25-Azavitamin D3, an inhibitor of vitamin D metabolism and action.

25-Azavitamin D3 inhibited both the bone calcium mobilization and intestinal calcium transport responses of rats to vitamin D3 but not to 25-hydroxyvitamin D3. Although 25-azavitamin D3 had no effect on the response of bone to 1alpha,25-dihydroxyvitamin D3, it did diminish the response of the intestine to that metabolite. 25-Azavitamin D3 increased liver vitamin D content and reduced the concentration of 25-hydroxyvitamin D3 required to inhibit the metabolism of vitamin D3 (75 and 200 microgram) were similar to the doses of 25-azavitamin D3 required to inhibit the action of vitamin D3 in vivo (50 and 150 microgram). 25-Azavitamin D3 is thus a vitamin D antagonist, acting for the most part via inhibition of the liver 25-hydroxylation of vitamin D3.


25-Azavitamin
DI inhibited both the bone calcium mobilization and intestinal calcium transport responses of rats to vitamin D1 but not to t&hydroxyvitamin DS. Although 25-azavitamin Da had no effect on the response of bone to la,2&dihydroxyvitamin Da, it did diminish the response of the intestine to that metabolite. 25 The hypercalcemia of a variety of disorders of calcium metabolism is the result of excessive intestinal transport of calcium or demineralization of bone (1,2). Since both of these physiological processes require vitamin D (3, 4), an antagonist of vitamin D action should effectively lower the hypercalcemia. It is now known that the biological activity of vitamin Ds in intestine and bone is the result of metabolic conversion to the hormone lcr,25-dihydroxyvitamin DS (5,6). A consequence of the obligatory nature of the two-step metabolic activation of vitamin D is that inhibitors of vitamin D metabolism should block the expression of the biological responses to vitamin D3. The metabolism of vitamin D3 is sequential; it involves hydroxylation of vitamin DB at C-25 (primarily in the liver) to form 25-hydroxyvitamin DB and then hydroxylation of the latter at C-l in the kidney to produce la,25-(OH)~D3~ (7). Of the two alternatives, inhibition of the 25-hydroxylase is more desirable since it precludes the potential accumulation of 25-OH-D3 which can directly give rise to responses in bone and intestine when present in a much higher than normal concentration (8,9). Toward this end, we have prepared a variety of side chain-modified analogs of vitamin D as potential 25-hydroxylase inhibitors (10). We wish to report that one of these compounds, 25 Column recoveries were 76 -C 12% (mean + SD., n = 26).
Metabolite levels (Tables  II, III, and IV) are expressed as a percentage of the total administered dose present in the organ examined. These values were readily obtained for liver samples, since the entire organ was extracted.
The total serum volume of a rat (in milliliters) was approximated to be 3% of the total body weight in grams (17). This estimation and knowledge of the volume of serum extracted permitted calculation of the serum metabolite levels in the desired units.

RESULTS
The effect of 25-N-D3 on the biological activity of vitamin Ds, 25-OH-D3, and la,25-(OH)zD3 was tested in the rat. Vitamin D-deficient animals, maintained on a low calcium diet, received graded doses of 25-N-D3 by intrajugular injection (Dose 1). Two hours after this dose, each rat was administered a physiological dose of either vitamin D3 (50 ng), 25-OH-D3 (25 ng), ln,25-(OH)2D3 (10 ng), or the vehicle (ethanol) by the same route (Dose 2). Two physiological responses dependent upon vitamin D were assayed 20 h after the second dose. The mobilization of bone calcium was indirectly observed as a rise in serum calcium concentration.
Intestinal calcium transport was measured using an everted duodenal segment. The results are summarized in Table I -20  chromatograms  of the metabolites extracted from serum and  liver samples obtained 12 h after the vitamin DS dose, whereas  Tables II and III summarize the metabolite levels in the serum and liver of rats at all time points examined. The chromatographic profiles of the serum and liver extracts from animals given radiolabeled vitamin Da (Fig. 1, top panel)       this chromatographic system.) The analogous profiles from rats predosed with 25-N-D, (Fig. 1, bottompanel) showed the same radioactive peaks, but serum 25-OH-D:3 content was much lower and liver vitamin D:j content was much higher than for the ethanol-dosed controls. Serum 25-OH-D3 levels were depressed by 25-N-Da at all times examined (Table II). The largest difference, an &fold decrease in serum 25-OH-D3, was observed for the earliest sample (4 h after vitamin Ds dose). Serum 25-OH-D3 did increase with time in the rats treated with 25-N-D3, but it never approached the level of normal controls. The liver vitamin Ds content was higher in the rats receiving 25-N-D, at all times examined (Table III). In the animal receiving 25-N-Dz, 39% of the dose remained in the liver as unchanged vitamin Ds (4 h after vitamin D3 dose). The liver 25-OH-D3 concentration (Table III) was only slightly depressed by 25-N-D3. Neither the liver (Table III) nor the serum (Table II) levels of vitamin D esters were appreciably altered by predosing the rats with 25-N-Ds.
In a second series of experiments, rats were given graded doses of 25-N-D3 2 h before a dose of [1,2-"HIvitamin Ds. All animals were killed 16 h after the vitamin D dose and serum and liver were analyzed for radioactive vitamin D and its metabolites.
The results listed in Table IV show that low doses of 25-N-D3 (10 or 25 pg) caused a slight reduction in serum 25-OH-D, content (compared to controls), while higher doses (75 or 200 pg) resulted in appreciably lower levels of serum 25-OH-Dg, demonstrating effective inhibition of the conversion of vitamin D3 to 25-OH-D:1. In each case, the inhibition of vitamin DS metabolism was accompanied by elevated vitamin D3 levels in the liver (Table IV). DISCUSSION

