EP0737070A1 - Vitamin d 3? analogues and pathway to mediate disorders - Google Patents

Vitamin d 3? analogues and pathway to mediate disorders

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Publication number
EP0737070A1
EP0737070A1 EP95906111A EP95906111A EP0737070A1 EP 0737070 A1 EP0737070 A1 EP 0737070A1 EP 95906111 A EP95906111 A EP 95906111A EP 95906111 A EP95906111 A EP 95906111A EP 0737070 A1 EP0737070 A1 EP 0737070A1
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Prior art keywords
vitamin
analog
analogues
composition
cells
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German (de)
French (fr)
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EP0737070A4 (en
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Anthony W. Norman
William H. Okamura
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University of California
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University of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates generally to the vitamin D endocrine 15 system. More particularly, the present invention relates to a method for controlling genomic and nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 vitamin D 3 [1 ⁇ ,25-(OH) 2 D 3 ].
  • Vitamin D 3 is a secosteroid which is responsible for a wide variety of biological responses in higher animals. These biological responses include maintenance of calcium homeostasis, immunomodulation and selected cell differentiation. Vitamin D 3 , itself, is biologically inert. However, metabolism of vitamin D 3 to metabolites such as 1 ⁇ ,25-(OH) 2 D 3 results in the formation
  • 1 ⁇ ,25-(OH) 2 D 3 has been shown to be present in 30 different tissues and it belongs to the same super family of proteins which includes receptors for the steroid hormones, retinoic acid and thyroxine (1 ,4,5).
  • genomic responses it has become apparent that a subset of biological responses are mediated by 1 ⁇ ,25-(OH) 2 D 3 via a nongenomic mechanism (3,6).
  • These biological responses include the rapid stimulation of intestinal Ca 2+ transport known as transcaltachia (7-9).
  • Transcaltachia involves the opening of Ca 2+ channels (10).
  • nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 D 3 include opening of voltage- gated Ca 2+ channels in rat osteosarcoma cells (11 ,12) as well as other rapid effects in kidney (13), liver (14), parathyroid cells (15) and intestine (16).
  • analogues of 1 ⁇ ,25-(OH) 2 D 3 are effective in controlling nongenomic cellular responses which are mediated by 1cr,25-(OH) 2 D 3 .
  • specific analogues are selected which function as either antagonists or agonists of the nongenomic cellular response.
  • the cellular responses mediated by 1 ⁇ ,25-(OH) 2 D 3 can be controlled.
  • Transcaltachia is a particular nongenomic cellular response which can be controlled in accordance with the present invention.
  • Vitamin D analogues which can be used as agonists of transcaltachia include 1 ⁇ ,25- (OH) 2 -previtamin D 3 .
  • 1 ?,25- (OH) 2 vitamin D 3 [1 ?,25-(OH) 2 D 3 ] is used as an antagonist.
  • control procedures provided in accordance with the present invention are effective in limiting or increasing transcaltachia and other nongenomic responses of 1 ⁇ ,25-(OH) 2 D 3 which are exhibited by a variety of cells.
  • the present invention is useful in controlling nongenomic responses both in vivo and in vitro.
  • fifteen analogues are provided which are effective in controlling either genomic or nongenomic cellular responses. The fifteen analogues are described in the following detailed description. The above described and many other features and attendant advan ⁇ tages of the present invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.
  • FIG. 1A shows the metabolic scheme for producing vitamin D 3 including the formation of previtamin D 3 .
  • FIG. 1 B shows the two conformations of the hormonally active form of vitamin D 3 , namely 1 ⁇ ,25-(OH) 2 D 3 .
  • FIG. 2 shows the equilibrium relationship between 1 ,25-(OH) 2 -pre- vitamin D and 1 ⁇ ,25-(OH) 2 D 3 forms.
  • FIG. 3 depicts the structures of 1 ⁇ ,25-(OH) 2 D 3 , 1/?,25-(OH) 2 D 3 and other related analogues.
  • FIG. 4 depicts the structures of 1 ⁇ ,25-(OH) 2 D 3 , 1£,25-(OH) 2 D 3 and other related analogues.
  • FIG. 5A depicts the effect of 1 ,25-(OH) 2 -d 5 -pre-D 3 and 1 ,25-(OH) 2 D 3 on the appearance of 45 Ca 2+ in the venous effluent of perfused duodena from vitamin D-replete chicks.
  • FIG. 5B shows the dose-response analysis of 1 ,25-(OH) 2 D 3 and 1 ,25- (OH) 2 -d 5 -pre-D 3 in stimulating transcaltachia in the perfused duodena.
  • the experimental conditions were as described in FIG. 5A. Values are the mean ⁇ S.E. at 30 minutes for three to five duodena perfused with each concentration of agonist. * p ⁇ 0.02; **p ⁇ 0.01 ; ***p ⁇ 0.005 with respect to duodena not exposed to agonist.
  • FIG. 6 shows the result of evaluation of 45 Ca 2+ uptake in osteosarcoma cells stimulated by 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -pre-D 3 .
  • the left inset displays the 45 Ca 2+ uptake properties of the cells in resting buffer (R), stimulating buffer (S), and when exposed to the optimal con ⁇ centration of 1 ,25-(OH) 2 D 3 , designated as C.
  • the ROS 17/2.8 cells were assayed for 45 Ca 2+ as described in Example 1. In all cases, additions of the secosteroids were made to cells exposed to the resting buffer.
  • 1 ,25-(OH) 2 D 3 stimulated 45 Ca 2+ uptake with a maximum response occurring between 1.0 and 5.0 nM (see Re. 28). Data points represent the mean of triplicate measurements ⁇ S.D.
  • FIG. 7A shows the affinity of the 1 ,25-(OH) 2 D 3 nuclear receptor from pig intestinal mucosa for 1 ,25-(OH) 2 D 3 (•), 1 ,25-(OH) 2 -d 5 -pre-D 3 ( ⁇ ), and
  • FIG. 7B shows the determination of the RCI for the chick intestinal receptor and the pig receptor for 1 ,25-(OH) 2 D 3 (analog C), 1 ,25-(OH) 2 -d 5 - pre-D 3 (analog HF), and 1 ,25-(OH) 2 -d 5 -D 3 (analog HG).
  • FIG. 8 shows the affinity of purified human DBP for 1 ,25-(OH) 2 D 3 (o),
  • FIG. 9 shows the effect of 1 ,25-(OH) 2 D 3 and its analogues on the serum concentration of osteocalcin after a single 400-ng intramuscular injection in vitamin D-deficient chicks. •, 1 ,25-(OH) 2 D 3 ; ⁇ , 1 ,25-(OH) 2 -d 5 -
  • FIG. 10A shows the effect of chronic administration to mice of 1 ,25-
  • mice were administered the indicated daily intraperitoneal dose of the indicated secosteroid for 7 days. Values represent the mean ⁇ S.E. (six mice/group). * p ⁇ 0.01 in comparison with the control group.
  • FIG. 10B shows the effect of chronic administration to mice of 1 ,25-
  • mice were administered the indicated daily intraperitoneal dose of the indicated secosteroid for 7 days. Values represent the mean ⁇ S.E. (six mice/group). *p ⁇ 0.01 in comparison with the control group.
  • FIG. 11 shows the effect of 1 ,25-(OH) 2 D 3 and its analogues in MG-63 cells on the inhibition of proliferation (FIG. 11 A) and induction of osteocalcin
  • FIG. 11 B •, 1 ,25-(OH) 2 D 3 ; ⁇ , 1 ,25-(OH) 2 -d 5 -pre-D 3 ; A , 1 ,25-(OH) 2 -d 5 -D 3 .
  • FIG. 12 shows the effect of 1 ,25-(OH) 2 D 3 and its analogues on differentiation of HL-60 cells.
  • the differentiating effect was evaluated by nitro blue tetrazolium (NBT) reduction. •, 1 ,25-(OH) 2 D 3 ; ⁇ , 1 ,25-(OH) 2 -d 5 -
  • FIG. 13 shows the results of screening of the four A-ring diastereomers of 1 ⁇ ,25-(OH) 2 D 3 in the transcaltachia assay.
  • the effect of each analog on the appearance of 45 Ca 2+ (5 //Ci/ml) in GBSS was vascularly perfused (25°C) for the first 20 minutes with control medium (GBSS containing 0.125% bovine serum albumin and 0.05 ⁇ of ethanol/ml) and then at time zero with the indicated concentration of the stipulated analog or control medium.
  • Values are the mean ⁇ S.E. for five duodena within each experimental group. •, indicated analog; o, control.
  • FIG. 14 depicts the antagonistic action of 1 ?,25-(OH) 2 D 3 -stimulated intestinal 45 Ca 2+ transport activity in the perfused chick duodena.
  • FIG. 15 depicts the results of the evaluation of the ability of 1 ?,25- (OH) 2 D 3 to inhibit the agonist actions of 1 ⁇ ,25-(OH) 2 D 3 on transcaltachia.
  • the 1 ?,25-(OH) 2 D 3 (HL) was presented to the perfused duodenum either in advance or simultaneously with 1 ⁇ ,25-(OH) 2 D 3 at varying concentrations.
  • the data presented represent the mean ⁇ S.E. from four or five duodena. •, analog HL and C [1 ⁇ ,25-(OH) 2 D 3 ]; o, control.
  • FIG. 15A the 1 ?,25-(OH) 2 D 3 (HL) was presented to the perfused duodenum either in advance or simultaneously with 1 ⁇ ,25-(OH) 2 D 3 at varying concentrations.
  • the data presented represent the mean ⁇ S.E. from four or five duodena. •, analog HL and C [1 ⁇ ,25-(OH) 2 D 3 ];
  • FIG. 16 shows the inhibition of 5 Ca 2+ uptake in osteosarcoma cells stimulated by 1 ⁇ ,25-(OH) 2 D 3 (C) by 1/?,25-(OH) 2 D 3 (HL).
  • the left side displays the 45 Ca 2+ uptake properties of the cells in resting buffer (/ ⁇ ?), stimulating buffer (S), and when exposed to the optimal concentration of 1 ⁇ ,25-(OH) 2 D 3 designated as C.
  • the concentration of HL is the upper value and C the lower value.
  • the ROS 17/2.8 cells were assayed for 45 Ca 2+ as described in Example 2. In all cases, additions of the secosteroids were made to cells exposed to the resting buffer.
  • FIG. 17 shows the dose-response effects of 1 ⁇ ,25-(OH) 2 D 3 , 1 ?,25- (OH) 2 D 3 , and 1 ⁇ ,25-(OH) 2 -3-epi-D 3 on ICA (FIG. 17A) and BCM (FIG. 17B) in the vitamin D-deficient chick.
  • the analogues and 1 ⁇ ,25-(OH) 2 D 3 were given intramuscularly to vitamin D-deficient chicks 12 hours before assay; the control D 3 was 48 hours before assay.
  • Results are expressed as mean ⁇ S.E. of groups of five to seven chicks. Each assay included a negative control, open bar (-D) and a positive control of vitamin D 3 3.25 mnol, solid black bar; the difference between these two groups was significant at p ⁇ 0.01. A detailed evaluation was carried out for the four diastereomers in separate bioassays; the results are summarized in Table I.
  • FIG. 18 shows the effect of the four A-ring diastereomers in MG-63 cells on the induction of osteocalcin.
  • Both 1 ⁇ ,25-(OH) 2 D 3 and 1 ?,25-(OH) 2 D 3 were evaluated independently of one another for their ability to induce osteocalcin (FIG. 18A).
  • the ability of 10,25-(OH) 2 D 3 to antagonize the actions of 1 ⁇ ,25-(OH) 2 D 3 on induction of osteocalcin was assessed (FIG. 18B).
  • the concentration of 1 ?,25-(OH) 2 D 3 was held constant at 10 "9 -10 "7 M, whereas the concentration of
  • 1 ⁇ ,25-(OH) 2 D 3 was varied from 10 "11 to 10 ° M. V, 10 "9 M 1/?,25-(OH) 2 D 3 + varying concentrations of 1 ⁇ ,25-(OH) 2 D 3 ; D , 1 ?,25-(OH) 2 D 3 , 10 "8 M + varying concentrations of 1 ⁇ ,25-(OH) 2 D 3 ; ⁇ , 1 ?,25-(OH) 2 D 3 , 10 "7 M + varying concentrations of 1 ⁇ ,25-(OH) 2 D 3 .
  • 1 ⁇ ,25-(OH) 2 D 3 (•) and 1 ?,25-(OH) 2 D 3 (o) were evaluated alone, i.e. in the absence of the other secosteroid. For details, see Example 2. The data presented are from a representative experiment; three experiments were conducted.
  • FIG. 19 shows the effect of 1 ⁇ ,25-(OH) 2 D 3 and 1 ?,25-(OH) 2 D 3 on dif ⁇ ferentiation of HL-60 cells.
  • the differentiating effect of the secosteroids was evaluated by NBT reduction as described in Example 2. The data presented are from a representative experiment that was repeated twice.
  • FIG. 20 shows the effects of 1cr,25-(OH) 2 D 3 A-ring analogues on keratinocyte differentiation.
  • Human skin keratinocytes were grown in tissue culture in 96-well plates as described in Example 2.
  • the A-ring analogues were added at the indicated concentrations, and then the rate of cell proliferation was assessed for 3 hours by the addition of [ 3 H]thymidine.
  • FIG. 21 sets forth the structural formulas for analogues GE, GF, HS, IB, JD, JM, JN, JO and JP in accordance with the present invention.
  • FIG. 22 sets forth the structural formulas for analogues JR, JS, JV, JW, JX and JY in accordance with the present invention.
  • FIG. 23 depicts the synthesis pathway for making analogues GE and
  • FIGS. 24(A) and (B) show the results of testing of analogues JM and JN, respectively, as set forth in Example 6.
  • augmented 5 Ca 2+ transport in duodenal loops was vascularly perfused with 1 ⁇ ,25(OH) 2 D 3 , or 1 ⁇ ,25(OH) 2 -7-dehydrocholesterol (JM), or 1 ⁇ ,25(OH) 2 -lumisterol 3 45 Ca 2+ (5 //Ci/ml of buffer), and vascularly perfused with control medium for the first 20 minutes with collection of the venous effluent occurring at 2-minute intervals during the final 10 minutes to establish basal transport rates.
  • duodena were then either re-exposed to control medium containing the vehicle ethanol (0.005%, final concentration) through the celiac artery, or vascularly perfused with 300 pM agonist, or 650 pM agonist.
  • the venous effluent was again collected at 2 minute intervals for liquid scintillation spectrophotometry.
  • FIGS. 25(A) and (B) show the results of testing of analogues JM and JN, respectively, as set forth in Example 6.
  • the tests involved dose- response analyses of JM and JN as agonists for transcaltachia.
  • Duodena which were perfused as described in FIG. 24 with vehicle or a range of JM or JN concentrations. Normalized transport after 40 minutes of perfusion is depicted for the indicated concentrations of (A) JM; (B) JN.
  • FIG. 26 is a chart showing the various diseases which may be treated using the analogous of the present invention.
  • FIG. 27 is a schematic representation of the synthesis of analogue IB.
  • the present invention provides methods for controlling transcaltachia and other nongenomic cellular responses which are mediated by 1 ⁇ ,25- (OH) 2 D 3 .
  • Activation of the nongenomic cellular response is accomplished in accordance with the present invention by treating cells with 1 ,25-(OH) 2 - previtamin D 3 (compound BC in FIG. 2).
  • the 1 ,25-(OH) 2 -previtamin D 3 has been found to be an agonist of transcaltachia as is described in detail in Example 1.
  • Another aspect of the present invention involves antagonizing the nongenomic responses mediated by 1 ,25-(OH) 2 D 3 by treating cells with 1/?,25-(OH) 2 D 3 as described in Example 2.
  • Treatment with 1 ⁇ ,25-(OH) 2 -previtamin D 3 or 1 ?,25-(OH) 2 D 3 can be accomplished as set forth in the examples, i.e. by direct injection or perfusion of the analog in an appropriate pharmaceutical carrier.
  • the dosage levels are preferably similar to the dosage levels described in the examples.
  • the type of cells treated can be any of those which are known to undergo nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 D 3 .
  • the cells may be treated either in vivo or in vitro.
  • the preferred nongenomic mediated response which can be con- trolled in accordance with the present invention is transcaltachia which involves opening of the Ca 2+ channels in the intestines.
  • Control of caltachia is preferably accomplished by selecting the desired agonist or antagonist analog described above and introducing it by way of perfusion into the intestine.
  • previtamin D 3 is effective in stimulating two nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 D 3 .
  • transcaltachia in isolated perfused chicken duodenum is stimulated and Ca + channel opening in rat osteogenic sarcoma cells is also stimulated.
  • FIG. 1A shows the metabolic scheme for production of vitamin D 3 .
  • the provitamin, 7-dehydrocholesterol, present in the skin is converted by ultraviolet irradiation into the secosteroid vitamin D 3 .
  • Previtamin D 3 is in thermal equilibrium with vitamin D 3 ; the conversion involves a [1 ,7]-sigmatropic shift, i.e. the intramolecular migration of a hydrogen from carbon-19 to carbon-9.
  • the resulting product vitamin D 3 is a conformationally mobile molecule with respect to the orientation of the A ring in relation to the C/D ring structure.
  • the seco-B ring can assume one of two conformations as a consequence of rotation about the carbon 6-7 single bond; in the 6-s-c/s orientation the A ring is related to the C/D rings as in the conventional steroid orientation, referred to here as the "steroid-like conformation" and when the conformation is in the 6-s-trans orientation, the A ring is present in an "extended conformation.”
  • FIG 1 B depicts two orientations of 1 ,25-(OH) 2 D 3 .
  • 1 ,25-(OH) 2 D 3 has free rotation about the single bond between carbon-6 and carbon-7; accordingly it can assume in solution the steroid-like conformation (6-s-c/s) or the extended conformation (6-s-frans) orientation.
  • Analogues HF and 1 ,25-(OH) 2 -d 5 -D 3 were synthesized according to the method of Curtin and Okamura (19).
  • 1 ,25-(OH) 2 -d 5 - pre-D 3 which had been stored at -60°C for about 1 year with occasional warming to ambient temperatures for withdrawal of samples for biological evaluation, the sample analyzed to be comprised of 4.4% of the vitamin and 95.6% of the previtamin form of analog HF.
  • the composition determinations were carried out by analytical high performance liquid chromatography on a normal phase column (Whatman Partisil column using 90% ethyl acetate, 10% hexane as solvent; 5 ml/min flow rate) using a Waters photodiode array detector.
  • a separate comparison using cut and weigh integrated peak areas was used as a cross-check, and the overall agreement was estimated to be ⁇ 0.7%.
  • the retention times were as follows: 1 ,25-(OH) 2 -d 5 -D 3 , about 18 minutes; 1 ,25-(OH) 2 -d 5 -pre-D 3 , 24.5 minutes. These retention times are essentially identical to those of the undeuteriated forms of these two secosteroids.
  • the human promyelocytic leukemia cell line (HL-60) and the MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD).
  • One-day-old RIR chicks were housed in a windowless room and raised on a vitamin D-replete diet for 1 week followed by a vitamin D- deficient diet (Hope Farms, Woerden, The Netherlands) for the next 5 weeks. After a total of 6 weeks, they were divided into groups and received a single intramuscular injection of 400 ng of 1 ,25-(OH) 2 D 3 or analogues HF or HG solubilized in 10:10:80 v/v/v ethanol, Tween 80, NaCI, 0.9%.
  • Serum osteocalcin was measured by radioimmunoassay using specific anti-chick antisera raised against these chick proteins.
  • Mice, strain NMRI were fed a normal diet (Hope Farms) for 40-60 days. They received a daily subcutaneous dose of 1 ,25-(OH) 2 D 3 or analogues HF and HG for 7 days.
  • Serum Ca 2+ was determined via atomic absorption spectrophotometry and serum osteocalcin levels via radioimmunoassay.
  • the pig intestinal mucosa was obtained from a normal 20-kg pig under Ketalar anesthesia. The mucosa was scraped and stored at -80°C until time of preparation of the 1 ,25-(OH) 2 D 3 nuclear receptor (see below).
  • the ROS 17/2.8 cells obtained from Merck, Sharp and Dohme (West Point, PA) were cultured in Dulbecco's modified Eagle's medium: Ham's F- 12 medium 1 :1 containing 10% fetal calf serum (GIBCO-BRL). The medium was supplemented with 1.1 mM CaCI 2 as described (24). For 45 Ca 2+ uptake experiments, cells were seeded at a density of 30,000 cells/ml into 3.5-cm dishes and grown to approximately 50% confluence. Calcium Uptake Assays ROS 17/2.8 cells were assayed for Ca 2+ uptake using procedures described previously (20). Assays were standardized to 1 minute, which preliminary experiments demonstrated to be within the interval of linear uptake.
  • Intestinal 45 Ca 2+ Transport Measurements of 45 Ca 2+ transport were carried out in perfused chick duodena as described previously (21-23). Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml/100 g), and the duodenal loop was surgically exposed. Blood vessels branching off from the celiac artery were lighted before cannulation of the celiac artery itself and simultaneous initiation of vascular perfusion. The duodenal loop was then excised and, after cannulation of the celiac vein, placed between layers of saline-moistened cheesecloth at 24°C.
  • the arterial perfusion was initiated during cannulation with modified Grey's balanced salt solution (GBSS) modified to contain 0.9 mM CaCI 2 and oxygenated with 95% 02 and 5% CO 2 at a flow rate of 2 ml/min.
  • GBSS Grey's balanced salt solution
  • An auxiliary pump was used for the introduction of vehicle (ethanol) or test substances plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min.
  • the intestinal lumen was then flushed and filled with GBSS containing 45 Ca 2+ (5 /Ci/MI) but without bicarbonate or glucose.
  • a basal transport rate was established by perfusion with control medium for 20 minutes after the lumen was filled with 45 Ca 2+ .
  • the tissue was then exposed to 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -pre-D 3 or reexposed to control medium for an additional 40 minutes.
  • the vascular perfusate was collected at 2-minute intervals during the last 10 minutes of the basal and during the entire treatment period.
  • Duplicate 100-//I aliquots were taken for determination of the 5 Ca 2+ levels by liquid scintillation spectrometry. The results are expressed as the ratio of the 45 Ca 2+ appearing in the 40-minute test period over the average initial basal transport period.
  • each analog to compete with [ 3 H]1,25-(OH) 2 D 3 for binding to the chick intestinal nuclear receptor for 1 ,25-(OH) 2 D 3 was carried out under in vitro conditions according to standard procedures (24,25).
  • increasing concentrations of nonradioactive 1 ,25-(OH) 2 D 3 or the test analog are incubated with a fixed saturating amount of [ 3 H]1 ,25-(OH) 2 D 3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [ 3 H]1 ,25-(OH) 2 D 3 was then calculated and plotted as a function of the relative concentration of the analog and [ 3 H]1 ,25-(OH) 2 D 3 .
  • the plots give linear curves characteristic for each analog, the slopes of which are equal to the analog's competitive index value (24).
  • the competitive index value for each analog was then normalized to a standard curve obtained with nonradioactive 1 ,25-(OH) 2 D 3 as the competing steroid and placed on a linear scale of relative competitive index (RCI), where the RCI of 1 ,25-(OH) 2 D 3 by definition is 100. Binding to the 1 ,25-(OH) 2 D 3 receptor was determined in mucosa obtained from a vitamin D-replete pig.
  • Frozen (- 80°C) duodenal mucosa was sonicated in 4 volumes of buffer (0.5 M Tris- HCI, 0.5 M KCl, 5 mM dithiothreitol, 10 mM Na 2 MoO 4 , 1.5 mM EDTA, pH 7.5). The high speed supernatant was then incubated with 0.2 nM [ 3 H]1 ,25- (OH) 2 D 3 and increasing concentrations of nonradioactive 1 ,25-(OH) 2 D 3 or its analogues in a final volume of 0.3 ml overnight at 25°C followed by 5 minutes at 4°C. Phase separation was then obtained by the addition of cold dextran-coated charcoal.
  • Binding of the 1 ,25-(OH) 2 D 3 and its analogues to hDBP was performed at 4°C essentially as described previously (26).
  • [ 3 H]1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 D 3 or its analogues were added in 5 ⁇ of ethanol into glass tubes and incubated with hDBP (0.18 ⁇ u) in a final volume of 1 ml (0.01 M Tris-HCl, 0.154 M NaCI, pH 7.4) for 4 h at 4°C. Phase separation was then obtained by the addition of 0.5 ml of cold dextran-coated charcoal.
  • HL-60 cells were seeded at 1.2 x 10 5 cells/ml, and 1 ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer).
  • RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer).
  • the MG-63 cells were seeded at 5 x 10 3 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 ⁇ of Dulecco's modified Eagle's medium containing 2% of heat-inactivated charcoal-treated fetal calf serum, and 1 ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%). After 72 hours of culture in a humidified atmosphere of 5% CO 2 in air at 37°C the inhibition of proliferation by [ 3 H] thymidine incorporation and measurement in the medium of osteocalcin concentration using a homologous human radioimmunoassay were performed (26).
  • Nitro Blue Tetrazolium Reduction Assay Superoxide production was assayed by nitro blue tetrazolium- reducing activity as described previously (26).
  • HL-60 cells at 1.0 x 10 5 cells/ml were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/ml) and nitro blue tetrazolium (2 mg/ml) and incubated for 30 minutes at 37°C. The percentage of cells containing black formazan deposits were determined using a hemacytometer.
  • This example compares the biological profile of the two deuteriated analogues, 1 ,25-(OH) 2 d 5 -pre-D 3 (HF) and 1 ,25-(OH) 2 -d 5 -D 3 (HG) in relation to 1 ,25-(OH) 2 D 3 .
  • the structures of the analogues are given in FIG. 2.
  • the 1 ,25-(OH) 2 -d 5 -pre-D 3 , analog HF, is kinetically suppressed (21) in its previtamin form (because of a primary deuterium kinetic isotope effect) and thus can function as an analog only of the 6-s-c/s form of 1 ,25-(OH) 2 D 3 (FIG. 1).
  • FIG. 5A shows the results of an evaluation of the relative ability of 1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 -d 5 -pre-D 3 to stimulate the nongenomic biological response of transcaltachia.
  • Vascular perfusion with the physiological concentration of 60 pM 1 ,25-(OH) 2 -d 5 -pre-D 3 for 34 minutes yielded a 4.5-fold increase in 45 Ca + transport over control levels.
  • the stimulatory effect of both secosteroids on 45 Ca 2+ becomes significant within 2-8 minutes as observed previously (7,9).
  • FIG. 5B shows the dose- responsive relationship for each secosteroid in terms of its ability to stimulate transcaltachia.
  • the analog 1 ,25-(OH) 2 -d 5 -pre-D 3 was able to stimulate transcaltachia significantly at a dose of 10 pM, and the maximal response was attained at 60 pM secosteroid.
