Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1alpha,25-dihydroxyvitamin D(3)*.

A hapten derivative of EB1089 [1(R),3(S),25-trihydroxy-26,27-dimethyl-9,10-seco-24-homocholesta-5(Z),7(E),10(19),22(E),24(E)-pentaene], a side-chain analog of 1alpha,25-dihydroxyvitamin D(3), was synthesized for raising antibodies with a high specificity for EB1089. The A-ring moiety of EB1089 was replaced in the hapten by a linker for conjugation to a protein. Three polyclonal antibodies were obtained by immunizing rabbits with a BSA-conjugate of the hapten. The antibodies were characterized for titer, avidity and specificity using an enzyme immunoassay with covalently bound EB1089. The three antibodies had similar binding profiles and were highly selective for EB1089 and its metabolites over the naturally occurring vitamin D metabolites. Cross-reactivities with 25-hydroxyvitamin D(3), the most abundant vitamin D metabolite in serum, were in the range 0.01-0.2% relative to EB1089.

Novel side chain analogs of 1alpha,25-dihydroxyvitamin D(3): design and synthesis of the 21,24-methano derivatives.

The syntheses of the new 21,24-methano derivatives of 1alpha,25-dihydroxyvitamin D(3) [viz. 1(S),3(R)-dihydroxy-17(R)-(1',4'-cis-(4'-(1'-hydroxy-1'-methylethyl)-cyclo-hexyl))-9,10-seco-androsta-5(Z),7(E),10(19)-triene (MC 2108) and its (1',4'-trans)-isomer (MC 2110)] are described. The key step is the establishment, by Diels-Alder reaction on a CD-ring side chain diene intermediate prepared from vitamin D(2), of a 1,4-disubstituted cyclohexene moiety in the side chain. Hydrogenation to a 1:1 mixture of cis and trans cyclohexane derivatives and separation of the two series at a stage prior to the standard Horner-Wittig coupling with the (Hoffmann-La Roche) ring-A building block were other important steps in the syntheses of the target analogs. The relative configurations of intermediates were assigned by NMR spectroscopy. MC 2108 and MC 2110 are of interest as conformationally locked side chain derivatives to probe the receptor interactions of not only the parent vitamin D hormone but also its biologically active symmetrical 'double side chain' analog [21-(3'-hydroxy-3'-methylbutyl)-9,10-seco-cholesta-5(Z),7(E),10(19)-triene-1(S),3(R),25-triol (MC 2100)], 'both' side chains of which can formally be traced out in the new analogs. The preferred conformations, inferred from an analysis of (13)C-NMR characteristics, notably the chemical shift of C-17 in a series of analogs, to have the tertiary alcohol (1'-hydroxy-1'-methylethyl) substituent equatorial on the cyclohexane chair, are confirmed by molecular modeling.

Potent suppression of the parathyroid glands by hydroxylated metabolites of dihydrotachysterol(2).

