(Received for publication, August 25, 1995; and in revised form, January 10, 1996)
From the
Using four cultured cell models representing liver,
keratinocyte, and osteoblast, we have demonstrated that the vitamin D
analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and
side chain truncated metabolites. Four of these metabolic products have
been rigorously identified by high pressure liquid chromatography,
diode array spectrophotometry, and gas chromatography-mass spectrometry
analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as
the cleaved molecules, hexanor-1,20-dihydroxyvitamin D
and hexanor-20-oxo-1
-hydroxyvitamin D
.
Comparison with chemically synthesized standards has revealed the
stereochemistry of the biological products. Although differences exist
in the amounts of products formed with the different cell types, it is
apparent that 22-oxacalcitriol is subject to metabolism by both vitamin
D-inducible and noninducible enzymes. Time course studies suggest that
the truncated 20-alcohol is derived from a side chain hydroxylated
molecule via a hemiacetal intermediate and the 20-oxo derivative is
likely formed from the 20-alcohol. Biological activity measurements of
the metabolites identified in our studies are consistent with the view
that these are catabolites and that the biological activity of
22-oxacalcitriol is due to the parent compound. These results are also
consistent with recent findings of others that the biliary excretory
form of 22-oxacalcitriol is a glucuronide ester of the truncated
20-alcohol.
It is now firmly established that the hormonal form of vitamin
D, 1,25-dihydroxyvitamin D
(1
,
25-(OH)
D
or calcitriol) (
)has potent
cell differentiating/anti-proliferative activities in addition to its
role in calcium homeostasis(1) . This has led researchers in
both universities and the pharmaceutical industry to search for
so-called ``noncalcemic'' vitamin D analogs with accentuated
differentiating/anti-proliferative properties and reduced ability to
cause hypercalcemia(2, 3) . 22-Oxacalcitriol (OCT) (
)was an early analog developed for this purpose and
contains a 22-oxygen atom that replaces the 22-carbon of calcitriol.
OCT binds to the chicken vitamin D receptor (VDR) with an approximately
8 times lower affinity than
1
,25-(OH)
D
(4) . However, OCT is 10
times more effective than 1
,25-(OH)
D
in suppressing cell growth and inducing differentiation of the
mouse myelocytic leukemic cell, WEHI-3, in vitro(5) .
OCT also possesses an enhanced in vivo immunomodulatory
potency in mice that is 50 times higher than that of
1
,25-(OH)
D
(6) . In contrast, OCT
has reduced calcemic activity in vivo (- that of 1
,
25-(OH)
D
), both in terms of mobilizing calcium
from bone and in stimulating intestinal calcium transport in vitamin
D-deficient and normal rats(7, 8) . The mechanisms
responsible for these differences in biological activity remain
unclear, but factors such as cellular uptake and intracellular
metabolism could contribute to these differences.
OCT binds poorly to the vitamin D binding protein (DBP) and is transported in the plasma bound by lipoproteins (chylomicrons and low density lipoprotein) in vivo(9) , and this leads to an unusual distribution pattern with a degree of concentration of the vitamin D analog in parathyroid tissue(10) . In contrast, little is known about OCT metabolism except that it appears to be excreted as a glucuronide conjugate in the bile, possibly a derivative of a truncated version of OCT(10) . Furthermore, there have been suggestions that a metabolite with a truncated side chain may be formed in bovine parathyroid cell cultures in vitro(11) , although details of this have yet to be published.
In these studies we set out to provide convincing physicochemical identification for metabolites generated in a variety of cultured cell models, namely the human hepatoma lines HepG2 and Hep3B(12) , the rat osteosarcoma cell line UMR-106(13) , and the human keratinocyte cell lines HPK1A and HPK1A-ras(14, 15) . Because these cell models mimic vitamin D metabolism found in vivo, we expected to observe the same metabolites found and in some cases tentatively identified by others(10, 11) . In addition, our objective was to study the rate of OCT metabolism in various cell lines in order to gauge the involvement of vitamin D-inducible catabolic pathways as compared with more general metabolizing systems. Our results support the concept that OCT is subject to extensive metabolism in a variety of tissues that leads to side chain truncated forms excreted in the bile.
Figure 1:
HPLC of extract of HPK1A-ras cells incubated with 10 µM OCT for 48 h. Peaks
showing the vitamin D chromophore are numbered 1-7. A shows the no cell control experiment. B shows the
extract from HPK1A-ras cells. the HPLC conditions were
Zorbax-SIL (3 µ; 6.2 80 mm); HIM 91:7:2, 1.0
ml/min.
Figure 2: A, mass spectrum of pertrimethylsilylated peak 2. B, mass spectrum of pertrimethylsilylated oxime derivative of peak 2.
