Effects of alendronate on plasma calcium levels, urinary calcium excretion, and bone resorption markers in normal rats: comparison with elcatonin, synthetic eel calcitonin.

In normal rats given alendronate (0.01-6.25 mg/kg) or elcatonin (synthetic eel calcitonin; 0.32-8.0 U/kg), changes in urinary calcium (Ca), pyridinoline (Pyr), and deoxypyridinoline (D-Pyr) excretion during the hypocalcemic response were assessed. Although the lower doses (0.01-0.25 mg/kg) of alendronate did not influence plasma Ca, the high doses (1.25-6.25 mg/kg) significantly decreased plasma Ca by the third day after single iv administration. In these groups, urinary Ca excretion did not show any significant change, but urinary Pyr and D-Pyr excretion decreased significantly at high doses. The hypocalcemic effect lasted for only 1 or 2 days (2-3 days after injection) even at high doses of alendronate. In the group receiving elcatonin (8.0 U/kg, twice daily), a significant decrease in plasma Ca was evident as early as 1 day after the start of administration. This was accompanied by a marked increase in urinary Ca excretion in the early stage without a significant decrease in urinary Pyr or D-Pyr excretion, and the suppression of bone resorption was more pronounced in the late phase of treatment with elcatonin. These results suggest that alendronate decreases plasma Ca chiefly by suppressing bone resorption, whereas elcatonin decreases plasma Ca by inhibiting bone resorption and accelerating Ca excretion. The present data show that both alendronate and elcatonin inhibit bone resorption and exert an antihypercalcemic effect, but the mechanism of action is different in the two drugs.

Antagonistic Actions in Vivo of (23S)-25-Dehydro-1alpha-Hydroxyvitamin D(3-)26,23-Lactone on Calcium Metabolism Induced by 1alpha,25-Dihydroxyvitamin D(3).

The vitamin D analog, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647), is an antagonist of the 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells. To clarify whether TEI-9647 could function as an antagonist of 1alpha,25(OH)(2)D(3) in vivo, we investigated in vitamin D-deficient (-D) rats the effects of single doses of TEI-9647 on several parameters of calcium metabolism modulated by 1alpha,25(OH)(2)D(3). TEI-9647 (50 microgram/kg iv) acting alone slightly, but significantly, stimulated intestinal calcium transport (ICA) and bone calcium mobilization (BCM) only at 8 h, but not at 24 h. In contrast, TEI-9647 dose-dependently inhibited ICA and BCM stimulated by an iv dose of 0.25 microgram/kg 1alpha,25(OH)(2)D(3) after 24 h, but not after 8 h. With respect to serum PTH levels, the administration of either TEI-9647, 50 microgram/kg, or 1alpha,25(OH)(2)D(3), 0.25 microgram/kg, began to decrease the circulating levels by 4 h, which reached a nadir 24 h after administration. But, when TEI-9647 and 1alpha,25(OH)(2)D(3) were simultaneously administered to -D rats, the TEI-9647 dose-dependently reversed the inhibition of PTH secretion caused by 1alpha,25(OH)(2)D(3), 0.25 microgram/kg, at 8 and 24 h after the treatment. In separate experiments, the daily iv administration of 20 microgram/kg of TEI-9647 alone to +D rats for 2 weeks resulted in no significant changes in the prevailing serum Ca(2+) concentration. But doses of 1-20 microgram/kg of TEI-9647 in combination with 0.5 microgram/kg of 1alpha,25(OH)(2)D(3), for 2 weeks, dose-dependently and significantly suppressed the serum calcium concentration increase caused by the 1alpha,25(OH)(2)D(3). Collectively, these results show that TEI-9647 acting alone displays in vivo weak agonistic actions, but when administered in combination with 1alpha,25(OH)(2)D(3), is a potent antagonist of three genomic-mediated calcium metabolism parameters. We conclude that TEI-9647 can also function as an antagonist of 1alpha,25(OH)(2)D(3) in vivo in the rat.

Phosphorylation of smg p21B in rat peritoneal mast cells in association with histamine release inhibition by dibutyryl-cAMP.