BACKGROUND: Dihydrotachysterol(2), a licensed pharmaceutical, is hydroxylated to 25-hydroxydihydrotachysterol(2) (25(OH)DHT(2)) and 1 alpha,25-dihydroxydihydrotachysterol(2) (1 alpha,25(OH)(2)DHT(2)) in man. We have compared the biological activity of these metabolites with calcitriol and the 'non-calcaemic' analogue, 22-oxacalcitriol (OCT) in bovine parathyroid cell cultures and in rats. METHODS: The effect of each sterol on parathyroid hormone (PTH) secreted by primary bovine parathyroid cells was measured. High-performance liquid chromotography and gas chromotography-mass spectrometry were used to investigate in vitro 25(OH)DHT(2) metabolism. Rats were given a single intraperitoneal injection or five daily injections of each sterol, and changes in ionized calcium and PTH were measured. RESULTS: In vitro, all sterols suppressed PTH significantly. Calcitriol and OCT were of similar potency, but 1 alpha, 25(OH)(2)DHT(2) and 25(OH)DHT(2) required higher concentrations to suppress PTH equally. We were unable to detect metabolism of 25(OH)DHT(2) to 1 alpha,25(OH)(2)DHT(2) in vitro. In rats, a single dose of 0.5 microg/rat of calcitriol increased ionized calcium at 30 and 40 h (statistically significant at 48 h). 50 microg of OCT and 1 alpha,25(OH)(2)DHT(2) did not cause significant hypercalcaemia at 48 h, although 1 alpha,25(OH)(2)DHT(2) caused hypercalcaemia at 30 h. In contrast, 50 microg of 25(OH)DHT(2) caused hypercalcaemia at 48 h but not at 30 h. Five daily doses of 0.001 microg/rat of calcitriol caused a significant rise in calcium and a 50% fall in PTH. OCT and 1 alpha,25(OH)(2)DHT(2) at 0.025 and 0.5 microg/rat respectively caused similar suppression of PTH but without hypercalcaemia. CONCLUSION: 1 alpha,25(OH)(2)DHT(2) and 25(OH)DHT(2) are potent suppressors of PTH in vitro and in vivo. 25(OH)DHT(2) may be active by virtue of its pseudo-1 alpha-hydroxyl group. Hypercalcaemia caused by a single dose of 1 alpha,25(OH)(2)DHT(2) appeared to be more transient than calcitriol. Five daily doses of 1 alpha, 25(OH)(2)DHT(2) and OCT could achieve 50% suppression of PTH without significant increments in ionized calcium. In contrast, suppression of PTH by calcitriol was associated with significant increments in ionized calcium. These data suggest that like OCT, 1 alpha, 25(OH)(2)DHT(2) can dissociate calcaemic actions from parathyroid-suppressing actions in a manner that may be therapeutically useful.

Evidence for the activation of vitamin D compounds in the skin by side-chain hydroxylation.

KH 1060 is the 20-epi-22-oxa-24a-homo-26,27-dimethyl analogue of the natural hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). We have previously shown that after topical application in hairless mice both KH 1060 and 1 alpha,25(OH)2D3 cause epidermal hyperproliferation. MC 1582 differs from KH 1060 by the lack of hydroxyl group in the side chain which is required for receptor binding. We found that MC 1582 strongly stimulates epidermal hyperplasia in hairless mice after topical application in vivo, approaching in potency KH 1060. A similar, although much weaker response was also obtained with 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3). Since only the vitamin D compounds which possess hydroxyl groups both in the position 1 alpha and in the side chain, bind to the vitamin D receptor, we suggest that a local metabolism of MC 1582 and 1 alpha(OH)D3 takes place in the skin to the active, side-chain-hydroxylated species, probably to KH 1060 and 1 alpha,25(OH)2D3. This study suggests that 1 alpha hydroxylated prodrugs may be of use in the dermatological treatment of the future.

The vitamin D analog, KH1060, is rapidly degraded both in vivo and in vitro via several pathways: principal metabolites generated retain significant biological activity.

Vitamin D analogs are valuable drugs with established and potential uses in hyperproliferative disorders. Lexacalcitol (KH1060) is over 100 times more active than 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3], as judged by in vitro antiproliferative and cell differentiating assays. The underlying biochemical reasons for the increased biological activity of KH1060 are unknown, but are thought to include 1) metabolic considerations in addition to explanations based upon 2) enhanced stability of KH1060-liganded transcriptional complexes. In this study we explored the in vivo and in vitro metabolism of KH1060. We established by physicochemical techniques the existence of multiple side-chain hydroxylated metabolites of KH1060, including 24-, 24a-, 26-, and 26a-hydroxylated derivatives as well as side-chain truncated forms. KH1060 metabolism could be blocked by the cytochrome P450 inhibitor, ketoconazole. KH1060 was not an effective competitor of C24 oxidation of 1alpha,25-(OH)2D3. Certain hydroxylated metabolites of KH1060 retained significant biological activity in vitamin D-dependent reporter gene systems (chloramphenicol acetyltransferase). Likewise, those metabolites accumulating in the target cell culture models in metabolism studies, particularly 24a-hydroxy-KH1060 and 26-hydroxy-KH1060, retained biological activities superior to those of 1alpha,25-(OH)2D3 in native gene expression systems in vitamin D target cells (osteopontin and P450cc24). We conclude that KH1060 is rapidly metabolized by a variety of cytochrome P450-mediated enzyme systems to products, many of which retain significant biological activity in vitamin D-dependent assay systems. These results provide an explanation for the considerable biological activity advantage displayed by KH1060 compared with 1alpha,25-(OH)2D3 in various in vitro assay systems.

