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26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3: A highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 229, No. 1, February 15, pp. 348-354, 1984
26,26,26,27,27,27-Hexafluoro-I ,25dihydroxyvitamin DS: A Highly Potent, Long-Lasting Analog of 1,25Dihydroxyvitamin D3 YOKO TANAKA,
HECTOR F. DELUCA,’ YOSHIRO AND NOBUO IKEKAWAt
KOBAYASHI,*
Department
of Biochemistry, College of Agricultural and Lge Sciences, University of Wisconsin-Madison, Mad&m, Wisconsin 5.9706; *Tokyo College of Pharmacy, Hachioji, Tokyo, Japan; and of Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo 152, Japan tDepartment
Received June 24, 1983, and in revised form October 13, 1983
A new fluoro analog of 1,25-dihydroxyvitamin Ds, i.e., 26,26,26,27,27,27-hexalluoro1,25-dihydroxyvitamin Da, has been compared with the native hormone, 1,25dihydroxyvitamin Ds, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25dihydroxyvitamin DS. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.
D to its function, 25-fluorovitamin D3 (5), 1-fluorovitamin D3 (6), 24,24-difluoro-25hydroxy-vitamin D3 (24,24-F2-25-OH-D3) (7), and 26,26,26,27,27,27-hexafluoro-25hydroxyvitamin D3 (26,27-Fs-25-OH-D3) (8) have been prepared. The latter two compounds have also been converted to their la-hydroxy derivatives (9, 10). The 24,24F2-1,25-(OH)2D3 proved to be about 10 times more active than 1,25-(OH)2D3 (11). However, the 26,26,26,27,27,27-hexafluoro1,25-dihydroxyvitamin D3 (26,27-FG-1,25(OH)ZDB) has not yet been tested for biological activity, although 26,27-FE-25OH-D, was found equally as active as 25 OH-D3 (12). This report demonstrates that 26,2’7-FG-1,25-(OH)2D3 is 5 to 10 times more active than 1,25-(OH)2D3 in the rat and its activity is long lasting.
The demonstration that 1,25-dihydroxyvitamin D3 (1,25-(OH),D3)2 is a metabolically active form of vitamin D and the utility of this hormone in treating bone disease has prompted a great interest in biopotent analogs (1,2). For example, a potent analog used commercially is la-hydroxyvitamin D3 (IQ-OH-Ds) (3,4). Because of an interest in developing tools to study the importance of the various hydroxylations of vitamin ) No reprints of this paper are available from the authors. Author to whom all correspondence should be addressed. * Abbreviations used: 25.OH-D3, 25-hydroxyvitamin DB; 1,25-(OH)*D3, 1,25-dihydroxyvitamin D,; 24,24F,24,24-difluoro-1,25-dihydroxyvitamin 1,25-(OHM&. DS; 26,27-Fs-1,25-(OH),D3, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3. 0003-9861/84 $3.00 Capyrlyht All rights
C 1984 by Academic Press. Inc. of reproduction in any form reserved.
348
26,26,26,27,27,27-HEXAFLUORO-1,25-DIHYDROXYVITAMIN MATERIALS
AND
METHODS
Vitamin D related compounds 26,27-FB-1,25-(0H)zDa was synthesized by Kobayashi et al (lo), while 1,25(OH)aDI was a gift of the Hoffmann-La Roche Company (Nutley, N. J.). [26,27-sH]-25-OH-Da and [26,273H]-1,25-(OH)zD3 (specific activity 160 Ci/mmol) were synthesized by the method described previously (13, 14). Rats. Weanling male rats were purchased from the Holtzman Company (Madison, Wise.) and fed either a low-phosphorus (O.l%), high-calcium (1.2%),vitamin D-deficient diet (15) or a low-calcium (O.O2%),adequate phosphorus (0.3%), vitamin D-deficient diet (16) for 3 weeks except in case of Table I. In this case, weanling rats were fed the low-phosphorus, high-calcium diet for 1 week before they were used in the experiment. of compounda The designated comAdministration pound was dissolved in a O.l-ml mixture of propylene glycoi-95% ethanol (95:5) and administered subcutaneously daily for biopotency determination (Fig. 1 and Table I). For the study of duration of action, the compound was dissolved in 0.05 ml of 95% ethanol and administered intrajugularly once (Figs. 2 and 3, Tables II and IV). The relative oral effectiveness of the compounds was examined by dissolving them in 0.1 ml of cotton seed/soybean oil (Wesson) and administering them directly into the stomach with polyethylene tubing. In all of the experiments, rats in the control group received the appropriate amount of vehicle by the indicated route. Determinations of serum inmganic phosphcrrus and calcium. Serum was obtained by centrifugation of clotted blood. Inorganic phosphorus was determined by the calorimetric method of Chen et al. (17), while calcium was determined in the presence of 0.1% lanthanum chloride by means of a Perkin-Elmer atomic absorption spectrometer Model 403. Measurement of antirachitic activity. Seven days after dose of a compound (Table II) or 7 days after the first of daily doses of a compound (Fig. 1, Table I), rats were killed and their radii and ulnae were removed. The new calcification in the epiphyseal plate was stained with a silver nitrate solution and scored by the line test method as described in the United States Pharmacopoeia (18). Measurement c$bvne ash. After the connective tissue was removed, femurs were extracted successively with 100% ethanol and 100% diethyl ether for 24 h each using a Soxhlet extractor. Fat-free bones were dried in a 100°C oven for 24 h and ashed in a muffle furnace at 650” for 24 h. Mcwmrwnent of intestinal calcium transport. Calcium transport activity was measured in everted duodenal sacs as described by Martin and DeLuca (19). Dk~i<~emtmt of [26,27-“Hj-25-OH-D3 frm rat plusjr~~tein by l.&(OH),D, or %,27-F,-1,25-(OH)SD3. Graded amounts of either 1,25-(OH),D, or 26,27-Fc1,25-(OH),D3 were dissolved in 0.05 ml of 95% ethanol.
Da
349
Triplicates of each sample were assayed for displacement of [3H]-25-OH-Da from rat plasma binding protein by unlabeled compound as described by Shepard and DeLuca (20). Displacement of [26,27-3Hf1,25-(OH)&3 fmm chick intestinal cytosol binding protein by 1,255-(OH)& w 26,27-F,-1,25-(OH)&. Graded amounts of either 1,25(OH)zDa or 26,27-Fs-1,25-(OH)aDa were dissolved in 0.05 ml of 95% ethanol. Triplicate determinations of displacement of [3H]-1,25-(OH)z-Da from chick intestinal cytosol binding protein by unlabeled compound was carried out as described by Shepard et al (21). Statistical analysis. Statistical analysis was performed by Student’s t test.