  • the dose-response for 1 ,25-(OH) 2 -d 5 -pre-D 3 is biphasic (7-9).
  • 1 ,25-(OH) 2 D 3 is active at the low concentration of 25 pM, and the maximum stimulation is achieved over the range of 60-650 pM 1 ,25-(OH) 2 D 3 (FIG. 5B).
  • the typical biphasic dose response is apparent.
  • FIG. 6 shows the results of the evaluation of the ability of 1 ,25-(OH) 2 - d 5 -pre-D 3 to stimulate 5 Ca 2+ uptake into ROS 17/2.8 cells.
  • concentration range of 1-10 x 10 "9 M 1 ,25-(OH) 2 -d 5 -pre-D 3 produced a maximum uptake of 45 Ca 2+ within 1 minute of the application of the secosteroids. Previous studies have established that this is the range of maximum response to 1 ,25-(OH) 2 D 3 (15,27).
  • the voltage-gated Ca 2+ channel can either be opened by exposure to appropriate agonists (vitamin D analogues or the dihydropyridine BAY K- 8644 (15)) or by depolarization of the cell membrane, which is achieved by the 132 mM external KCl (stimulating buffer; see "Experimental Proce ⁇ dures”).
  • appropriate agonists vitamin D analogues or the dihydropyridine BAY K- 8644 (15)
  • depolarization of the cell membrane which is achieved by the 132 mM external KCl (stimulating buffer; see "Experimental Proce ⁇ dures”).
  • Such stimulated uptake of 45 Ca 2+ in the presence of depolarizing extracellular solutions is characteristic of cells expressing voltage-gated Ca 2+ channels, and the level of this stimulation is directly related to the concentration of the Ca 2+ channels on the cell surface.
  • the maximum influx of 45 Ca 2+ which can be achieved (see inset of FIG. 6) occurs in the presence of high external K + .
  • the level of 45 Ca 2+ uptake occurring in low K + represents the basal uptake, which is a reflection of the Ca 2+ permeability of the resting membrane.
  • S/R the maximum ratio of (stimulated)/( resting), S/R is approximately 2.4-fold, which represents 100% Ca 2+ channel opening.
  • Both the analog 1 ,25-(OH) 2 -d 5 -pre- D 3 and 1 ,25-(OH) 2 D 3 achieve a 2-fold stimulation of 45 Ca 2+ uptake over that which occurs in the low K + environment.
  • FIG. 7 shows the results of the determination of the RCI for binding to the intestinal nuclear 1 ,25-(OH) 2 D 3 receptor from both the chick and pig, as determined under in vitro conditions.
  • the steroid that is kinetically repressed in the previtamin form has a reduction in its RCI from 90-100% to 12-14%, which shows that the nuclear 1 ,25-(OH) 2 D 3 receptor can discriminate between the previtamin 6-s-c/s and the vitamin D form, which may exist either as the 6-s-c/s or 6-s-trans forms, with the latter predominating.
  • the principal carrier of vitamin D secosteroids in the blood compart ⁇ ment is the plasma DBP.
  • This protein has a binding domain that tightly binds its ligand with a K,, of 5 x 10 "9 M and 5 x 10 "8 M for 25-(OH)D 3 and 1 ,25-(0H) 2 D 3 , respectively (29); thus the affinity of any ligand for DBP will effectively determine its "free" concentration in the plasma and perhaps influence its relative availability to target cells.
  • FIG. 7-10 show the evaluation of the biological efficacy of the previtamin form of the pentadeuteriated analog of 1 ,25-(OH) 2 D 3 under in vivo conditions as well as in cultured cells.
  • FIG. 9 shows the levels of serum osteocalcin which are achieved after a single intramuscular injection of vitamin D-deficient chick with 400 ng of either 1 ,25-(OH) 2 D 3 , 1 ,25-(OH) 2 -d 5 -D 3 , or 1,25-(OH) 2 -d 5 -pre-D 3 .
  • 1 ,25-(OH) 2 D 3 has been shown to induce via interaction with a nuclear 1 ,25-(OH) 2 D 3 receptor present in bone osteoblast cells the de novo biosynthesis of osteocalcin; small amounts of the osteocalcin are released into the blood (as a consequence of bone remodeling) where it may be conveniently determined via a radioimmunoassay (26). It is apparent that the 1 ,25-(OH) 2 -d 5 -pre-D 3 , when administered as a single dose under in vivo conditions, has little ability to interact effectively with the nuclear 1 ,25-(OH) 2 D 3 receptor to induce osteocalcin.
  • both 1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 -d 5 -D 3 caused a significant increase in the plasma levels of osteocalcin.
  • treatment with the deuteriated analog resulted in a consistently higher induction of the plasma osteocalcin levels.
  • FIG. 10 shows the levels of serum Ca 2+ and osteocalcin achieved after 1 week of daily treatment with doses of 1 ,25-(OH) 2 D 3 or the analogues 1 ,25-(OH) 2 -d 5 -pre-D 3 and 1 ,25-(OH) 2 -d 5 -D 3 .
  • Both 1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 -d 5 -D 3 were virtually equipotent in regard to evaluation of serum Ca 2+ and osteocalcin.
  • both of these responses are mediated by the nuclear 1 ,25-(OH) 2 D 3 receptor.
  • the 1 ,25-(OH) 2 -d 5 -pre-D 3 had only approximately 1% of the activity of 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -D 3 .
  • the 1 ,25-(OH) 2 -d 5 -D 3 was indistinguishable from 1 ,25-(OH) 2 D 3 in its ability to induce osteocalcin and displayed approximately 90% of the activity of 1 ,25-(OH) 2 D 3 in terms of its ability to inhibit cell proliferation.
  • the differentiation of HL-60 cells was markedly enhanced by the pre ⁇ sence of 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -D 3 (FIG. 12).
  • the 1 ,25- (OH) 2 -d 5 -pre-D 3 displayed only 1 -4% of the potency of 1 ,25-(OH) 2 D 3 , which again shows that the HL-60 nuclear 1 ,25-(OH) 2 D 3 receptor does not effi ⁇ ciently bind this ligand.
  • the biological profile of 1 ,25-(OH) 2 - d 5 -pre-D 3 has been compared with that of the pair of rapidly interconverting 6-s conformers of 1 ,25-(OH) 2 D 3 (FIGS. 1 and 2).
  • the example demonstrates that two nongenomic biological systems are fully responsive to the 1 ,25-(OH) 2 -d 5 -pre-D 3 analog. Both the process of transcaltachia as studied in the isolated perfused chick duodenum (FIG. 5) and the process of Ca 2+ channel opening in the rat osteogenic sarcoma cell line, ROS 17/2.8 cells (FIG.
  • FIG. 8 indicates that both the pig and chick intestinal 1 ,25-(OH) 2 D 3 nuclear receptors discriminate against the previtamin form of the secosteroid.
  • the RCI of 1 ,25-(OH) 2 -d 5 -pre-D 3 for binding to the chick intestinal receptor was 10% and for the pig intestinal receptor was 4%. Accordingly, the nuclear 1 ,25-(OH) 2 D 3 receptor's ligand binding domain favors the 6-s-trans conformer (extended steroid conformation) over the 6-s- c/s (steroid-like conformation).
  • FIGS. 9-12 show the evaluation of 1,25-(OH) 2 -d 5 -pre-D 3 , 1 ,25-(OH) 2 -d 5 -D 3 , and 1 ,25-(OH) 2 D 3 in four systems that all generate biological effects via a nuclear receptor- mediated regulation of gene transcription.
  • FIG. 9-12 show the evaluation of 1,25-(OH) 2 -d 5 -pre-D 3 , 1 ,25-(OH) 2 -d 5 -D 3 , and 1 ,25-(OH) 2 D 3 in four systems that all generate biological effects via a nuclear receptor- mediated regulation of gene transcription.
  • FIG. 9 shows the in vivo measurement of serum osteocalcin levels, after a single dose of 1 ,25-(OH) 2 - d 5 -pre-D 3 to chicks.
  • FIG. 10 shows the results of daily administration of 1 ,25-(OH) 2 -d 5 -pre-D 3 on serum Ca 2+ and osteocalcin levels in mice.
  • FIG. 11 shows, using MG-63 cells in cell culture, the inhibition of cell proliferation and the induction of osteocalcin.
  • FIG. 12 shows, using HL-60 cells in culture, the inhibition of cell proliferation. The relative inability of analog 1 ,25-(OH) 2 -d 5 -pre-D 3 (less than 2% for osteocalcin induction in vivo, FIG.
  • mice 7 days (mice, in FIG. 10), and 96 h (MG-63 cells, in FIG. 11 ; HL-60 cell, in FIG. 12), and if the vitamin form, 1 ,25-(OH) 2 d- 5 -D 3 had been generated there would have been ample time for appearance of detectable manifestations of the various genomic responses.
  • the results from the chronic dosing of mice with 1 ,25-(OH) 2 -d 5 -pre-D 3 show that the previtamin form is subject to metabolic clearance before it has had an opportunity to isomerize thermally into the biologically active 1 ,25-(OH) 2 d- 5 -D 3 .
  • the previtamin D form of 1 ,25- (OH) 2 D 3 is effective as an agonist of the nongenomic receptor for 1 ,25- (OH) 2 D 3 and therefore may be used to initiate biological responses which utilize the nongenomic receptor.
  • White Leghorn cockerels (Lakeview Farms, Lakeview, CA) were obtained on the day of hatch and maintained on a vitamin D-supplemented diet (1.2% calcium and 0.7% phosphorous; O.H. Kruse Grain and Milling, Ontario, CA) for 5-6 weeks to prepare normal vitamin D 3 -replete chicks. All experiments employing animals were approved by the University of California-Riverside Chancellor's Committee on Animals in Research. The human promyelocytic leukemia cell line (HL-60) and the human osteoblast MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD).
  • Both uptake solutions contained 12.5 /Ci/ml 45 Ca + (Du Pont-New England Nuclear) and the concentrations of vitamin D agonists indicated in FIG. 13. Uptake was terminated by aspiration of the labeling solution, followed by three washes with ice-cold resting buffer. Cell-associated 45 Ca 2+ was extracted by a 2- hour incubation with 0.5 M NaOH and measured by liquid scintillation counting. It was found that 5 Ca 2+ uptake by monolayer cultures of ROS 17/2.8 cells was density-dependent. Maximal uptake rates were consis ⁇ tently found for cultures that were between 50 and 80% confluent.
  • Intestinal 45 Ca 2+ Transport Measurements of 45 Ca 2+ transport were carried out in perfused chick duodena as described previously (32-34). Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml/100 g), and the duodenal loop was surgically exposed. Blood vessels branching off from the celiac artery were lighted before cannulation of the celiac artery itself and simultaneous initiation of vascular perfusion. The duodenal loop was then excised and, after cannulation of the celiac vein, placed between layers of saline-moistened cheesecloth at 24°C.
  • the arterial perfusate consisted of Grey's balanced salt solution (GBSS) modified to contain 0.9 mM CaCI 2 and oxygenated with 95% 0 2 and 5% CO 2 at a flow rate of 2 ml/min.
  • GBSS Grey's balanced salt solution
  • An auxiliary pump was used for the introduction of vehicle (ethanol) or test substances plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min.
  • the intestinal lumen was then flushed and filled with GBSS containing 5 Ca 2+ (5 Ci/MI) but without bicarbonate or glucose and perfused at a flow rate of 0.2 ml/min.
  • a basal transport rate was established by perfusion with control medium for 20 minutes after the lumen was filled with 45 Ca 2+ .
  • the tissue was then exposed to 1 ⁇ ,25-(OH) 2 D 3 or 1 ?,25-(OH) 2 D 3 or reexposed to control medium for an additional 40 minutes.
  • the vascular perfusate was collected at 2-minute intervals during the last 10 minutes of the basal period and during the entire treatment period.
  • Duplicate 100- /I aliquots were taken for determination of the 45 Ca 2+ levels by liquid scintillation spectrometry. The results are expressed as the ratio of the 5 Ca 2+ appearing in the 40-minute test period over the average initial basal transport period.
  • each analog to compete with [ 3 H]1 ⁇ ,25-(OH) 2 D 3 for binding to the chick intestinal nuclear receptor for 1 ⁇ ,25-(OH) 2 D 3 was carried out in vitro according to the procedure set forth in Example 1.
  • increasing concentrations of nonradioactive 1 ,25-(OH) 2 D 3 or the test analog were incubated with a fixed saturating amount of [ 3 H]1 ⁇ ,25-(OH) 2 D 3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [ 3 H]1 ⁇ ,25-(OH) 2 D 3 was then calculated and plotted as a function of the relative concentration of the analog and [ 3 H]1cr,25-(OH) 2 D 3 .
  • Such plots give linear curves characteristic for each analog, the slopes of which are equal to the analog's competitive index value (25).
  • the competitive index value for each analog is then normalized to a standard curve obtained with nonradioactive 1 ⁇ ,25-(OH) 2 D 3 as the competing steroid and placed on a linear scale of relative competitive index (RCI), where the RCI of 1 ⁇ ,25- (OH) 2 D 3 by definition is 100.
  • HL-60 cells were seeded at 1.2 x 10 5 cells/ml, and 1 ⁇ ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer Mannheim). After 4 days of culture in a humidified atmosphere of 5% CO 2 in air at 37°C the dishes were shaken to loosen any adherent cells, and all cells were then assayed for differentiation by NBT reduction assay and for proliferation by [ 3 H]thymidine incorporation.
  • the MG-63 cells were seeded at 5 x 10 3 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 ⁇ of Dulecco's modified Eagle's medium containing 2% of heat-inactivated charcoal-treated fetal calf serum, and 1 ⁇ ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%).
  • Human skin keratinocytes were isolated and cultured using a modifi ⁇ cation of the method of Kitano and Okada (36). Briefly, skin from biopsies of patients with breast tumors was cut into pieces measuring 3-5 mm and soaked overnight at 4°C in a solution of dispase (20 Boehringer units/ml). The epidermis was peeled from the dermis, washed with calcium- and magnesium-free phosphate-buffered saline, and incubated and shaken in a 0.25% trypsin solution for 10 minutes at room temperature. The reaction was then stopped by the addition of phosphate-buffered saline containing 10% fetal calf serum.
  • the cells were collected after centrifugation at 4°C for 10 minutes at 800 rpm. After an additional washing with phosphate- buffered saline, the pellet was suspended in culture medium into 25-cm 2 primaria flasks from Becton Dickinson. The keratinocytes were cultivated at 37°C in an atmosphere of 5% CO 2 in air. A few hours later the medium was replaced by a new one. The medium (keratinocyte medium from Life Technologies, Inc., containing epidermal growth factor (5 ng/ml), bovine pituitary extract (35-50 /g/ml), and antibiotics) was renewed every other day until confluence.
  • epidermal growth factor 5 ng/ml
  • bovine pituitary extract 35-50 /g/ml
  • antibiotics was renewed every other day until confluence.
  • keratinocytes were cultured in 96-well plates and after 24 hours were treated with various concentrations of the vitamin D analogues followed by pulse labeling with 1 ⁇ C ⁇ of [ 3 H]thymidine for 3 hours. Cultures were washed three times with phosphate-buffered saline and twice with 10% (v/v) ice-cold trichloroacetic acid. Cells were solubilized with 1 M NaOH and the radioactivity determined via liquid scintillation measurement. Statistics Statistical evaluation of the data was performed in the same manner as Example 1.
  • This example provides a comparison of the biological profile of the three A-ring diastereomers of 1 ⁇ ,25-(OH) 2 D 3 with respect to their ability to act as agonists for nongenomic and genomic responses of various components of the vitamin D endocrine system.
  • the structures of these secosteroids are presented in FIGS. 3 and 4.
  • the two asymmetric centers are located at carbons-1 and -3.
  • the orientation of the two hydroxyl groups on the A-ring of the naturally occurring hormone 1 ⁇ ,25-(OH) 2 D 3 are 1 ⁇ and 3/?.
  • transcaltachia A biological response of 1 ⁇ ,25-(OH) 2 D 3 which has been shown to occur via a nongenomic mechanism is the rapid hormonal stimulation of intestinal Ca 2+ , termed transcaltachia (22).
  • FIG. 13 presents the results showing the relative ability of the four A-ring diastereomers of the secosteroid hormone to stimulate transcaltachia.
  • the optimal agonist is the naturally occurring hormone 1 ⁇ ,25-(OH) 2 D 3 (designated C).
  • the onset of stimulation of 45 Ca + transport occurs within 4 minutes of introduction of the hormone.
  • the dose-response curve for transcaltachia is biphasic, with a maximal stimulation occurring at 650 pM 1 ⁇ ,25-(OH) 2 D 3 (21 ,22).
  • FIG. 14 illustrates the ability of 1/?,25-(OH) 2 D 3 to block the action of 1 ⁇ ,25-(OH) 2 D 3 to stimulate transcaltachia.
  • the duodena are simulta ⁇ neously perfused with both 1 ?,25-(OH) 2 D 3 and 1 ⁇ ,25-(OH) 2 D 3 the characteri- stic stimulation of transcaltachia was absent; this shows that 1 ?,25-(OH) 2 D 3 can function as an antagonist of 1 ⁇ ,25-(OH) 2 D 3 to control nongenomic responses in accordance with the present invention.
  • FIG. 14 illustrates the ability of 1/?,25-(OH) 2 D 3 to block the action of 1 ⁇ ,25-(OH) 2 D 3 to stimulate transcaltachia.
  • FIG. 15A there is presented an evaluation of the different concen ⁇ trations of 1 ?,25-(OH) 2 D 3 which are effective at inhibiting 300 pM 1 ⁇ ,25- (OH) 2 D 3 -stimulated transcaltachia.
  • FIG. 15B summarizes the dose response of the inhibition of 1 ⁇ ,25-(OH) 2 D 3 -stimulated transcaltachia by varying concentrations of 1 ?,25-(OH) 2 D 3 . It is apparent that a concentration as low as 60 pM 1/?,25-(OH) 2 D 3 can inhibit 300 pM 1 ⁇ ,25-(OH) 2 D 3 .
  • FIG. 16 presents an evaluation of the ability of 1 ?,25-(OH) 2 D 3 to function as an agonist or antagonist of 45 Ca + uptake into ROS 17/2.8 cells.
  • this response occurs as a consequence of the ability of 1 ⁇ ,25-(OH) 2 D 3 or its analogues to open dihydropyridine-sensitive Ca 2+ channels via a nongenomic mechanism (20).
  • 1 ,25-(OH) 2 D 3 is the most potent agonist in this system.
  • Concentrations as high as 10 "8 M 1 ?,25-(OH) 2 D 3 is the most potent agonist in this system.
  • FIG. 17 presents the ICA and BCM results for 1 ⁇ ,25-(OH) 2 D 3 , 1 ?,25-(OH) 2 D 3 (HL) and 1 ⁇ ,25-(OH) 2 -3-epi-D 3 (HJ).
  • Table I summarizes the ICA and BCM results for all four diastereomers.
  • the most potent stimulator of ICA and BCM was the reference compound 1 ⁇ ,25-(OH) 2 D 3 ; the activity produced by 100 pmol of 1 ⁇ ,25-(OH) 2 D 3 was set to 100% for both ICA and BCM. Then, the dose of the comparison analogues required to achieve a biological response of either ICA or BCM equivalent to the 100-pmol dose of 1 ⁇ ,25-(OH) 2 D 3 was calculated and converted to a percentage.
  • the analog 1 ⁇ ,25-(OH) 2 D-3-epi- 3 was the only diastereomer to have detectable ICA or BCM, which was only 1.5-2.8% of that of the reference 1cr,25-(OH) 2 D 3 .
  • the two diastereomers, 1 ?,25-(OH) 2 D 3 and 1 ?,25-(OH) 2 D-3-epi-D 3 had less than 0.1% ICA and BCM.
  • the alteration of the orientation of either the 3 ?-hydroxyl group or the 1 ⁇ -hydroxyl group greatly diminishes the biological activity in the vitamin D- deficient chick in vivo.
  • results presented for the bioassay ICA and BCM are derived from dose- response studies like that presented in FIG. 6. The results are expressed in terms of the dose of analog required to achieve an ICA or BCM response equivalent to that achieved by a 100-pmol dose of 1 ⁇ ,25-(OH) 2 D 3 , calculated as a percentage, i.e. [1 ⁇ ,25-(OH) 2 D 3 dose]/[analog dose] x 100.
  • Table I also summarizes the relative ability of the four diastereomers to bind in vitro to the chick intestinal 1 ⁇ ,25-(OH) 2 D 3 nuclear receptor as well as to the DBP.
  • the 1 ⁇ ,25-(OH) 2 D 3 nuclear receptor is the presumed media ⁇ tor of genomic responses to 1or,25-(OH) 2 D 3 in vivo.
  • 1 ⁇ ,25-(OH) 2 D 3 is the reference compound, and its RCI is by definition 100%. Inversion of the orientation of the 3 ?-hydroxyl to 3 ⁇ -hydroxyl, as in analog 1 ⁇ ,25-(OH) 2 -3- epi-D 3 , resulted only in a reduction of RCI to 24%.
  • the reference analog has been defined to be 1 ⁇ ,25-(OH) 2 D 3 ; but it is important to realize that the optimal ligand for DBP is 25-(OH)D 3 and that is has an RCI of 66,700. If the RCI of 25-(OH)D 3 were set to 100%, then the RCI for 1 ⁇ ,25-(OH) 2 D 3 would only be 0.15%; this is a reflection of the fact that the presence of an ⁇ -hydroxyl on carbon-1 results in a marked reduction in affinity of the ligand for DBP. Inversion of the 1 ⁇ -hydroxyl to the 1/?
  • calbindin-D28k levels present in the chick intestine 12 hours after dosing vitamin D-deficient chicks with either 1 ⁇ ,25-(OH) 2 D 3 alone or in the presence of 1 ? ,25-(OH) 2 D 3 were determined via enzyme-linked immunosorbent assay, as described under "Experimental Procedures.”
  • FIG. 18A results are presented describing the potency of the four diastereomers to induce osteocalcin in MG-63 cells; in addition, the ability of 1/?,25-(OH) 2 D 3 to function as an antagonist of 1 ⁇ ,25-(OH) 2 D 3 - induced osteocalcin is presented (FIG. 18B).
  • 1 ⁇ ,25-(OH) 2 D 3 is a potent agonist for osteocalcin in the MG-63 cell line (35); half-maximal induction occurs at a concentration of 3.8 x 10 *9 M 1 ⁇ ,25-(OH) 2 D 3 (FIG. 18A).
  • 1 ⁇ ,25-(OH) 2 D 3 is a potent stimulator of HL-60 cell differentiation; the half-maximal concentration was 1.5 x 10 "8 M. The concentration of 1 ?,25-(OH) 2 D 3 which achieved half- maximal stimulation of cell differentiation was 2.5 x 10 ⁇ 7 M, some 10 times higher. Again there was no evidence that 1 ?,25-(OH) 2 D 3 could antagonize the cell differentiation actions of 1 ⁇ ,25-(OH) 2 D 3 .
  • FIG. 20 presents the evaluation of the potencies of 1 ⁇ ,25-(OH) 2 D 3 and the three A-ring diastereomers in inhibiting the proliferation of human keratinocytes.
  • the relative order of potency was 1 ⁇ ,25-(OH) 2 D 3 , 1 ⁇ ,25-(OH) 2 -3-epi-D 3 , 1 ?,25-(OH) 2 D 3 , and 1 ?,25-(OH) 2 -3-epi-D 3 , 1 :6.2:27:75.
  • the most potent inhibitor of cell proliferation was 1 ⁇ ,25-(OH) 2 D 3
  • the least potent was 1/?,25-(OH) 2 D 3 .
  • the potential of 1/?,25-(OH) 2 D 3 to antagonize 1 ⁇ ,25-(OH) 2 D 3 -mediated inhibition of keratinocyte was tested.
  • This example demonstrates the biological profile of the four A-ring diastereomers of the hormonally active form of vitamin D 3 (see FIG. 3). Only 1 ⁇ ,25-(OH) 2 D 3 is known to occur naturally in biologically systems. The only difference in structure of these four compounds is the orientation of the hydroxyl groups on carbons-1 and -3.
  • the nuclear 1 ⁇ ,25-(OH) 2 D 3 receptor's ligand binding domain clearly prefers the 1 ⁇ , 3 ⁇ orientation of the naturally occurring hormone and that it can also discern differences among the three other A- ring diastereomers (see the RCI values of Table I).
  • the correct orientation of the hydroxyl on carbon-1 is more critical than the orientation of the hydroxyl on carbon-3.
  • inversion of the 1 ⁇ -hydroxyl to the . ⁇ orientation results in a change in RCI from 100 to 0.8%
  • inversion of the 3 .-hydroxyl to a 3 ⁇ -hydroxyl only results in a reduction from 100 to 24%.
  • DBP is the principal plasma transport protein for vitamin D metabolites; the ligand with highest affinity is 25-(OH)D 3 ; however, it also binds 1 ⁇ ,25-(OH) 2 D 3 , 24R,25-(OH) 2 D 3 , and the parent vitamin D 3 with a significant affinity.
  • the addition of a 1 ⁇ -hydroxyl to 25-(OH)D 3 results in a 666-fold reduction in RCI (3).
  • 1 ⁇ ,25-(OH) 2 D 3 has the lowest RCI; this implies that 1 ⁇ ,25-(OH) 2 D 3 would have, under in vivo circumstances, the highest "free” concentration or greatest "availability" of the four diastereomers.
  • ICA and BCM responses occur under in vivo conditions and both likely represent a response to an integrated set of components that respond to vitamin D ligands, it is not known which proportion of the responding elements is comprised of genomic and nongenomic responses. However, both, ICA and BCM responses can be blocked by administration of actinomycin D, an inhibitor of DNA-directed RNA synthesis (38,39), and a good correlation between binding to the nuclear 1 ⁇ ,25-(OH) 2 D 3 receptor and ICA and BCM has been shown (3).
  • hormone response element(s) associated with the nongenomic response of transcaltachia clearly have a ligand specificity different from that of the nuclear 1 ⁇ ,25-(OH) 2 D 3 receptor (compare with RCI results in Table I).
  • the putative transcaltachic membrane response element (40) is more tolerant of the presence of 3 ⁇ -hydroxyl.
  • this example shows that the analog 10,25-(OH) 2 D 3 (HL) is a potent antagonist of both 1 ⁇ ,25-(OH) 2 D 3 - stimulated transcaltachia (FIG. 15) and rat osteoblast 5 Ca 2+ uptake (FIG. 16).
