only at 8 hr. by Compounds 1 and 2 [ Table 6b].
Compound 2 at 12.5, 25 and 50 M markedly elevated proline incorporation whereas Compounds 1 and 3 had no effect or actually reduced incorporation at 12.5 tM. [Table 6c].
Non-collagen incorporation of proline was not affected by the higher concentrations of any of the agents [ Table 6d]. TNF at i00 ng/ml at 5 hr. caused a 49% increase of proline incorporation into collagen but at i0 ng/ml had no affect at 90 min. or 5 hr. nor was there any effect on the non-collagen incorporation.      12 nmoles p-nitrphenol released/ml medium; 39 nmo+/-es p-nitrophenol released/ml medium; 40 nmoles p-nitrophenol released/ml mediu; * p<0.0001 F'ol. 3, No. I, 1996 The Effects of Amine-Carboxyborane Related Derivatives       (18,150 DPM/mg protein);* p<0.0001;ND = not determined

Discussion
The destructive phase of bone osteoporosis is initiated by cytokines which are chemical cell communicators released by invading macrophages after being stimulated. Early bone resorption effects are evoked by TNF whereas later effects are regulated by IL-I and then IL-6 and IL- 8. Bone resorption is divided into two phases" phase I involves inorganic demineralization of cortical bone which is a process initiated by influxing macrophages, PMNs, fibroblasts and osteoclasts, etc. and phase II is the organic phase where the bone matrix collagen is digested by proteolytic and hydrolytic enzymes to release hydroproline. Drug therapy should involve blocking the early resorption events as well as the acceleration of the reconstruction of bone.
In vivo studies with the amine-carboxyboranes at 8 mg/kg/day suggest that the calcium uptake and increase in bone collagen deposition had occurred improving the tensile strength of the bone after 14 days treatment 2 The present studies have demonstrated that aminecarboxyboranes block TNF and IL-I release from the types of cells which invade the bone surface to begin the resorption process. Further, these studies showed that these agents also block early adhesion of these migratory cells to the bone cells from 90 min. to 5 hr. and macrophage adhesion is blocked at 2 and 5 hr. at higher concentration of the agents.
Blockage of the adhesion of these cell to the bone surface should reduce the hydrolytic and proteolytic enzymes as well as the release of cytokines into the area of the bone surface.
Previous studies had demonstrated that the amine-carboxyboranes reduce the activity of macrophages lysosomal and proteolytic enzyme activites with IC-50 values -10 -6 M. [I].
Furthermore the amine-carboxyboranes at 8