RESULTS
Initially, the ability of 26,27-F6-1,25(OH)2D3 to calcify rachitic bone was tested in comparison with 1,25-(OH)2D3 (Fig. 1, Table I). Rats that had been fed a lowphosphorus, high-calcium, vitamin D-deficient diet (rachitogenic diet) for 3 weeks developed a severe hypophosphatemia, enlarged epiphyseal plate, and a low content of bone ash. The increase in serum inorganic phosphorus concentration and new calcification in epiphyseal plate in these rats were measured in response to graded doses of either compound given subcutaneously daily for 7 days. As shown in Fig. lA, both compounds increased serum inorganic phosphorus levels but the fluoro analog was clearly more active than 1,25(OH)ZD3. Since 4 to 5 times more 1,25(OH)zD3 is required to produce the same level of response as the fluoro analog, it appears that the fluoro analog is 5 times more active than 1,25-(OH)zD3 in this response. Likewise, 26,2’7-FG-1,25-(OH)zD3 was 5 to 10 times more effective than 1,25(OH)zD3 in mineralization of epiphyseal plate cartilage as shown in Fig. 1B. Calcification stimulated by 13 pmol/day of the fluoro analog was actually unscorable by the line test method employed while the calcification stimulated by an equivalent dose of 1,25-(OH)zD3 was barely apparent. Another experiment was performed to compare the activity of the two compounds in increasing femur ash. As shown in Table I, the fluoro analog was considerably more active than 1,25-(OH)zD3. When the two compounds were given to
350
TANAKA
26-
(A)
03) 26.2%F,-1.25~tOH&,’ I
F&
I
2-
8 I.. 0 ”
,
1 IO pmoleshy
,
, , , , ,,, loo
FIG. 1. Serum inorganic phosphorus and epiphyseal plate calcification responses to 26,27-FG-1,25-(0H)aDa. Weanling rata were fed a rachitogenic diet for 3 weeks. They were then given indicated doses of either compound dissolved in 0.10 ml of a mixture of 95% ethanolpropylene glycol(595) subcutaneously daily for 7 days. Rata in a control group received the vehicle. (A) Serum inorganic phosphorus concentration. (B) Epiphyseal plate calcification. Each point represents a mean value from six rats while the bar represents standard deviation from the mean. *, Significantly different from a value achieved with an equivalent dose of 1,25(OH)rDa; P < 0.001.
very young rats, the marked difference in potency was revealed in the elevation of serum inorganic phosphorus concentration (Table II). Either at 48 or 72 h after a single dose, the fluoro analog maintained a high serum phosphorus level above that in control animals or that in animals given the same dose of 1,25-(OH)zD3. This finding suggested that 26,27-F6-1,25-(OH)2D3 may persist longer and have a long-lasting action. Thus, the temporal increase of serum inorganic phosphorus concentration in response to a single intrajugular dose of 26,27-F6-1,25-(OH)2D3 was carried out in rachitic rats. The phosphorus level increased in response to 1,25-(OH)zD3 1 day
ET AL.
after the dose but declined virtually to the control level within 2 days (Fig. 2), whereas the serum phosphorus level in response to 26,2’7-F6-1,25-(OH)zD3 was much higher at 1 day and persisted at least 4 days. We have previously shown that a single dose of 1,25-(OH),D3 given to the rachitic rats 7 days prior to sacrifice is not capable of curing rickets, presumably because an elevated serum inorganic phosphorus level in response to a single dose of the metabolite did not endure for even 2 days (15). A single dose of the fluoro analog given 7 days earlier gave marked calcification of the epiphyseal plate and a sustained elevation of serum phosphorus in clear contrast to 1,25-(OH)zD3 (Table III). Serum inorganic phosphorus concentration was also markedly elevated in the case of even the low dose of the fluoro analog, while 1,25-(OH)zD3 had little effect at the seventh day after dosing. This finding of a long-lasting activity of 26,27-FG-1,25-(OH)zD3 in the elevation of serum inorganic phosphorus concentration prompted us to examine the extent and duration of the action of the fluoro analog on bone calcium mobilization and intestinal calcium transport. Feeding a low-calcium, vitamin D-deficient diet for 3 weeks resulted in severe hypocalcemia. An increase in serum calcium in these rats in response to vitamin D or its metabolites is presumed to result from bone calcium mobilization. A shown in Fig. 3, increased serum calcium level in response to 1,25(OH)zD3 declined after 2 days, whereas the elevated level in response to the fluoro analog was maintained at least 4 days and the maximum response achieved was significantly greater than that achieved with 1,25-( OH)2D3. Since the maximum response was achieved with either compound at the 24h time point, comparison of biopotencies of these compounds in the stimulation of the intestinal calcium transport and bone calcium mobilization in hypocalcemic rats was carried out 24 h after a dose of either compound (Table IV). Intestinal calcium transport becomes saturated at the 65pmol dose level with both compounds in both experiments. However, the same
26,26,26,27,27,27-HEXAFLUORO-1,25-DIHYDROXYVITAMIN TABLE
351
Da
I
SERUM INORGANIC PHOSPHORUS, BONE ASH ACCUMULATION, AND EPIPHYSEAL PLATE CALCIFICATION RESPONSES TO DAILY DOSES OF 26,27-F6-1,25-(OH)2D3 OR 1,25-(OH)zD3
Compound given
Dose level (pmol/day)
None 26,27-F‘6-1,25-(OH)2D3
Serum inorganic phosphorus (mg/lOO ml)
-
3.0 4.3 6.8 3.5 5.3
6.5 65 6.5 65
1,25-(OH)aD3
f f -c 2 f
0.7” 0.8 0.4* 0.5 0.7”
Bone ash (md 34.6 35.5 47.8 37.9 41.7
f 4.5 f 7.0
f 1.8d f 6.0 f 1.5=
Epiphyseal calcification
plate (units)
Of0 3.0 f. 0.5d >6 1.5 f 0.5e 5.0 f 0.5
Note. Weanling male rats were fed a rachitogenic diet for 3 weeks. They were then given either 6.5 or 65 pmol of either 26,27-Fs-1,25-(OH),D, or 1,25-(OH)aD3 dissolved in a 0.1 ml of 95% ethanol/propylene glycol (5195) subcutaneously daily for 7 days. Rats in a control group were given the vehicle in the same manner. Each group had five to six rats. a Standard deviation from the mean. * Significantly different from c, P < 0.005. d Significantly different from e, P < 0.001.
transport level is achieved by 6.5 pmol of the fluoro derivative or with 65 pmol of 1,25-(OH)zD3. Elevation of serum calcium concentration in response to 650 pmol of 26,2’7-FG-1,25-(OH)aD3 was significantly greater than that in response to an equivalent dose of 1,25-(OH)aD3 by a factor of 10.