  • 1/?,25-(OH) 2 D 3 and 1 ⁇ ,25-(OH) 2 D 3 were perfused simultaneously and, even in some circumstances when the intestine was preexposed only to the 1 ?,25-(OH) 2 D 3 for 8 minutes and then followed by perfusion with only 1 ⁇ ,25-(OH) 2 D 3 , there was a clear inhibition by exposure of the intestine to 1 ?,25-(OH) 2 D 3 .
  • This example demonstrates that the analog of 1 ⁇ ,25-(OH) 2 D 3 is able to function as an antagonist of a biological response stimulated by 1 ⁇ ,25-(OH) 2 D 3 .
  • analogues of vitamin D 3 have been synthesized and demonstrated to be active in controlling genomic and/or nongenomic responses in the vitamin D endocrine system.
  • the fifteen analogues are set forth in FIGS. 21 and 22 and tabulated in Table III.
  • the analogues may be administered in the same manner as the previously described two analogues. They are effective in controlling a wide variety of responses within the vitamin D endocrine system including the genomic mechanisms which are controlled by mechanisms similar to that of other steroid or steroid like hormones (e.g. estradiol, testosterone, stanolone, progesterone, cortisol, aldosterone, retinoic acid and thyroxine).
  • the analogues are useful in treating a variety of diseases associated with malfunction of the vitamin D endocrine system including skin conditions (e.g. psoriasis), bone conditions (e.g. osteoporosis, venal osteodystrophy), and oncologic diseases such as breast, colon and prostate cancers and leukemia, induction of hey proteins like nerve growth factor and other brain proteins which may be involved in Alzheimer's disease.
  • skin conditions e.g. psoriasis
  • bone conditions e.g. osteoporosis, venal osteodystrophy
  • oncologic diseases such as breast, colon and prostate cancers and leukemia, induction of hey proteins like nerve growth factor and other brain proteins which may be involved in Alzheimer's disease.
  • the various diseases associated with vitamin D metabolism are set forth in FIG. 26.
  • the analogues are useful in treating and diagnosing this group of diseases. The synthesis and usefulness of these analogues will be further described in the following examples.
  • Example 3
  • analogues GE and GF were prepared from the known A-ring phosphine oxide 10(41) and the appropriate (CD) ketone 14.
  • Grundmann's ketone 11(42) readily available from the ozonolysis of vitamin D 3 , was selectively oxidized at C-25 to alcohol 12 as previously described (43). Epimerization of the latter to the c/s-fused hydrindanone 13 was accomplished with base.
  • the crude mixture consisted of a 71/29 ratio of 13/12 whereas a 49% yield (66% based on recovered 12) of purified 13 was actually isolated by HPLC(44,45,46).
  • Intestinal calcium absorption (ICA) and bone calcium mobilization (BCM) were measured in vivo to compare analogues GE and GF to 1 ⁇ ,25- (OH) 2 -D 3 (3) in the vitamin D deficient chick system previously described(37).
  • the results in this standard rachitic chick assay can be reported as the percentage of activity observed for ICA and BCM in comparison to standard doses of 1 ⁇ ,25-(OH) 2 -D 3 (47).
  • the two analogues GE and GF exhibited 3.9% and ⁇ 0.1%, respectively of the activity as compared to 1 ⁇ ,25-(OH) 2 -D 3 . Similar results ( ⁇ 0.01% and 2%, respectively) were obtained with respect to the BCM determination.
  • the GE and GF analogues were evaluated in vitro in terms of their ability to bind to the chick intestinal nuclear receptor.
  • the analogues were evaluated in terms of their chick intestinal receptor relative competitive indices (RCIs) wherein the value for 1 ⁇ ,25-(OH) 2 -D 3 is 100 by definition (48).
  • the RCI values for GE and GF were 15.0 ⁇ 2.0 and 1.6 ⁇ 0.9, respectively.
  • the lack of in vivo calcemic activity observed for GE is somewhat at variance with its RCI value of 15. It is believed that GE binds to the chick intestinal receptor without inducing its necessary activation, which is required of steroid hormone receptors prior to stimulation of transcription.
  • the human DBP RCI values for analogues GE and GF were 12.1 ⁇ 2.1 and 2.2 ⁇ 0.7, respectively.
  • the above test results show that GE and GF are useful respectively for biological responses involving the nuclear VDR (GE) and regulation of gene transcription (GE) and the membrane VDR anovated with nongenomic rapid actions (GF).
  • BCM BCM Mobilization
  • ICA and BCM were determined in vivo in vitamin D deficient chicks as described previously (37, 47). Twelve hours before assay, the chicks, which had been placed on a zero-calcium diet 48 hours before assay, were injected intramuscularly with the vitamin D metabolite or analogue in 0.1 mL of ethanol/1 ,2-propanediol (1 :1 , v/v) or with vehicle. At the time of assay, 4.0 mg of 40 Ca 2+ + 5 ⁇ C ⁇ of 45 Ca 2+ (New England Nuclear) were placed in the duodenum of the animals anesthetized with ether. After 30 min, the birds were decapitated and the blood collected.
  • the radioactivity content of 0.2 mL of serum was measured in a liquid scintillation counter (Beckman LS8000) to determine the amount of 45 Ca 2+ absorbed (which is a measure of ICA).
  • BCM activity was estimated from the increase of total serum calcium as measured by atomic absorption spectrophotometry.
  • Chick Intestinal Receptor Steroid Competition Assay A measure of competitive binding to the chick intestinal 1 ⁇ ,25- (OH) 2 -D 3 receptor was performed by using the hydroxylapatite batch assay (48). Increasing amounts of non radioactive 1 ⁇ ,25-(OH) 2 -D 3 or analogue were added to a standard amount of [ 3 H]-1 ⁇ ,25-(OH) 2 -D 3 and incubated with chick intestinal cytosol.
  • the relative competitive index (RCI) for the analogues was determined by plotting the percent maximum 1 ⁇ ,25-(OH) 2 - [ 3 H]-D 3 bound x 100 on the ordinate versus [competitor]/[1 ⁇ ,25-(OH) 2 -[ 3 H]- D 3 ] on the abscissa.
  • the slope of the line obtained for a particular analogue is divided by the slope of the line obtained for 1 ⁇ ,25-(OH) 2 -D 3 ; multiplication of this value by 100 gives the RCI value.
  • the RCI for 1 ⁇ ,25- (OH) 2 -D 3 is 100.
  • DBP human vitamin D binding protein
  • Example 4 Synthesis and Biological Activity of 1 ⁇ , 18,25-(OH 3 )-D 3 (HS) and 1 ⁇ ,25-Dihydroxy-.rat.s-isotachysterol (1 ,25-fratvs-lso-T) (JD) HS is prepared as follows: The protected alcohol precursor of HS was first prepared as follows: 18-Acetoxy-25-[(trimethylsilyl)oxy]-1 ⁇ -[(fetf-Butyldimethylsilyloxy)
  • Vitamin D 3 tert-butyldimethylsilyl] ether (Compound A) was prepared according to the procedure described in Maynard et al., J. Org. Chem., 1992, v. 57, pp. 3214-17.
  • JD was synthesized by preparing and reacting precursors A-D as follows:
  • precursor D (4.5 mg, 43% yield).
  • the analytical data for precursor D is 1 H-NMR: ⁇ 0.88 (3H, C 18 -CH 3 , s), 0.96 (3H, C 21 -CH 3 , d, J -6.6 Hz), 1.21 (6H, C 2627 -2CH 3 , s), 1.69 (3H, C 19 -CH 3 , s), 2.52 (1 H, dd, J - 16.5 Hz, 4.2 Hz), 4.07 (1 H, C 3 -H, m), 4.19 (1 H, C H, br s), 5.78 and 5.92 (2H, C 6 -H and C7-H, AB pattern, J - 12.1 Hz).
  • JJV (100% EtOH) ⁇ max 256 nm (e 11 ,000); ⁇ mn
  • a solution of precursor D (7.5 mg, 0.0180 mmol) was dissolved in ether (1 ml) under argon.
  • the solution was then condensed to leave a crude oily residue.
  • the crude residue was subjected to HPLC (Rainin Microsorb, 5 ⁇ m silica, 10 mm x 25 cm, 11% isopropanol/hexanes) to produce JD (3.9 mg, 52%).
  • HS and JD will have the same biological activity as described in Example 2.
  • HS biological responses will mimic 1 ,25(OH) 2 D 3 in that it can assume both 6-s-cis and 6-s-trans.
  • IB was prepared according to the procedure set forth in FIG. 27. In step 1 of the synthesis, 3-iodobenzoic acid is refluxed for 14 hours in 80 ml
  • step 3 the product of step 2 was reacted with 55 mg OH, 183 mg pyridinium chlorochromate (PDC), 12 mg pyridinium trifluoroacetate (PTFA) and 100 ml CH 2 CI 2 according to the procedure set forth in S.A. Barrack et al., J. Org. Chem. 1988, 53, 1790. The reaction was carried out at room temperature for 5 hours. The resulting black mixture was filtered and washed with CH 2 CR 2 and extracted with ethyl acetate to produce pale yellow oil. This oil was flash chromatographed to produced C 22 H 26 O 3 .
  • PDC pyridinium chlorochromate
  • PTFA pyridinium trifluoroacetate
  • step 4 the product of step 3 was reacted with 70 mg phosphine oxide, 82 ⁇ n-Butyl, 35 mg of the CD ketone in 2 ml of a solution of THF.
  • the n-Butyl was added dropwise to the solution of phosphine oxide in THF.
  • the resulting orange colored solution was stirred at -78°C for 10 minutes and the CD ring ketone in THF was added dropwise.
  • the reaction mixture was stirred at -78°C for 4 hours. At this point the solution turned pale yellow.
  • the solution was quenched with H 2 O, extracted with ethyl acetate and dried over Na 2 SO 4 .
  • the solvent was vacuum evaporated and the resulting product purified by flash chromatography.
  • 10 mg of the product of step 4 reacted with 53 ⁇ MeLi in
  • step 6 10 mg of the product of step 5 was dissolved in 1 ml THF.
  • Example 6 Synthesis and Biological Activity of 1 ⁇ ,25-(OH) 2 -7-DHC (JM) and 1 ⁇ ,25-(OH) 2 -Lumisterol 3 (JN).
  • JM and JN are closed B-ring analogues which both stimulate transcaltachia while neither competes with 1 ⁇ ,25(OH) 2 D 3 for binding to either the nuclear vitamin D receptor (N-VDR) or the serum vitamin D transport protein (DBP).
  • N-VDR nuclear vitamin D receptor
  • DBP serum vitamin D transport protein
  • JM [1 ⁇ ,25-(OH) 2 -7-Dehydrocholesterol] and JN [1 ⁇ ,25-(OH) 2 -7-Lumi- sterol 3 ] were synthesized as follows.
  • a solution of the known 1 ⁇ ,25-(OH) 2 - previtamin D 3 (120 mg) in methanol was irradiated (Hanovia 450 watt medium pressure mercury lamp, pyrex filter, ⁇ > 300 nm) for 3 hours at room temperature.
  • JN The identifying characteristics of JN are: 1 H-NMR (CDCI 3 ): ⁇ 0.61 (3H, C 18 -CH 3 , s), 0.78 (3H, C 19 -CH 3 , s), 0.91 (3H, C 21 -CH 3 , d, J -5.2 (Hz), 1.21 (6H, C 2627 -CH 3) s), 2.50 (2H, m), 4.10 (1 H, H,, dd, J -9.2 Hz, 4.8 Hz), 4.14 (1 H, H3, dd, J - 3.0 Hz, 3.0 Hz), 5.45 (1 H, H 6or7 , m), 5.75 (1 H, H 7or6 , dd, J - 5.1 Hz, 1.7 Hz).
  • auxiliary pump was used for the introduction of vehicle (0.005% ethanol v/v, final concentration) or test analogues plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min.
  • vehicle 0.005% ethanol v/v, final concentration
  • test analogues plus albumin 0.125% w/v final concentration
  • the intestinal loop was then excised and the lumen flushed and filled with GBSS (lacking NaHCO 3 and glucose) containing 45 Ca 2+ (5 /Ci/ml).
  • the lumenal solution was renewed constantly at a rate of 0.25 ml/min to insure a steady concentration of 45 Ca 2+ at the brush border of the epithelia.
  • the intestinal preparation at 27°C was kept moist under layers of saline-dampened cheese cloth.
  • duodenum was perfused with control medium (vehicle) for 20 min after filling the lumen with 45 Ca 2+ to establish the basal transport rate.
  • the tissue was then either exposed to the test analog or continued on vehicle for an additional 40 minutes.
  • the competitive index value for each analog is then normalized to a standard curve obtained with nonradioactive 1 ⁇ ,25(OH) 2 D 3 as the competitive steroid and placed on a linear scale of Relative Competitive lndex(s) (RCI), where the RCI of 1 ⁇ ,25(OH) 2 D 3 is by definition 100.
  • RCI Relative Competitive lndex(s)
  • the relative ability of vitamin D analogues to bind to the plasma transport protein, the vitamin D binding protein (DBP) were carried out in a similar fashion (52). JM and JN differ in the fixed orientations of the two A-ring hydroxyls;
  • JM is 1 ⁇ -axial and 3 ?-equatorial
  • JN is 1 ⁇ -equatorial and 3 ?-axial.
  • JM and JN are analogues of the 6-s-c/s form of 1 ⁇ ,25(OH) 2 D 3 ; they cannot exist in the extended 6-s-trans conformation.
  • FIGS. 24A and 24B illustrate the appearance of 5 Ca 2+ in the venous effluent mediated by the two different concentrations of analogues, JM and JN, respectively, vehicle control (ethanol only) and 650 pM 1 ⁇ ,25(OH) 2 D 3 as positive control.
  • the efficacy of 300 pM JM in initiating transcaltachia is not significantly greater than the control, and the response elicited at 650 pM JM is only 60% of that induced by the natural metabolite.
  • Perfusion with JN produced a stimulation nearly identical to that of 1 ⁇ ,25(OH) 2 D 3 with JN achieving only a slightly lower ratio of transport 45 Ca 2+ than that achieved by 1 ⁇ ,25(OH) 2 D 3 .
  • FIGS. 25A and 25B present the dose response curves for JM and JN, respectively.
  • Each bar represents the 40 minute data point of FIGS. 25A and 25B which is taken as the maximum response elicited by the analog at that concentration.
  • Analog JN eventually reaches the 4-fold plateau at 1300 pM which is the equivalent of the maximum stimulation achieved by 1 ⁇ ,25(OH) 2 D 3 at 650 pM.
  • the 5 Ca 2+ transport ratio for analog JM at 650 pM peaked at 2.5 and was not further increased as a consequence of increasing the JM concentration of 1300 pM.
  • JM and JN are useful in controlling nongenomic mechanisms, such as transcaltachia. These two analogues can be used to act as agonists for nongenomic responses as previously described.
  • Example 7 Synthesis and Biological Activity of (9 ⁇ ,10 ⁇ )-and (9/?, 10/?)- 1 ⁇ ,25-Dihydroxy-7-dehydrocholesterol-1 ⁇ ,25(OH) 2 -Pyrocalciferol (JO) and 1 ⁇ ,25(OH) 2 -lsopropylcalciferol 3 (JP).
  • JO and JP were prepared according to the following procedures: An argon flushed solution of 1 ⁇ ,25-(OH) 2 -previtamin D 3 (54.2 mg) dis ⁇ solved in DMF (15 mL) containing a drop of 2,4,6-trimethylpyridine was heated in a screw cap vial (156°C) for 18 hours.
  • Both JO and JP will have the same nongenomic actions achieved by analogues JM and JN since JO and JP locked in the 6-s-cis conformation.
  • Example 8 Synthesis and Biological Activity of (1S, 3R, 6S)-7,19-Retro- 1 ,25-(OH) 2 -D 3 (JV) and (1S, 3R, 6S)-7,19-Retro-1 ,25-(OH) 2 -D 3 (JW)
  • JV The analog JV was synthesized as follows:
  • (1S,3R,6S)-1,3-Di(tert-butyldimethylsilyloxy)-25-trimethylsilyloxy- 9,10-secocholesta-5(10),6,7-triene (A) is a starting material that was prepared first as follows: Freshly purified 1 ,2-diiodoethane (412 mg, 1.46 mmol) and samarium metal (286 mg, 1.90 mmol) were dried under vacuum and suspended in 4 mL THF under an argon atmosphere. This solution was stirred for 2 hours until it became deep blue.
  • the product was purified by flash chromatography (silica gel, 2% EtOAc/hexanes) followed by HPLC (2% EtOAc/hexanes, Rainin Dynamax column, 8 mL/min flow rate) to afford vinylallene A (0.3085 g, 75.5%).
  • the product was identified by 1 H-NMR analysis. This material is more stable as the triol.
  • JW which is also known as (1S,3R,6R)-1,3,25-Trihydroxy-9,10- secocholesta-5(10),6,7-triene was isolated from the above solution as follows: A solution of (6S/6R)-vinylallenes JV and JW (2.6 mg, 0.0062 mmol, -92:8 ratio of 6S:6R) in methanol-d 4 (1 mL) was prepared in a quartz NMR tube. The solution was saturated with argon for 30 minutes and then the NMR tube was capped and then irradiated with ultraviolet light from a Hanovia 450 watt medium pressure lamp for 30 minutes.
  • Example 9 Synthesis and Biological Activity of 1 ,25-(OH) 2 7, 8-c/s-D 3 (JR) and 1 ,25-(OH) 2 -5,6-fra ⁇ s-7,8-c/s-D 3 (JS). JR was synthesize from JV as follows:
  • Acetone was removed under reduced pres ⁇ sure and the product was purified by flash chromatography (silica gel, 80% EtOAc/hexanes) followed by separation by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4.0 mL/min flow rate) to afford three components in the following order of elution: major product JR (17.0 mg, 86.4%), recovered starting material JV (1.4 mg, 7.1%), and a minor amount of cis- isotachysterol (1.5 mg, 7.6%).
  • the first separation (80% ethyl acetate/ hexanes, Rainin Microsorb column, 4 mL/min flow rate) gave two fractions, each of which was subjected to NMR analysis.
  • Fraction I contained products A, B, and C and fraction II contained products B, C, and D.
  • JR and JS were determined by the chick intestinal receptor steroid competition assay which has been described in the preceding examples. Both JR and JS significantly suppress the ability of the natural hormone to bind receptor. These results show that JR and JS are also useful for regulating nongenomic mechanisms such as transcaltachia.
  • Example 10 Synthesis and Biological Activity of 22-(p-Hydroxyphenyl)- 23,24,25,26,27,pentanor-D 3 (JX) and 22-(m-Hydroxyphenyl-23,24,25,26,27- pentanor-D 3 (JY)
  • the A-ring phosphine oxide (48 mg, 0.11 mmol) in dry THF (1.8 mL) was cooled to -78°C and n-butyllithium (1.5 M in hexanes, 0.074 mL, 0.11 mmol) was added dropwise via a syringe.
  • the resulting deep red solution was stirred for 10 minutes and then treated with a solution of the appropriate CD-ring ketone (28 mg, 0.070 mmol) in dry THF (0.6 mL) via cannula.
  • the mixture was stirred 2 hours at -78°C, warmed to room temperature and quenched with water (5 mL).
  • the aqueous layer was separated and extracted with EtOAc (3 x 5 mL).
  • the protected vitamin (19.2 mg, 0.03 mmol) in dry THL (1 mL) was placed under argon and TBAF (1 M in THF, 0.30 mn, 0.30 mmol) was added dropwise. After stirring 18 hours, the solvent was partially evaporated and diluted with water (5 mL). After extracting the aqueous layer with EtOAc (3 x 5 mL), the combined organic layers were washed with brine and dried over Na 2 SO 4 . The residue was purified by HPLC (20% EtOAc/hexanes) and after vacuum drying afforded 2.8 mg (23%) of JY.
  • Vitamin D Gene Regulation, Structure-Function Analysis and Clinical Application (Norman, A.W., Bouillon, R., and Thomasset, M., eds) pp. 146-154, Walter de Gruyter, Berlin.

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Abstract

Methods for controlling genomic and nongenomic cellular responses which are mediated by 1α,25-(OH)2 vitamin D3[1α,25-(OH)2D3]. The method involves treating cells which exhibit nongenomic responses to 1α,25-(OH)2D3 with the vitamin C3 analogues 1,25(OH)2-previtamin D3 or 1β,25-(OH)2 vitamin D3 [1β,25-(OH)2D3]. The 1,25(OH)2-previtamin D3 functions as an agonist to promote the cellular response while the 1β,25-(OH)2D3 functions as an antagonist. Transcaltachia is one of the nongenomic responses which is affected and can be controlled by administration of 1,25(OH)2-previtamin D3 or 1β,25-(OH)2D3. Fifteen additional analogues are disclosed which may be used to control genomic and/or nongenomic cellular responses.

Description

VITAMIN D3 ANALOGUES AND PATHWAY TO MEDIATE DISORDERS
Λ
BACKGROUND OF THE INVENTION
This is a continuation-in-part of copending application Serial No.
08/173,561 which was filed on December 23, 1993. This invention was made with Government support under Grant Nos. DK-09012 and DK-
10 16,595, awarded by the National Institute of Health. The Government has certain rights in this invention.
li Field of the Invention.
The present invention relates generally to the vitamin D endocrine 15 system. More particularly, the present invention relates to a method for controlling genomic and nongenomic cellular responses which are mediated by 1σ,25-(OH)2 vitamin D3 [1σ,25-(OH)2D3].
. Description of Related Art.
20 The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and identified in the appended bibliography.
25 Vitamin D3 is a secosteroid which is responsible for a wide variety of biological responses in higher animals. These biological responses include maintenance of calcium homeostasis, immunomodulation and selected cell differentiation. Vitamin D3, itself, is biologically inert. However, metabolism of vitamin D3 to metabolites such as 1σ,25-(OH)2D3 results in the formation
30 of biologically active compounds which are responsible for the wide array of biological responses which are observed as part of the vitamin D endocrine system. 1σ,25-(OH)2D3 generates many biological responses by interaction with nuclear receptors. The result of this interaction with nuclear receptors is the regulation of gene transcription (1 ,2,3). The nuclear receptor for
1σ,25-(OH)2D3 has been shown to be present in 30 different tissues and it belongs to the same super family of proteins which includes receptors for the steroid hormones, retinoic acid and thyroxine (1 ,4,5). In addition to the above-mentioned genomic responses, it has become apparent that a subset of biological responses are mediated by 1σ,25-(OH)2D3 via a nongenomic mechanism (3,6). These biological responses include the rapid stimulation of intestinal Ca2+ transport known as transcaltachia (7-9). Transcaltachia involves the opening of Ca2+ channels (10). Other nongenomic cellular responses which are mediated by 1σ,25-(OH)2D3 include opening of voltage- gated Ca2+ channels in rat osteosarcoma cells (11 ,12) as well as other rapid effects in kidney (13), liver (14), parathyroid cells (15) and intestine (16).
SUMMARY OF THE INVENTION
In accordance with the present invention, it was discovered that certain analogues of 1σ,25-(OH)2D3 are effective in controlling nongenomic cellular responses which are mediated by 1cr,25-(OH)2D3. As a feature of the present invention, specific analogues are selected which function as either antagonists or agonists of the nongenomic cellular response. As a result, the cellular responses mediated by 1σ,25-(OH)2D3 can be controlled.
Transcaltachia is a particular nongenomic cellular response which can be controlled in accordance with the present invention. Vitamin D analogues which can be used as agonists of transcaltachia include 1σ,25- (OH)2-previtamin D3. When a decrease in transcaltachia is desired, 1 ?,25- (OH)2 vitamin D3 [1 ?,25-(OH)2D3] is used as an antagonist.
The control procedures provided in accordance with the present invention are effective in limiting or increasing transcaltachia and other nongenomic responses of 1σ,25-(OH)2D3 which are exhibited by a variety of cells. The present invention is useful in controlling nongenomic responses both in vivo and in vitro. As another feature of the present invention, fifteen analogues are provided which are effective in controlling either genomic or nongenomic cellular responses. The fifteen analogues are described in the following detailed description. The above described and many other features and attendant advan¬ tages of the present invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the metabolic scheme for producing vitamin D3 including the formation of previtamin D3.
FIG. 1 B shows the two conformations of the hormonally active form of vitamin D3, namely 1σ,25-(OH)2D3. FIG. 2 shows the equilibrium relationship between 1 ,25-(OH)2-pre- vitamin D and 1σ,25-(OH)2D3 forms.
FIG. 3 depicts the structures of 1σ,25-(OH)2D3, 1/?,25-(OH)2D3 and other related analogues.
FIG. 4 depicts the structures of 1σ,25-(OH)2D3, 1£,25-(OH)2D3 and other related analogues.
FIG. 5A depicts the effect of 1 ,25-(OH)2-d5-pre-D3 and 1 ,25-(OH)2D3 on the appearance of 45Ca2+ in the venous effluent of perfused duodena from vitamin D-replete chicks. Each duodenum, filled with 45Ca2+ (5 //Ci/ml) in GBSS, was vascularly perfused (25°C) for the first 20 minutes with control medium (GBSS containing 0.125% bovine serum albumin and 0.05 μ\ of ethanol/ml) and then at time zero either 60 pwi 1 ,25-(OH)2-d5-pre-D3, 60 pM 1 ,25-(OH)2-d5-pre-D3, or control medium. Values are the mean ±
S.E. for five duodena within each experimental group. •, 1 ,25-(OH)2D3; Δ,
1 ,25-(OH)2-d5-pre-D3; D, control. FIG. 5B shows the dose-response analysis of 1 ,25-(OH)2D3 and 1 ,25- (OH)2-d5-pre-D3 in stimulating transcaltachia in the perfused duodena. The experimental conditions were as described in FIG. 5A. Values are the mean ± S.E. at 30 minutes for three to five duodena perfused with each concentration of agonist. *p < 0.02; **p < 0.01 ; ***p < 0.005 with respect to duodena not exposed to agonist.
FIG. 6 shows the result of evaluation of 45Ca2+ uptake in osteosarcoma cells stimulated by 1 ,25-(OH)2D3 or 1 ,25-(OH)2-d5-pre-D3. The left inset displays the 45Ca2+ uptake properties of the cells in resting buffer (R), stimulating buffer (S), and when exposed to the optimal con¬ centration of 1 ,25-(OH)2D3, designated as C. The ROS 17/2.8 cells were assayed for 45Ca2+ as described in Example 1. In all cases, additions of the secosteroids were made to cells exposed to the resting buffer. 1 ,25-(OH)2D3 stimulated 45Ca2+ uptake with a maximum response occurring between 1.0 and 5.0 nM (see Re. 28). Data points represent the mean of triplicate measurements ± S.D.
FIG. 7A shows the affinity of the 1 ,25-(OH)2D3 nuclear receptor from pig intestinal mucosa for 1 ,25-(OH)2D3 (•), 1 ,25-(OH)2-d5-pre-D3 (Δ), and
1 ,25-(OH)2-d5-D3 ( A ). Data presented are mean ± S.D. of three experiments.