Treatment of peak 2 with sodium
borodeuteride gave two product peaks, one of which co-migrated with
peak 4 (peak 2 (untreated) = 10.77 min; peak 2 treated with
NaBD product 1 = 12.88 min and product 2 =
14.94 min; peak 4 (untreated) = 14.91 min using Zorbax-SIL, HIM,
91:7:2 1 ml/min). When the NaBD
reduction product of peak 2
was further derivatized with N-trimethylsilylimidazole, the
pertrimethylsilylated product gave a molecular ion of m/z 549 (Fig. 3B) corresponding to the addition of one
deuterium atom to the original molecule and fragments at m/z 459(549 - 90), 418 (549 - 131 due to
loss of a fragment containing carbons C-2,3,4), 369(549 - 90
- 90), and a base fragment of m/z 118
corresponding to cleavage of the C-17-C-20 bond. This spectrum is
very similar to that of peak 4 and is consistent with the reduction of
the 20-ketone to a mixture of monodeuterated 20-alcohols.
Figure 3: A, mass spectrum of pertrimethylsilylated peak 4. B, mass spectrum of pertrimethylsilylated derivative of the sodium borodeuteride reduction product of peak 2.
Final
proof of the identity of peak 2 as the 20-ketone came from formation of
an oxime derivative. When the oxime was further derivatized with N-trimethylsilylimidazole, it gave a pertrimethylsilylated
derivative with a molecular ion of m/z 503 (Fig. 2B). Major fragment at 413
(M-90), 382 (M
-OCH
-90),
370 (M
-131 due to loss of a fragment containing
carbons C-2,3,4), and 292
(M
-OCH
-90-90) are all consistent
with a 20-oxime derivative. We conclude from HPLC, GC-MS, borohydride
reduction, and oxime formation that peak 2 is the truncated 20-ketone.
Figure 4: A, mass spectrum of pertrimethylsilylated peak 6. B, mass spectrum of pertrimethylsilylated derivative of the n-butyl boronate ester of peak 6.
The n-butylboronate derivative of peak 6 was formed, again suggesting vicinal hydroxyls at C-24 and C-25. The pertrimethylsilylated n-butyl boronate of peak 6 gave the mass spectrum (Fig. 4B) with the predicted molecular ion of m/z 644 and a fragmentation pattern particularly m/z = 169 due to cleavage of the oxygen O-22-C-23 bond consistent with a 24-hydroxylated precursor. Finally, peak 6 was shown to co-chromatograph with chemically synthesized 24R-OH-OCT on HPLC. We conclude on the basis of extensive evidence that peak 6 is 24R-hydroxy-OCT.
Figure 5: A, mass spectrum of pertrimethylsilylated peak 7. B, mass spectrum of pertrimethylsilylated derivative of the n-butyl boronate ester of peak 7. C, mass spectrum of pertrimethylsilylated derivative of the sodium periodate cleavage product of peak 7.
On GC-MS, the periodate cleavage product of peak 7 gave no
discernible molecular ion, but the fragmentation pattern featured large
fragments at m/z 456 (M - 90)
and 366 (M
- 90 - 90) and m/z 415 (M
- 131 due to loss of a fragment
containing carbons C-2,3,4), data consistent with the molecular ion
being extrapolated to be m/z 546 (Fig. 5C). This is consistent with a molecule truncated
between C-25 and C-26 (C-27) and containing a C-25 ketone. Further
confirmation of the peak 7 being a molecule with vicinal hydroxyls came
from formation of an n-butylboronate derivative. As with peak
6 the pertrimethylsilylated version of this was subjected to GC-MS and
gave a molecular ion of m/z 644 (Fig. 5B). In addition to the distinctive fragment at m/z 169 found with the n-butylboronate,
pertrimethylsilylated derivative of 24-hydroxy-OCT, we observed a
distinctive fragment at m/z 141 due to cleavage of
the C-24-C-25 bond and loss of a fragment containing the C-25,
C-26, and C-27 butylboronate ester group. Finally, peak 7 was shown to
co-chromatograph with chemically synthesized (25R)-26-OH-OCT
on HPLC. We conclude from these data that peak 7 must be
(25R)-26-hydroxy-OCT.
Figure 6: Time course of OCT metabolism in HPK1A-ras cells.
Figure 7: HPLC of extract of other cell lines incubated with 10 µM OCT for 48 h. A, HPK1A cells. B, UMR-106 cells. The HPLC conditions are as described in the Fig. 1legend.