TABLE
It has been demonstrated that the introduction of a fluorine atom(s) allylic to a hydroxyl enhances activities of steroid hormones (22). Although the reason for the enhancement is not fully understood, it is
II
SERUM INORGANIC PHOSPHORUS RESPONSE TO A SINGLE DOSE OF 26,27-F‘6-1,25-(OH)ZD3 OR 1,25-(OH)aD3
Serum inorganic phosphorus (mg/lOO ml) Compound given
48 h
72 h
None 26,27-F6-1,25-(OH)2D3 1,25-(OH)aD3
3.0 f 0.2” (4) 9.0 + 0.8* (4) 3.4 f 0.7 (7)
2.4 f 0.6 (5) 7.1 f 0.6* (7) 3.2 + 0.5 (7)
Note. Weanling male rats were fed a rachitogenic diet for 1 week. They were then injected with 650 pmol of either compound dissolved in a 0.05 ml of 95% ethanol intrajugularly either 48 or 72 h prior to measurement. Rats in a control group were injected the vehicle. The number of rats in each group is shown in parentheses. a Standard deviation from the mean. ‘Significantly different from the control, P < 0.001.
Day after dose
FIG. 2. Serum inorganic phosphorus response to 26,27-F,-1,25-(OH)zD30r 1,25-(OH)aD3. Weanling rats were fed a rachitogenic diet for 3 weeks. They were then injected with 650 pmol of either compound dissolved in 0.05 ml of 95% ethanol intrajugularly, while rats in the control group received the vehicle. Each point represents a mean value from six rats + standard deviation from the mean. *, Significantly different from a value in response to 1,25-(OH)*D3 at the corresponding time point; P < 0.001.
352
TANAKA
ET AL.
TABLE SERUM INORGANIC
III
PHOSPHORUS AND EPIPHYSEAL PLATE CALCIFICATION RESPONSES 7 DAYS AFTER A SINGLE ORAL DOSE OF 26,27-F’6-1,25-(OH)2D3 OR 1,25-(OH)*D3
Compound given
Dose level (pm4
Serum inorganic phosphorus (mg/lOO ml)
-
3.0 f 0.4”
325 650 650
4.4 f o.s* 5.0 f 1.2* 3.7 + 1.0
None 26,27-F’6-1,25-(OH)2D3 1,25-(OH)*D3
Line test score (units) Of0 4.0 f 0.5” >6.0” 1.5 f 0.5
Note. Weanling male rats were fed a rachitogenic diet for 3 weeks. They were then given either 325 or 650 pmol of 26,27-FG-1,25-(0H)aDa or 650 pmol of 1,25-(OH)aDa dissolved in 0.1 ml of cotton seed oil by gastric tube. Seven days before measurement, rats in the control group received vehicle. Each group had six rats. a Standard deviation from the mean. * Significantly different from the control level, P < 0.005. ’ Significantly different from the control level, P < 0.001.
assumed that the high electronegativity of fluorine atom in a fluorinated compound increases receptor interaction by changing the properties of the adjacent functional hydroxyl. We therefore examined whether or not a mechanism for enhanced biological
7 t
I
Ic
“,
1 ,#‘I\:
26,27-F,-1,25-(OH),D,
1.25-(OH&D,
I
“\\-
-1
activity of 26,27-FG-1,25-(OH)zD3 is an enhanced binding ability of the compound for either rat plasma vitamin D binding protein or chick intestinal cytosol receptor protein for 1,25-(OH)aD3. As shown in Fig. 4A, 26,27-FG-1,25-(OH)aD3 was found to be a slightly weaker binder to the rat plasma protein than 1,25-(OH)aD3. To the chick intestinal cytosol receptor protein for 1,25(OH)aDa, binding ability of the fluoro analog was found to be about three times less than that of 1,25-(OH)aDa (Fig. 4B). It is therefore unlikely that enhanced biological activity of 26,27-Fs-1,25-(OH)zD3 is the result of enhanced transport of the compound or the result of increased interaction of the compound with the receptor protein. DISCUSSION
01
I I 1234567
I
I
I
I
I
Day after dose FIG. 3. Serum calcium response to either 26,27-F61,25-(OH)*Da or 1,25-(OH)eD,. Weanling rats were fed a low calcium vitamin D-deficient diet for 3 weeks. They were then injected with 650 pmol of either eompound dissolved in 0.05 ml of 95% ethanol intrajugularly or ethanol only. Each point represents a mean value from six rats f standard deviation from the mean. *, Significantly different from the value in response to 1,25-(OH)zD3 at the corresponding time point; P < 0.001.
The 26,27-Fs-1,25-(OH)aD3 is clearly much more biologically active than the native hormone, 1,25-(OH)aDa in the rat. Thus, it, like 24,25-Fz-1,25-(OH)aD3, may be of considerable medical interest for therapeutic reasons. However, the reason for its increased biopotency cannot be clearly discerned at this stage. The best indication is that its activity in elevating blood phosphorus or blood calcium of vitamin D-deficient rats fed a low-phosphorus or low-calcium diet is of much longer duration than that of an equal dose of 1,25-
26,26,26,27,27,27-HEXAFLUORO-1,25-DIHYDROXYVITAMIN TABLE
353
D3
IV
INCREASEININTESTINALCALCIUMTRANSPORTANDSERUMCALCIUM CONCENTRATIONINRESPONSETO 26,27-Fe-1,25-(OH),D, OR1,25-(OH)aDs Intestinal calcium transport (Ca serosal/Ca mucosal) Compound given None 26,27-F-1,25-(OH)aDs
1,25-(OH)aD3
Dose (pmol) 6.5 65 650 6.5 65 650
Experiment 4.3 f 0.7” 6.4 k 0.9* 6.9 +- 1.4 4.4 k 0.6” 5.5 * 1.0
I
Experiment 3.2 f 0.6 8.2 f 1.6 10.0 +- 1.2 7.3 t 2.5 7.4 k 2.4
Serum calcium (mg/lOO ml) II
Experiment 4.7 25 0.3 5.0 + 0.3 6.4 f 0.4 5.0 f 0.3 5.9 f 0.3
I
Experiment
II
4.5 + 0.2 5.8 f 0.6d 7.0 f 0.1” 4.7 -+ 0.3f 6.2 t 0.7”
Note. Weanling rats were fed a low-calcium, vitamin D-deficient diet for 3 weeks. They were then given either compound dissolved in a 0.05 ml of 95% ethanol intrajugularly 24 h prior to sacrifice. Rats in a control group were injected with the vehicle. Each group had five to six rats. o Standard deviation from the mean. *Significantly different from ‘, P < 0.005. d Significantly different from ‘; P < 0.005. e Significantly different from 8, P < 0.025.