FIG. 7B shows the determination of the RCI for the chick intestinal receptor and the pig receptor for 1 ,25-(OH)2D3 (analog C), 1 ,25-(OH)2-d5- pre-D3 (analog HF), and 1 ,25-(OH)2-d5-D3 (analog HG). The RCI values are indicated in the figure and represent the mean and where available ± S.E. (n = 5) and were calculated as described in Example 1.
FIG. 8 shows the affinity of purified human DBP for 1 ,25-(OH)2D3 (o),
1 ,25-(OH)2-d5-pre-D3 (Δ), and 1 ,25-(OH)2-d5-D3 ( A ). Data presented are mean ± S.D. (n = 2). FIG. 9 shows the effect of 1 ,25-(OH)2D3 and its analogues on the serum concentration of osteocalcin after a single 400-ng intramuscular injection in vitamin D-deficient chicks. •, 1 ,25-(OH)2D3; Δ, 1 ,25-(OH)2-d5-
pre-D3; A , 1 ,25-(OH)2-d5-D3. Values represent mean ± S.E. (eight birds/ group).
FIG. 10A shows the effect of chronic administration to mice of 1 ,25-
(OH)2D3 and its analogues 1 ,25-(OH)2-d5-pre-D3 and 1 ,25-(OH)2-d5-D3 on serum Ca2+. The mice were administered the indicated daily intraperitoneal dose of the indicated secosteroid for 7 days. Values represent the mean ± S.E. (six mice/group). *p < 0.01 in comparison with the control group.
FIG. 10B shows the effect of chronic administration to mice of 1 ,25-
(OH)2D3 and its analogues 1 ,25-(OH)2-d5-pre-D3 and 1 ,25-(OH)2-d5-D3 on osteocalcin. The mice were administered the indicated daily intraperitoneal dose of the indicated secosteroid for 7 days. Values represent the mean ± S.E. (six mice/group). *p < 0.01 in comparison with the control group.
FIG. 11 shows the effect of 1 ,25-(OH)2D3 and its analogues in MG-63 cells on the inhibition of proliferation (FIG. 11 A) and induction of osteocalcin
(FIG. 11 B). •, 1 ,25-(OH)2D3; Δ, 1 ,25-(OH)2-d5-pre-D3; A , 1 ,25-(OH)2-d5-D3.
The data presented are from a representative experiment that was repeated twice.
FIG. 12 shows the effect of 1 ,25-(OH)2D3 and its analogues on differentiation of HL-60 cells. The differentiating effect was evaluated by nitro blue tetrazolium (NBT) reduction. •, 1 ,25-(OH)2D3; Δ, 1 ,25-(OH)2-d5-
pre-D3; and A , 1 ,25-(OH)2-d5-D3. The data presented are from a representative experiment that was repeated twice.
FIG. 13 shows the results of screening of the four A-ring diastereomers of 1σ,25-(OH)2D3 in the transcaltachia assay. The effect of each analog on the appearance of 45Ca2+ (5 //Ci/ml) in GBSS, was vascularly perfused (25°C) for the first 20 minutes with control medium (GBSS containing 0.125% bovine serum albumin and 0.05 μ\ of ethanol/ml) and then at time zero with the indicated concentration of the stipulated analog or control medium. Values are the mean ± S.E. for five duodena within each experimental group. •, indicated analog; o, control.
FIG. 14 depicts the antagonistic action of 1 ?,25-(OH)2D3-stimulated intestinal 45Ca2+ transport activity in the perfused chick duodena. Each duodenum, filled with 45Ca2+ (5 Ci/ml) jn GBSS, was vascularly perfused (25°C) for the first 20 minutes with control medium (GBSS containing 0.125% bovine serum albumin and 0.05% μ\ of ethanol/ml) and then with either 300 pM 1σ,25-(OH)2D3 (analog C) or 300 pM 1 ?,25-(OH)2D3 (analog HL) alone or in combination at 300 pM. Values are the mean ± S.E. for four or five duodena per group.
FIG. 15 depicts the results of the evaluation of the ability of 1 ?,25- (OH)2D3 to inhibit the agonist actions of 1σ,25-(OH)2D3 on transcaltachia. In FIG. 15A, the 1 ?,25-(OH)2D3 (HL) was presented to the perfused duodenum either in advance or simultaneously with 1σ,25-(OH)2D3 at varying concentrations. The data presented represent the mean ± S.E. from four or five duodena. •, analog HL and C [1σ,25-(OH)2D3]; o, control. In FIG. 15B, dose-response relationship of 1 ?,25-(OH)2D3 inhibiting the stimulation of transcaltachia by 300 pM 1σ,25-(OH)2D3. Data represent the ratio of treated to basal values ± S.E. extracted from a time-course plot (as in FIG. 15A) at 32 minutes.
FIG. 16 shows the inhibition of 5Ca2+ uptake in osteosarcoma cells stimulated by 1σ,25-(OH)2D3(C) by 1/?,25-(OH)2D3 (HL). The left side displays the 45Ca2+ uptake properties of the cells in resting buffer (/■?), stimulating buffer (S), and when exposed to the optimal concentration of 1σ,25-(OH)2D3 designated as C. In each treatment containing both HL and C, the concentration of HL is the upper value and C the lower value. The ROS 17/2.8 cells were assayed for 45Ca2+ as described in Example 2. In all cases, additions of the secosteroids were made to cells exposed to the resting buffer. 1 ,25-(OH)2D3 stimulated 45Ca2+ uptake with a maximum response occurs between 1.0 and 5.0 nM. Data points represent the mean of triplicate measurements ± S.D. FIG. 17 shows the dose-response effects of 1σ,25-(OH)2D3, 1 ?,25- (OH)2D3, and 1σ,25-(OH)2-3-epi-D3 on ICA (FIG. 17A) and BCM (FIG. 17B) in the vitamin D-deficient chick. The analogues and 1σ,25-(OH)2D3 were given intramuscularly to vitamin D-deficient chicks 12 hours before assay; the control D3 was 48 hours before assay. Results are expressed as mean ± S.E. of groups of five to seven chicks. Each assay included a negative control, open bar (-D) and a positive control of vitamin D3 3.25 mnol, solid black bar; the difference between these two groups was significant at p <0.01. A detailed evaluation was carried out for the four diastereomers in separate bioassays; the results are summarized in Table I.
FIG. 18 shows the effect of the four A-ring diastereomers in MG-63 cells on the induction of osteocalcin. Both 1σ,25-(OH)2D3 and 1 ?,25-(OH)2D3 were evaluated independently of one another for their ability to induce osteocalcin (FIG. 18A). •, 1σ,25-(OH)2D3; o, 1/?,25-(OH)2D3 (HL); D, 1/?,25-(OH)2-3-epi-D3 (HH). In addition, the ability of 10,25-(OH)2D3 to antagonize the actions of 1σ,25-(OH)2D3 on induction of osteocalcin was assessed (FIG. 18B). In this example, the concentration of 1 ?,25-(OH)2D3 was held constant at 10"9-10"7 M, whereas the concentration of
1σ,25-(OH)2D3 was varied from 10"11 to 10 ° M. V, 10"9 M 1/?,25-(OH)2D3 + varying concentrations of 1 σ,25-(OH)2D3; D , 1 ?,25-(OH)2D3, 10"8 M + varying concentrations of 1σ,25-(OH)2D3; Δ, 1 ?,25-(OH)2D3, 10"7 M + varying concentrations of 1σ,25-(OH)2D3. Also 1σ,25-(OH)2D3 (•) and 1 ?,25-(OH)2D3 (o) were evaluated alone, i.e. in the absence of the other secosteroid. For details, see Example 2. The data presented are from a representative experiment; three experiments were conducted.
FIG. 19 shows the effect of 1σ,25-(OH)2D3 and 1 ?,25-(OH)2D3 on dif¬ ferentiation of HL-60 cells. The differentiating effect of the secosteroids was evaluated by NBT reduction as described in Example 2. The data presented are from a representative experiment that was repeated twice. • , 1σ,25-(OH)2D3; o, 10,25-(OH)2D3; V, 1σ,25-(OH)2D3, 10'9 M + varying concentrations of 1/?,25-(OH)2D3; Δ, 1σ,25-(OH)2D3, 10"8 M + varying concentrations of 1£,25-(OH)2D3; D, 1σ,25-(OH)2D3, 10"7 M + varying concentrations of 1 ?,25-(OH)2D3.
FIG. 20 shows the effects of 1cr,25-(OH)2D3 A-ring analogues on keratinocyte differentiation. Human skin keratinocytes were grown in tissue culture in 96-well plates as described in Example 2. The A-ring analogues were added at the indicated concentrations, and then the rate of cell proliferation was assessed for 3 hours by the addition of [3H]thymidine. •,
[1σ,25-(OH)2D3; D, 1/. ,25-(OH)2D3; Δ, 1 ?,25-(OH)2-3-epi-D3; O , 1σ,25-(OH)2-3-epi-D3.
FIG. 21 sets forth the structural formulas for analogues GE, GF, HS, IB, JD, JM, JN, JO and JP in accordance with the present invention.
FIG. 22 sets forth the structural formulas for analogues JR, JS, JV, JW, JX and JY in accordance with the present invention. FIG. 23 depicts the synthesis pathway for making analogues GE and
GF.
FIGS. 24(A) and (B) show the results of testing of analogues JM and JN, respectively, as set forth in Example 6. In the testing, augmented 5Ca2+ transport in duodenal loops was vascularly perfused with 1σ,25(OH)2D3, or 1σ,25(OH)2-7-dehydrocholesterol (JM), or 1σ,25(OH)2-lumisterol3 45Ca2+ (5 //Ci/ml of buffer), and vascularly perfused with control medium for the first 20 minutes with collection of the venous effluent occurring at 2-minute intervals during the final 10 minutes to establish basal transport rates. The duodena were then either re-exposed to control medium containing the vehicle ethanol (0.005%, final concentration) through the celiac artery, or vascularly perfused with 300 pM agonist, or 650 pM agonist. The venous effluent was again collected at 2 minute intervals for liquid scintillation spectrophotometry. The results during the treated phase were normalized to average basal transport within each duodena. Values represent mean ± SEM (or n = 4 in each group, (A) perfusion with JM; (B) perfusion with JN. Included in each graph are both vehicle control and 650 pM 1σ,25(OH)2D3 as positive control.
FIGS. 25(A) and (B) show the results of testing of analogues JM and JN, respectively, as set forth in Example 6. The tests involved dose- response analyses of JM and JN as agonists for transcaltachia. Duodena which were perfused as described in FIG. 24 with vehicle or a range of JM or JN concentrations. Normalized transport after 40 minutes of perfusion is depicted for the indicated concentrations of (A) JM; (B) JN.
FIG. 26 is a chart showing the various diseases which may be treated using the analogous of the present invention.
FIG. 27 is a schematic representation of the synthesis of analogue IB.
DETAILED DESCRIPTION The present invention provides methods for controlling transcaltachia and other nongenomic cellular responses which are mediated by 1σ,25- (OH)2D3. Activation of the nongenomic cellular response is accomplished in accordance with the present invention by treating cells with 1 ,25-(OH)2- previtamin D3 (compound BC in FIG. 2). The 1 ,25-(OH)2-previtamin D3 has been found to be an agonist of transcaltachia as is described in detail in Example 1.
Another aspect of the present invention involves antagonizing the nongenomic responses mediated by 1 ,25-(OH)2D3 by treating cells with 1/?,25-(OH)2D3 as described in Example 2. Treatment with 1σ,25-(OH)2-previtamin D3 or 1 ?,25-(OH)2D3 can be accomplished as set forth in the examples, i.e. by direct injection or perfusion of the analog in an appropriate pharmaceutical carrier. The dosage levels are preferably similar to the dosage levels described in the examples. The type of cells treated can be any of those which are known to undergo nongenomic cellular responses which are mediated by 1σ,25-(OH)2D3. The cells may be treated either in vivo or in vitro. For example, the preferred nongenomic mediated response which can be con- trolled in accordance with the present invention is transcaltachia which involves opening of the Ca2+ channels in the intestines. Control of caltachia is preferably accomplished by selecting the desired agonist or antagonist analog described above and introducing it by way of perfusion into the intestine.
The following two examples provide further details with respect to the present invention and the use of previtamin D3 or 1/?,25-(OH)2D3 to alter nongenomic responses which are mediated by 1σ,25-(OH)2D3.
Example 1 —
The following example demonstrates that previtamin D3 is effective in stimulating two nongenomic cellular responses which are mediated by 1σ,25-(OH)2D3. Specifically the example shows that transcaltachia in isolated perfused chicken duodenum is stimulated and Ca+ channel opening in rat osteogenic sarcoma cells is also stimulated.
FIG. 1A shows the metabolic scheme for production of vitamin D3. The provitamin, 7-dehydrocholesterol, present in the skin is converted by ultraviolet irradiation into the secosteroid vitamin D3. Previtamin D3 is in thermal equilibrium with vitamin D3; the conversion involves a [1 ,7]-sigmatropic shift, i.e. the intramolecular migration of a hydrogen from carbon-19 to carbon-9. The resulting product vitamin D3 is a conformationally mobile molecule with respect to the orientation of the A ring in relation to the C/D ring structure. As indicated in the bottom of this figure, the seco-B ring can assume one of two conformations as a consequence of rotation about the carbon 6-7 single bond; in the 6-s-c/s orientation the A ring is related to the C/D rings as in the conventional steroid orientation, referred to here as the "steroid-like conformation" and when the conformation is in the 6-s-trans orientation, the A ring is present in an "extended conformation." FIG 1 B depicts two orientations of 1 ,25-(OH)2D3. 1 ,25-(OH)2D3 has free rotation about the single bond between carbon-6 and carbon-7; accordingly it can assume in solution the steroid-like conformation (6-s-c/s) or the extended conformation (6-s-frans) orientation.
45Ca2+ was obtained from Du Pont-New England Nuclear. 1 ,25-
(OH)2D3 was obtained from Hoffman La Roche (Nutley, NJ). [Methyl- 3H]Thymidine (2 Ci/mmol) was purchased from Amersham Corp. Cell culture media were purchased from GIBCO. Penicillin and streptomycin were from Boehringer (Mannheim, Germany). 4-nitro blue tetrazolium was obtained from Sigma. Human plasma vitamin D-binding protein (DBP) was prepared by affinity chromatography as described previously (17,18). Chemical Synthesis of 1,25-(OH)2-9, 14, 19, 19, 19- d5-pre-D3 (Analog HF)
Analogues HF and 1 ,25-(OH)2-d5-D3 (analog HG) were synthesized according to the method of Curtin and Okamura (19). When 1 ,25-(OH)2-d5- pre-D3, which had been stored at -60°C for about 1 year with occasional warming to ambient temperatures for withdrawal of samples for biological evaluation, the sample analyzed to be comprised of 4.4% of the vitamin and 95.6% of the previtamin form of analog HF. A similar sample of 1 ,25-(OH)2- d5-pre-D3 maintained at the same temperature without occasional warming or a freshly synthesized sample was, by comparison, found to be comprised of 1.2% of the vitamin form and 98.8% of the preform. The composition determinations were carried out by analytical high performance liquid chromatography on a normal phase column (Whatman Partisil column using 90% ethyl acetate, 10% hexane as solvent; 5 ml/min flow rate) using a Waters photodiode array detector. The electronically integrated peak area was the average of two values, one obtained at 260 nm and the other at 266 nm (1 ,25-(OH)2-d5-D3D E = 17,200; 1 ,25-(OH)2-d5-pre-D3 E = 7,200). A separate comparison using cut and weigh integrated peak areas was used as a cross-check, and the overall agreement was estimated to be ± 0.7%. The retention times were as follows: 1 ,25-(OH)2-d5-D3, about 18 minutes; 1 ,25-(OH)2-d5-pre-D3, 24.5 minutes. These retention times are essentially identical to those of the undeuteriated forms of these two secosteroids. Animals and Cells White Leghorn cockerels (Lakeview Farms, Lakeview, CA) were obtained on the day of hatch and maintained on a vitamin D-supplemented diet (1.0% calcium and 1.0% phosphorous; O.H. Kruse Grain and Milling, Ontario, CA) for 5-6 weeks to prepare normal vitamin D3-replete chicks. All experiments employing animals were approved by the University of California-Riverside Chancellor's Committee on Animals in Research.
The human promyelocytic leukemia cell line (HL-60) and the MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD). One-day-old RIR chicks were housed in a windowless room and raised on a vitamin D-replete diet for 1 week followed by a vitamin D- deficient diet (Hope Farms, Woerden, The Netherlands) for the next 5 weeks. After a total of 6 weeks, they were divided into groups and received a single intramuscular injection of 400 ng of 1 ,25-(OH)2D3 or analogues HF or HG solubilized in 10:10:80 v/v/v ethanol, Tween 80, NaCI, 0.9%. Blood was obtained from the wing vein at 2, 4, 6, 9, 12, 16, 24, and 36 h after the time of administration of the secosteroid. Serum osteocalcin was measured by radioimmunoassay using specific anti-chick antisera raised against these chick proteins. Mice, strain NMRI, were fed a normal diet (Hope Farms) for 40-60 days. They received a daily subcutaneous dose of 1 ,25-(OH)2D3 or analogues HF and HG for 7 days. Serum Ca2+ was determined via atomic absorption spectrophotometry and serum osteocalcin levels via radioimmunoassay. The pig intestinal mucosa was obtained from a normal 20-kg pig under Ketalar anesthesia. The mucosa was scraped and stored at -80°C until time of preparation of the 1 ,25-(OH)2D3 nuclear receptor (see below).
The ROS 17/2.8 cells obtained from Merck, Sharp and Dohme (West Point, PA) were cultured in Dulbecco's modified Eagle's medium: Ham's F- 12 medium 1 :1 containing 10% fetal calf serum (GIBCO-BRL). The medium was supplemented with 1.1 mM CaCI2 as described (24). For 45Ca2+ uptake experiments, cells were seeded at a density of 30,000 cells/ml into 3.5-cm dishes and grown to approximately 50% confluence. Calcium Uptake Assays ROS 17/2.8 cells were assayed for Ca2+ uptake using procedures described previously (20). Assays were standardized to 1 minute, which preliminary experiments demonstrated to be within the interval of linear uptake. Culture medium was aspirated and the cells washed with room temperature Hanks' buffered saline and then incubated 1 minute with either "resting buffer" (containing, in mM concentration, 132 NaCI, 5 KCl, 1.3 MgCI2, 1.2 CaCI2, 10 glucose, and 25 Tris-HCl, pH 7.4) or "stimulating buffer" (containing, in mM concentration, 5 NaCI, 132 KCl, 1.3 MgCI2, 1.2 CaCI2, 10 glucose, and 25 Tris-HCl, pH 7.4). Both uptake solutions contained 12.5 /yCi/ml 5Ca2+ (Du Pont-New England Nuclear) and the concentrations of vitamin D agonists indicated in FIG. 2. Uptake was terminated by aspiration of the labeling solution, followed by three washes with ice-cold resting buffer. Cell-associated 45Ca2+ was extracted by a 2-h incubation with 0.5 M NaOH and measured by liquid scintillation counting. It was found that 45Ca2+ uptake by monolayer cultures of ROS 17/2.8 cells was density-dependent. Maximal uptake rates were consistently found for cultures that were between 50 and 80% confluent.
Intestinal 45Ca2+ Transport (Transcaltachia) Measurements of 45Ca2+ transport were carried out in perfused chick duodena as described previously (21-23). Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml/100 g), and the duodenal loop was surgically exposed. Blood vessels branching off from the celiac artery were lighted before cannulation of the celiac artery itself and simultaneous initiation of vascular perfusion. The duodenal loop was then excised and, after cannulation of the celiac vein, placed between layers of saline-moistened cheesecloth at 24°C. The arterial perfusion was initiated during cannulation with modified Grey's balanced salt solution (GBSS) modified to contain 0.9 mM CaCI2 and oxygenated with 95% 02 and 5% CO2 at a flow rate of 2 ml/min. An auxiliary pump was used for the introduction of vehicle (ethanol) or test substances plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min. The intestinal lumen was then flushed and filled with GBSS containing 45Ca2+ (5 /Ci/MI) but without bicarbonate or glucose. A basal transport rate was established by perfusion with control medium for 20 minutes after the lumen was filled with 45Ca2+. The tissue was then exposed to 1 ,25-(OH)2D3 or 1 ,25-(OH)2-d5-pre-D3 or reexposed to control medium for an additional 40 minutes. The vascular perfusate was collected at 2-minute intervals during the last 10 minutes of the basal and during the entire treatment period. Duplicate 100-//I aliquots were taken for determination of the 5Ca2+ levels by liquid scintillation spectrometry. The results are expressed as the ratio of the 45Ca2+ appearing in the 40-minute test period over the average initial basal transport period.
Ligand Binding Studies
The relative ability of each analog to compete with [3H]1,25-(OH)2D3 for binding to the chick intestinal nuclear receptor for 1 ,25-(OH)2D3 was carried out under in vitro conditions according to standard procedures (24,25). In this assay, increasing concentrations of nonradioactive 1 ,25-(OH)2D3 or the test analog are incubated with a fixed saturating amount of [3H]1 ,25-(OH)2D3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [3H]1 ,25-(OH)2D3 was then calculated and plotted as a function of the relative concentration of the analog and [3H]1 ,25-(OH)2D3. The plots give linear curves characteristic for each analog, the slopes of which are equal to the analog's competitive index value (24). The competitive index value for each analog was then normalized to a standard curve obtained with nonradioactive 1 ,25-(OH)2D3 as the competing steroid and placed on a linear scale of relative competitive index (RCI), where the RCI of 1 ,25-(OH)2D3 by definition is 100. Binding to the 1 ,25-(OH)2D3 receptor was determined in mucosa obtained from a vitamin D-replete pig. Frozen (- 80°C) duodenal mucosa was sonicated in 4 volumes of buffer (0.5 M Tris- HCI, 0.5 M KCl, 5 mM dithiothreitol, 10 mM Na2MoO4, 1.5 mM EDTA, pH 7.5). The high speed supernatant was then incubated with 0.2 nM [3H]1 ,25- (OH)2D3 and increasing concentrations of nonradioactive 1 ,25-(OH)2D3 or its analogues in a final volume of 0.3 ml overnight at 25°C followed by 5 minutes at 4°C. Phase separation was then obtained by the addition of cold dextran-coated charcoal.
Binding of the 1 ,25-(OH)2D3 and its analogues to hDBP was performed at 4°C essentially as described previously (26). [3H]1 ,25-(OH)2D3 and 1 ,25-(OH)2D3 or its analogues were added in 5 μ\ of ethanol into glass tubes and incubated with hDBP (0.18 μu) in a final volume of 1 ml (0.01 M Tris-HCl, 0.154 M NaCI, pH 7.4) for 4 h at 4°C. Phase separation was then obtained by the addition of 0.5 ml of cold dextran-coated charcoal.
Culture Conditions for HL-60 and MG-63 Cells HL-60 cells were seeded at 1.2 x 105 cells/ml, and 1 ,25-(OH)2D3 or its analogues were added in ethanol (final concentration <0.2%) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer). After 4 days of culture in a humidified atmosphere of 5% CO2 in air at 37°C the dishes were shaken to loosen any adherent cells, and all cells were then assayed for differentiation by nitro blue tetrazolium reduction assay and for proliferation by [3H] thymidine incorporation. The MG-63 cells were seeded at 5 x 103 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 μ\ of Dulecco's modified Eagle's medium containing 2% of heat-inactivated charcoal-treated fetal calf serum, and 1 ,25-(OH)2D3 or its analogues were added in ethanol (final concentration <0.2%). After 72 hours of culture in a humidified atmosphere of 5% CO2 in air at 37°C the inhibition of proliferation by [3H] thymidine incorporation and measurement in the medium of osteocalcin concentration using a homologous human radioimmunoassay were performed (26). Nitro Blue Tetrazolium Reduction Assay Superoxide production was assayed by nitro blue tetrazolium- reducing activity as described previously (26). HL-60 cells at 1.0 x 105 cells/ml were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/ml) and nitro blue tetrazolium (2 mg/ml) and incubated for 30 minutes at 37°C. The percentage of cells containing black formazan deposits were determined using a hemacytometer.
Statistics
Statistical evaluation of the data was performed by Student's test for unpaired observations.
This example compares the biological profile of the two deuteriated analogues, 1 ,25-(OH)2d5-pre-D3 (HF) and 1 ,25-(OH)2-d5-D3 (HG) in relation to 1 ,25-(OH)2D3. The structures of the analogues are given in FIG. 2. The 1 ,25-(OH)2-d5-pre-D3, analog HF, is kinetically suppressed (21) in its previtamin form (because of a primary deuterium kinetic isotope effect) and thus can function as an analog only of the 6-s-c/s form of 1 ,25-(OH)2D3 (FIG. 1). Nongenomic Actions — FIG. 5A shows the results of an evaluation of the relative ability of 1 ,25-(OH)2D3 and 1 ,25-(OH)2-d5-pre-D3 to stimulate the nongenomic biological response of transcaltachia. Vascular perfusion with the physiological concentration of 60 pM 1 ,25-(OH)2-d5-pre-D3 for 34 minutes yielded a 4.5-fold increase in 45Ca + transport over control levels. The stimulatory effect of both secosteroids on 45Ca2+ becomes significant within 2-8 minutes as observed previously (7,9). FIG. 5B shows the dose- responsive relationship for each secosteroid in terms of its ability to stimulate transcaltachia. The analog 1 ,25-(OH)2-d5-pre-D3 was able to stimulate transcaltachia significantly at a dose of 10 pM, and the maximal response was attained at 60 pM secosteroid. As had been noted before for all secosteroids that have been evaluated with regard to their transcaltachic activity, the dose-response for 1 ,25-(OH)2-d5-pre-D3 is biphasic (7-9). 1 ,25-(OH)2D3 is active at the low concentration of 25 pM, and the maximum stimulation is achieved over the range of 60-650 pM 1 ,25-(OH)2D3 (FIG. 5B). Also the typical biphasic dose response is apparent. It is apparent from FIG. 5B that there is no significant difference in the potency of analog 1 ,25-(OH)2-d5-pre-D3, which is locked in the pre-form with that of 1 ,25-(OH)2D3, which suggests that the signal transducing agent or receptor for transcaltachia is capable of responding to a secosteroid in the 6-s-c/s conformation.