Metabolism of the vitamin D analog EB1089 by cultured human cells: redirection of hydroxylation site to distal carbons of the side-chain.

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3' (E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) is a novel synthetic analog of 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D3] with potential for use in the treatment of hyperproliferative disorders. It has an altered side-chain structure compared to 1,25-(OH)2D3, featuring 26,27 dimethyl groups, insertion of an extra carbon atom (24a) at C-24, and two double bonds at C-22,23 and C-24,24a. In vitro metabolism of EB1089 was studied in a human keratinocyte cell model, HPK1A-ras, previously shown to metabolize 1,25-(OH)2D3. Four metabolites were formed, all of which possessed the same UV chromophore as EB1089, indicating the retention of the side-chain conjugated double bond system. Two metabolites were present in sufficient quantities to identify them as 26-hydroxy EB1089 (major product) and 26a-hydroxy EB1089 (minor product), based on mass spectral analysis and cochromatography with synthetic standards. Similar metabolites were generated in vivo and using a liver postmitochondrial fraction in vitro (Kissmeyer et al., companion paper). Studies with the human hepatoma Hep G2 gave rise to 2 isomers of 26-hydroxy EB1089. Studies using ketoconazole, a general cytochrome P450 inhibitor, implicated cytochrome P450s in the formation of the EB1089 metabolites. COS-1 transfection cell experiments using vectors containing CYP27 and CYP24 suggest that these cytochrome P450s are probably not involved in 26- or 26a-hydroxylation of EB1089. Other experiments that examined the HPK1A-ras metabolism of related analogs containing only a single side-chain double bond: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1' (E)-en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (MC1473; double bond at C-22,23) and 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-3'(E)-en-1'-yl)-9, 10-secopregna-5(Z),7(E),10(19)-triene (MC1611; double bond at C-24,24a) revealed that the former compound was subject to 24-hydroxylation and the latter compound was mainly 23-hydroxylated. Metabolism experiments involving EB1089, MC1473, and MC1611 in competition with [1 beta-3H]1,25-(OH)2D3 in HPK1A-ras confirmed that CYP24 is probably not involved in the metabolism of EB1089 whereas, in the case of MC1473 and MC1611, it does appear to carry out side-chain hydroxylation. Our interpretation is that the conjugated double bond system in the side-chain of EB1089 is responsible for directing the target cell hydroxylation to the distal positions, C-26 and C-26a. We conclude that EB1089 is slowly metabolized via unique in vitro metabolic pathways, and that these features may explain the relative stability of EB1089 compared to other analogs in vivo.

Different mechanisms of hydroxylation site selection by liver and kidney cytochrome P450 species (CYP27 and CYP24) involved in vitamin D metabolism.

A series of homologated 1 alpha-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 molecules with one to three extra carbons in the side chain were used to examine the substrate preferences and hydroxylation site selection mechanisms of the liver vitamin D3-25-hydroxylase (CYP27) and the target cell 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Cultured and transfected cell models, used as sources of these hydroxylases, gave 23-, 24-, 25-, and 27-hydroxylated metabolites which were identified by their high performance liquid chromatography and GC-MS characteristics. Lengthening the side chain is tolerated by each cytochrome P450 isoform such that 25-hydroxylation or 24-hydroxylation continues to occur at the same rate as in the native side chain, while the site of hydroxylation remains the same for the liver enzyme in that CYP27 continues to hydroxylate at C-25 and C-27 (minor) despite the two-carbon-atom extension. Somewhat surprising is the finding that C-24 and C-23 (minor) hydroxylations also do not change as the side chain is extended by as much as three carbons. We conclude that CYP24 must be directed to its hydroxylation site(s) by the distance of carbon 24 from the vitamin D ring structure and not as in CYP27 by the distance of the hydroxylation site from the end of the side chain.

Trends in tobacco use.