(OH)2D3. This might be best explained by slower metabolism and excretion of the tluoro analog. However, no direct evidence is yet available on this point. In this respect, it is interesting that the 26,27-FG1,25-(OH),D3 is one-third as active as 1,25(OH)zD3 in binding to the chick intestinal receptor and slightly less active in binding to the rat serum vitamin D transport protein. It would appear that increased affinity for the receptor cannot be the reason for increased biological activity. The biopotency of the fluoro analog is between 5 and 10 times that of 1,25(OH)zD3 itself in both bone and intestinal activities. This is remarkably similar to the potency of 24,24-Fz-1,25-(OH)2D3 (11). Thus, in terms of activity, it seems there is little difference between the two analogs. However, a comparison of the present results and those for 24,24-F,-1,25-(OH)zD3 (11) suggests that the activity of the 26,27Fs-1,25-(OH)2D3 is more prolonged than that of 24,24-Fz-1,25-(OH)zD3. Thus far, the side-chain fluoro analogs of 1,25-(OH)2D3 having lluoro groups on the 24 and 26 carbons have approximately equal biological activity. Both have fluoro groups on car-
bons immediately adjacent to an hydroxyl. Similarly, the 2@F-lo-hydroxyvitamin D3 is more active than la-OH-D3 (23). Of considerable interest is whether a fluoro substitution on a position not adjacent to a binding hydroxyl but in a position that might block metabolism is more active than 1,25-(OH)zD3. Thus, 23,23-F,-1,25(OH)zD3 would be such an analog of considerable interest. The present results and those previously obtained with fluoro derivatives of vitamin D have added new insight into the functional metabolism of vitamin D. Certainly, blocking l- and 25-hydroxylations with fluoro groups markedly reduced biological activity of vitamin D compounds (5, 6), while 24- and 26-fluoro substitutions do not reduce activity. These results show that land 25-hydroxylations are required for function while 24- and 26-hydroxylations are not (12, 24). Since the 24,24-F2-1,25and 26,27-Fs-1,25-(OH)2D, are (OH)& more active than the natural hormone, the fluoro compounds can also be of considerable use as tools in unraveling the metabolism and mechanism of action of vitamin D,.
TANAKA
ET AL.
W 4.
5.
6.
7.
8.
9.
10.
I 0.1
1
11.
FIG. 4. (A) Displacement of [26,27-3H]-25-OH-D3 from rat plasma vitamin D binding protein by 26,27F,-1,25-(OH),D, or 1,25-(OH)zD3. The compounds were dissolved in 0.05 ml of 95% ethanol and added to diluted rat plasma together with [26,27-‘HI-25OH-D3. Each point represents the mean value of triplicate determinations +- standard deviation from the mean. (B) Displacement of [26,27-3H]-1,25-(OH)zD, from the chick intestinal cytosol receptor protein for 1,25(OH),4 by 26,27-F,-1,25-(OH),D, or 1,25-(OH),D3. The compounds were dissolved in a 0.05 ml of 95% ethanol and added to the cytosol receptor together with [26,273H]-1,25-(OH)zD3. Each point represents the mean value of triplicate determination rt standard deviation from the mean.
12.
OLt
1 0.01
Unlabeled compound/tube
(pmdes)
ACKNOWLEDGMENTS We thank Dean Faber for his excellent technical assistance. This work was supported by a Program Project Grant AM-14881 from the National Institutes of Health, an NSF U. S./Japan Cooperative Grant RMPC-0163, and by the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation. REFERENCES 1. DELUCA, H. F., AND SCHNOES, H. K. (1976) Annu. Rev. Biochem. 45, 631-661. 2. DELUCA, H. F. (1979) in Proceedings of the 31st Postgraduate Assembly of the Endocrine Society, pp. X4-X27. 3. PIERIDES, A. M., KERR, N. S., ELLIS, H. A., PEART,
K. M., O’RIORDAN, L. H., AND DELUCA, H. F. (1976) Clin Nephrol. 5, 189-196. DELUCA, H. F., KERR, N. S., STANBURY, S. W., NORDIN, B. E. C., AND MARTIN, T. J. (1977) Clin Endocrinol 7, (suppl.) lS-17s. NAPOLI, J. L., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1978) Biochemistry 17, 2387-2392. NAPOLI, J. L., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1979) Biochemistry 18, 1641-1645. KOBAYASHI, Y., TAGUCHI, T., TERADA, T., OSHIDA, J., MORISAKI, M., AND IKEKAWA, N. (1979) Tetrahedron L&t. 22,2023-2026. KOBAYASHI, Y., TAGUCHI, T., KAMURA, N., IKEKAWA, N., AND OSHIDA, J. (1980) J. Chem SOC Chem Commun 10,459-460. TANAKA, Y., DELUCA, H. F., SCHNOES, H. K., IKEKAWA, N., AND KOBAYASHI, Y. (1980) Arch B&hem. Biophys. 199, 473-478. KOBAYASHI, Y., TAGUCHI, T., MITSUHASHI, S., EGUCHI, T., OSHIMA, E., AND IKEKAWA, N. (1982) Chem Pharm. Bull 30.4297-4303. OKAMOTO, S., TANAKA, Y., DELUCA, H. F., KoBAYASHI, Y., AND IKEKAWA, N. (1983) ~mer. J. Physiol 244, E159-163. TANAKA, Y., PAHUJA, D. N., WICHMANN, J. K., DELUCA, H. F., KOBAYASHI, Y., TAGUCHI, T., AND IKEKAWA, N. (1982) Arch. B&hem. Biophys.
218,134-141. 13. NAPOLI, J. L., FIVIZZANI, M. A., HAMSTRA, A. J.,
14.
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22. 23.
24.
SCHNOES,H. K., ANDDELUCA, H. F. (1979) Ad Biochem. 96,481-488. NAPOLI, J. L., MELLON, W. S., FIVIZZANI, M. A., SCHNOES,H. K., ANDDELUCA, H. F. (1980) Biochemistry 19, 2515-2521. TANAKA, Y., AND DELUCA, H. F. (1974) Proc. Natl. Acad. Sci. USA 71, 1040-1044. SUDA, T., DELUCA, H. F., AND TANAKA, Y. (1970) J. Nutr. 100, 1049-1052. CHEN, P. A., JR., TORIBARA, T. Y., AND WARNER, H. (1956) Anal Chem. 28,1756-1758. United States Pharmacopoeia (1955) 15th revision, p. 889, Mack, Easton, Pa. MARTIN, D. L., AND DELUCA, H. F. (1969) Amer. J. Physiol. 216, 1351-1359. SHEPARD, R. M., AND DELUCA, H. F. (1980) Arch. B&hem. Biophys. 202,43-53. SHEPARD, R. M., HORST, R. L., HAMSTRA, A. J., ANDDELUCA, H. F. (19’79) B&hem. .I 182,5569. GOLDMAN, P. (1969) Science 164, 1123-1130. DELUCA, H. F., IKEKAWA, N., TANAKA, Y., MORISAKI, M., AND OSHIDA, J. (1981) United States Patent 4,254,045, March 3, 1981. TANAKA, Y., DELUCA, H. F., KOBAYASHI, Y., TAGUCHI, T., IKEKAWA, N., ANDMORISAKI, M. (1979) J. Biol Chem. 254, 7163-7167.