FIG. 6 shows the results of the evaluation of the ability of 1 ,25-(OH)2- d5-pre-D3 to stimulate 5Ca2+ uptake into ROS 17/2.8 cells. The concentration range of 1-10 x 10"9 M 1 ,25-(OH)2-d5-pre-D3 produced a maximum uptake of 45Ca2+ within 1 minute of the application of the secosteroids. Previous studies have established that this is the range of maximum response to 1 ,25-(OH)2D3 (15,27). The voltage-gated Ca2+ channel can either be opened by exposure to appropriate agonists (vitamin D analogues or the dihydropyridine BAY K- 8644 (15)) or by depolarization of the cell membrane, which is achieved by the 132 mM external KCl (stimulating buffer; see "Experimental Proce¬ dures"). Such stimulated uptake of 45Ca2+ in the presence of depolarizing extracellular solutions is characteristic of cells expressing voltage-gated Ca2+ channels, and the level of this stimulation is directly related to the concentration of the Ca2+ channels on the cell surface. Thus, the maximum influx of 45Ca2+ which can be achieved (see inset of FIG. 6) occurs in the presence of high external K+. The level of 45Ca2+ uptake occurring in low K+ represents the basal uptake, which is a reflection of the Ca2+ permeability of the resting membrane. In growth phase ROS 17/2.8 cells the maximum ratio of (stimulated)/( resting), S/R is approximately 2.4-fold, which represents 100% Ca2+ channel opening. Both the analog 1 ,25-(OH)2-d5-pre- D3 and 1 ,25-(OH)2D3 achieve a 2-fold stimulation of 45Ca2+ uptake over that which occurs in the low K+ environment. Again there is no significant difference in the biological activity of 1 ,25-(OH)2d5-pre-D3, which is locked in the previtamin form as compared with the 1 ,25-(OH)2D3, which as a facile interchange between the steroid-like conformation and the extended steroid conformation.
Ligand Binding Studies — FIG. 7 shows the results of the determination of the RCI for binding to the intestinal nuclear 1 ,25-(OH)2D3 receptor from both the chick and pig, as determined under in vitro conditions. The calculated RCI for the chick and pig, respectively, is as follows: 1 ,25-(OH)2D3 (RCI = 100% and 100%), 1 ,25-(OH)2-d5-D3 (RCI = 92% and 67%), and for 1 ,25-(OH)2-d5-pre-D3 (RCI = 10% and 4%). Thus there is no effect of species difference in binding of these three secosteroids to the intestinal nuclear receptor. The steroid that is kinetically repressed in the previtamin form, the 1 ,25-(OH)2-d5-pre-D3, has a reduction in its RCI from 90-100% to 12-14%, which shows that the nuclear 1 ,25-(OH)2D3 receptor can discriminate between the previtamin 6-s-c/s and the vitamin D form, which may exist either as the 6-s-c/s or 6-s-trans forms, with the latter predominating.
The principal carrier of vitamin D secosteroids in the blood compart¬ ment is the plasma DBP. This protein has a binding domain that tightly binds its ligand with a K,, of 5 x 10"9 M and 5 x 10"8 M for 25-(OH)D3 and 1 ,25-(0H)2D3, respectively (29); thus the affinity of any ligand for DBP will effectively determine its "free" concentration in the plasma and perhaps influence its relative availability to target cells. In FIG. 8 is presented the competition curves of 1 ,25-(OH)2D3, 1,25-(OH)2-d5-D3, and 1,25-(OH)2-d5- pre-D3, respectively, with [3H]1 ,25-(OH)2D3 for binding to human DBP as determined under in vitro conditions. The calculated RCI for 1 ,25-(OH)2D3 is 100%, 95% for 1 ,25-(OH)2-d5-D3, and 7% for 1 ,25-(OH)2-d5-pre-D3. This result suggests that the DBP ligand binding domain has a preference for the extended (6-s-trans conformation) steroid conformation of vitamin D3 secosteroids as compared with the steroidal (6-s-c/s) conformation. Thus freezing 1 ,25-(OH)2-d5-pre-D3 in the previtamin form reduces the RCI from 100 to 7%. This reduced affinity for DBP means effectively that there will be a higher free concentration of 1 ,25-(OH)2-d5-pre-D3 as compared with 1 ,25-(OH)2D3 or 1 ,25-(OH)2-d5-D3. Genomic Actions — FIGS. 7-10 show the evaluation of the biological efficacy of the previtamin form of the pentadeuteriated analog of 1 ,25-(OH)2D3 under in vivo conditions as well as in cultured cells. FIG. 9 shows the levels of serum osteocalcin which are achieved after a single intramuscular injection of vitamin D-deficient chick with 400 ng of either 1 ,25-(OH)2D3, 1 ,25-(OH)2-d5-D3, or 1,25-(OH)2-d5-pre-D3. 1 ,25-(OH)2D3 has been shown to induce via interaction with a nuclear 1 ,25-(OH)2D3 receptor present in bone osteoblast cells the de novo biosynthesis of osteocalcin; small amounts of the osteocalcin are released into the blood (as a consequence of bone remodeling) where it may be conveniently determined via a radioimmunoassay (26). It is apparent that the 1 ,25-(OH)2-d5-pre-D3, when administered as a single dose under in vivo conditions, has little ability to interact effectively with the nuclear 1 ,25-(OH)2D3 receptor to induce osteocalcin. In contrast, both 1 ,25-(OH)2D3 and 1 ,25-(OH)2-d5-D3 caused a significant increase in the plasma levels of osteocalcin. Although identical does of 1 ,25-(OH)2D3 and 1 ,25-(OH)2d5-D3 were administered, treatment with the deuteriated analog resulted in a consistently higher induction of the plasma osteocalcin levels.
FIG. 10 shows the levels of serum Ca2+ and osteocalcin achieved after 1 week of daily treatment with doses of 1 ,25-(OH)2D3 or the analogues 1 ,25-(OH)2-d5-pre-D3 and 1 ,25-(OH)2-d5-D3. Both 1 ,25-(OH)2D3 and 1 ,25-(OH)2-d5-D3 were virtually equipotent in regard to evaluation of serum Ca2+ and osteocalcin. In contrast 1 ,25-(OH)2-d5-pre-D3 was only able to elevate serum Ca2+ at a dose (10 //g/kg/day) that was 20 times higher than the dose of 1 ,25-(OH)2D3 and 1 ,25-(OH)2-d5-D3 (5 /g/kg/day), which achieved a significant elevation of these parameters above the base line. An even higher dose of 1 ,25-(OH)2-d5-pre-D3 (50 / g/kg/day) was required to elevate the osteocalcin levels significantly above that of the control group. Since a single dose of 1 ,25-(OH)2-d5-pre-D3 did not elevate serum Ca2+ in chicks (FIG. 9), it is likely that the elevation of serum Ca2+ and osteocalcin which resulted in the mice has occurred as a consequence of the slow thermal conversion, in vivo, of 1 ,25-(OH)2-d5-pre-D3 to 1,25-(OH)2D3. 1 ,25-(OH)2D3 and its two analogues HF and HG were evaluated in MG-63 cells with respect to inhibition of proliferation as assessed by [3H]thymidine incorporation (FIG. 11 A) and induction of human osteocalcin (FIG. 11B). Both of these responses are mediated by the nuclear 1 ,25-(OH)2D3 receptor. In both assays, the 1 ,25-(OH)2-d5-pre-D3 had only approximately 1% of the activity of 1 ,25-(OH)2D3 or 1 ,25-(OH)2-d5-D3. In addition, the 1 ,25-(OH)2-d5-D3 was indistinguishable from 1 ,25-(OH)2D3 in its ability to induce osteocalcin and displayed approximately 90% of the activity of 1 ,25-(OH)2D3 in terms of its ability to inhibit cell proliferation. These results are consistent with the interpretation that the previtamin form of 1 ,25-(OH)2D3 is not able to efficiently interact with the nuclear 1 ,25-(OH)2D3 receptor.
The differentiation of HL-60 cells was markedly enhanced by the pre¬ sence of 1 ,25-(OH)2D3 or 1 ,25-(OH)2-d5-D3 (FIG. 12). In contrast the 1 ,25- (OH)2-d5-pre-D3 displayed only 1 -4% of the potency of 1 ,25-(OH)2D3, which again shows that the HL-60 nuclear 1 ,25-(OH)2D3 receptor does not effi¬ ciently bind this ligand.
In the above described example, the biological profile of 1 ,25-(OH)2- d5-pre-D3 has been compared with that of the pair of rapidly interconverting 6-s conformers of 1 ,25-(OH)2D3 (FIGS. 1 and 2). The example demonstrates that two nongenomic biological systems are fully responsive to the 1 ,25-(OH)2-d5-pre-D3 analog. Both the process of transcaltachia as studied in the isolated perfused chick duodenum (FIG. 5) and the process of Ca2+ channel opening in the rat osteogenic sarcoma cell line, ROS 17/2.8 cells (FIG. 6), respond with equivalent potency to both 1 ,25-(OH)2-d5-pre-D3 and 1 ,25-(OH)2D3. Thus the responsiveness of the signal transduction process for these two nongenomic systems occurs in two species, the rat and chick, and in two different vitamin D target organs, the intestine and bone. The present example demonstrates the inability of the analog 1 ,25-
(OH)2-d5-pre-D3 to interact with the nuclear 1 ,25-(OH)2D3 receptor and support genomic responses. FIG. 8 indicates that both the pig and chick intestinal 1 ,25-(OH)2D3 nuclear receptors discriminate against the previtamin form of the secosteroid. The RCI of 1 ,25-(OH)2-d5-pre-D3 for binding to the chick intestinal receptor was 10% and for the pig intestinal receptor was 4%. Accordingly, the nuclear 1 ,25-(OH)2D3 receptor's ligand binding domain favors the 6-s-trans conformer (extended steroid conformation) over the 6-s- c/s (steroid-like conformation). Further it was found that the presence of the five deuterium atoms on 1 ,25-(OH)2D3 as in 1 ,25-(OH)2-d5-D3 did not signi¬ ficantly alter the RCI (89.5% for chick and 67% for the pig).
The relative inability of the nuclear 1 ,25-(OH)2D3 receptor to effectively bind the 6-s-c/s (steroid-like conformation) of 1 ,25-(OH)2D3, as studied by the analog, 1 ,25-(OH)2-d5-pre-D3, shows that the latter compound would not be an efficient mediator of genomic responses. FIGS. 9-12 show the evaluation of 1,25-(OH)2-d5-pre-D3, 1 ,25-(OH)2-d5-D3, and 1 ,25-(OH)2D3 in four systems that all generate biological effects via a nuclear receptor- mediated regulation of gene transcription. FIG. 9 shows the in vivo measurement of serum osteocalcin levels, after a single dose of 1 ,25-(OH)2- d5-pre-D3 to chicks. FIG. 10 shows the results of daily administration of 1 ,25-(OH)2-d5-pre-D3 on serum Ca2+ and osteocalcin levels in mice. FIG. 11 shows, using MG-63 cells in cell culture, the inhibition of cell proliferation and the induction of osteocalcin. FIG. 12 shows, using HL-60 cells in culture, the inhibition of cell proliferation. The relative inability of analog 1 ,25-(OH)2-d5-pre-D3 (less than 2% for osteocalcin induction in vivo, FIG. 9; less than 1% for inhibition of proliferation in MG-63 cells, FIG. 11 A; approximately 2% for osteocalcin induction in MG-63 cells, FIG. 11 B; and approximately 10% in promoting differentiation of HL-60 cells), in comparison with 1 ,25-(OH)2D3 and 1 ,25-(OH)2d-5-D3, to effect the appropr¬ iate generation of a genomic response is consistent with the data of FIG. 8 reporting its low RCI for the nuclear receptor.
In addition, the very low ability of 1 ,25-(OH)2-d5-pre-D3 when admini- stered daily for 7 days to mice (FIG. 10) to elevate serum Ca + and osteocalcin levels, demonstrates that 1 ,25-(OH)2-d5-pre-D3 has a low ability to activate these genome-dependent responses. The above results also show that the previtamin form, 1 ,25-(OH)2-d5-pre-D3, is not subject to some metabolic transformation that converts it to the vitamin form, i.e. 1 ,25-(OH)2d-5-D3. The time duration of each of these assays was 40 h (chicks, FIG. 9); 7 days (mice, in FIG. 10), and 96 h (MG-63 cells, in FIG. 11 ; HL-60 cell, in FIG. 12), and if the vitamin form, 1 ,25-(OH)2d-5-D3 had been generated there would have been ample time for appearance of detectable manifestations of the various genomic responses. In fact the results from the chronic dosing of mice with 1 ,25-(OH)2-d5-pre-D3 (FIG. 10) show that the previtamin form is subject to metabolic clearance before it has had an opportunity to isomerize thermally into the biologically active 1 ,25-(OH)2d-5-D3.
The above example demonstrates that the previtamin D form of 1 ,25- (OH)2D3 is effective as an agonist of the nongenomic receptor for 1 ,25- (OH)2D3 and therefore may be used to initiate biological responses which utilize the nongenomic receptor.
Example 2 —
In this example, the use of 1 ?,25-(OH)2D3 as an antagonist to control nongenomic actions of 1σ,25-(OH)2D3 is described. The chemicals used in this example were obtained from the same source and prepared in the same manner as described in Example 1. Chemical synthesis of the three A-ring diastereo-isomers of 1cr,25-(OH)2D3 was achieved by the methods described by Muralidharan et al (30).
Animals and Cells
White Leghorn cockerels (Lakeview Farms, Lakeview, CA) were obtained on the day of hatch and maintained on a vitamin D-supplemented diet (1.2% calcium and 0.7% phosphorous; O.H. Kruse Grain and Milling, Ontario, CA) for 5-6 weeks to prepare normal vitamin D3-replete chicks. All experiments employing animals were approved by the University of California-Riverside Chancellor's Committee on Animals in Research. The human promyelocytic leukemia cell line (HL-60) and the human osteoblast MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD).
The rat osteosarcoma (ROS) 17/2.8 cells obtained from Merck, Sharp and Dohme (West Point, PA) were cultured in Dulbecco's modified Eagle's medium: Ham's F-12 medium 1 :1 containing 10% fetal calf serum (Life Technologies, Inc.). The medium was supplemented with 1.1 mM CaCI2 as described (37). For 45Ca2+ uptake experiments, cells were seeded at a density of 30,000 cells/ml into 3.5-cm dishes and grown to approximately 50% confluence.
Calcium Uptake Assays ROS 17/2.8 cells were assayed for Ca2+ uptake using procedures described previously (31). Assays were standardized to 1 minute, which preliminary experiments demonstrated to be within the interval of linear uptake. Culture medium was aspirated and the cells washed with room temperature Hanks' buffered saline and then incubated 1 minute with either "resting buffer" (containing 132 mM NaCI, 5 mM KCl, 1.3 mM MgCI2, 1.2 mM CaCI2, 10 mM glucose, and 25 mM Tris-HCl, pH 7.4) or stimulating buffer 5 mM NaCI, 132 mM KCl, 1.3 mM MgCI2, 1.2 mM CaCI2, 10 mM glucose, and 25 mM Tris-HCl, pH 7.4). Both uptake solutions contained 12.5 /Ci/ml 45Ca + (Du Pont-New England Nuclear) and the concentrations of vitamin D agonists indicated in FIG. 13. Uptake was terminated by aspiration of the labeling solution, followed by three washes with ice-cold resting buffer. Cell-associated 45Ca2+ was extracted by a 2- hour incubation with 0.5 M NaOH and measured by liquid scintillation counting. It was found that 5Ca2+ uptake by monolayer cultures of ROS 17/2.8 cells was density-dependent. Maximal uptake rates were consis¬ tently found for cultures that were between 50 and 80% confluent. Intestinal 45Ca2+ Transport (Transcaltachia) Measurements of 45Ca2+ transport were carried out in perfused chick duodena as described previously (32-34). Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml/100 g), and the duodenal loop was surgically exposed. Blood vessels branching off from the celiac artery were lighted before cannulation of the celiac artery itself and simultaneous initiation of vascular perfusion. The duodenal loop was then excised and, after cannulation of the celiac vein, placed between layers of saline-moistened cheesecloth at 24°C. The arterial perfusate consisted of Grey's balanced salt solution (GBSS) modified to contain 0.9 mM CaCI2 and oxygenated with 95% 02 and 5% CO2 at a flow rate of 2 ml/min. An auxiliary pump was used for the introduction of vehicle (ethanol) or test substances plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min. The intestinal lumen was then flushed and filled with GBSS containing 5Ca2+ (5 Ci/MI) but without bicarbonate or glucose and perfused at a flow rate of 0.2 ml/min. A basal transport rate was established by perfusion with control medium for 20 minutes after the lumen was filled with 45Ca2+. The tissue was then exposed to 1σ,25-(OH)2D3 or 1 ?,25-(OH)2D3 or reexposed to control medium for an additional 40 minutes. The vascular perfusate was collected at 2-minute intervals during the last 10 minutes of the basal period and during the entire treatment period. Duplicate 100- /I aliquots were taken for determination of the 45Ca2+ levels by liquid scintillation spectrometry. The results are expressed as the ratio of the 5Ca2+ appearing in the 40-minute test period over the average initial basal transport period.
Ligand Binding Studies
The relative ability of each analog to compete with [3H]1σ,25-(OH)2D3 for binding to the chick intestinal nuclear receptor for 1σ,25-(OH)2D3 was carried out in vitro according to the procedure set forth in Example 1. In this assay, increasing concentrations of nonradioactive 1 ,25-(OH)2D3 or the test analog were incubated with a fixed saturating amount of [3H]1σ,25-(OH)2D3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [3H]1σ,25-(OH)2D3 was then calculated and plotted as a function of the relative concentration of the analog and [3H]1cr,25-(OH)2D3. Such plots give linear curves characteristic for each analog, the slopes of which are equal to the analog's competitive index value (25). The competitive index value for each analog is then normalized to a standard curve obtained with nonradioactive 1σ,25-(OH)2D3 as the competing steroid and placed on a linear scale of relative competitive index (RCI), where the RCI of 1σ,25- (OH)2D3 by definition is 100.
Culture Conditions for HL-60 and MG-63 Cells HL-60 cells were seeded at 1.2 x 105 cells/ml, and 1σ,25-(OH)2D3 or its analogues were added in ethanol (final concentration <0.2%) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer Mannheim). After 4 days of culture in a humidified atmosphere of 5% CO2 in air at 37°C the dishes were shaken to loosen any adherent cells, and all cells were then assayed for differentiation by NBT reduction assay and for proliferation by [3H]thymidine incorporation. The MG-63 cells were seeded at 5 x 103 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 μ\ of Dulecco's modified Eagle's medium containing 2% of heat-inactivated charcoal-treated fetal calf serum, and 1σ,25-(OH)2D3 or its analogues were added in ethanol (final concentration <0.2%). After 72 hours of culture in a humidified atmosphere of 5% CO2 in air at 37°C the inhibition of proliferation by [3H]thymidine incorporation and genomic induction of osteocalcin determined via measurement in the medium of osteocalcin concentrations using a homologous human radioimmunoassay (35). NBT Reduction Assay Superoxide production was assayed by NBT-reducing activity as described previously (35). HL-60 cells at 1.0 x 105 cells/ml were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/ml) and NBT (2 mg/ml) and incubated for 30 minutes at 37°C. The percentage of cells containing black formazan deposits were determined using a hemacytometer.
Human skin keratinocytes were isolated and cultured using a modifi¬ cation of the method of Kitano and Okada (36). Briefly, skin from biopsies of patients with breast tumors was cut into pieces measuring 3-5 mm and soaked overnight at 4°C in a solution of dispase (20 Boehringer units/ml). The epidermis was peeled from the dermis, washed with calcium- and magnesium-free phosphate-buffered saline, and incubated and shaken in a 0.25% trypsin solution for 10 minutes at room temperature. The reaction was then stopped by the addition of phosphate-buffered saline containing 10% fetal calf serum. The cells were collected after centrifugation at 4°C for 10 minutes at 800 rpm. After an additional washing with phosphate- buffered saline, the pellet was suspended in culture medium into 25-cm2 primaria flasks from Becton Dickinson. The keratinocytes were cultivated at 37°C in an atmosphere of 5% CO2 in air. A few hours later the medium was replaced by a new one. The medium (keratinocyte medium from Life Technologies, Inc., containing epidermal growth factor (5 ng/ml), bovine pituitary extract (35-50 /g/ml), and antibiotics) was renewed every other day until confluence. Next the keratinocytes were cultured in 96-well plates and after 24 hours were treated with various concentrations of the vitamin D analogues followed by pulse labeling with 1 μC\ of [3H]thymidine for 3 hours. Cultures were washed three times with phosphate-buffered saline and twice with 10% (v/v) ice-cold trichloroacetic acid. Cells were solubilized with 1 M NaOH and the radioactivity determined via liquid scintillation measurement. Statistics Statistical evaluation of the data was performed in the same manner as Example 1.
This example provides a comparison of the biological profile of the three A-ring diastereomers of 1σ,25-(OH)2D3 with respect to their ability to act as agonists for nongenomic and genomic responses of various components of the vitamin D endocrine system. The structures of these secosteroids are presented in FIGS. 3 and 4. The two asymmetric centers are located at carbons-1 and -3. The orientation of the two hydroxyl groups on the A-ring of the naturally occurring hormone 1σ,25-(OH)2D3 are 1σ and 3/?.
A biological response of 1σ,25-(OH)2D3 which has been shown to occur via a nongenomic mechanism is the rapid hormonal stimulation of intestinal Ca2+, termed transcaltachia (22). FIG. 13 presents the results showing the relative ability of the four A-ring diastereomers of the secosteroid hormone to stimulate transcaltachia. The optimal agonist is the naturally occurring hormone 1σ,25-(OH)2D3 (designated C). The onset of stimulation of 45Ca + transport occurs within 4 minutes of introduction of the hormone. We have shown previously that the dose-response curve for transcaltachia is biphasic, with a maximal stimulation occurring at 650 pM 1σ,25-(OH)2D3 (21 ,22). It is also apparent that 1σ,25-(OH)2-3-epi-D3 (HH) is partially able to stimulate transcaltachia. In contrast 300 pM 1 ?,25-(OH)2D3 (HL) is unable to stimulate transcaltachia at this concentration. In other data, not shown, the concentration of 1 ?,25-(OH)2D3 has been increased to 900 pM with no consistently detectable stimulation of transcaltachia.
FIG. 14 illustrates the ability of 1/?,25-(OH)2D3 to block the action of 1σ,25-(OH)2D3 to stimulate transcaltachia. When the duodena are simulta¬ neously perfused with both 1 ?,25-(OH)2D3 and 1 σ,25-(OH)2D3 the characteri- stic stimulation of transcaltachia was absent; this shows that 1 ?,25-(OH)2D3 can function as an antagonist of 1σ,25-(OH)2D3 to control nongenomic responses in accordance with the present invention. In FIG. 15A there is presented an evaluation of the different concen¬ trations of 1 ?,25-(OH)2D3 which are effective at inhibiting 300 pM 1σ,25- (OH)2D3-stimulated transcaltachia. FIG. 15B summarizes the dose response of the inhibition of 1σ,25-(OH)2D3-stimulated transcaltachia by varying concentrations of 1 ?,25-(OH)2D3. It is apparent that a concentration as low as 60 pM 1/?,25-(OH)2D3 can inhibit 300 pM 1σ,25-(OH)2D3.
FIG. 16 presents an evaluation of the ability of 1 ?,25-(OH)2D3 to function as an agonist or antagonist of 45Ca + uptake into ROS 17/2.8 cells. As originally described by Caffrey and Farach-Carson, this response occurs as a consequence of the ability of 1σ,25-(OH)2D3 or its analogues to open dihydropyridine-sensitive Ca2+ channels via a nongenomic mechanism (20). 1 ,25-(OH)2D3 is the most potent agonist in this system. Concentrations as high as 10"8 M 1 ?,25-(OH)2D3 is the most potent agonist in this system. Concentrations as high as 10"8 M 1 ?,25-(OH)2D3 were ineffective in stimulating 45Ca + uptake. However when 1 σ,25-(OH)2D3 and 1 ?,25-(OH)2D3 were added simultaneously to the ROS 17/2.8 cells, there was a complete inhibition of the 1σ,25-(OH)2D3-mediated uptake of the 45Ca2+.
The relative ability of the four A-ring diastereomers to generate within 12 hours the biological responses of intestinal Ca2+ absorption (ICA) and bone Ca2+ mobilization (BCM) in the vitamin D-deficient chick, in vivo, was determined. FIG. 17 presents the ICA and BCM results for 1σ,25-(OH)2D3, 1 ?,25-(OH)2D3 (HL) and 1σ,25-(OH)2-3-epi-D3 (HJ). A portion of Table I summarizes the ICA and BCM results for all four diastereomers. The most potent stimulator of ICA and BCM, as expected, was the reference compound 1σ,25-(OH)2D3; the activity produced by 100 pmol of 1σ,25-(OH)2D3 was set to 100% for both ICA and BCM. Then, the dose of the comparison analogues required to achieve a biological response of either ICA or BCM equivalent to the 100-pmol dose of 1σ,25-(OH)2D3 was calculated and converted to a percentage. The analog 1σ,25-(OH)2D-3-epi-3 was the only diastereomer to have detectable ICA or BCM, which was only 1.5-2.8% of that of the reference 1cr,25-(OH)2D3. The two diastereomers, 1 ?,25-(OH)2D3 and 1 ?,25-(OH)2D-3-epi-D3 had less than 0.1% ICA and BCM. The alteration of the orientation of either the 3 ?-hydroxyl group or the 1σ-hydroxyl group greatly diminishes the biological activity in the vitamin D- deficient chick in vivo.
TABLE I
Relative effects of 1α,25-(OH)2D3 and its A-ring diastereomers upon components of the vitamin D endocrine system
Compound Name Analog Relative competitive data3 Bioassay" Code
DBP Intestine ICA BCM
1σ,25-(OH)2D3 C 100 100 100 100
1σ,25-(OH)2-3-epi-D3 HJ 800 24 ± 4.5 2.8 1.5
1/. ,25-(OH)2D3 HL 6,570 0.22 ± 0.01 <0.1 <0.1
1 . ,25-(OH)2-3-epi-D3 HH 449 0.80 ± 0.07 <0.1 <0.1 a The results are expressed as the percentage activity for the relative competitive index for DBP and intestine in comparison with 1σ,25-(OH)2D3 (RCI = 100%). b The results presented for the bioassay ICA and BCM are derived from dose- response studies like that presented in FIG. 6. The results are expressed in terms of the dose of analog required to achieve an ICA or BCM response equivalent to that achieved by a 100-pmol dose of 1σ,25-(OH)2D3, calculated as a percentage, i.e. [1σ,25-(OH)2D3 dose]/[analog dose] x 100.