The production and use of tobacco have been accepted parts of the American life-style for over 500 years. Tobacco use in the United States has gone through many stages over the years. The present article reviews the trends of tobacco use over the last 100 years and the public health strategies recently initiated to control tobacco use and promote public health. Finally, suggestions regarding the dentist's role in limiting tobacco use and promoting public health are presented.

Increased biological activity of 20-epi-1,25-dihydroxyvitamin D3 is due to reduced catabolism and altered protein binding.

The 20-epi series of vitamin D3 analogs has been shown to be made up of more potent inducers of cell differentiation than calcitriol in vitro. Using 20-epi-1 alpha,25-dihydroxyvitamin D3 (MC 1288), we attempted to rationalize this increased biological activity by examining several parameters including the binding affinity of the analog for the plasma binding globulin (DBP) and the target cell vitamin D receptor (VDR), as well as attempting to measure the rate at which MC 1288 is metabolized. MC 1288 was found to be metabolized 36 times more slowly than its epimer 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3), forming several metabolites which were analogous to metabolites of 1,25-(OH)2D3 formed in the side chain oxidation pathway. Bovine thymus VDR bound MC 1288 with five times greater affinity than calcitriol, while rat plasma DBP did not bind MC 1288 even at a concentration of 50 microM, 5000 times the B50 of 25-OH-D3, the ligand used in the assay. Using a vitamin D-inducible growth hormone gene reporter system we were able to demonstrate that MC 1288 induces human growth hormone (hGH) activity 30-fold more efficiently than 1,25-(OH)2D3 in the presence of fetal calf serum (FCS), while the analog is only 10 times more efficient than 1,25-(OH)2D3 in the absence of FCS. We therefore conclude that MC 1288 is more biologically active than calcitriol in vitro due to a combination of factors: the increased VDR binding affinity, the decreased DBP binding affinity, and the decreased rate of metabolism. As with other analogs of vitamin D, the altered protein binding and decreased catabolism of MC 1288 may be important in pharmaceutical applications such as a topical treatment for psoriasis or skin cancer.

In vitro metabolism of the anti-psoriatic vitamin D analog, calcipotriol, in two cultured human keratinocyte models.

Calcipotriol (MC903) is a side chain analog of the vitamin D hormone calcitriol, containing a 22-23 double bond, a 24(S)-hydroxyl function, and carbons 25, 26, and 27 incorporated into a cyclopropane ring which has been developed for treating psoriasis. The in vitro metabolism of calcipotriol was studied in two keratinocyte cell models, HPK1A and HPK1A-ras. Calcipotriol was initially converted into the 24-ketone (MC1046) and its 22,23-hydrogenated derivative (MC1080), metabolites observed in osteosarcoma, kidney, and hepatoma cell lines. We also observed the formation of further metabolites, identified as the two 23-hydroxylated derivatives of MC1080 (MC1439 and MC1441), the two 23,24-dihydroxylated compounds (MC1575 and 1577), and the side chain-cleaved compounds, tetranor-1,23-(OH)2D3 and calcitroic acid, the end products of catabolism of calcitriol. These findings suggest that calcitriol and calcipotriol may share catabolic enzymes. The biological activity of each of the principal metabolites of calcipotriol, assessed using a growth hormone reporter gene transcriptional activation system and a vitamin D receptor assay, was found to be lower than that of calcipotriol. If the extensive in vitro metabolism of calcipotriol is also found in normal and psoriatic keratinocytes in vivo, then this may explain the lack of systemic calcemic activity of topically applied drug.

A dialogue on analogues Newer vitamin-D drugs for use in bone disease, psoriasis, and cancer.

Many new analogues of the vitamin-D hormone, 1 alpha,25-dihydroxy-vitamin D(3) [1 alpha,25-(OH)(2)D(3); calcitriol], have emerged that can mimic its various actions in classic calcium transport systems and/or in the regulation of cell proliferation and cell differentiation. Though some of these analogues have accentuated activity in cell differentiation assays in vitro, they lack appreciable "calcemic" activity in vivo, leading to the name "noncalcemic analogues." Several of these analogues are promising candidates for use in treatment of psoriasis and in tumor suppression, one of them, calcipotriol, being already widely approved for the former indication. New generations of calcemic analogues with altered pharmacokinetics are appearing for use in secondary hyperparathyroidism and osteoporosis. We believe that the selective properties of both types of analogues stem from altered receptor binding, blood protein binding, and rate of catabolism.