26,26,26,27,27,27-Hexafluoro-I ,25dihydroxyvitamin DS: A Highly Potent, Long-Lasting Analog of 1,25Dihydroxyvitamin D3 YOKO TANAKA,
HECTOR F. DELUCA,’ YOSHIRO AND NOBUO IKEKAWAt
KOBAYASHI,*
Department
of Biochemistry, College of Agricultural and Lge Sciences, University of Wisconsin-Madison, Mad&m, Wisconsin 5.9706; *Tokyo College of Pharmacy, Hachioji, Tokyo, Japan; and of Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo 152, Japan tDepartment
Received June 24, 1983, and in revised form October 13, 1983
A new fluoro analog of 1,25-dihydroxyvitamin Ds, i.e., 26,26,26,27,27,27-hexalluoro1,25-dihydroxyvitamin Da, has been compared with the native hormone, 1,25dihydroxyvitamin Ds, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25dihydroxyvitamin DS. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.
D to its function, 25-fluorovitamin D3 (5), 1-fluorovitamin D3 (6), 24,24-difluoro-25hydroxy-vitamin D3 (24,24-F2-25-OH-D3) (7), and 26,26,26,27,27,27-hexafluoro-25hydroxyvitamin D3 (26,27-Fs-25-OH-D3) (8) have been prepared. The latter two compounds have also been converted to their la-hydroxy derivatives (9, 10). The 24,24F2-1,25-(OH)2D3 proved to be about 10 times more active than 1,25-(OH)2D3 (11). However, the 26,26,26,27,27,27-hexafluoro1,25-dihydroxyvitamin D3 (26,27-FG-1,25(OH)ZDB) has not yet been tested for biological activity, although 26,27-FE-25OH-D, was found equally as active as 25 OH-D3 (12). This report demonstrates that 26,2’7-FG-1,25-(OH)2D3 is 5 to 10 times more active than 1,25-(OH)2D3 in the rat and its activity is long lasting.
The demonstration that 1,25-dihydroxyvitamin D3 (1,25-(OH),D3)2 is a metabolically active form of vitamin D and the utility of this hormone in treating bone disease has prompted a great interest in biopotent analogs (1,2). For example, a potent analog used commercially is la-hydroxyvitamin D3 (IQ-OH-Ds) (3,4). Because of an interest in developing tools to study the importance of the various hydroxylations of vitamin ) No reprints of this paper are available from the authors. Author to whom all correspondence should be addressed. * Abbreviations used: 25.OH-D3, 25-hydroxyvitamin DB; 1,25-(OH)*D3, 1,25-dihydroxyvitamin D,; 24,24F,24,24-difluoro-1,25-dihydroxyvitamin 1,25-(OHM&. DS; 26,27-Fs-1,25-(OH),D3, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3. 0003-9861/84 $3.00 Capyrlyht All rights
C 1984 by Academic Press. Inc. of reproduction in any form reserved.
348
26,26,26,27,27,27-HEXAFLUORO-1,25-DIHYDROXYVITAMIN MATERIALS
AND
METHODS
Vitamin D related compounds 26,27-FB-1,25-(0H)zDa was synthesized by Kobayashi et al (lo), while 1,25(OH)aDI was a gift of the Hoffmann-La Roche Company (Nutley, N. J.). [26,27-sH]-25-OH-Da and [26,273H]-1,25-(OH)zD3 (specific activity 160 Ci/mmol) were synthesized by the method described previously (13, 14). Rats. Weanling male rats were purchased from the Holtzman Company (Madison, Wise.) and fed either a low-phosphorus (O.l%), high-calcium (1.2%),vitamin D-deficient diet (15) or a low-calcium (O.O2%),adequate phosphorus (0.3%), vitamin D-deficient diet (16) for 3 weeks except in case of Table I. In this case, weanling rats were fed the low-phosphorus, high-calcium diet for 1 week before they were used in the experiment. of compounda The designated comAdministration pound was dissolved in a O.l-ml mixture of propylene glycoi-95% ethanol (95:5) and administered subcutaneously daily for biopotency determination (Fig. 1 and Table I). For the study of duration of action, the compound was dissolved in 0.05 ml of 95% ethanol and administered intrajugularly once (Figs. 2 and 3, Tables II and IV). The relative oral effectiveness of the compounds was examined by dissolving them in 0.1 ml of cotton seed/soybean oil (Wesson) and administering them directly into the stomach with polyethylene tubing. In all of the experiments, rats in the control group received the appropriate amount of vehicle by the indicated route. Determinations of serum inmganic phosphcrrus and calcium. Serum was obtained by centrifugation of clotted blood. Inorganic phosphorus was determined by the calorimetric method of Chen et al. (17), while calcium was determined in the presence of 0.1% lanthanum chloride by means of a Perkin-Elmer atomic absorption spectrometer Model 403. Measurement of antirachitic activity. Seven days after dose of a compound (Table II) or 7 days after the first of daily doses of a compound (Fig. 1, Table I), rats were killed and their radii and ulnae were removed. The new calcification in the epiphyseal plate was stained with a silver nitrate solution and scored by the line test method as described in the United States Pharmacopoeia (18). Measurement c$bvne ash. After the connective tissue was removed, femurs were extracted successively with 100% ethanol and 100% diethyl ether for 24 h each using a Soxhlet extractor. Fat-free bones were dried in a 100°C oven for 24 h and ashed in a muffle furnace at 650” for 24 h. Mcwmrwnent of intestinal calcium transport. Calcium transport activity was measured in everted duodenal sacs as described by Martin and DeLuca (19). Dk~i<~emtmt of [26,27-“Hj-25-OH-D3 frm rat plusjr~~tein by l.&(OH),D, or %,27-F,-1,25-(OH)SD3. Graded amounts of either 1,25-(OH),D, or 26,27-Fc1,25-(OH),D3 were dissolved in 0.05 ml of 95% ethanol.
Da
349
Triplicates of each sample were assayed for displacement of [3H]-25-OH-Da from rat plasma binding protein by unlabeled compound as described by Shepard and DeLuca (20). Displacement of [26,27-3Hf1,25-(OH)&3 fmm chick intestinal cytosol binding protein by 1,255-(OH)& w 26,27-F,-1,25-(OH)&. Graded amounts of either 1,25(OH)zDa or 26,27-Fs-1,25-(OH)aDa were dissolved in 0.05 ml of 95% ethanol. Triplicate determinations of displacement of [3H]-1,25-(OH)z-Da from chick intestinal cytosol binding protein by unlabeled compound was carried out as described by Shepard et al (21). Statistical analysis. Statistical analysis was performed by Student’s t test.