IP3 formation and histamine release from rat peritoneal mast cells stimulated by compound 48/80 were dose-dependently inhibited by Bt2cAMP. These inhibitions were restored to the control level in the presence of H-8, a protein kinase A inhibitor. The 22 kDa protein in mast cells was revealed as a markedly phosphorylated protein by incubating with Bt2cAMP, and this phosphorylation was also diminished by H-8. The 22 kDa phosphoprotein of rat mast cells comigrated with phosphorylated smg p21B, purified from human platelets and phosphorylated by protein kinase A in cell-free system, in both one- and two-dimensional PAGE analysis. Moreover, 22 kDa protein in mast cells was identified as smg p21B by immunoblot analysis using an antibody against smg p21B. From the present study, it became clear that smg p21B is phosphorylated by means of protein kinase A system in rat peritoneal mast cells, and it was assumed that phosphorylated smg p21B plays some important role in the suppression of IP3 formation and histamine release from rat peritoneal mast cells.

Synthesis, biological evaluation, and conformational analysis of A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: unique activity profiles depending on the stereochemistry of the A-ring and at C-20.

All eight possible A-ring diastereomers of 2-methyl-1, 25-dihydroxyvitamin D(3) (2) and 2-methyl-20-epi-1, 25-dihydroxyvitamin D(3) (3) were convergently synthesized. The A-ring enyne synthons 19 were synthesized starting with methyl (S)-(+)- or (R)-(-)-3-hydroxy-2-methylpropionate (8). This was converted to the alcohol 14 as a 1:1 epimeric mixture in several steps. After having been separated by column chromatography, each isomer led to the requisite A-ring enyne synthons 19 again as 1:1 mixtures at C-1. Coupling of the resulting A-ring enynes 20a-h with the CD-ring portions 5a,b in the presence of a Pd catalyst afforded the 2-methyl analogues 2a-h and 3a-h in good yield. In this way, all possible A-ring diastereomers were synthesized. The synthesized analogues were biologically evaluated both in vitro and in vivo. The potency was highly dependent on the stereochemistry of each isomer. In particular, the alpha alpha beta-isomer 2g exhibited 4-fold higher potency than 1 alpha,25-dihydroxyvitamin D(3) (1) both in bovine thymus VDR binding and in elevation of rat serum calcium concentration and was twice as potent as the parent compound in HL-60 cell differentiation. Furthermore, its 20-epimer, that is, 20-epi-alpha alpha beta 3g, exhibited exceptionally high activities: 12-fold higher in VDR binding affinity, 7-fold higher in calcium mobilization, and 590-fold higher in HL-60 cell differentiation, as compared to 1 alpha,25-dihydroxyvitamin D(3) (1). Accordingly, the double modification of 2-methyl substitution and 20-epimerization resulted in unique activity profiles. Conformational analysis of the A-ring by (1)H NMR and an X-ray crystallographic analysis of the alpha alpha beta-isomer 2g are also described.

(23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone function as antagonists of vitamin D receptor-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3).

We synthesized various analogues of 1alpha,25-(OH)(2)D(3)-26,23-lactone and examined the effects of them on HL-60 cell differentiation using the evaluation system of the genomic action of 1alpha,25-(OH)(2)D(3). We found that (23S)- and (23R)-25-dehydro-1alpha-OH-D(3)-26,23-lactone (TEI-9647 and TEI-9648) strongly bound to the VDR, but did not induce HL-60 cell differentiation. Intriguingly, TEI-9647 and TEI-9648 did inhibit that induced by 1alpha,25-(OH)(2)D(3), whereas they did not suppress that caused by retinoic acid or TPA. On the contrary, the similar 25-dehydrated 24-dehydro analogues, TEI-D1807 and TEI-D1808, weakly but significantly induced HL-60 cell differentiation, never showing inhibitory effect on HL-60 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In other experiments, TEI-9647 and TEI-9648 markedly suppressed 25-OH-D(3)-24-hydroxylase gene expression induced by 1alpha,25-(OH)(2)D(3) in HL-60 cells. TEI-9647 also inhibited the heterodimer formation between VDR and RXRalpha, and the VDR interaction with co-activator SRC-1 according to the results obtained from the mammalian two-hybrid system in Saos-2 cells. Taking all these results into consideration, we reached a manifest conclusion that TEI-9647 and TEI-9648 are the specific and first antagonists of 1alpha,25-(OH)(2)D(3) action, specifically VDR-VDRE mediated genomic action.

Systematic studies on synthesis, structural elucidation, and biological evaluation of A-ring diastereomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) and 20-epi-2-methyl-1alpha,25-dihydroxyvitamin D(3).