Removable partial denture framework try-in.

Removable partial denture frameworks must fit accurately to function properly and not cause injury to remaining teeth or soft tissue. This article presents a logical method and rationale for seating a removable partial denture framework and a review of several disclosing media used in the try-in procedure.

New guidelines for preparation taper.

The purpose of this article is to provide new guidelines for minimally acceptable preparation taper by developing a theoretical mathematical foundation based on principles of resistance form. Yes and no are the possible answers to the question, "Does a preparation have resistance form?" This dual nature is useful because in graphing resistance form as a function of taper, potential tapers on the x axis are divided into two groups with an exact dividing point. Tapers less than this dividing point provide resistance form; tapers larger or equal do not provide resistance form. This dividing point makes a reasonable standard for minimally acceptable taper. Average tapers are defined as the limiting average taper and are mathematically determined to equal 1/2 arcsin (H/B), where H is the height of the preparation and B is the base. From this equation, the taper required to provide resistance form for an individual preparation can be calculated by using the preparation's height to base ratio. The equation can also be used to provide guidelines by tooth group. Dies of a prosthodontist were saved and sorted by group (incisors, canines, premolars, and molars). Measurements of 30 dies from each of these groups were used to calculate the average (H/B) ratios and standard deviations. Calculating the limiting average taper by using the average height-to-base ratio minus two standard deviations provides the dividing point taper that is acceptable for over 97% of the preparations. The values calculated are: 29 degrees for incisors, 33 degrees for canines, 10 degrees for premolars, and 8.4 degrees for molars.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo metabolism of the vitamin D analog, dihydrotachysterol. Evidence for formation of 1 alpha,25- and 1 beta,25-dihydroxy-dihydrotachysterol metabolites and studies of their biological activity.

Dihydrotachysterol (DHT), a reduced vitamin D analog in which the A-ring has been rotated through 180 degrees is a biologically active molecule which can be used to study the structural requirements for the calcemic and cell differentiating properties of the vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), as well as to investigate the specificity of the enzyme systems that catalyze the formation of this hormone. In this study we showed that dihydrotachysterol was metabolized in vivo into a significant polar metabolite observed on straight-phase high performance liquid chromatography (HPLC) which subsequently split into two peaks on reverse-phase HPLC. These two metabolites were identified by HPLC and gas chromatography-mass spectrometry techniques as 1 alpha,25-(OH)2DHT and 1 beta,25-(OH)2DHT. This pair of metabolites was formed from either DHT2 or DHT3. Standard 1 alpha,25-(OH)2DHTs were generated in vitro from chemically synthesized 1-hydroxydihydrotachysterol precursors using a liver hepatoma cell system. Both 1 alpha,25-(OH)2D2 and 1 alpha,25-(OH)2DHT3 showed a binding affinity to the mammalian vitamin D receptor only 50-100 less than 1 alpha,25-(OH)2D3 whereas 1 beta,25-(OH)2DHTs showed poor binding. On the other hand 1 beta,25-(OH)2DHT3 bound to the rat vitamin D transport protein (DBP) with stronger affinity than did 1 alpha,25-(OH)2DHT3. When tested in a COS-1 cell transfection assay system using a rat osteocalcin vitamin D responsive element coupled to a growth hormone reporter gene, 1 alpha,25-(OH)2DHT3 showed a biological activity only 10 times lower than 1 alpha,25-(OH)2D3. It is therefore suggested that 1 alpha,25-(OH)2DHT probably represents the metabolite of DHT responsible for some of its in vivo effects although we cannot rule out in vivo effects of other metabolites identified. Our studies suggest that 1 alpha,25-dihydroxylated DHTs represent a promising novel group of vitamin D analogs worthy of study for cell differentiation as well as calcemic properties.

Polar metabolites of dihydrotachysterol3 in the rat. Comparison with in vitro metabolites of 1 alpha,25-dihydroxydihydrotachysterol3.