RESULTS
Initially, the ability of 26,27-F6-1,25(OH)2D3 to calcify rachitic bone was tested in comparison with 1,25-(OH)2D3 (Fig. 1, Table I). Rats that had been fed a lowphosphorus, high-calcium, vitamin D-deficient diet (rachitogenic diet) for 3 weeks developed a severe hypophosphatemia, enlarged epiphyseal plate, and a low content of bone ash. The increase in serum inorganic phosphorus concentration and new calcification in epiphyseal plate in these rats were measured in response to graded doses of either compound given subcutaneously daily for 7 days. As shown in Fig. lA, both compounds increased serum inorganic phosphorus levels but the fluoro analog was clearly more active than 1,25(OH)ZD3. Since 4 to 5 times more 1,25(OH)zD3 is required to produce the same level of response as the fluoro analog, it appears that the fluoro analog is 5 times more active than 1,25-(OH)zD3 in this response. Likewise, 26,2’7-FG-1,25-(OH)zD3 was 5 to 10 times more effective than 1,25(OH)zD3 in mineralization of epiphyseal plate cartilage as shown in Fig. 1B. Calcification stimulated by 13 pmol/day of the fluoro analog was actually unscorable by the line test method employed while the calcification stimulated by an equivalent dose of 1,25-(OH)zD3 was barely apparent. Another experiment was performed to compare the activity of the two compounds in increasing femur ash. As shown in Table I, the fluoro analog was considerably more active than 1,25-(OH)zD3. When the two compounds were given to
350
TANAKA
26-
(A)
03) 26.2%F,-1.25~tOH&,’ I
F&
I
2-
8 I.. 0 ”
,
1 IO pmoleshy
,
, , , , ,,, loo
FIG. 1. Serum inorganic phosphorus and epiphyseal plate calcification responses to 26,27-FG-1,25-(0H)aDa. Weanling rata were fed a rachitogenic diet for 3 weeks. They were then given indicated doses of either compound dissolved in 0.10 ml of a mixture of 95% ethanolpropylene glycol(595) subcutaneously daily for 7 days. Rata in a control group received the vehicle. (A) Serum inorganic phosphorus concentration. (B) Epiphyseal plate calcification. Each point represents a mean value from six rats while the bar represents standard deviation from the mean. *, Significantly different from a value achieved with an equivalent dose of 1,25(OH)rDa; P < 0.001.
very young rats, the marked difference in potency was revealed in the elevation of serum inorganic phosphorus concentration (Table II). Either at 48 or 72 h after a single dose, the fluoro analog maintained a high serum phosphorus level above that in control animals or that in animals given the same dose of 1,25-(OH)zD3. This finding suggested that 26,27-F6-1,25-(OH)2D3 may persist longer and have a long-lasting action. Thus, the temporal increase of serum inorganic phosphorus concentration in response to a single intrajugular dose of 26,27-F6-1,25-(OH)2D3 was carried out in rachitic rats. The phosphorus level increased in response to 1,25-(OH)zD3 1 day
ET AL.
after the dose but declined virtually to the control level within 2 days (Fig. 2), whereas the serum phosphorus level in response to 26,2’7-F6-1,25-(OH)zD3 was much higher at 1 day and persisted at least 4 days. We have previously shown that a single dose of 1,25-(OH),D3 given to the rachitic rats 7 days prior to sacrifice is not capable of curing rickets, presumably because an elevated serum inorganic phosphorus level in response to a single dose of the metabolite did not endure for even 2 days (15). A single dose of the fluoro analog given 7 days earlier gave marked calcification of the epiphyseal plate and a sustained elevation of serum phosphorus in clear contrast to 1,25-(OH)zD3 (Table III). Serum inorganic phosphorus concentration was also markedly elevated in the case of even the low dose of the fluoro analog, while 1,25-(OH)zD3 had little effect at the seventh day after dosing. This finding of a long-lasting activity of 26,27-FG-1,25-(OH)zD3 in the elevation of serum inorganic phosphorus concentration prompted us to examine the extent and duration of the action of the fluoro analog on bone calcium mobilization and intestinal calcium transport. Feeding a low-calcium, vitamin D-deficient diet for 3 weeks resulted in severe hypocalcemia. An increase in serum calcium in these rats in response to vitamin D or its metabolites is presumed to result from bone calcium mobilization. A shown in Fig. 3, increased serum calcium level in response to 1,25(OH)zD3 declined after 2 days, whereas the elevated level in response to the fluoro analog was maintained at least 4 days and the maximum response achieved was significantly greater than that achieved with 1,25-( OH)2D3. Since the maximum response was achieved with either compound at the 24h time point, comparison of biopotencies of these compounds in the stimulation of the intestinal calcium transport and bone calcium mobilization in hypocalcemic rats was carried out 24 h after a dose of either compound (Table IV). Intestinal calcium transport becomes saturated at the 65pmol dose level with both compounds in both experiments. However, the same
26,26,26,27,27,27-HEXAFLUORO-1,25-DIHYDROXYVITAMIN TABLE
351
Da
I
SERUM INORGANIC PHOSPHORUS, BONE ASH ACCUMULATION, AND EPIPHYSEAL PLATE CALCIFICATION RESPONSES TO DAILY DOSES OF 26,27-F6-1,25-(OH)2D3 OR 1,25-(OH)zD3
Compound given
Dose level (pmol/day)
None 26,27-F‘6-1,25-(OH)2D3
Serum inorganic phosphorus (mg/lOO ml)
-
3.0 4.3 6.8 3.5 5.3
6.5 65 6.5 65
1,25-(OH)aD3
f f -c 2 f
0.7” 0.8 0.4* 0.5 0.7”
Bone ash (md 34.6 35.5 47.8 37.9 41.7
f 4.5 f 7.0
f 1.8d f 6.0 f 1.5=
Epiphyseal calcification
plate (units)
Of0 3.0 f. 0.5d >6 1.5 f 0.5e 5.0 f 0.5
Note. Weanling male rats were fed a rachitogenic diet for 3 weeks. They were then given either 6.5 or 65 pmol of either 26,27-Fs-1,25-(OH),D, or 1,25-(OH)aD3 dissolved in a 0.1 ml of 95% ethanol/propylene glycol (5195) subcutaneously daily for 7 days. Rats in a control group were given the vehicle in the same manner. Each group had five to six rats. a Standard deviation from the mean. * Significantly different from c, P < 0.005. d Significantly different from e, P < 0.001.
transport level is achieved by 6.5 pmol of the fluoro derivative or with 65 pmol of 1,25-(OH)zD3. Elevation of serum calcium concentration in response to 650 pmol of 26,2’7-FG-1,25-(OH)aD3 was significantly greater than that in response to an equivalent dose of 1,25-(OH)aD3 by a factor of 10.