All possible A-ring diastereomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) (2) and 20-epi-2-methyl-1alpha,25-dihydroxyvitamin D(3) (3) were synthesized by palladium-catalyzed coupling reaction of A-ring 'enyne' synthons with CD-ring portions. The A-ring synthons were rationally synthesized via a novel and practical route, starting with methyl (R)-(+)- and (S)-(-)-3-hydroxy-2-methyl-propionate, in good yields. X-ray crystallographic analysis of 2alpha-methyl-1alpha,25-dihydroxyvitamin D(3) (2b) and conformational analysis of the A-ring of 2alpha-methyl-(2b) and 2beta-methyl-1alpha,25-dihydroxyvitamin D(3) (2f) were carried out, and the results are described. All A-ring diastereomers (2 and 3), thus synthesized, were biologically evaluated both in vitro and in vivo. The biologic potency was highly dependent on the stereochemistry of the A-ring substituents. In particular, 2b showed 4-fold higher vitamin D receptor [VDR] binding activity than the natural hormone, and its 20-epimer (3b) exhibited exceptionally high activity, 12-fold more potent in VDR binding, 7-fold in calcium mobilization, and 590-fold in induction of human promyelocytic leukemia (HL-60) cell differentiation as compared with the natural hormone. Further, the 20-epi-2beta-Me-1beta, 3alpha(OH)(2) isomer (3g) had significant biologic potencies compared to the natural hormone despite having 1beta-OH configuration. The transcriptional activities on human osteocalcin gene promoter, including VDRE in transfected mammalian cells, were also evaluated. Finally, there was a clear contrast between the effects of the 2-methyl group on the HL-60 cell differentiation- and apoptosis-inducing activities of 2 and 3.

Opening of the mitochondrial permeability transition pore links mitochondrial dysfunction to insulin resistance in skeletal muscle.

Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.

Comparison of cytotoxic effects of bisphosphonates in vitro and in vivo.

We compared the cytotoxic effects of alendronate (ALN) and incadronate (YM175) on isolated rabbit osteoclasts in vitro and on rats in vivo. In the in vitro experiment, each bisphosphonate was added to the culture of isolated osteoclasts at the final concentration of 3 x 10(-5), 3 x 10(-4), or 3 x 10(-3) M, and the amount of creatine phosphokinase (CPK) released into the medium was taken as an index of cytotoxicity at 5, 10, and 24 hours after the treatment. Also viability of osteoclasts, measured in terms of trypan blue exclusion, was assessed at 24 hours after the treatment. In YM175-treated groups, CPK activity in the medium increased in a concentration-dependent manner with time, and phase-contrast microscopic observation revealed damaged cell membranes and nuclear deterioration in YM175-treated osteoclasts. As a result, the viability of the osteoclasts was decreased at the concentrations of 3 x 10(-4) and 3 x 10(-3) M. However, in the ALN-treated groups, neither CPK activity nor viability of isolated osteoclasts changed significantly compared with control levels even at 3 x 10(-3) M for up to 24 hours. In the in vivo experiment, each bisphosphonate was administered separately to normal rats (7 weeks old, Sprague-Dawley) by intravenous injection at 1, 5, or 25 nmol/kg. Two days after the injection, the animals were euthanized, and the plasma Ca concentration and total CPK activity were measured. In YM175-injected rats, the CPK activity increased at 25 mmol/kg, and a slight decrease in the plasma Ca level was seen at this dose. In contrast, in ALN-injected rats, CPK activity did not increase even at 5 or 25 mmol/kg, and the plasma Ca level did decrease significantly compared with controls. Isozyme analysis revealed that, not only was CPK activity increased in the BB type in YM175-injected rats, it was also increased in the MB and MM types. In conclusion, alendronate, unlike YM175, does not have any cytotoxic effects on osteoclasts either in vitro or in vivo.

Antiallergic profile of the novel H1-antihistaminic compound levocabastine.