Table I also summarizes the relative ability of the four diastereomers to bind in vitro to the chick intestinal 1 σ,25-(OH)2D3 nuclear receptor as well as to the DBP. The 1σ,25-(OH)2D3 nuclear receptor is the presumed media¬ tor of genomic responses to 1or,25-(OH)2D3 in vivo. Again 1σ,25-(OH)2D3 is the reference compound, and its RCI is by definition 100%. Inversion of the orientation of the 3 ?-hydroxyl to 3σ-hydroxyl, as in analog 1σ,25-(OH)2-3- epi-D3, resulted only in a reduction of RCI to 24%. However, when the 1σ- hydroxyl was changed in orientation to that of a 1 ?-hydroxyl, as in analogues 1i?,25-(OH)2D3 and 1/?,25-(OH)2-3-epi-D3, the RCI was dramatically reduced to values less than 1%. These results emphasize the critical role that the 1σ orientation of the hydroxyl on carbon-1 plays in achieving a high affinity of a ligand for the 1σ,25-(OH)2D3 nuclear receptor. The RCI values for binding to the DBP for the four diastereomers also emphasize the importance of the orientation of the hydroxyl groups on carbon-1 and carbon-3 of vitamin D secosteroids. Here the reference analog has been defined to be 1σ,25-(OH)2D3; but it is important to realize that the optimal ligand for DBP is 25-(OH)D3 and that is has an RCI of 66,700. If the RCI of 25-(OH)D3 were set to 100%, then the RCI for 1σ,25-(OH)2D3 would only be 0.15%; this is a reflection of the fact that the presence of an σ-hydroxyl on carbon-1 results in a marked reduction in affinity of the ligand for DBP. Inversion of the 1 σ-hydroxyl to the 1/? orientation, as in analogues HL and HH, results in a 65.7- and 4.4-fold increase, respectively, in the RCI values, showing that in the absence of a 1 σ-hydroxyl the analogues behave more like 1-deoxy analogues.
An evaluation was made whether 1 ?,25-(OH)2D3 could inhibit in vivo in the vitamin D-deficient chick, the genomic response of induction of the vitamin D-dependent calcium-binding protein, known as calbindin-D28k. It has been shown previously that calbindin-D28k induction in the chick intestine is a genomic event well correlated with the occupancy of the nuclear 1σ,25- (OH)2D3 receptor (41). The results summarized in Table II indicate that 1 ?,25-(OH)2D3, when administered either simultaneously or 2 hours before 1σ,25-(OH)2D3, cannot block the appearance of intestinal calbindin-D28k. In fact 1 ?,25-(OH)2D3 may have a weak agonist activity for this genomic response; administration of the large dose of 6.5 nmol of 1 ?.25-(OH)2D3 resulted at 12 hours in the appearance of a level of calbindin-D28k which was 50% of that induced by 0.65 nmol of 1σ,25-(OH)2D3. This is consistent with the fact that 1^,25-(OH)2D3 does have a finite, but low, RCI for the nuclear receptor (see Table I). TABLE II
Effect of 1β,25-(OH)2D3 on 1a,25-(OH)2D3 induction of calbindin-D28k in the chick intestine
The calbindin-D28k levels present in the chick intestine 12 hours after dosing vitamin D-deficient chicks with either 1σ,25-(OH)2D3 alone or in the presence of 1 ? ,25-(OH)2D3 were determined via enzyme-linked immunosorbent assay, as described under "Experimental Procedures."
Calbindin-
Group Dose D28k nmol ng/mg protein
-D 26 ± 1 1σ,25-(OH)2D3 0.65 1 ,180 ± 75 1σ,25-(OH)2D3 1.95 1 ,140 ± 80 1or,25-(OH)2D3 6.95 1 ,190 ± 60
1 ?,25-(OH)2D3 0.65 53 ± 20 1/?,25-(OH)2D3 1.95 280 ± 80
1/ ,25-(OH)2D3 6.5 510 ± 120
1/?,25-(OH)2D3 + 1σ,25-(OH)2D3 (6.5 + 0.65) 1 ,020 ± 70 1/?,25-(OH)2D3 + 1σ,25-(OH)2D3 (6.5 + 0.65) 1 ,080 ± 75 a The 1/?,25-(OH)2D3 and the 1σ,25-(OH)2D3 were added simultaneously. b The 1/?,25-(OH)2D3 was administered 2 hours in advance of the 1σ,25-(OH)2D3.
In FIG. 18, A and B, results are presented describing the potency of the four diastereomers to induce osteocalcin in MG-63 cells; in addition, the ability of 1/?,25-(OH)2D3 to function as an antagonist of 1σ,25-(OH)2D3- induced osteocalcin is presented (FIG. 18B). As has been reported previously, 1σ,25-(OH)2D3 is a potent agonist for osteocalcin in the MG-63 cell line (35); half-maximal induction occurs at a concentration of 3.8 x 10*9 M 1σ,25-(OH)2D3 (FIG. 18A). Using their ED50 concentrations, it can be calculated for the four diastereomers that their relative potencies are: 1σ,25-(OH)2D3 = 100%, 1σ,25-(OH)2-3-epi-D3 = 17%, 10,25-(OH)2D3 = 2%, and 1 ?,25-(OH)2-3-epi-D3 = 1.2%. These results parallel the RCI values for the nuclear 1σ,25-(OH)2D3 receptor for these four diastereomers which are tabulated in Table I. When both 1 ?,25-(OH)2D3 and 1σ,25-(OH)2D3 were presented simultaneously to the MG-63 cells (FIG. 18B), at 10"9-10"7 M 1 ?,25-(OH)2D3, there was no evidence of antagonist actions by 1/?,25-(OH)2D3. The ability of the two epimers, 1σ,25-(OH)2D3 and 1/?,25-(OH)2D3, to promote differentiation of HL-60 cells was assessed by NBT reduction; also, the ability of 1 ?,25-(OH)2D3 to function as an antagonist of 1σ,25-(OH)2D3- mediated cell differentiation was determined. The results are presented in FIG. 19. As has been reported many times previously, 1σ,25-(OH)2D3 is a potent stimulator of HL-60 cell differentiation; the half-maximal concentration was 1.5 x 10"8 M. The concentration of 1 ?,25-(OH)2D3 which achieved half- maximal stimulation of cell differentiation was 2.5 x 10~7 M, some 10 times higher. Again there was no evidence that 1 ?,25-(OH)2D3 could antagonize the cell differentiation actions of 1σ,25-(OH)2D3. FIG. 20 presents the evaluation of the potencies of 1σ,25-(OH)2D3 and the three A-ring diastereomers in inhibiting the proliferation of human keratinocytes. To achieve a 50% inhibition of proliferation, the relative order of potency was 1σ,25-(OH)2D3, 1σ,25-(OH)2-3-epi-D3, 1 ?,25-(OH)2D3, and 1 ?,25-(OH)2-3-epi-D3, 1 :6.2:27:75. Again the most potent inhibitor of cell proliferation was 1σ,25-(OH)2D3, and the least potent was 1/?,25-(OH)2D3. In separate experiments (data not presented) the potential of 1/?,25-(OH)2D3 to antagonize 1σ,25-(OH)2D3-mediated inhibition of keratinocyte was tested. Similar to the results presented in FIG. 18B for the MG-63 cell, 1 . ,25-(OH)2D3 when present at 5 x 10"8 M could not block the action of 1 x 10"9 M 1σ,25-(OH)2D3.
This example demonstrates the biological profile of the four A-ring diastereomers of the hormonally active form of vitamin D3 (see FIG. 3). Only 1σ,25-(OH)2D3 is known to occur naturally in biologically systems. The only difference in structure of these four compounds is the orientation of the hydroxyl groups on carbons-1 and -3.
It is apparent that the nuclear 1σ,25-(OH)2D3 receptor's ligand binding domain clearly prefers the 1σ, 3β orientation of the naturally occurring hormone and that it can also discern differences among the three other A- ring diastereomers (see the RCI values of Table I). The correct orientation of the hydroxyl on carbon-1 is more critical than the orientation of the hydroxyl on carbon-3. Thus inversion of the 1 σ-hydroxyl to the .β orientation results in a change in RCI from 100 to 0.8%, whereas inversion of the 3 .-hydroxyl to a 3σ-hydroxyl only results in a reduction from 100 to 24%.
Comparison of the RCI data for the intestinal nuclear receptor with those for the DBP of the four diastereomers clearly emphasizes the intrinsic preferences of these two ligand binding domains for vitamin D3 secosteroids. DBP is the principal plasma transport protein for vitamin D metabolites; the ligand with highest affinity is 25-(OH)D3; however, it also binds 1σ,25-(OH)2D3, 24R,25-(OH)2D3, and the parent vitamin D3 with a significant affinity. The addition of a 1 σ-hydroxyl to 25-(OH)D3 results in a 666-fold reduction in RCI (3). But it is still possible to compare the relative values of RCI between DBP and the nuclear 1σ,25-(OH)2D3 receptor for the four diastereomers (see Table I). Inversion of the 1 σ-hydroxyl to the .β orientation, as in analogues 1/?,25-(OH)2D3 (HL) and 1 ?,25-(OH)2-3-epi-D3 (HH), results in a 65.7- and 4.4-fold increase, respectively, in the DBP RCI values, suggesting that in the absence of a 1 σ-hydroxyl, the analogues behave more like 1-deoxy analogues. Of the four diastereomers, 1σ,25-(OH)2D3 has the lowest RCI; this implies that 1σ,25-(OH)2D3 would have, under in vivo circumstances, the highest "free" concentration or greatest "availability" of the four diastereomers. Evaluation of the four compounds in the standard bioassay system of the vitamin D-deficient chick, under in vivo conditions (37), indicated that only the naturally occurring hormone, 1σ,25-(OH)2D3, and 1σ,25-(OH)2-3-epi- D3 (analog HJ) had a significant ability to stimulate ICA and BCM (see FIG. 17 and Table I). Thus, 1σ,25-(OH)2-3-epi-D3 has 2.8 and 1.5% of the activity of 1σ,25-(OH)2D3 to stimulate ICA and BCM, which is consistent with a reduced affinity for the intestinal nuclear 1σ.25-(OH)2D3 receptor, RCI = 24%. The other two diastereomers, 1 ?,25-(OH)2D3 (HL) and 1 ?,25-(OH)2-3- epi-D3 (HH), were virtually devoid of ICA and BCM; again this is consistent with their poor affinities for the intestinal 1σ,25-(OH)2D3 nuclear receptor (RCI = 0.2 and 0.8 respectively). Since ICA and BCM responses occur under in vivo conditions and both likely represent a response to an integrated set of components that respond to vitamin D ligands, it is not known which proportion of the responding elements is comprised of genomic and nongenomic responses. However, both, ICA and BCM responses can be blocked by administration of actinomycin D, an inhibitor of DNA-directed RNA synthesis (38,39), and a good correlation between binding to the nuclear 1σ,25-(OH)2D3 receptor and ICA and BCM has been shown (3). Thus the low ICA and BCM of the three diastereomers, corre¬ lated with their low RCI values, emphasizes the high specificity of the nuclear receptor for 1σ,25-(OH)2D3 for the orientation of the hydroxyl group on carbons-1 and -3. The optimal orientation is clearly 1σ and 3β. The four diastereomers also showed a differential effect on inhibition of keratinocyte proliferation in culture (FIG. 20). The relative activities of 1σ,25-(OH)2D3 > 1σ,25-(OH)2-3-epi-D3 > 1/?,25-(OH)2D3 > 1/?,25-(OH)2-3-epi- D3 again emphasize the importance of the orientation of the 1σ- and 3β- hydroxyl groups in effecting the biological response. This example shows the biological activity of the four diastereomers with regard to their agonist activity for the nongenomic response of trans¬ caltachia, as assayed in the perfused chick duodenum. In contrast to the genomic responses of ICA and BCM (discussed above) where the three unnatural diastereomers were largely devoid of activity, two of the diastereomers were quite active in stimulating transcaltachia. 1σ,25-(OH)2- 3-epi-D3, when perfused at 300 pM, was approximately 80% as active as 1σ,25-(OH)2D3; and 1 7,25-(OH)2-3-epi-D3, 300 pM, was 20-30% as active. Only 1 ?,25-(OH)2D3 was without agonist activity. Thus the hormone response element(s) associated with the nongenomic response of transcaltachia clearly have a ligand specificity different from that of the nuclear 1σ,25-(OH)2D3 receptor (compare with RCI results in Table I). The putative transcaltachic membrane response element (40) is more tolerant of the presence of 3σ-hydroxyl.
In accordance with the present invention, this example shows that the analog 10,25-(OH)2D3 (HL) is a potent antagonist of both 1σ,25-(OH)2D3- stimulated transcaltachia (FIG. 15) and rat osteoblast 5Ca2+ uptake (FIG. 16). When 1/?,25-(OH)2D3 and 1σ,25-(OH)2D3 were perfused simultaneously and, even in some circumstances when the intestine was preexposed only to the 1 ?,25-(OH)2D3 for 8 minutes and then followed by perfusion with only 1σ,25-(OH)2D3, there was a clear inhibition by exposure of the intestine to 1 ?,25-(OH)2D3. This example demonstrates that the analog of 1σ,25-(OH)2D3 is able to function as an antagonist of a biological response stimulated by 1σ,25-(OH)2D3.
Based on the results presented in FIGS. 18 and 19 and Table II, it appears that the antagonistic actions of 1/?,25-(OH)2D3 are restricted to the nongenomic responses of Ca2+ channel opening. 1 ?,25-(OH)2D3 was not able to inhibit the genomic responses of induction of calbindin-D28k under in vivo conditions in the chick (Table II), induction of osteocalcin in MG-63 cells (FIG. 18), or inhibition of cell differentiation in HL-60 cells (FIG. 19).
In accordance with the present invention, fifteen analogues of vitamin D3 have been synthesized and demonstrated to be active in controlling genomic and/or nongenomic responses in the vitamin D endocrine system. The fifteen analogues are set forth in FIGS. 21 and 22 and tabulated in Table III. The analogues may be administered in the same manner as the previously described two analogues. They are effective in controlling a wide variety of responses within the vitamin D endocrine system including the genomic mechanisms which are controlled by mechanisms similar to that of other steroid or steroid like hormones (e.g. estradiol, testosterone, stanolone, progesterone, cortisol, aldosterone, retinoic acid and thyroxine). Some of the analogues, as indicated in Table III are effective in controlling nongenomic mechanisms. Also, some of the analogues, as indicated in Table III, like the reference 1σ,25(OH)2D3 are effective in controlling both genomic and nongenomic mechanisms. TABLE III
Analogues of 1α,25(OH)2D3
MODE OF ACTION
Analog Analog Code Abbreviated name Genomic Nongenomic
GE 14-Epi-1 ,25-(OH)2-D3 X X
GF 14-Epi-1 ,25-(OH)2-Pre-D3 X
HS 1σ,18,25-(OH)3-D3 X X
IB 23-(m-(Dimethylhydroxymethyl)phenyl)-22- X X yne-24,25,26,27-tetranor-1σ-OH-D3
JD 1σ,25-Dihydroxy-trans-lsotachysterol X X (1 ,25-trans-lso-T)
JM 1σ,25-(OH)2-7-DHC X
JN 1 σ,25-(OH)2-Lumisterol3 X
JO 1 σ,25-(OH)2-Pyrocalciferol3 X
JP 1σ,25-(OH)2-lsopyrocalciferol3 X
JR 1,25-(OH)2-7,8-cis-D3 X X
JS 1 ,25-(OH)2-5,6-trans-7,8-cis-D3 X X
JV (1S,3R,6S)-7,19-Retro-1,25-(OH)2-D3 X X
JW (1S,3R,6R)-7,19-Retro-1 ,25-(OH)2-D3 X X
JX 22-(p-Hydroxyphenyl)-23,24,25,26,27- X X pentanor-D3
JY 22-(m-Hydroxyphenyl)-23,24,25,26,27- X X pentanor-D3
The analogues are useful in treating a variety of diseases associated with malfunction of the vitamin D endocrine system including skin conditions (e.g. psoriasis), bone conditions (e.g. osteoporosis, venal osteodystrophy), and oncologic diseases such as breast, colon and prostate cancers and leukemia, induction of hey proteins like nerve growth factor and other brain proteins which may be involved in Alzheimer's disease. The various diseases associated with vitamin D metabolism are set forth in FIG. 26. The analogues are useful in treating and diagnosing this group of diseases. The synthesis and usefulness of these analogues will be further described in the following examples. Example 3 — Synthesis and biological activity of 14-Epi-1 ,25-(OH)2-D3 (GE) and 14-Ep-1 ,25-(OH2)-Pre-D3 (GF)
As set forth in FIG. 23, analogues GE and GF were prepared from the known A-ring phosphine oxide 10(41) and the appropriate (CD) ketone 14. Grundmann's ketone 11(42) readily available from the ozonolysis of vitamin D3, was selectively oxidized at C-25 to alcohol 12 as previously described (43). Epimerization of the latter to the c/s-fused hydrindanone 13 was accomplished with base. The crude mixture consisted of a 71/29 ratio of 13/12 whereas a 49% yield (66% based on recovered 12) of purified 13 was actually isolated by HPLC(44,45,46). Protection of the C-25 hydroxyl as the trimethylsilyl (TMS) ether afforded CD fragment 14 and the deprotection of the silyl ether with tetrabutylammonium fluoride (TBAF) provided the 1σ-hydroxylated 14-epi-vitamin GE. The GE analogues underwent smooth thermal [1 ,7]-sigmatropic hydrogen shifts at 80°C (benzene-d6) to afford the previtamin GF.
Intestinal calcium absorption (ICA) and bone calcium mobilization (BCM) were measured in vivo to compare analogues GE and GF to 1σ,25- (OH)2-D3 (3) in the vitamin D deficient chick system previously described(37). The results in this standard rachitic chick assay can be reported as the percentage of activity observed for ICA and BCM in comparison to standard doses of 1σ,25-(OH)2-D3 (47). With respect to the ICA determination, the two analogues GE and GF exhibited 3.9% and <0.1%, respectively of the activity as compared to 1σ,25-(OH)2-D3. Similar results (<0.01% and 2%, respectively) were obtained with respect to the BCM determination.
The GE and GF analogues were evaluated in vitro in terms of their ability to bind to the chick intestinal nuclear receptor. In this assay, the analogues were evaluated in terms of their chick intestinal receptor relative competitive indices (RCIs) wherein the value for 1σ,25-(OH)2-D3 is 100 by definition (48). The RCI values for GE and GF were 15.0 ± 2.0 and 1.6 ± 0.9, respectively. The lack of in vivo calcemic activity observed for GE is somewhat at variance with its RCI value of 15. It is believed that GE binds to the chick intestinal receptor without inducing its necessary activation, which is required of steroid hormone receptors prior to stimulation of transcription. This renders the 14-epi structural feature of GE a useful parameter as an antagonist of the steroid hormone, 1σ,25-(OH)2-D3. Analogues GE and GF were also subjected to a second in vitro steroid competition assay involving the human vitamin D binding protein (DBP) (49). In this assay, each analogue was evaluated in terms of its ability to bind to human DBP in comparison to the natural hormone 1σ,25- (OH)2-D3. Like the chick intestinal receptor assay results, the human DBP data are expressed as RCI values [the value for 1σ,25-(OH)2-D3 is 100 by definition]. It should be noted that human DBP has the highest affinity for the natural metabolite 25-OH-D3 (2, RCI = 66,800). The human DBP RCI values for analogues GE and GF were 12.1 ± 2.1 and 2.2 ± 0.7, respectively. The above test results show that GE and GF are useful respectively for biological responses involving the nuclear VDR (GE) and regulation of gene transcription (GE) and the membrane VDR anovated with nongenomic rapid actions (GF).
Details of the synthesis protocols are as follows: 14-Epi-1σ,25-dihydroxyvitamin D3 (GE). Referring to FIG. 23, to a stirred solution of 10 (67 mg, 0.11 mmol) in anhydrous THF (1.4 mL) at -78°C under argon was added n-butyllithium (74 μ , 0.12 mmol, 1.55 M solution in hexanes) to give a deep orange solution. After adding CD ketone 14 (27.1 mg, 0.076 mmol) in dry THF (0.46 mL), the solution was stirred for 3 hours at -78°C and then warmed to room temperature. After concentration, the residue dissolved in ether (3 mL) and washed with a saturated solution of NaHCO3 (3 mL) and brine (3 mL). After drying (MgSO4) and concentrating the ether solution, the crude residue was purified by flash chromatography to afford 48.2 mg (86% yield) of protected vitamin 15, which was treated with TBAF (0.79 mL, 0.79 mmol, 1 M solution in THF). After 3 hours, the solvent was removed and the crude residue dissolved in EtOAc (5 mL). The solution was washed (water, 3 mL; and brine, 3 mL), dried (Na2SO4), filtered and concentrated. Purification by HPLC (50% EtOAc/hexanes, Rainin Dynamax 60 A column) afforded after vacuum drying 11 mg (81 %) of analog GE. 1H-NMR (300 MHz): (CDCI3) δ 0.87 (3H, C21-CH3, d, J -6.4 Hz), 0.90 (3H, C18-CH3, s), 1.22 (6H, C2627-CH3, s), 2.31 (1 H, dd, J - 13.2 Hz, 7.2 Hz), 2.46 (1 H, br d, J - 14.3 Hz), 2.60 (1 H, dd, J - 13.3 Hz, 3.5 Hz), 4.23 (1H, H3, m), 4.44 (1H, H1 ? t, J -5.4 Hz), 5.00 (1 H, H1β> br s), 5.34 (1 H, H19, br s), 6.14 and 6.33 (2H, H67-AB pattern, d, J ~ 11.2 Hz).
14-Epi-1σ,25-dihydroxyprevitamin D3 (GF). Asolution of analog GE (4.9 mg, 0.012 mmol) in benzene-d6 (2 mL) was subjected to three freeze- thaw cycles under vacuum and then placed in a thermostated bath at 80°C. After 4 hours, the solution was cooled to room temperature and the vitamin/ previtamin ratio determined by 1H-NMR integration (-7:93). The solution was concentrated and purified by HPLC (100% EtOAc, Rainin Dynamax 60 A column) to afford, in order of elution, epi-vitamin 5 (0.3 mg) and epi- previtamin 5' (3.7 mg). 1H-NMR (300 MHz): (CDCI3) δ 0.91 (3H, C18-CH3, s), 0.94 (3H, C21-CH3, d, J -6.3 Hz), 1.22 (6H, C2627-CH3, s), 1.75 (3H, C19- CH3, br s), 2.55 (1H, br d, J - 16.6 Hz), 4.05 (1H, H3, m), 4.18 (1H, H1 ( br s), 5.65 (1 H, H9, m), 5.80 and 5.85 (H67, AB pattern, d, J - 12.5 Hz). De-A,B-25-hydroxycholestan-8-one (12). This compound was pre¬ pared using a procedure similar to that described in (43). To a solution of ketone 11 (1.0 g, 3.8 mmol) in CCI4 (15 mL), CH3CN (15 mL) and pH 7 aqueous buffer solution (19 mL, 0.05 M KH2PO4 and 0.05 M in NaOH) was added RuCI3 H2O (78.4 mg, 0.37 mmol) and NalO4 (2.83 g, 13.2 mmol). Upon vigorous stirring at 45°C, the black solution turned yellow. The heterogeneous suspension returned to its original black color within 5 days. The organic phase was separated and the aqueous layer extracted with CH2CI2 (4 x 20 mL). The combined organic layers were washed with brine, dried (Na2SO4), filtered and concentrated. Purification by flash chromatography (35% EtOAc/hexanes) afforded after vacuum drying 432 mg (40% yield) of 25-hydroxy-Grundmann's ketone as a colorless oil. 1H*; NMR (300 MHz): (CDCI3) δ 0.62 (3H, C18-CH3, s), 0.95 (3H, C21-CH3, d, J ~ 5.9 Hz), 1.20 (6H, C2627-CH3, s), 2.43 (1 H, dd, J ~ 11.6 Hz, 7.6 Hz). 13Cr NMR (75.5 MHz): (CDCI3) δ 12.4, 18.7, 19.0, 20.7, 24.0, 27.5, 29.1 , 29.3, 35.4, 36.2, 38.9, 40.9, 44.2, 49.9, 56.6, 61.9, 70.9, 212.1.
De-A,B-14-epi-25-hydroxy-8-cholestanone (13). To a solution of 1.0 M NaOMe in methanol (421 μl) was added 12 (99.9 mg, 0.356 mmol) in MeOH (120 μL) at room temperature under an argon atmosphere. After stirring for 48 hours, the ice cooled mixture was quenched with acetic acid (49 μL, 0.86 mmol) and diluted with water. The crude mixture was extracted with hexanes and the combined organic layers were washed with brine (3 x 10 mL), dried (Na2SO4), filtered and concentrated to yield a mixture consisting of -71% epi-isomer 13 and -29% starting material 12. Purification by HPLC (25 x 1 cm Rainin, silica gel, 35% EtOAc/hexanes) afforded in order of elution 49 mg of the epi-ketone (49%) and 25 mg (25%) of starting material. 1H-NMR (300 MHz): (CDCI3) δ 0.89 (1 H, C22-CH3, d, J -6.3 Hz), 1.02 (3H, C18-CH3, s), 1.18 (6H, C2627-CH3, s). 3C-NMR (75.5 MHz): (CDCI3) δ 19.0, 20.7, 21.1, 21.2, 23.0, 27.7, 29.1 , 29.2, 34.3, 36.0, 36.3, 40.1 , 44.2, 48.6, 50.4, 61.3, 70.9, 213.7.
De-A,B-14-epi-25-trimethylsilyloxy-8-cholestanone(14). N-(trime- thylsilyl)-imidazole (259.7 mg, 1.85 mmol) was added dropwise to a stirred solution of 13 (173.4 mg, 0.618 mmol) in dry THF (7 mL). The mixture was stirred for 4.5 hours under argon and was then passed directly through a short column of silica gel (15% EtOAc/hexanes). The eluent was concen¬ trated and subjected to flash chromatography (15% EtOAc/hexanes) to afford after vacuum drying 194.2 mg (89%) of the TMS protected alcohol 14 as a colorless oil. 1H-NMR (300 MHz): (CDCI3) δ 0.095 (9H, TMS methyl H's, s), 0.91 (3H, C21, d, J -6.3 Hz), 1.04 (3H, C18, s), 1.19 (6H, C2627, s). 13C-NMR (75.5 MHz): (CDCI3) δ 2.6, 19.0, 20.8, 21.2, 23.0, 27.7, 29.80, 29.83, 34.3, 36.0, 36.3, 40.1 , 45.1 , 48.6, 50.5, 61.3, 73.9, 213.4. Details of the tests showing biological activity are as follows: Intestinal Calcium Absorption (ICA) and Bone Calcium
Mobilization (BCM). ICA and BCM were determined in vivo in vitamin D deficient chicks as described previously (37, 47). Twelve hours before assay, the chicks, which had been placed on a zero-calcium diet 48 hours before assay, were injected intramuscularly with the vitamin D metabolite or analogue in 0.1 mL of ethanol/1 ,2-propanediol (1 :1 , v/v) or with vehicle. At the time of assay, 4.0 mg of 40Ca2+ + 5 μC\ of 45Ca2+ (New England Nuclear) were placed in the duodenum of the animals anesthetized with ether. After 30 min, the birds were decapitated and the blood collected. The radioactivity content of 0.2 mL of serum was measured in a liquid scintillation counter (Beckman LS8000) to determine the amount of 45Ca2+ absorbed (which is a measure of ICA). BCM activity was estimated from the increase of total serum calcium as measured by atomic absorption spectrophotometry.