When we compared metabolism of OCT at the same time, under the same experimental conditions (Fig. 8), we confirmed that OCT had virtually disappeared at the end of the 48-h period in HPK1A-ras cells, whereas considerable amounts remained in plates containing UMR-106 and HPK1A cells. The major metabolite found in cell cultures of UMR-106 and HPK1A was the 24-hydroxy-OCT (peak 6), whereas the truncated metabolites, peaks 4 and 2, appeared to be more important as the degree of metabolism increased (e.g. in HPK1A-ras). These data are consistent with the scheme shown in Fig. 9with monohydroxylated versions of OCT acting as intermediates to the truncated metabolites, peaks 4 and 2.
Figure 8: Amounts of metabolites formed from OCT in various cultured cell lines. The results are expressed as the total amount of product formed (and thus isolated) in µg/48 h. The amount of OCT remaining in the cultures was 1.4 µg for HPK1A-ras, 26 µg for HPK1A, and 28.5 µg for UMR106 and was not determined for Hep G2.
Figure 9: Proposed pathway of OCT metabolism in cultured vitamin D target cells in vitro.
Figure 10:
Biological activity of the metabolites of
OCT (relative to 1, 25-(OH)
D
). A,
VDR binding. B, DBP binding. C, inhibition of HL-60
cell growth. For clarity, only the chemically synthesized epimer
corresponding to the biologically generated metabolite is illustrated.
The biological activity of the other epimer was determined, and the
data are depicted numerically in Fig. 11.
,
1
,25-(OH)
D
;
, OCT;
,
24R-OH-OCT;
, (25R)-26-OH-OCT;
,
hexanor-20-oxo-1
-OH-D
;
,
hexanor-1
,20S-(OH)
D
.
Figure 11: Structures and biological activity data for chemically synthesized metabolites of OCT. The data are summarized for both stereochemical versions of each metabolite.
The measurements of the DBP-binding affinity (Fig. 10B) confirmed earlier observations (4, 9) that OCT showed a value 2 orders of magnitude
lower than 1,25-(OH)
D
and 4 orders of
magnitude lower than 25-OH-D
(data not shown). No
metabolite of OCT possessed a higher affinity for DBP than OCT.
In
the growth response study (Fig. 10C),
1,25-(OH)
D
suppressed cell growth of HL-60
in a dose-dependent manner when used at a concentration between 1
10
M and 1
10
M. Significant inhibition of growth could be seen at a
steroid concentration as low as 10
M 1
,25-(OH)
D
where the growth was
decreased to <40% of control. The suppression of cell growth
provided by OCT was virtually identical, whereas the suppressions
caused by the hexanor-20-oxo-compound (ED26) or the
hexanor-20S-hydroxy- compound (ED67) were greater than 2 orders of
magnitude less effective than 1
,25-(OH)
D
.
Other metabolites such as 24R-hydroxy-OCT (ED106) or
(25R)-26-hydroxy-OCT (ED141) showed slightly inferior
growth-inhibiting activity to OCT, with potencies between
and
that of 1
,25-(OH)
D
. The results of the
biological activity studies for all chemically synthesized epimers,
including biologically generated products of OCT, are summarized in Fig. 11.
We have described here the extensive metabolism of the
vitamin D analog, 22-oxacalcitriol in a variety of cultured cell lines,
including one hepatoma cell line and three vitamin D-target cell lines
previously shown to catabolize 1,25-(OH)
D
.
Our study is the first to rigorously identify any of the metabolic
products of OCT formed in vitro, and this was made possible by
the generation of microgram quantities of these metabolites. The novel
metabolites identified include two truncated versions of the OCT
molecule lacking carbons 23-27 and containing a 20-oxo or
20-hydroxyl group. The 20-hydroxy compound has been reported before in vitro, but identification was based solely upon comigration
with a chemically synthesized standard on HPLC(11) . Here we
were able to augment the preliminary identification afforded by HPLC
co-chromatography with GC-MS analysis and later comparison with
chemically synthesized standards. Furthermore, our work is also
consistent with the preliminary finding of a glucuronide conjugate of
the 20-hydroxy compound in rat bile following OCT
administration(10) . The 24R-OH-OCT and 26-OH-OCT in
the 25R-form identified here are novel, and their
stereochemistry is also established.