Levocabastine hydrochloride (R50 547, CAS79516-68-0) caused no inhibitory effect on the histamine release from rat peritoneal mast cells induced by compound 48/80, A23187 and concanavalin A. However, the drug inhibited histamine release from passively sensitized mast cells and passive peritoneal anaphylaxis in rats, though higher concentrations or doses were required. Moreover, levocabastine provided a relatively potent inhibitory effect on histamine release from lung pieces of actively sensitized guinea pigs exposed to antigen, and simultaneously the drug prevented a decrease in the cyclic AMP (cAMP) content. Levocabastine potently inhibited histamine-induced cutaneous reactions in rats and the drug also prevented histamine-induced contraction of isolated guinea pig ileum. Levocabastine did not induce any significant changes in platelet aggregation or in the contraction of guinea pig ileum induced by platelet activating factor (PAF). However, the drug inhibited eosinophil migration induced by PAF. The chemotaxis of neutrophils induced by N-formyl-methionyl-leucylphenylalanine (fMLP) was also inhibited by levocabastine in a dose-dependent fashion. Levocabastine has no influence on the order parameter tested with liposomes, suggesting that the drug provides no significant effect on the membrane fluidity of lipid bilayer. These results seem to indicate that the antiallergic effect of levocabastine is mainly dependent on its potent antihistaminic activity.

Synthesis and biological activity of 2-methyl-20-epi analogues of 1 alpha,25-dihydroxyvitamin D3.

Synthesis and biological evaluation of all eight possible A-ring diastereomers of 2-methyl-20-epi-1,25-dihydroxyvitamin D3 are described. Among the analogues synthesized. 2 alpha-methyl-20-epi-1 alpha,25-dihydroxyvitamin D3 exhibited exceptionally high potency. The double modification of 2-methyl substitution and 20-epimerization yielded analogues with unique activity profiles.

A novel and practical route to A-ring enyne synthon for 1 alpha,25-dihydroxyvitamin D3 analogs: synthesis of A-ring diastereomers of 1 alpha,25-dihydroxyvitamin D2 and 3-methyl-1,25-dihydroxyvitamin D3.

A novel and practical route to the A-ring enyne synthon (2), which can be versatile for a variety of A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 (1), was developed. This novel method led to an improved synthesis of the A-ring diastereomers of 1, the compounds 13-15, and synthesis of the new analogs, 2-methyl-1,25-dihydroxyvitamin D3 (4) with its all possible diastereomers. The biological evaluation of the 2-methyl analogs showed the alpha alpha beta-isomer to be more potent than 1.

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs on 25-hydroxyvitamin-D(3)-24-hydroxylase gene expression induced by 1alpha,25-dihydroxy-vitamin D(3) in human promyelocytic leukemia (HL-60) cells.

We have demonstrated that 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs, (23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647, TEI-9648, respectively), inhibit HL-60 cell differentiation induced by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], but not differentiation caused by all-trans retinoic acid (D. Miura et al., 1999, J. Biol. Chem. 274, 16392). To assess whether the antagonistic actions of TEI-9647 and TEI-9648 in HL-60 cells are related to 1alpha,25(OH)(2)D(3) breakdown, we investigated their effects on catabolism of 1alpha,25(OH)(2)D(3). In HL-60 cells, the C-24 but not the C-23 side-chain oxidation pathway of 1alpha,25(OH)(2)D(3) has been reported. Here we demonstrate that 1alpha,25(OH)(2)D(3) was metabolized both to 24,25,26,27-tetranor-1alpha,23-(OH)(2)D(3) and 1alpha,25(OH)(2)D(3)-26,23-lactone; thus HL-60 cells constitutively possess both the 24- and the 23-hydroxylases. Metabolism of 1alpha, 25(OH)(2)D(3) was strongly suppressed by 10(-7) M TEI-9647 or 10(-6) M TEI-9648. 1alpha,25(OH)(2)D(3) alone slightly induced 24-hydroxylase gene expression by 8 h with full enhancement by 24-48 h; this induction was inhibited by 10(-6) M TEI-9647 and 10(-6) M TEI-9648 (86.2 and 31.9%, respectively) 24 h after treatment. However, analogs of TEI-9647 and TEI-9648 without the 25-dehydro functionality induced 24-hydroxylase gene expression. These results indicate that TEI-9647 and TEI-9648 clearly mediate their stereoselective antagonistic actions independent of their actions to block the catabolism of 1alpha,25(OH)(2)D(3). Therefore, TEI-9647 and TEI-9648 appear to be the first antagonists specific for the nuclear 1alpha,25(OH)(2)D(3) receptor-mediated genomic actions of 1alpha,25(OH)(2)D(3) in HL-60 cells.