TABLE
It has been demonstrated that the introduction of a fluorine atom(s) allylic to a hydroxyl enhances activities of steroid hormones (22). Although the reason for the enhancement is not fully understood, it is
II
SERUM INORGANIC PHOSPHORUS RESPONSE TO A SINGLE DOSE OF 26,27-F‘6-1,25-(OH)ZD3 OR 1,25-(OH)aD3
Serum inorganic phosphorus (mg/lOO ml) Compound given
48 h
72 h
None 26,27-F6-1,25-(OH)2D3 1,25-(OH)aD3
3.0 f 0.2” (4) 9.0 + 0.8* (4) 3.4 f 0.7 (7)
2.4 f 0.6 (5) 7.1 f 0.6* (7) 3.2 + 0.5 (7)
Note. Weanling male rats were fed a rachitogenic diet for 1 week. They were then injected with 650 pmol of either compound dissolved in a 0.05 ml of 95% ethanol intrajugularly either 48 or 72 h prior to measurement. Rats in a control group were injected the vehicle. The number of rats in each group is shown in parentheses. a Standard deviation from the mean. ‘Significantly different from the control, P < 0.001.
Day after dose
FIG. 2. Serum inorganic phosphorus response to 26,27-F,-1,25-(OH)zD30r 1,25-(OH)aD3. Weanling rats were fed a rachitogenic diet for 3 weeks. They were then injected with 650 pmol of either compound dissolved in 0.05 ml of 95% ethanol intrajugularly, while rats in the control group received the vehicle. Each point represents a mean value from six rats + standard deviation from the mean. *, Significantly different from a value in response to 1,25-(OH)*D3 at the corresponding time point; P < 0.001.
352
TANAKA
ET AL.
TABLE SERUM INORGANIC
III
PHOSPHORUS AND EPIPHYSEAL PLATE CALCIFICATION RESPONSES 7 DAYS AFTER A SINGLE ORAL DOSE OF 26,27-F’6-1,25-(OH)2D3 OR 1,25-(OH)*D3
Compound given
Dose level (pm4
Serum inorganic phosphorus (mg/lOO ml)
-
3.0 f 0.4”
325 650 650
4.4 f o.s* 5.0 f 1.2* 3.7 + 1.0
None 26,27-F’6-1,25-(OH)2D3 1,25-(OH)*D3
Line test score (units) Of0 4.0 f 0.5” >6.0” 1.5 f 0.5
Note. Weanling male rats were fed a rachitogenic diet for 3 weeks. They were then given either 325 or 650 pmol of 26,27-FG-1,25-(0H)aDa or 650 pmol of 1,25-(OH)aDa dissolved in 0.1 ml of cotton seed oil by gastric tube. Seven days before measurement, rats in the control group received vehicle. Each group had six rats. a Standard deviation from the mean. * Significantly different from the control level, P < 0.005. ’ Significantly different from the control level, P < 0.001.
assumed that the high electronegativity of fluorine atom in a fluorinated compound increases receptor interaction by changing the properties of the adjacent functional hydroxyl. We therefore examined whether or not a mechanism for enhanced biological
7 t
I
Ic
“,
1 ,#‘I\:
26,27-F,-1,25-(OH),D,
1.25-(OH&D,
I
“\\-
-1
activity of 26,27-FG-1,25-(OH)zD3 is an enhanced binding ability of the compound for either rat plasma vitamin D binding protein or chick intestinal cytosol receptor protein for 1,25-(OH)aD3. As shown in Fig. 4A, 26,27-FG-1,25-(OH)aD3 was found to be a slightly weaker binder to the rat plasma protein than 1,25-(OH)aD3. To the chick intestinal cytosol receptor protein for 1,25(OH)aDa, binding ability of the fluoro analog was found to be about three times less than that of 1,25-(OH)aDa (Fig. 4B). It is therefore unlikely that enhanced biological activity of 26,27-Fs-1,25-(OH)zD3 is the result of enhanced transport of the compound or the result of increased interaction of the compound with the receptor protein. DISCUSSION
01
I I 1234567
I
I
I
I
I
Day after dose FIG. 3. Serum calcium response to either 26,27-F61,25-(OH)*Da or 1,25-(OH)eD,. Weanling rats were fed a low calcium vitamin D-deficient diet for 3 weeks. They were then injected with 650 pmol of either eompound dissolved in 0.05 ml of 95% ethanol intrajugularly or ethanol only. Each point represents a mean value from six rats f standard deviation from the mean. *, Significantly different from the value in response to 1,25-(OH)zD3 at the corresponding time point; P < 0.001.
The 26,27-Fs-1,25-(OH)aD3 is clearly much more biologically active than the native hormone, 1,25-(OH)aDa in the rat. Thus, it, like 24,25-Fz-1,25-(OH)aD3, may be of considerable medical interest for therapeutic reasons. However, the reason for its increased biopotency cannot be clearly discerned at this stage. The best indication is that its activity in elevating blood phosphorus or blood calcium of vitamin D-deficient rats fed a low-phosphorus or low-calcium diet is of much longer duration than that of an equal dose of 1,25-
26,26,26,27,27,27-HEXAFLUORO-1,25-DIHYDROXYVITAMIN TABLE
353
D3
IV
INCREASEININTESTINALCALCIUMTRANSPORTANDSERUMCALCIUM CONCENTRATIONINRESPONSETO 26,27-Fe-1,25-(OH),D, OR1,25-(OH)aDs Intestinal calcium transport (Ca serosal/Ca mucosal) Compound given None 26,27-F-1,25-(OH)aDs
1,25-(OH)aD3
Dose (pmol) 6.5 65 650 6.5 65 650
Experiment 4.3 f 0.7” 6.4 k 0.9* 6.9 +- 1.4 4.4 k 0.6” 5.5 * 1.0
I
Experiment 3.2 f 0.6 8.2 f 1.6 10.0 +- 1.2 7.3 t 2.5 7.4 k 2.4
Serum calcium (mg/lOO ml) II
Experiment 4.7 25 0.3 5.0 + 0.3 6.4 f 0.4 5.0 f 0.3 5.9 f 0.3
I
Experiment
II
4.5 + 0.2 5.8 f 0.6d 7.0 f 0.1” 4.7 -+ 0.3f 6.2 t 0.7”
Note. Weanling rats were fed a low-calcium, vitamin D-deficient diet for 3 weeks. They were then given either compound dissolved in a 0.05 ml of 95% ethanol intrajugularly 24 h prior to sacrifice. Rats in a control group were injected with the vehicle. Each group had five to six rats. o Standard deviation from the mean. *Significantly different from ‘, P < 0.005. d Significantly different from ‘; P < 0.005. e Significantly different from 8, P < 0.025.
