WO1997045440A1 - Cytostatic sterols - Google Patents
Cytostatic sterols Download PDFInfo
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- WO1997045440A1 WO1997045440A1 PCT/SE1997/000936 SE9700936W WO9745440A1 WO 1997045440 A1 WO1997045440 A1 WO 1997045440A1 SE 9700936 W SE9700936 W SE 9700936W WO 9745440 A1 WO9745440 A1 WO 9745440A1
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- cells
- cholesten
- cholesterol
- dihydroxy
- hydroxy
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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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
- A61K31/56—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
Definitions
- the present invention relates to the use of cholesterol derivatives with an oxo group and a conjugated double bond in the steroid nucleus and a hydroxyl group in the side-chain as inhibitors of cell growth and/or as inducers of death in abnormal cells.
- ⁇ cholesterols which have shown some cytostatic activity include the compound 7 ⁇ -hydroxycholesterol with the formula:
- X is a straight or branched, hydroxy substituted C 1 -C 15 hydrocarbon chain, for use in medicine.
- a preferred group of compounds within the scope of the invention has the formula IA:
- Particularly preferred compounds of Formula I include 7 ⁇ ,25-dihydroxy-4-cholesten-3-one, 7 ⁇ ,25- dihydroxy-4-cholesten-3-one and 7 ⁇ ,27-dihydroxy-4-cholesten-3-one.
- the enantiomer 7 ⁇ ,25-dihydroxy-4-cholesten-3-one does not appear to have been previously described in the literature and thus a further aspect of the invention provides this compound, preferably in substantially pure form, for instance > 75%, preferably > 90% and most preferably > 95% enantiomerically pure.
- the invention further provides pharmaceutical compositions comprising the compounds of the invention, preferably those within Formula IA, in admixture with a physiologically acceptable diluent or pharmaceutical carrier.
- cancers which may or may not be virally transformed
- Representative cancers include sarcomas, such as soft tissue sarcoma, myeloproliferative tumours such as leukaemia, glioblastoma, pancreatic, ovarian and adenocystic cancers.
- sarcomas such as soft tissue sarcoma
- myeloproliferative tumours such as leukaemia, glioblastoma, pancreatic, ovarian and adenocystic cancers.
- psoriasis a disorder in which there is a loss of control of normal epidermal turnover. Increased mitosis of epidermal cells results in thickening of the epidermis and the production of imperfect keratin scales.
- the invention also provides a method for the treatment of conditions associated with rapidly growing cells comprising the administration of an effective amount of a compound of formula I, or more preferably Formula IA to a human or animal in need thereof.
- a preferred aspect of the invention provides the above compounds for use in the preparation of a medicament or in methods for the treatment of breast carcinoma or colonic carcinoma in a human or animal.
- a particularly preferred aspect of the invention provides use of the above groups of compounds for use in the preparation of a medicament or in methods for the treatment of malignant melanoma cells.
- the compounds of the invention include the corresponding pharmaceutically accceptable derivatives as are known in the steroid art and which release the respective compound of Formula I in vivo.
- Suitable derivatives include ethers and esters of the hydroxy groups and/or derivatives of the oxo group.
- esters and ethers include glycosides, such as the hexoses described in the abovementioned WO 91 11452, and bis-hemisuccinates, phosphates, glycoside phosphates, silyl ethers, acetate, formate and other fatty esters, such as the oleate, glucuronides, phosphodiesters, cyclodext ⁇ ns, such as 2-hydroxy- ⁇ -cyclodextrin and the like as are known in the steroid art.
- Other derivatives include the corresponding oximes and pharmaceutically acceptable salts.
- the compounds of the invention are subject to mtracellular metabolism and such active metabolites are within the scope of the invention.
- 7,25-dihydroxycholest-4-en-3-ones may be metabolized to the corresponding cholestenoic acids.
- the administration to a mal, including humans, of such active metabolites for the indications specified above is thus to be regarded as an aspect of the invention.
- the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, powders, solution, suspensions, or emulsions.
- the compounds may be administered as injectable dosages of a solution, suspension or emulsion of the compound in a physiologically acceptable diluent with a pharmaceutical earner which can be a sterile liquid such as water, alcohols, oils and other acceptable organic solvents, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
- a pharmaceutical earner which can be a sterile liquid such as water, alcohols, oils and other acceptable organic solvents, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
- the compounds can also be administered in the form of a depot injection or implant preparation which may be formulated in such manner as to permit a sustained release of the active ingredient.