1σ,25-(OH)2-D3 Chick Intestinal Receptor Steroid Competition Assay. A measure of competitive binding to the chick intestinal 1σ,25- (OH)2-D3 receptor was performed by using the hydroxylapatite batch assay (48). Increasing amounts of non radioactive 1σ,25-(OH)2-D3 or analogue were added to a standard amount of [3H]-1σ,25-(OH)2-D3 and incubated with chick intestinal cytosol. The relative competitive index (RCI) for the analogues was determined by plotting the percent maximum 1σ,25-(OH)2- [3H]-D3 bound x 100 on the ordinate versus [competitor]/[1σ,25-(OH)2-[3H]- D3] on the abscissa. The slope of the line obtained for a particular analogue is divided by the slope of the line obtained for 1σ,25-(OH)2-D3; multiplication of this value by 100 gives the RCI value. By definition, the RCI for 1σ,25- (OH)2-D3 is 100.
25-OH-D3 Human Vitamin D Binding Protein Steroid Competition Assay. A steroid competition assay for the human vitamin D binding protein (DBP) was carried out using human DBP (Gc-Globulin, Sigma, St. Louis, MO) as the binding protein. DBP (0.015 mg) in 0.8 μL 0.1 M barbital buffer, pH 8.6, and [3H]-25-OH-D3 (0.9 pmol, 20 Ci/mmol, Amersham, Arlington Heights, IL), and nonradioactive competitor (the test analogue or standard 25-OH-D3 in an appropriate range of concentrations) in 0.1 mL ethanol were incubated in duplicate in conical tubes on ice. After 2 h, 0.2 ml charcoal-dextran slurry (4 g charcoal, 0.4 g dextran in 200 mL barbital buffer) was added. The incubation was continued for another 30 min on ice, and the tubes were centrifuged for 10 min at 900 x g. The tritium, representing [3H]-25-OH-D3 bound to DBP, was determined in aliquots of the supernatant fluid by liquid scintillation measurement. At least triplicate assays were carried out on each analogue. The data was plotted as [competitor]/[3H]-25-OH-D3] vs. 1/[% maximum bound]. The relative competitive index or RCI was calculated as [slope of competitor]/[slope for 25-OH-D3] x 100 as described previously (49). Such plots yield linear transformations characteristic for each analogue, the slopes of which are equal to the analogues competitive index value. Note that although each analogue was assayed in competition with [3H]-25-OH-D3, the data are expressed as relative to the binding of 1σ,25-(OH)2-D3, with its RCI set to 100. Thus, the RCI of 1σ,25(OH)2-D3 = 100, and the RCI for 25-OH-D3 = 66,800.
Example 4 — Synthesis and Biological Activity of 1 σ, 18,25-(OH3)-D3(HS) and 1σ,25-Dihydroxy-.rat.s-isotachysterol (1 ,25-fratvs-lso-T) (JD) HS is prepared as follows: The protected alcohol precursor of HS was first prepared as follows: 18-Acetoxy-25-[(trimethylsilyl)oxy]-1 σ-[(fetf-Butyldimethylsilyloxy)
Vitamin D3 tert-butyldimethylsilyl] ether (Compound A) was prepared according to the procedure described in Maynard et al., J. Org. Chem., 1992, v. 57, pp. 3214-17.
Compound A (139 mg, 1.8 mmol) was dissolved in anhydrous ethyl ether (0.2 mL) and was added dropwise to a solution of LiAIH4 (21 mg, 5.4 mmol) in ether (0.5 mL). The reaction mixture was stirred for 30 minutes, by which time the solution had become viscous and an additional 0.2 mL of ether was added. After stirring for 20 minutes, the reaction mixture was quenched with ethyl acetate (1 mL) and then filtered through a sintered glass funnel. The grey solid was washed with ethyl acetate (5 mL) and the combined filtrate concentrated. The crude residue was purified by flash chromatography (20% ethyl acetate/hexanes) to afford after vacuum drying 102 mg (78%) of the protected alcohol precursor. The analytical data for the precursor is: H-NMR (300 MHz): (CDCI3) δ 0.06 (12H, Si-CH3, s), 0.10 (9H, TMS, s), 0.87 (9H, t-Bu, s), 0.89 (9H, t-Bu, s), 1.04 (3H, C21- CH3, d, J -6.3 Hz), 1.20 (6H, C2627-CH3, s), 0.9-2.5 (remaining ring and side chain hydrogens, series of m), 2.88 (1 H, br d, J - 11.8 Hz), 3.44 (1H, H18, d, J - 11.5 Hz), 3.53 (1 H, H18, d, J - 11.5 Hz), 4.18 (1 H, H3, m), 4.37 (1 H, W,, m), 4.84 (1 H, H19, br s), 5.18 (1 H, H19, br s), 6.04 and 6.22 (2H, H67 AB pattern, d, J - 11.1 Hz). 13C-NMR (75.5 MHz): (CDCI3) δ -5.1 , -4.8,
-4.7, 2.6, 18.1 , 18.2, 19.3, 20.7, 22.0, 23.9, 25.8, 25.9, 27.6,
28.8, 29.8, 30.0, 35.7, 36.1 , 36.6, 44.8, 45.3, 46.0, 49.7, 55.3,
56.9, 61.5, 67.5, 72.0, 74.1 , 111.3, 118.1 , 122.8, 135.9, 141.0, 148.3. IR: (CCI4) v 3500 (OH, br), 2960 (C-H, s), 2930 (C-H, s), 2860 (C-H, m), 1650 (w), 1470 (w), 1360 (w), 1250 (s),
1090 (s), 1045 (s), 910 (m), 840 (s) cm"1. UV: (95% EtOH) .max 264 nm (e 18,000): Λmin 232 nm (β 10,900). Anal, calcd. for C42H80O4Si3: 68.79; H, 11.00. Found: C, 68.74; H, 11.17. HS was prepared by adding tetra-n-butylammonium fluoride (2.16 L, 0.216 mmol, 1 M in THF) to a solution of the protected alcohol precursor (18.1 mg, 0.024 mmol) in anhydrous THF (2 mL). The mixture was stirred for 20 hours at room temperature, then concentrated to dryness. The resulting crude material was directly flash chromatographed through a short column of silica gel (EtOAc) and then purified by HPLC (Rainin Dynamax, 1.0 x 25 cm, 8 μm silica column, EtOAc) to give after vacuum drying HS (7 mg, 70%) as a white foam.
The analytical data for HS is:
1 H-NMR (300 MHz): (CD3OD) δ 1.07 (3H, C21-CH3, d, J -6.4 Hz), 1.16 (6H, C2627-CH3, s), 1.0-2.2 (remaining ring and side chain hydrogens, series of m), 2.24 (1H, dd, J - 13.2 Hz, 7.2
Hz), 2.51 (2H, br d, J - 13.0 Hz), 2.91 (1 H, br d, J - 11.2 Hz), 3.30 (solvent peak, methyl group of CD3OD), 3.35 (2H, H18, d, J - 11.8 Hz), 3.41 (1 H, H18, d, J - 11.8 Hz), 4.10 (1 H, H3, m), 4.34 (1 H, H1 f t, J - 5.6 Hz), 4.87 (1 H, H19, s), 4.88 (solvent peaks, OH group of CD3OD), 5.28 (1 H, H1B, s), 6.06 and 6.32 (2H, H67, AB pattern d, J - 11.1 Hz). JUV: (95% EtOH) λmax 264 nm (e 18,100); λmn 230 nm (e 10,300). HRMS: m/z 432.3242 (calcd. for C27H44O4, 432.3241). MS: m/z 432 (1 ,
M), 414 (4, M - H2O), 396 (1 , M - 2H2O), 257 (2), 171 (3), 152 (1 , A-ring fragment due to C78 cleavage), 134 (8, 152 - H2O), 105 (6), 91 (10), 79 (17), 69 (20), 59 (base). JD was synthesized by preparing and reacting precursors A-D as follows:
(1S,3R,6S)-1,3-Di(tert-butydimethylsilyloxy)-25-trimethylsilyloxy- 9,10-secocholesta-5(10),6,7-triene (A). Freshly purified 1 ,2-diiodoethane (412 mg, 1.46 mmol) and samarium metal (286 mg, 1.90 mmol) were dried under vacuum and suspended in 4 mL THF under an argon atmosphere. This solution was stirred for 2 hours until it became deep blue. A solution of propargyl benzoate (477 mg, 0.570 mmol) and Pd(PPh3)4 (65.8 mg. 0.037 mmol) in 6 mL THF was added via cannula. Freshly distilled isopropanol (0.5 mL) was added and the solution was stirred under a positive argon atmosphere for 14 hours. Saturated aqueous Na2CO3 (2 mL) was added to quench the reaction. The organic layer was diluted with ether and then the mixture was washed with Na2CO3 (3 x 10 mL), dried with MgSO4 and concentrated. The product was purified by flash chromatography (silica gel, 2% EtOAc/hexanes) followed by HPLC (2% EtOAc/hexanes, Rainin Dynamax column 8, mL. min flow rate) to form A. (1S,3R,6S)-1,3,25-Trihydroxy-9,10-secocholesta-5(10),6,7-triene
(B). To the vinylallene A (0.1054 g, 0.1469 mmol) was added TBAF (1 M in THF, 1.6 ml, 1.6 mmol). The solution was stirred under an argon atmosphere for 19 hours. Water (1 mL) was added and the solution stirred 30 minutes. The mixture was extracted with ether (3 x 15 mL) and the ether extracts washed with brine (1 x 10 mL) and dried (MgSO4). The concentrated residue was subjected to flash chromatography (silica gel, 80% EtOAc/hexanes) followed by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4 mL/min flow rate) to afford purified deprotected vinylallene B together with its 6R-diastereomer (46.1 mg, 75.3% total yield) in a -92:8 ratio by NMR integration. By shave-recycle HPLC separation, pure B could be obtained and characterized by spectroscopic analysis. 1ϋ NMR: δ 0.74 (3H, C18-CH3, s), 0.95 (3H, C21-CH3, d, J -6.4 Hz), 1.22 (6H, C26,27-CH3, s), 1.87 (3H, C19-CH3, s), 2.29 (1H, br d, J - 13.2 Hz), 2.62 (1 H, br dd, J - 16.5 Hz, 4.5 Hz), 4.11-4.20 (1 H, C3-H, m, W-27.8 Hz), 4.23 (1 H, C1-H, br m W-8.6 Hz), 6.14 (1 H, C6-H, dd, J -4.1 Hz). UV: (100% EtOH) λmax 242 nm (e 24,300), 234 nm (e 23,500). 7,8-cis-1σ,25-Dihydroxyvitamin D3, (C). To the vinylallene B (19.7 mg, 0.047 mmol) and (np) (CO)3 Cr (14.7 mg, 0.0557 mmol) in a 10 mL flask with a stir bar was added 1 mL of acetone (distilled from CaS04). After deoxygenation of the mixture by four freeze-pump-thaw cycles, the solution was stirred at 40°C under a positive pressure of argon for 4 hours. Acetone was removed under reduced pressure and the product was purified by flash chromatography (silica gel, 80% EtOAc/hexanes) followed by separation by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4.0 mL/min flow rate) to afford three components in the following order of elution: major product C (17.0 mg, 86.4%), recovered starting precursor B (1.4 mg, 7.1%), and a minor product (1.5 mg, 7.6%).
1σ,25-Dihydroxy-cis-isotachysterol (D). To an NMR tube was added precursor C (10.4 mg, 0.0250 mmol) in 1 mL of acetone-d6. The NMR tube was covered with foil and flushed with argon, and then heated at 57°C. At time intervals of 0, 23, 85, 177, 454 and 1099 min, the reaction mixture was monitored by NMR analysis. Formation of the thermal product 35 was observed to occur with a t1/2 of - 1130 min, by NMR integration. No other products were observed. After 18.5 hours, the solvent was removed and the product readily separated from starting material by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4 mL/min flow rate) to obtain pure precursor D (4.5 mg, 43% yield). The analytical data for precursor D is 1H-NMR: δ 0.88 (3H, C18-CH3, s), 0.96 (3H, C21-CH3, d, J -6.6 Hz), 1.21 (6H, C2627-2CH3, s), 1.69 (3H, C19-CH3, s), 2.52 (1 H, dd, J - 16.5 Hz, 4.2 Hz), 4.07 (1 H, C3-H, m), 4.19 (1 H, C H, br s), 5.78 and 5.92 (2H, C6-H and C7-H, AB pattern, J - 12.1 Hz). JJV: (100% EtOH) λmax 256 nm (e 11 ,000); λmn 230 nm (e 7,500).
A solution of precursor D (7.5 mg, 0.0180 mmol) was dissolved in ether (1 ml) under argon. A solution of iodine dissolved in ether (0.49 mM, 150 μl) was added and the solution was stirred in the presence of room lights for 8 hours under a positive pressure of argon. The solution was then condensed to leave a crude oily residue. The crude residue was subjected to HPLC (Rainin Microsorb, 5 μm silica, 10 mm x 25 cm, 11% isopropanol/hexanes) to produce JD (3.9 mg, 52%).
The analytical data for JD is 1H-NMR (300 MHz): (CDCI3) δ 0.91 (3H, C18-CH3, s), 0.97 (3H, C21-CH3, d, J -7.6 Hz), 1.22 (6H, C2627-CH3, s), 1.92 (3H, C19-CH3, s), 0.79-2.79 (remaining ring and side chain hydrogens, various m), 4.16 (1H, H3, m), 4.25 (1H, H1 ( brs), 6.47 (1H, H6or7, d, J - 16.0 Hz), 6.54 (1 H, H7or6, d, J - 16.0 Hz). 13C-NMR (75.5 MHz): (CDCI3) δ 16.4, 18.2, 19.0, 20.8, 24.2, 25.9, 27.0, 29.25, 29.32, 34.6, 35.2, 36.2, 37.0, 40.7, 44.1 , 44.4, 56.2, 64.2, 71.1 , 71.5, 122.9, 124.4, 128.5, 129.5, 130.7, 151.3. UV: (100% EtOH) λmax 292 nm (e 30,800); λsh 302 nm (e 23,100), 282 (e 25,600). HRMS: (FAB) m/z 416.3302 (calcd. for C27H44O3, 416.3292). MS: (70ev El) m/z 416 (5, M), 398 (13, M-H2O), 380 (9, M-2H2O), 365 (6), 347 (4), 287 (4), 269 (7), 251 (27), 236 (7), 197 (17), 181 (9), 129 (24), 105 (39), 91 (48), 81 (25), 69 (43), 59 (100, base).
HS and JD will have the same biological activity as described in Example 2. HS biological responses will mimic 1 ,25(OH)2D3 in that it can assume both 6-s-cis and 6-s-trans.
Example 5 — Synthesis and Biological Properties of 23-(m- (Dimethylhydroxymethyl)-22-yne-24,25,26,27-tetranor-1σ-OH-D3(IB)
IB was prepared according to the procedure set forth in FIG. 27. In step 1 of the synthesis, 3-iodobenzoic acid is refluxed for 14 hours in 80 ml
MeoH and 3 ml of H2SO4. The resulting product (C8H7O2l) is recrystallized from an H2O/hexane mixture. In step 2, C8H7O2l is reacted with C14H22O in the presence of PPh3 PdCI2, Cul and Et2NH. The resulting C22H28O3 was obtained by flash chromatography using a solvent of 20 percent ethyl acetate in hexane.
In step 3, the product of step 2 was reacted with 55 mg OH, 183 mg pyridinium chlorochromate (PDC), 12 mg pyridinium trifluoroacetate (PTFA) and 100 ml CH2CI2 according to the procedure set forth in S.A. Barrack et al., J. Org. Chem. 1988, 53, 1790. The reaction was carried out at room temperature for 5 hours. The resulting black mixture was filtered and washed with CH2 CR2 and extracted with ethyl acetate to produce pale yellow oil. This oil was flash chromatographed to produced C22H26O3.
In step 4, the product of step 3 was reacted with 70 mg phosphine oxide, 82 μ\ n-Butyl, 35 mg of the CD ketone in 2 ml of a solution of THF. The n-Butyl was added dropwise to the solution of phosphine oxide in THF. The resulting orange colored solution was stirred at -78°C for 10 minutes and the CD ring ketone in THF was added dropwise. The reaction mixture was stirred at -78°C for 4 hours. At this point the solution turned pale yellow. The solution was quenched with H2O, extracted with ethyl acetate and dried over Na2SO4. The solvent was vacuum evaporated and the resulting product purified by flash chromatography. In step 5, 10 mg of the product of step 4 reacted with 53 μ\ MeLi in
2 ml THF. To the solution of the step 4 product in THF at room temperature was added MeLi dropwise. The mixture was stirred at room temperature for 1 hour, quenched with H2O. The resulting product was washed with H2O and dried. In step 6, 10 mg of the product of step 5 was dissolved in 1 ml THF.
60 μ\ TBAF (1 m in THF) was added dropwise and the solution stirred at room temperature overnight. The resulting mixture was passed through a short volume of AI2O3 (neutral) and extracted with ethyl acetate. The resulting product was identified as IB by NMR and UV. IB will mimic the genomic and nongenomic biological activity of
1σ,25(OH)2D3 because it can assume the 6-s-cis and 6-s-trans conforma¬ tion. Example 6 — Synthesis and Biological Activity of 1σ,25-(OH)2-7-DHC (JM) and 1σ,25-(OH)2-Lumisterol3 (JN).
JM and JN are closed B-ring analogues which both stimulate transcaltachia while neither competes with 1σ,25(OH)2D3 for binding to either the nuclear vitamin D receptor (N-VDR) or the serum vitamin D transport protein (DBP).
JM [1σ,25-(OH)2-7-Dehydrocholesterol] and JN [1σ,25-(OH)2-7-Lumi- sterol3] were synthesized as follows. A solution of the known 1σ,25-(OH)2- previtamin D3 (120 mg) in methanol was irradiated (Hanovia 450 watt medium pressure mercury lamp, pyrex filter, λ > 300 nm) for 3 hours at room temperature. The solution was concentrated and subjected to HPLC (Rainin Microsorb, 5 μm silica, 10 mm x 25 cm, 11% isopropanol/hexanes) to afford in order of elution JM (9.1 mg, 7.6%), JN (15.0 mg, 12%) and starting 1σ,25-(OH)2-previtamin D3 (10.6 mg, 8.8%). The identifying characteristics of JM are: 1H-NMR (CDCI3): δ 0.63
(3H, C18-CH3, s), 0.95 (3H, C19-CH3, s), 0.96 (3H, C21-CH3, d, J -5.6 Hz), 1.22 (6H, C2627-CH3, s), 2.35 (1 H, apparent t, J - 12.7 Hz), 2.55 (1 H, d with fine structure, J - 14.2 Hz), 2.70 (1H, m), 3.77 (1 H, H1 f br s), 4.07 (1 H, H3, m), 5.38 (1 H, H6or7, ddd, J -5.5 Hz, 2.8 Hz, 2.8 Hz), 5.73 (1 H, H7or6, dd, J -5.5 Hz, 2.2 Hz). 13C-NMR (CDCI3): δ 11.9, 16.3, 18.8, 20.8, 20.9, 23.0, 28.1 , 29.2, 29.4, 36.1 , 36.4, 38.0, 38.5, 39.2, 40.0, 43.1 , 44.4, 54.7, 55.8, 65.5, 71.1 , 73.0, 115.2, 122.1 , 141.4 UV: (100% EtOH) λmax 294 nm (e 13,400), 272 nm (e 12,800); Λmin 290 nm (e 7,800), 278 nm (e 11 ,500); λsh 264 nm (<r 9,600). HRMS: (Cl, CH4) m/z 417.3365 (calcd. for C27H44O3 plus H, 417.3370). MS: (Cl, CH4) m/z 417 (28, MH), 400 (67), 381 (31), 354 (11), 338 (6), 323 (6), 297 (4), 267 (4), 251 (8), 225 (10), 211 (10), 197 (11), 171 (19), 157 (15), 119 (12), 105 (15), 91 (14), 81e (14), 69 (27), 59 (base).
The identifying characteristics of JN are: 1H-NMR (CDCI3): δ 0.61 (3H, C18-CH3, s), 0.78 (3H, C19-CH3, s), 0.91 (3H, C21-CH3, d, J -5.2 (Hz), 1.21 (6H, C2627-CH3) s), 2.50 (2H, m), 4.10 (1 H, H,, dd, J -9.2 Hz, 4.8 Hz), 4.14 (1 H, H3, dd, J - 3.0 Hz, 3.0 Hz), 5.45 (1 H, H6or7, m), 5.75 (1 H, H7or6, dd, J - 5.1 Hz, 1.7 Hz). 13C-NMR (CDCI3); δ IΛ, 18.3, 18.5, 20.9, 21.7, 22.6, 28.8, 29.2, 29.4, 29.7, 36.2, 37.5, 38.9, 39.5, 41.4, 43.9, 44.4, 46.2, 49.5, 57.3, 66.2, 71.1 , 75.8, 115.5, 123.6, 137.2, 142.2 UV: (100% EtOH) λmax 282 nm (e 6,900), 274 nm (e 7,300); λsh 294 nm (e 3,900), 264 nm (e 5,900). HRMS: m/z (Cl, CH4) 417.3365 (calcd. for C27H44O3 plus H, 417.3370), MS (Cl, CH4): m/z 417 (86, MH), 400 (base), 382 (60), 366 (13), 343 (8), 325 (6), 311 (5), 287 (15), 269 (13), 251 (9), 227 (13), 213 (9), 174 (46), 157 (21), 143 (14), 119 (7), 105 (8), 95 (8), 81 (8), 69 (14), 59 (38). The biological assays were conducted as follows: White Leghorn cockerels (Hyline International, Lakeview, CA) were obtained on the day of hatch and maintained on a vitamin D supplemented diet (O.H. Kruse Grain & Milling, Ontario, CA) for 4-5 weeks (400-600 gm). When vitamin D deficient chicks were employed, they were raised for 4 weeks on a rachitogenic diet (50).
Intestinal 45Ca2+ Transport Measurements (Transcaltachia)
Measurement of Ca2+ transport (transcaltachia) was carried out in perfused chick duodena essentially as previously described (6, 8). The chicks were anesthetized with chloropent (0.3 ml/100 gm) and the duodenal loop exposed. Three pairs of blood vessels branching off from the celiac artery were ligated prior to cannulation of the celiac artery itself. The arterial perfusion was initiated with Gey's Balanced Salt Solution (GBSS) modified to contain 0.9 mM CaCI2 and oxygenated with 95% O2/5% CO2 at a flow rate of 2 ml/min. An auxiliary pump was used for the introduction of vehicle (0.005% ethanol v/v, final concentration) or test analogues plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min. The intestinal loop was then excised and the lumen flushed and filled with GBSS (lacking NaHCO3 and glucose) containing 45Ca2+ (5 /Ci/ml). The lumenal solution was renewed constantly at a rate of 0.25 ml/min to insure a steady concentration of 45Ca2+ at the brush border of the epithelia. The intestinal preparation at 27°C was kept moist under layers of saline-dampened cheese cloth. Each duodenum was perfused with control medium (vehicle) for 20 min after filling the lumen with 45Ca2+ to establish the basal transport rate. The tissue was then either exposed to the test analog or continued on vehicle for an additional 40 minutes. The venous effluent was collected at 2 minute intervals during basal and treatment periods and assayed for 45Ca + activity via liquid scintillation. The results are expressed as the ratio of the 45Ca2+ appearing at each interval of the treated phase (40 minutes = 20 data points) over the average basal rate (initial 20 minutes) (6, 51). Receptor and DBP Binding The relative ability of an analog to compete with [3H]-1σ,25(OH)2D3 for binding to the chick intestinal nuclear receptor was carried out in vitro according to the previously described procedures (25). In this assay, increasing concentrations of nonradioactive 1σ,25(OH)2D3 or the JM or JN analog were incubated with a fixed saturating amount of [3H]-1σ,25(OH)2D3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [3H]-1σ,25(OH)2D3 was then calculated and plotted as a function of the relative concentration of the analog and [3H]-1σ,25(OH)2D3. The competitive index value for each analog is then normalized to a standard curve obtained with nonradioactive 1σ,25(OH)2D3 as the competitive steroid and placed on a linear scale of Relative Competitive lndex(s) (RCI), where the RCI of 1σ,25(OH)2D3 is by definition 100. The relative ability of vitamin D analogues to bind to the plasma transport protein, the vitamin D binding protein (DBP) were carried out in a similar fashion (52). JM and JN differ in the fixed orientations of the two A-ring hydroxyls;
JM is 1σ-axial and 3 ?-equatorial, JN is 1σ-equatorial and 3 ?-axial. Also it is apparent that JM and JN are analogues of the 6-s-c/s form of 1σ,25(OH)2D3; they cannot exist in the extended 6-s-trans conformation. The results of the above biological tests are summarized as follows: Transcaltachia
FIGS. 24A and 24B illustrate the appearance of 5Ca2+ in the venous effluent mediated by the two different concentrations of analogues, JM and JN, respectively, vehicle control (ethanol only) and 650 pM 1σ,25(OH)2D3 as positive control. The efficacy of 300 pM JM in initiating transcaltachia is not significantly greater than the control, and the response elicited at 650 pM JM is only 60% of that induced by the natural metabolite. Perfusion with JN, however, produced a stimulation nearly identical to that of 1σ,25(OH)2D3 with JN achieving only a slightly lower ratio of transport 45Ca2+ than that achieved by 1σ,25(OH)2D3.
FIGS. 25A and 25B present the dose response curves for JM and JN, respectively. Each bar represents the 40 minute data point of FIGS. 25A and 25B which is taken as the maximum response elicited by the analog at that concentration. Analog JN eventually reaches the 4-fold plateau at 1300 pM which is the equivalent of the maximum stimulation achieved by 1σ,25(OH)2D3 at 650 pM. The 5Ca2+ transport ratio for analog JM at 650 pM peaked at 2.5 and was not further increased as a consequence of increasing the JM concentration of 1300 pM. N-VDR & DBP Binding
The results of these tests are summarized in Table IV. Neither JM nor JN significantly competes with 1σ,25(OH)2D3 for binding to the nuclear receptor. Other studies have correlated very poor N-VDR binding to lack of genomic activity (7,8,11), so both of these analogues are ineffective gene regulators.