Data summarized
in Table I (Table I). This inhibition is not expected, given the inability of 25-N-D3 to depress the calcium transport response to 25-OH-D3, a metabolite which acts via metabolism to lcu,25-(OH)2Da. The apparent paradox may be the result of the different time course of response of 25-OH-D3 and la,25-(OH)BDs, and thus it might be possible to demonstrate an inhibition of the intestinal response to 25-OH-D,? by later and more frequent administration of 25-N-Dz. This rationalization is most speculative, and the explanation must await the results of further investigation.
In any case, the inhibition of the intestinal response to 1~~,25-(0H)~D~ by 25-N-D3 is of considerable interest. It demonstrates direct antagonism toward the hormonal form of vitamin D-l~u,25-(0H)~D~. Furthermore, this antagonism is selective since no effect on the bone response to 1~~,25-(0H)~D~ is observed. 25-N-D:1 is the fist known vitamin D analog to possess either of these properties.
Other agents are known to antagonize the expression of vitamin D activity. Glucocorticoids have been shown to inhibit the intestinal calcium transport response to vitamin D3 (la), 25-OH-D3 (19), and la,25-(OH)2D3 (20). Neither the conversion of vitamin D3 to 25-OH-D3 (19) nor of 25-OH-D3 to la,25-(OH)zDa (21) was effected by glucocorticoid treatment; further metabolism of 1~~,25-(0H)~D3 may be accelerated, however (22). The calcium transport response of rats to either vitamin DB or 25-OH-Da is depressed by prior treatment with anticon-v&ants (23). Recent data suggests that this effect is the result of inactivation of vitamin D metabolites by an anticonvulsantinduced liver enzyme system (24,25). The presence of a rachitic factor in plants has been demonstrated (26,27), but the identity of the substance is not known. Finally, 24-nor-25-OH-D3 (28) and 19S-19-hydroxy-10(19)-dihydrovitamin DS (29) have been reported to possess anti-vitamin D activity. Like 25-N-Ds, 24-nor-25-OH-D3 did not impair the bone or intestinal responses to 25OH-D3 (28), suggesting that the liver is the site of action for both substances. However, 24nor-25-OH-D3 has also been reported to be an active analog of vitamin D (30)' and thus the inhibitory nature of the compound is unclear.
In summary, we have found that 25-N-D3 antagonizes both the bone and intestinal responses of vitamin D-deficient animals to vitamin DB. Since 25-N-Da does not inhibit the action of 25-OH-Ds, and since it causes a build-up of liver vitamin DB and a reduction of serum 25-OH-Ds, the antagonism of the biological responses in rats to vitamin D results from an inhibition of 25-hydroxylation of vitamin D. Furthermore, 25 N-D3 depresses the intestinal calcium transport response in animals to 1~1,25(0H)~D~, presumably the result of a direct inhibition of the activation (or assembly) of the calciumtranslocating machinery. Due to these inhibitory effects, 25-N-D3 may prove useful clinically in reducing the hypercalcemia of a variety of human disorders such as primary hyperparathyroidiim, hypercalcemia of malignancy (pseudohyperparathyroidism), vitamin D toxicity, idiopathic hypercalcemia of infancy, sarcoidosis, and tertiary hyperparathyroidism.