The metabolism of 25-hydroxydihydrotachysterol3 (25-OH-DHT3) to more polar metabolites was investigated in vivo in the rat and compared with the in vitro metabolism of 1 alpha,25-dihydroxy-DHT3 (1 alpha,25-(OH)2DHT3) in the osteosarcoma cell line UMR 106. Rats were given 2 mg of DHT3 in divided doses at 0 and 6 hr. Plasma was collected 24 hr after the initial dose, extracted, separated, and polar metabolites purified by HPLC. A number of polar metabolites were formed in vivo with mass spectrometric characteristics which suggested that they were derived from a previously isolated metabolite of 25-OH-DHT3, T3/H. Of these, four were isolated and identified as 24-oxo-T3/H, 24-hydroxy-T3/H, 26-hydroxy-T3/H and the 26,23-lactone of T3/H. In view of the identification of T3/H as a mixture of 1 alpha- and 1 beta-hydroxylated 25-OH-DHT3, osteosarcoma cells (UMR 106) were incubated with chemically synthesized 1 alpha,25-(OH)2DHT3 in an attempt to determine from which component of the T3/H mixture these metabolites were derived. Again, more polar metabolites were formed and five of these were isolated by lipid extraction, purified by HPLC and identified as 24-oxo-1 alpha,25-(OH)2DHT3, 1 alpha,23,25-(OH)3DHT3, 24-oxo-1 alpha,23,25-(OH)3DHT3, 1 alpha,24,25-(OH)3DHT3 and 1 alpha,25,26-(OH)3DHT3. Three of the in vitro metabolites were similar to those found in rat plasma but only two of these metabolites were available in sufficient amounts to allow comparison. The chromatographic characteristics, using HPLC and gas chromatography, of these two pairs of metabolites (24-oxo and 24-hydroxy) were examined and it was demonstrated that they were not the same. It is therefore suggested that the polar metabolites formed in vivo are in fact metabolites of the T3/Hb component (1 beta,25-(OH)2DHT3) rather than the T3/Ha component (1 alpha,25-(OH)2DHT3). Supporting evidence for this suggestion was obtained when a small quantity of 1 beta,25-(OH)2DHT3, obtained from chemically synthesized 1 beta-OH-DHT3 by incubation with Hep 3B cells, was further incubated in the osteosarcoma UMR 106 system. Preliminary studies indicated that the putative 24-oxo and 24-hydroxy metabolites formed from 1 beta,25-(OH)2DHT3 had chromatographic and mass spectral properties almost indistinguishable from those of corresponding metabolites of T3/H formed in vivo. All the metabolites formed in vivo and in vitro are components of two metabolic pathways described previously for 25-hydroxyvitamin D3 and also for 25-OH-DHT3.

The metabolism of dihydrotachysterols: renal side chain and non-renal nuclear hydroxylations in vivo and in vitro.

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT2 and DHT3. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT2) was demonstrated in human plasma after administration of oral DHT2. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT3 in vitro showed that 25-hydroxy-DHT3 was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT3-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT3. The osteosarcoma cell line metabolized 25-OH-DHT3 in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1 alpha,25-dihydroxy-DHT3. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1 alpha,25-dihydroxy-DHT whereas compound Hb is possibly 1 beta,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1 alpha,25-dihydroxy-DHT is very effective in displacing radiolabelled 1 alpha,25-dihydroxyvitamin-D3 and is thus most likely to be the calcaemic metabolite of DHT.

Metabolism of a cyclopropane-ring-containing analog of 1 alpha-hydroxyvitamin D3 in a hepatocyte cell model. Identification of 24-oxidized metabolites.

MC969 is an analog of the calcemic drug 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3) in which carbons 25,26, and 27 in the side chain are incorporated into a cyclopropane ring. Metabolites of MC 969 were generated in an in vitro human hepatocyte cell model, Hep 3B. The identity of the metabolites was established by comigration on HPLC with authentic standards, and by mass spectrometry of native and chemically modified metabolites. Unequivocal identification of the 24-keto- and the two epimeric 24-alcohol metabolites is provided. No 25-hydroxylated metabolites were detected. In competition studies, MC 969 was able to inhibit 25-hydroxylation of tritiated vitamin D3 more effectively than 1 alpha-OH-D3 itself, indicating that the vitamin D3-25-hydroxylase may be responsible for generation of one or more of the metabolites observed.