TABLE IV Relative Competitive Index (RCI)
For Analogues of 1α,25(OH)2D3
RCI RCI
Analog DBP N-VDR
1σ,25(OH)2D3 100 100
25(OH)D3 66,800 0.15 ± 0.05
HJ 800 24 ± 4.5
HF 8.6 ± 0.1 10 ± 6
JM < 0.1 0.12 ± 0.05
JN < 0.1 1.8 ± 0.5 The above results show that both JM and JN are useful in controlling nongenomic mechanisms, such as transcaltachia. These two analogues can be used to act as agonists for nongenomic responses as previously described.
Example 7 — Synthesis and Biological Activity of (9σ,10σ)-and (9/?, 10/?)- 1σ,25-Dihydroxy-7-dehydrocholesterol-1σ,25(OH)2-Pyrocalciferol (JO) and 1σ,25(OH)2-lsopropylcalciferol3 (JP).
JO and JP were prepared according to the following procedures: An argon flushed solution of 1σ,25-(OH)2-previtamin D3 (54.2 mg) dis¬ solved in DMF (15 mL) containing a drop of 2,4,6-trimethylpyridine was heated in a screw cap vial (156°C) for 18 hours. The cooled solution was then concentrated and the crude mixture was purified by HPLC (Rainin Microsorb, 5 //m silica, 10 mm x 25 cm, 11% isopropanol/hexanes) to afford in order of elution the 1 ,25-isopyrocholecalciferol (7.3 mg, 13.5%), 1,25- pyrocholecalciferol (20.1 mg, 37.1%) and 1 ,25-vitamin D3 (2.1 mg, 3.9%). Analysis of the crude mixture by 1H-NMR spectroscopy showing the ratio of pyro to isopyro isomers to be 3:1.
The spectral data for JO is 1H-NMR (300 MHz): (CDCI3) δ 0.53 (3H, C18-CH3, s), 0.90 (3H, C21-CH3, d, J -6.0 Hz), 1.02 (3H, C19-CH3, s), 1.21 (6H, C2627-CH3, s), 0.80-2.05 (remaining ring and side chain hydrogens, various m), 2.15 (1H, dd, J - 12.6 Hz, 7.6 Hz), 2.26 (1 H, d with fine structure, J - 6.1 Hz), 2.54 (1H, br d, J -6.1 Hz), 4.16 (1H, H3, dddd, J -2.8 Hz, 2.8Hz, 2.8 Hz, 2.8 Hz), 4.31 (1H, H,, dd, J - 12.0 Hz, 4.6 Hz), 5.34 (1 H, H6or7, d, J -5.7 Hz), 5.61 (1H, H7or6, dd, J - Hz, 2.5 Hz). 13C-NMR (75.5 MHz): (CDCI3) 6 12.2, 17.4, 18.7, 20.8, 20.9, 26.0, 28.5, 29.2, 29.4, 29.7, 36.2, 36.4, 37.6, 38.0, 41.1 , 44.4, 48.7, 50.6, 56.4, 57.6, 66.7, 66.9, 71.1 , 111.7, 121.1 , 134.8, 140.1. UV: (100% EtOH) ylmax 286 nm (e 9,400), 276 nm (e 9,300); Λmjn 280 nm (e 8,800); λsh 296 nm (e 5,700), 266 nm (e 7,000). HRMS: (Cl, CH4) m/z 417.3366 (calcd. for C27H44O3 plus H, 417.3370). MS: (Cl, CH4) m/z 417 (49, MH), 400 (base), 382 (54), 364 (9), 343 (4), 326 (4), 312 (3), 287 (4), 269 (4), 251 (4), 227 (6), 213 (4), 197 (6), 157 (12), 143 (8), 111 (9), 95 (13), 81 (17), 69 (24), 59 (85).
The spectral data for JP is 1H-NMR (300 MHz): (CDCI3) δ 0.65 (3H, C18-CH3, s), 0.92 (3H, C21-CH3, d, J -5.3 Hz), 1.21 (6 H, C2627-CH3, s), 1.30 (3H, C19-CH3, s), 0.80-2.08 (remaining ring and side chain hydrogens, various m), 2.39-2.66 (3H, overlapping m), 3.71 (1 H, H1 t dd, J -2.8 Hz, 2.8 Hz), 3.94 (1 H, H3, dddd, J - 10.9 Hz, 10.9 Hz, 5.5 Hz, 5.5 Hz), 5.34 (1 H, H6or7, ddd, J -5.5 Hz, 2.7 Hz, 2.7 Hz), 5.95 (1 H, H7or6, d, J -5.5 Hz). 13C NMR (75.5 MHz: (CDCI3) δ 18.3, 18.6, 20.4, 20.9, 22.4, 26.1 , 28.8, 29.2, 29.3, 29.7, 36.1 , 37.5, 39.2, 41.2, 42.0, 43.5, 44.4, 49.2, 57.3, 69.8, 71.1 , 74.5, 115.2, 122.8, 135.5, 142.8. UV: (100% EtOH) λmax 286 nm (e 7,800), 278 nm (e 7,900); λsh 296 nm (e 6,500). HRMS: (Cl, CH4) m/z 417.3351 (calcd. for C27H44O3 plus H, 417.3370). MS: (Cl, CH4) m/z 417 (36, MH), 400 (base), 382 (51), 364 (12), 342 (4), 312 (3), 288 (6), 270 (10), 252 (10), 215 (9), 197 (6), 171 (11), 157 (7), 143 (5), 123 (6), 105 (13), 91 (8), 81 (8), 69 (17), 59 (40).
Both JO and JP will have the same nongenomic actions achieved by analogues JM and JN since JO and JP locked in the 6-s-cis conformation.
Example 8 - Synthesis and Biological Activity of (1S, 3R, 6S)-7,19-Retro- 1 ,25-(OH)2-D3 (JV) and (1S, 3R, 6S)-7,19-Retro-1 ,25-(OH)2-D3 (JW) The analog JV was synthesized as follows:
(1S,3R,6S)-1,3-Di(tert-butyldimethylsilyloxy)-25-trimethylsilyloxy- 9,10-secocholesta-5(10),6,7-triene (A) is a starting material that was prepared first as follows: Freshly purified 1 ,2-diiodoethane (412 mg, 1.46 mmol) and samarium metal (286 mg, 1.90 mmol) were dried under vacuum and suspended in 4 mL THF under an argon atmosphere. This solution was stirred for 2 hours until it became deep blue. A solution of propargyl benzoate (477 mg, 0.570 mmol) and Pd(PPh3)4 (65.8 mg, 0.037 mmol) in 6 mL THF was added via cannula. Freshly distilled isopropanol (0.5 mL) was added and the solution was stirred under a positive argon atmosphere for 14 hours. Saturated aqueous Na2CO3 (2 mL) was added to quench the reaction. The organic layer was diluted with ether and then the mixture was washed with Na2CO3 (3 x 10 mL), dried with MgSO4 and concentrated. The product was purified by flash chromatography (silica gel, 2% EtOAc/hexanes) followed by HPLC (2% EtOAc/hexanes, Rainin Dynamax column, 8 mL/min flow rate) to afford vinylallene A (0.3085 g, 75.5%). The product was identified by 1H-NMR analysis. This material is more stable as the triol. 1H-NMR: δ 0.06 (6H, Si-2CH3, s), 0.10 (9H, Si-3CH3, s), 0.11 (6H, Si-2CH3, s), 0.73 (3H, C21-CH3, s), 0.89 (9H, Si-tBu, s), 0.91 (9H, Si-tBu, s), 0.94 (3H, C18-CH3, d, J -6.5 Hz), 1.20 (6H, C2627-CH3, s), 1.76 (3H, C19-CH3, s), 4.09-4.13 (1 H, C3-H, m, overlapping CrH), 4.17 (1 H, C H, br distorted singlet), 6.13 (1 H, C6-H, dd, J -3.9 Hz, 3.9 Hz).
(1S,3R,6S)-1 ,3,25-Trihydroxy-9,10-secocholesta-5(10),6,7-triene (JV) was then prepared as follows: To the vinylallene A (0.1054 g, 0.1469 mmol) was added TBAF (1 M in THF, 1.6 mL, 1.6 mmol). The solution was stirred under an argon atmosphere for 19 hours. Water (1 mL) was added and the solution stirred 30 minutes. The mixture was extracted with ether (3 x 15 mL) and the ether extracts washed with brine (1 x 10 mL) and dried (MgSO4). The concentrated residue was subjected to flash chromatography (silica gel, 80% EtOAc/hexanes) followed by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4 mL/min flow rate) to afford purified deprotected vinylallene JV together with its 6R-diastereomer JW (46.1 mg, 75.3% total yield) in a —92:8 ratio by NMR integration. By shave-recycle HPLC separa¬ tion, pure JV could be obtained and characterized by spectroscopic analysis. 1H-NMR: δ 0.74 (3H, C18-CH3, s), 0.95 (3H, C21-CH3, d, J - 6.4 Hz), 1.22 (6H, C2627-CH3, s), 1.87 (3H, C19-CH3, s), 2.29 (1 H, br d, J - 13.2 Hz), 2.62 (1 H, br dd, J - 16.5 Hz, 4.5 Hz), 4.11-4.20 (1 H, C3-H, m, W-27.8 Hz), 4.23 (1 H, CrH, br m W-8.6 Hz), 6.14 (1 H, C6-H, dd, J -4.1 Hz, 4.1 Hz). UV: (100% EtOH) λmax 242 nm (e 24,300), 234 nm {e 23,500).
JW which is also known as (1S,3R,6R)-1,3,25-Trihydroxy-9,10- secocholesta-5(10),6,7-triene was isolated from the above solution as follows: A solution of (6S/6R)-vinylallenes JV and JW (2.6 mg, 0.0062 mmol, -92:8 ratio of 6S:6R) in methanol-d4 (1 mL) was prepared in a quartz NMR tube. The solution was saturated with argon for 30 minutes and then the NMR tube was capped and then irradiated with ultraviolet light from a Hanovia 450 watt medium pressure lamp for 30 minutes. Integration of the C18-Me signals in the NMR spectrum revealed a - 50:50 mixture of the two isomers. Solvent was removed and the products separated by HPLC (11% isopropanol/hexanes, Rainin Microsorb column, 6 mL/min, flow rate). Taking a front cut of the overlapping peaks gave pure (6R)- vinylallene JW (0.9 mg, 35%). This product was identified and characterized through spectroscopic analysis. 1H-NMR: δ 0.65 (3H, C18- CH3, s), 0.94 (3H, C21-CH3, d, J -6.4 Hz), 1.21 (6H, C2627-2CH3, s), 1.87 (3H, C19-CH3, br s), 2.28 (1H, br d, J - 13.0 Hz), 2.52 (1 H, dd, J ~ 16.3 Hz, 5.0 Hz), 4.12 (1H, C3-H, m, W-30.0 Hz, overlapping), 4.20 (1 H, CrH, br s), 6.10 (1 H, C6-H, dd, J -3.2 Hz, 3.2 Hz). UV: (100% EtOH) 242 nm (e 22,300), 234 nm (e 22,100). The biological activity of JV and JW will mimic those achieved by
1 ,25(OH)2D3.
Example 9 — Synthesis and Biological Activity of 1 ,25-(OH)27, 8-c/s-D3 (JR) and 1 ,25-(OH)2-5,6-fraπs-7,8-c/s-D3 (JS). JR was synthesize from JV as follows:
To the vinylallene JV (19.7 mg, 0.047 mmol) and (np)(CO)3Cr (14.7 mg, 0.0557 mmol) in a 10 mL flask with a stir bar was added 1 mL of ace¬ tone (distilled from CaSO4). After deoxygenation of the mixture by four freeze-pump-thaw cycles, the solution was stirred at 40°C under a positive pressure of argon for 4 hours. Acetone was removed under reduced pres¬ sure and the product was purified by flash chromatography (silica gel, 80% EtOAc/hexanes) followed by separation by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4.0 mL/min flow rate) to afford three components in the following order of elution: major product JR (17.0 mg, 86.4%), recovered starting material JV (1.4 mg, 7.1%), and a minor amount of cis- isotachysterol (1.5 mg, 7.6%). The NMR analysis of JR provided the following results: 1H-NMR: δ 0.64 (3H, C18-CH3, s), 0.95 (3H, C21-CH3, δ, J -6.4 Hz), 1.22 (6H, C2627-2CH3, s), 2.24 (1 H, dd, J - 12.4 Hz, 9.0 Hz), 2.55 (1 H, dd, J - 12.5 Hz, 3.4 Hz), 4.17 (1 H, C3-H, dddd, J -4.2 Hz, 4.2 Hz, 4.2 Hz, 4.2 Hz), 4.42 (1 H, C H, br s), 5.01 (1 H, C19-H, br s), 5.32 (1 H, C19- H, br s), 6.20 and 6.54 (2H, C6-H and C7-H, AB pattern, J - 11.5 Hz). UV: (100% EtOH) λmax 266 nm (e 15,000); _._.._ 228 nm (e 9,300).
5,6-frans-7,8-c/s-1σ,25-Dihydroxyvitamin D3(JS)wassynthesized by Iodine Catalyzed Cis-Trans Isomerization of JR. The procedure was as follows: To 7,8-c/s analog JR ( -5.0 mg, 0.012 mmol) was added a solu¬ tion of 0.1 mol % l2 in ether (1 mL, 9.8 x 10"6 M). The solution was stirred under an argon atmosphere for 30 min, the solvent removed and the products separated by HPLC. The first separation (80% ethyl acetate/ hexanes, Rainin Microsorb column, 4 mL/min flow rate) gave two fractions, each of which was subjected to NMR analysis. Fraction I contained products A, B, and C and fraction II contained products B, C, and D. By 1H-NMR analysis of the two fractions and their weights, it was estimated that the four products, A, B, C, and D, were obtained in an overall -8:32:42:18 ratio. The four products were identified from the 1H-NMR analysis as the following geometric isomers: A, 5,6-c/s-7,8-frans; B, 5,6- trans-7 ,8-trans; C, 5,6-trans-7 ,8-c/s (JS); and D, 5,6-c/s-7,8-c/s (JR). Repeated purification by HPLC (11% EtOAc/hexanes, Rainin Microsorb column, 6 mL/min flow rate) eventually afforded pure (JS) (0.5 mg; color¬ less, viscous oil), suitable for spectroscopic characterization. 1H-NMR: δ 0.66 (3H, C18-CH3, s), 0.96 (3H, C21-CH3, d, J -6.3 Hz), 1.22 (6H, C2627- 2CH3, s), 2.78 (1H, dd, J - 12.9 Hz, 2.7 Hz), 4.20-4.28 (1 H, C3-H, m, W-26 Hz), 4.45-4.52 (1 H, C H, m, W-23 Hz), 4.95 (1H, C19-H, br s), 5.05 (1 H, C19-H, br s), 6.15 and 6.75 (2H, C6-H and C7-H, AB pattern, δ, J - 11.8 Hz). UV: (100% EtOH) λma 27 '4 nm (e 17,400); Λmin 234 nm (e 5,500).
The biological activity of JR and JS were determined by the chick intestinal receptor steroid competition assay which has been described in the preceding examples. Both JR and JS significantly suppress the ability of the natural hormone to bind receptor. These results show that JR and JS are also useful for regulating nongenomic mechanisms such as transcaltachia.
Example 10 — Synthesis and Biological Activity of 22-(p-Hydroxyphenyl)- 23,24,25,26,27,pentanor-D3(JX) and 22-(m-Hydroxyphenyl-23,24,25,26,27- pentanor-D3 (JY)
The A-ring phosphine oxide (48 mg, 0.11 mmol) in dry THF (1.8 mL) was cooled to -78°C and n-butyllithium (1.5 M in hexanes, 0.074 mL, 0.11 mmol) was added dropwise via a syringe. The resulting deep red solution was stirred for 10 minutes and then treated with a solution of the appropriate CD-ring ketone (28 mg, 0.070 mmol) in dry THF (0.6 mL) via cannula. The mixture was stirred 2 hours at -78°C, warmed to room temperature and quenched with water (5 mL). The aqueous layer was separated and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The crude residue was purified by rapid filtration through a short silica gel column (20% EtOAc/hexanes) to afford 20.1 mg (46%) of the protected vitamin. The latter (20.1 mg, 0.0315 mmol) in THF (1 mL) was placed under argon and TBAF (0.32 mL, 1 M in THF, 0.32 mmol) was added dropwise. After stirring for 18 hours, the solvent was partially evaporated and the residue diluted with water (5 ml). After extracting the aqueous layer with EtOAc (3 x 5 mL), the combined organic layers were washed with brine and dried with Na2SO4. The residue was then purified by HPLC (20% EtOAc/hexanes) to afford, after vacuum drying, 4.7 mg (36% of JX.
The spectral data for JX is as follows: H-NMR (300 MHz): (CDCI3) δ 0.57 (3H, C18-Me), 0.81 (3H, H21, d, J -6.4 Hz), 1.2-1.5 (remaining ring and side chain hydrogens, series of m), 2.58 (dd, J - 13.0 Hz, 3.0 Hz), 2.83 (dd, J - 13.1 Hz, 3.0 Hz), 3.96 (1H, H3, m), 4.83 (1H, H19, br s), 5.06 (1 H, H19, br s), 6.05 (1 H, d, J - 11.2 Hz), 6.24 (1 H, d, J - 11.2 Hz), 6.74 (2H, Ar-H35, d, J - 8.4 Hz), 7.00 (2H, Ar-H26, d, J -8.3 Hz). UV: (100% EtOH) -4max 266 nm (e 20,600); λmm 240 nm (e 15,000). HRMS: m/z 406.2855 (calcd. for C28H38O2, 406.2873). MS: m/z 406 (23, M), 388 (3), 373 (11), 347 (35),
299 (4), 281 (5), 253 (45), 239 (3), 211 (5), 197 (5), 158 (14), 136 (29, A-ring fragment due to C78 cleavage), 118 (30, m/z 136-H2O), 107 (base), 91 (20), 81 (16), 67 (10), 55 (17). The A-ring phosphine oxide (70 mg, 0.154 mmol) in dry THF (2.8 mL) was cooled to -78°C under argon and n-butyllithium (1.5 M in hexanes, 0.100 mL, 0.154 mmol) was added via a syringe. The solution was stirred 10 minutes and then treated dropwise with a solution of the appropriate CD- ring ketone (41 mg, 0.102 mmol) in dry THF (0.85 mL). The mixture was stirred 2 hours at -78°C and then allowed to warm to room temperature over 1 hour. The solvent was partially evaporated and then quenched with 5 mL water. The aqueous layer was separated and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude residue was purified by rapid filtration through a short silica gel column (20% EtOAc/hexanes) to yield 19.2 mg (29% of the protected vitamin. The protected vitamin (19.2 mg, 0.03 mmol) in dry THL (1 mL) was placed under argon and TBAF (1 M in THF, 0.30 mn, 0.30 mmol) was added dropwise. After stirring 18 hours, the solvent was partially evaporated and diluted with water (5 mL). After extracting the aqueous layer with EtOAc (3 x 5 mL), the combined organic layers were washed with brine and dried over Na2SO4. The residue was purified by HPLC (20% EtOAc/hexanes) and after vacuum drying afforded 2.8 mg (23%) of JY. The spectral data for JY is as follows: 1H-NMR (300 MHz): (CDCI3) δ 0.58 (3H, H18-CH3, s), 0.83 (3H, H20-CH3, d, J -6.5 Hz), 1.2-1.5 (remaining ring and side chain hydrogens, series of m), 2.58 (1 H, dd, J - 13.0 Hz, 3.3 Hz), 2.85 (2H, H22, m), 3.97 (1 H, H3, m), 4.83 (1 H, H19, s), 5.07
(1H, H19, s), 6.06 (1H, H67, AB pattern, d, J - 11.2 Hz), 6.24 (1 H, H67, AB pattern, d, J - 11.2 Hz), 6.63 (1H, Ar H, s), 6.64 (1 H, Ar H, d, J -7.4 Hz), 6.71 (1H, Ar H, d, J -7.52 Hz), 7.13 (1H, Ar H, dd, J - 15.45 Hz, 7.8 Hz). HRMS: m/z 406.2872 (calcd. for C28H38O2, 406.2873). MS: m/z 406 (44), 373 (14),
347 (7), 299 (6), 271 (9), 253 (7), 211 (12), 176 (20), 158 (30), 136 (23), 118 (54), 107 (35), 91 (23), 79 (22), 67 (12), 55 (11). The biological activity of JX and JY will mimic that of 1σ,25(OH)2D3 since these two analogues can assume both the 6-s-cis and 6-s-trans conformation.
Having thus described exemplary embodiments of the present inven¬ tion, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternations, adaptations and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.
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Claims

CLAIMS What is Claimed is:
1. A method for controlling genomic or nongenomic cellular responses of a cell which are mediated by 1σ,25-(OH)2 vitamin D3, said method comprising the step of treating said cell with an amount of a vitamin D analog selected from the group consisting of previtamin D3, 1 ?,25-(OH)2 vitamin D3, 14-Epi-1 ,25-(OH)2-D3 (GE), 14-Epi-1 ,25(OH)2-Pre-D3 (GF), 1 σ, 18,25-(OH)3-D3(HS),23-(m-(Dimethylhydroxymethyl)phenyl)-22-yne- 24,25,26,27-tetranor-1σ-OH-D3 (IB), 1σ,25-Dihydroxy-trans-lsotachysterol (1 ,25-trans-lso-T) (JD), 1σ,25-(OH)2-7-DHC (JM), 1σ,25-(OH)2-Lumisterol3 (J N ) , 1 σ , 25- ( O H )2- Py ro ca l cife ro l3 (J O ) , 1 σ, 25- ( O H )2- lsopyrocalciferol3(JP),1 ,25-(OH)2-7,8-cis-D3(JR),1 ,25-(OH)2-5,6-trans-7,8-cis- D3 (JS), (1S,3R,6S)-7,19-Retro-1 ,25-(OH)2-D3 (JV), (1S,3R,6R)-7,19-Retro- 1,25-(OH)2-D3(JW),22-(p-Hydroxyphenyl)-23,24,25,26,27-penanor-D3(JX) and 22-(m-Hydroxyphenyl)-23,24,25,26,27-penanor-D3(JY), wherein the amount of said vitamin D analog used to treat said cell is sufficient to measurably effect at least one genomic or nongenomic cellular response which is medi¬ ated by 1σ,25-(OH)2 vitamin D3.
2. A method according to claim 1 , wherein said nongenomic cellular response which is mediated by 1σ,25-(OH)2 vitamin D3 is transcaltachia.
3. A method according to claim 1 wherein said cell is located in vivo.
4. A method according to claim 1 wherein said cell is located in vitro.
5. A method according to claim 1 wherein said cell is located in intestinal tissue.
6. A method according to claim 1 wherein said vitamin D analog is an agonist of said nongenomic cellular response mediated 1σ,25-(OH)2D3.
7. A method according to claim 1 wherein said vitamin D analog is an antagonist of said nongenomic cellular response mediated 1σ,25-(OH)2 vitamin D3.
8. A method according to claim 6 wherein said vitamin D analog is previtamin D3.
9. A method according to claim 7 wherein said vitamin D analog is 1 ?,25-(OH)2 vitamin D3.
10. A composition of matter comprising a vitamin D analog selected from the group consisting of 14-Epi-1 ,25-(OH)2-D3 (GE), 14-Epi-1 ,25-(OH)2- P r e - D 3 ( G F ) , 1 σ , 1 8 , 2 5 - ( O H ) 3 - D 3 ( H S ) , 2 3 - ( m - (Dimethylhydroxymethyl)phenyl)-22-yne-24,25,26,27-tetranor-1σ-OH-D B), 1σ,25-Dihydroxy-trans-lsotachysterol(1 ,25-trans-lso-T)(JD), 1σ,25-(OH)2-7- DHC (JM), 1σ,25-(OH)2-Lumisterol3 (JN), 1σ,25-(OH)2-Pyrocalciferol3 (JO), 1σ,25-(OH)2-lsopyrocalciferol3 (JP), 1 ,25-(OH)2-7,8-cis-D3 (JR), 1 ,25-(OH)2- 5,6-trans-7,8-cis-D3 (JS), (1S,3R,6S)-7,19-Retro-1 ,25-(OH)2-D3 (JV), (1 S,3R,6R)-7,19-Retro-1 ,25-(OH)2-D3 (JW), 22-(p-Hydroxyphenyl)- 23,24,25,26,27-pentanor-D3(JX)and22-(m-Hydroxyphenyl)-23,24,25,26,27- pentanor-D3 (JY).
11. A composition of matter according to claim 10 wherein said analog is 14-Epi-1 ,25-(OH)2-D3 (GE).
12. A composition of matter according to claim 10 wherein said analog is 14-Epi-1 ,25-(OH)2-Pre-D3 (GF).
13. A composition of matter according to claim 10 wherein said analog is 1σ,18,25-(OH)3-D3 (HS).
14. A composition of matter according to claim 10 wherein said analog is 23-(m-(Dimethylhydroxymethyl)phenyl)-22-yne-24,25,26,27- tetranor-1σ-OH-D3 (IB).
15. A composition of matter according to claim 10 wherein said analog is 1σ,25-Dihydroxy-trans-lsotachysterol (1 ,25-trans-lso-T) (JD).
16. A composition of matter according to claim 10 wherein said analog is 1σ,25-(OH)2-7-DHC (JM).
17. A composition of matter according to claim 10 wherein said analog is 1σ,25-(OH)2-Lumisterol3 (JN).
18. A composition of matter according to claim 10 wherein said analog is 1σ,25-(OH)2-Pyrocalciferol3 (JO).
19. A composition of matter according to claim 10 wherein said analog is 1σ,25-(OH)2-lsopyrocalciferol3 (JP).
20. A composition of matter according to claim 10 wherein said analog is 1 ,25-(OH)2-7,8-cis-D3 (JR).
21. A composition of matter according to claim 10 wherein said analog is 1 ,25-(OH)2-5,6-trans-7,8-cis-D3 (JS).
22. A composition of matter according to claim 10 wherein said analog is (1S,3R,6S)-7,19-Retro-1 ,25-(OH)2-D3 (JV).
23. A composition of matter according to claim 10 wherein said analog is (1S,3R,6R)-7,19-Retro-1 ,25-(OH)2-D3 (JW). 24. A composition of matter according to claim 10 wherein said analog is 22-(p-Hydroxyphenyl)-23,
24,25,26,27-pentanor-D3 (JX).
25. A composition of matter according to claim 10 wherein said analog is 22-(m-Hydroxyphenyl)-23,24,25,26,27-pentanor-D3 (JY).
EP95906111A 1993-12-23 1994-12-23 Vitamin d 3? analogues and pathway to mediate disorders Withdrawn EP0737070A4 (en)

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US24938594A 1994-05-25 1994-05-25
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CA2179288A1 (en) 1995-06-29

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