The finding of both intact and
truncated versions of OCT in cell extracts evokes the obvious question
of how is OCT cleaved? Our venture into the metabolic fate of an oxygen
containing molecule was not without precedent. In a previous study, we
examined the metabolic fate of 24-oxa-1-OH-D
in the
same hepatoma cell line HepG2 (23) . In that case, we also
found cleaved molecules, although the ultimate products were
23-substituted and lacked the carbon atoms C-25, C-26, and C-27 of the
starting material. Thus, in the cases of both 22-oxacalcitriol and
24-oxa-1
-hydroxyvitamin D
, the molecule is cleaved
just distal to the oxygen atom. In the case of
24-oxa-1
-OH-D
, we hypothesized that the enzymatic
hydroxylation of a carbon vicinal to the oxygen atom would give rise to
an unstable hemiacetal linkage that would undergo spontaneous breakdown
to the C-23 substituted products(23) . If we extrapolate from
this result to the case of OCT we would predict that C-23 hydroxylation
would have to occur in order to generate an unstable hemiacetal and
truncated products retaining the 22-oxa group. The metabolites that
were observed, namely 24-OH-OCT and 26-OH-OCT, would appear to be
either precursors to this 23-hydroxylated intermediate or are
by-products of the metabolic machinery. It is interesting to note that
a 24-hydroxylated version of 1
,25-(OH)
D
precedes a 23-hydroxylated metabolite in the catabolic sequence
for the natural hormone during its conversion to calcitroic acid
observed in a variety of target cell systems(19, 24) .
Alternatively, others have identified 26-hydroxylated derivatives of
25-OH-D
(25) and 1
,25-(OH)
D
(26) and a 26-hydroxylated metabolite is observed as an
intermediate during formation of the 26,23-lactone, but this is
postulated to occur after and not prior to
23-hydroxylation(27) . It remains to be proven if either
24-OH-OCT or 26-OH-OCT is a precursor to the 20-hydroxy and 20-oxo
derivatives also found here.
Only in the case of the keratinocyte
cell lines (HPK1A-ras and HPK1A) did we find appreciable
quantities of the 20-oxo-truncated derivative of OCT. It is possible
that the pattern of OCT metabolism that we observed, including the
formation of the 20-oxo derivative, resulted from the induction of the
catabolic enzymes caused by the high substrate concentrations that we
employed. Interestingly, we observed the same pattern of metabolites
including the 20-hydroxy and 20-oxo-truncated derivatives when
[2-
H]OCT (12 Ci/mmol) was incubated at low
nanomolar concentrations with HPK1A-ras cells (data not
shown). The formation of the 20-oxo derivative could also be due to the
different redox state of these cells compared with the other cell lines
used. On the other hand, this may provide evidence of the role of one
compound as an intermediate in the formation of the other (see Fig. 9). The time course study carried out suggests that the
20-hydroxy compound predates the 20-oxo derivative and thus may be
converted to the latter in this culture system.
The biological
activities of the metabolites identified here (where possible together
with their epimeric forms; see Fig. 11) provides evidence that
the in vivo properties of OCT can probably be ascribed to the
parent compound and not to its principal products. In all cases,
metabolites of OCT were inferior to OCT (and
1,25-(OH)
D
) in all parameters measured (i.e. VDR binding, DBP binding, and growth suppression of
HL-60). This was particularly marked for the 20-oxo and 20-hydroxy
derivatives where truncation of the side chain of OCT lowered
biological activity by more than 2 orders of magnitude. A rough
correlation between the strength of calf thymus VDR binding and the
ability of the vitamin D analog to inhibit growth of HL-60 cells was
still discernible. As with other 22-oxa analogs studied in this field (e.g. 28), the calcium-transporting activity of the
metabolites reported here remains unknown.
In the work described
here, we observed OCT metabolism in both vitamin D target cells,
namely, bone and keratinocyte, as well as a tissue involved in the
activation of vitamin D, namely liver. It has been previously shown
that like 1,25-(OH)
D
metabolism(19) , OCT metabolism is vitamin D-inducible in
cultured bovine parathyroid cells (11) and normal human
peripheral monocytes (29) . The result that hepatoma cells are
also able to form truncated OCT metabolites is interesting given that
liver cells are thought to be devoid of the vitamin D receptor and the
vitamin D-inducible catabolic enzymes (e.g. cytochrome P-450
CYP24)(30, 31) . However, small modifications of the
vitamin D side chain have been shown to result in much more rapid
hepatic metabolism and indeed 24-hydroxylation and 24-oxidation of
certain analogs (e.g. calcipotriol)(32, 12, 15) . Thus, it is
possible that in addition to being susceptible to attack by vitamin
D-inducible enzymes in classical vitamin D target cells, OCT is also
attacked by more general enzymes in the liver leading to cleavage of
the side chain by similar or slightly different mechanisms. It is clear
from the studies presented here that OCT is more efficiently degraded
by vitamin D-target cells than by hepatoma cells, and this may be
important to the in vivo distribution and biological activity
of the drug (33) as well as to the endogenous
ligand(34) .
Lastly, our finding of truncated versions of OCT would seem to open the door to study the origin of the conjugates found in bile in vivo following OCT administration to rats(10) . The free 20-oxo or 20-hydroxy compounds identified here would likely be transported to the liver prior to conjugation to glucuronic acid in the hepatocyte, but this remains to be proven.