(OH)2D3. This might be best explained by slower metabolism and excretion of the tluoro analog. However, no direct evidence is yet available on this point. In this respect, it is interesting that the 26,27-FG1,25-(OH),D3 is one-third as active as 1,25(OH)zD3 in binding to the chick intestinal receptor and slightly less active in binding to the rat serum vitamin D transport protein. It would appear that increased affinity for the receptor cannot be the reason for increased biological activity. The biopotency of the fluoro analog is between 5 and 10 times that of 1,25(OH)zD3 itself in both bone and intestinal activities. This is remarkably similar to the potency of 24,24-Fz-1,25-(OH)2D3 (11). Thus, in terms of activity, it seems there is little difference between the two analogs. However, a comparison of the present results and those for 24,24-F,-1,25-(OH)zD3 (11) suggests that the activity of the 26,27Fs-1,25-(OH)2D3 is more prolonged than that of 24,24-Fz-1,25-(OH)zD3. Thus far, the side-chain fluoro analogs of 1,25-(OH)2D3 having lluoro groups on the 24 and 26 carbons have approximately equal biological activity. Both have fluoro groups on car-
bons immediately adjacent to an hydroxyl. Similarly, the 2@F-lo-hydroxyvitamin D3 is more active than la-OH-D3 (23). Of considerable interest is whether a fluoro substitution on a position not adjacent to a binding hydroxyl but in a position that might block metabolism is more active than 1,25-(OH)zD3. Thus, 23,23-F,-1,25(OH)zD3 would be such an analog of considerable interest. The present results and those previously obtained with fluoro derivatives of vitamin D have added new insight into the functional metabolism of vitamin D. Certainly, blocking l- and 25-hydroxylations with fluoro groups markedly reduced biological activity of vitamin D compounds (5, 6), while 24- and 26-fluoro substitutions do not reduce activity. These results show that land 25-hydroxylations are required for function while 24- and 26-hydroxylations are not (12, 24). Since the 24,24-F2-1,25and 26,27-Fs-1,25-(OH)2D, are (OH)& more active than the natural hormone, the fluoro compounds can also be of considerable use as tools in unraveling the metabolism and mechanism of action of vitamin D,.
TANAKA
ET AL.
W 4.
5.
6.
7.
8.
9.
10.
I 0.1
1
11.
FIG. 4. (A) Displacement of [26,27-3H]-25-OH-D3 from rat plasma vitamin D binding protein by 26,27F,-1,25-(OH),D, or 1,25-(OH)zD3. The compounds were dissolved in 0.05 ml of 95% ethanol and added to diluted rat plasma together with [26,27-‘HI-25OH-D3. Each point represents the mean value of triplicate determinations +- standard deviation from the mean. (B) Displacement of [26,27-3H]-1,25-(OH)zD, from the chick intestinal cytosol receptor protein for 1,25(OH),4 by 26,27-F,-1,25-(OH),D, or 1,25-(OH),D3. The compounds were dissolved in a 0.05 ml of 95% ethanol and added to the cytosol receptor together with [26,273H]-1,25-(OH)zD3. Each point represents the mean value of triplicate determination rt standard deviation from the mean.
12.
OLt
1 0.01
Unlabeled compound/tube
(pmdes)
ACKNOWLEDGMENTS We thank Dean Faber for his excellent technical assistance. This work was supported by a Program Project Grant AM-14881 from the National Institutes of Health, an NSF U. S./Japan Cooperative Grant RMPC-0163, and by the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation. REFERENCES 1. DELUCA, H. F., AND SCHNOES, H. K. (1976) Annu. Rev. Biochem. 45, 631-661. 2. DELUCA, H. F. (1979) in Proceedings of the 31st Postgraduate Assembly of the Endocrine Society, pp. X4-X27. 3. PIERIDES, A. M., KERR, N. S., ELLIS, H. A., PEART,
K. M., O’RIORDAN, L. H., AND DELUCA, H. F. (1976) Clin Nephrol. 5, 189-196. DELUCA, H. F., KERR, N. S., STANBURY, S. W., NORDIN, B. E. C., AND MARTIN, T. J. (1977) Clin Endocrinol 7, (suppl.) lS-17s. NAPOLI, J. L., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1978) Biochemistry 17, 2387-2392. NAPOLI, J. L., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1979) Biochemistry 18, 1641-1645. KOBAYASHI, Y., TAGUCHI, T., TERADA, T., OSHIDA, J., MORISAKI, M., AND IKEKAWA, N. (1979) Tetrahedron L&t. 22,2023-2026. KOBAYASHI, Y., TAGUCHI, T., KAMURA, N., IKEKAWA, N., AND OSHIDA, J. (1980) J. Chem SOC Chem Commun 10,459-460. TANAKA, Y., DELUCA, H. F., SCHNOES, H. K., IKEKAWA, N., AND KOBAYASHI, Y. (1980) Arch B&hem. Biophys. 199, 473-478. KOBAYASHI, Y., TAGUCHI, T., MITSUHASHI, S., EGUCHI, T., OSHIMA, E., AND IKEKAWA, N. (1982) Chem Pharm. Bull 30.4297-4303. OKAMOTO, S., TANAKA, Y., DELUCA, H. F., KoBAYASHI, Y., AND IKEKAWA, N. (1983) ~mer. J. Physiol 244, E159-163. TANAKA, Y., PAHUJA, D. N., WICHMANN, J. K., DELUCA, H. F., KOBAYASHI, Y., TAGUCHI, T., AND IKEKAWA, N. (1982) Arch. B&hem. Biophys.
218,134-141. 13. NAPOLI, J. L., FIVIZZANI, M. A., HAMSTRA, A. J.,
14.
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24.
SCHNOES,H. K., ANDDELUCA, H. F. (1979) Ad Biochem. 96,481-488. NAPOLI, J. L., MELLON, W. S., FIVIZZANI, M. A., SCHNOES,H. K., ANDDELUCA, H. F. (1980) Biochemistry 19, 2515-2521. TANAKA, Y., AND DELUCA, H. F. (1974) Proc. Natl. Acad. Sci. USA 71, 1040-1044. SUDA, T., DELUCA, H. F., AND TANAKA, Y. (1970) J. Nutr. 100, 1049-1052. CHEN, P. A., JR., TORIBARA, T. Y., AND WARNER, H. (1956) Anal Chem. 28,1756-1758. United States Pharmacopoeia (1955) 15th revision, p. 889, Mack, Easton, Pa. MARTIN, D. L., AND DELUCA, H. F. (1969) Amer. J. Physiol. 216, 1351-1359. SHEPARD, R. M., AND DELUCA, H. F. (1980) Arch. B&hem. Biophys. 202,43-53. SHEPARD, R. M., HORST, R. L., HAMSTRA, A. J., ANDDELUCA, H. F. (19’79) B&hem. .I 182,5569. GOLDMAN, P. (1969) Science 164, 1123-1130. DELUCA, H. F., IKEKAWA, N., TANAKA, Y., MORISAKI, M., AND OSHIDA, J. (1981) United States Patent 4,254,045, March 3, 1981. TANAKA, Y., DELUCA, H. F., KOBAYASHI, Y., TAGUCHI, T., IKEKAWA, N., ANDMORISAKI, M. (1979) J. Biol Chem. 254, 7163-7167.