- the compounds can be administered in the form of an unguent, cream, ointment, or a lotion.
- Fig. 1 outlines a simplified scheme of the metabolism of LDL (low density lipoprotein) cholesterol and major autooxidation products of fibroblasts and the formation of potent HMG-CoA reductase suppressors.
- the names of the steroids are listed in Table I.
- major reactions of sterols are I, 27-hydroxylation, II, 7 ⁇ -hydroxylation; III, 3-oxidation with isomerization of the 5-double bond; and IV, oxidation to a 27-carboxy group. Hydrolyzed LDL cholesterol is metabolized via these reactions (filled arrows).
- Oxidation of a 7 ⁇ -hydroxy group (V) to a ketone is also observed. Minor reactions noted are shown by broken arrows. The formation of 7 ⁇ ,25-dihydroxy-4-cholesten- 3-one by 25-hydroxylation of sterols (not shown) was observed under specific conditions. Reactions I, II and III were obstructed in virus-transformed fibroblasts displaying a defective suppression of HMG-CoA reductase by LDL cholesterol and autooxidation products of cholesterol. Sterol metabolites with an apparently normal suppressive effect also in transformed cells are indicated in frame.
- Fig. 2 shows structures of the three naturally occurring cholesterol derivatives that were potent suppressors of HMG-CoA reductase also in transformed fibroblasts.
- the sterols 7 ⁇ ,27-dihydroxy-4-cholesten-3-one (I), 7 ⁇ ,25-dihydroxy-4-cholesten-3- one (II), and 27-hydroxy-7-oxo-cholesterol (III) did not seem to require further metabolism in order to suppress HMG-CoA reductase in human fibroblasts.
- Common to these sterols is the presence of an oxo group with a conjugated double bond in the steroid nucleus and a distal hydroxyl group in the side chain.
- the sterols are drawn in such a way that their apparent structural similarities are illustrated.
- 27-hydroxylated 3 ⁇ -hydroxy-5 ⁇ -cholest-8(14)-en-15-one (IV) is also shown.
- Fig. 3 discloses gas chromatographic-mass spectrometic GC/MSanalysis of neutral oxysterols isolated from the medium after incubating normal fibroblasts with Iipoproteins.
- Normal human fibroblasts protein contents 1.1 mg, dish size: 143 cm
- FCS fetal calf serum
- Choesterol concentration: 1,2 mM cholesterol
- Fragment ion current chromatograms characteristic of the trimethylsilyl ethers of oxysterols were constructed by the computer from mass spectra taken every 2h during the analysis and for the pupose of illustration the intensities of the ions (m/z) were multiplied by appropriate factors.
- the principal sterols indicated by the numbers are listed in Table I.
- the equivalent of about 0.2 ml of medium was injected onto a Finnigan SSQ 710 instrument housing a 25-m fused- silica column coated with methyl silicone, and the oven temperature was programmed from 185 till 280"C at a rate of 5°C x min " ' .
- Fig. 4 relates to HPLC analyses of ⁇ -labeled 7 ⁇ ,27-dihydroxy-4-cholesten-3-one and 7 ⁇ -hydroxy-3-oxo-4-cholcstcnoic acid isolated from the medium of normal human fibroblasts.
- the cells (0.7 mg of protein) were incubated for 68 h with LDL (4% FCS) labeled with [ 3 H]cholesteryl oleate, and the medium was then taken for analysis by HPLC.
- LDL 4% FCS
- [ 3 H]cholesteryl oleate labeled with [ 3 H]cholesteryl oleate
- Fig 5. shows effects of LDL on HMG-CoA reductase in normal (O) and virustransformed (•) human fibroblasts.
- Activities of HMG-CoA reductase in the fibroblasts were determined after incubation for 24 h with media containing diffferent concentrations of FCS. In the absence of FCS, media contained 10% LDS (lipoprotein deficient serum). All cells were preincubated for 24 h in media containing 10% LDS. The concentrations of cholesterol in FCS and LDS were 1.2 and 0.1 mM, respectively.
- the control activities of HMG-CoA reductase in normal and transformed cells were 72% and 101 pmol/min/mg of protein, respectively.
- Fig. 6 discloses time-response curves for the LDL-induced production of 7 ⁇ ,27- dihydroxy-4-cholesten-3-one (A), 27-hydroxy-7-oxocholesterol (•), and 7 ⁇ ,25- dihydroxy-4-cholestn-3-one (I), and suppression of HMG-CoA reductase (O) in normal human fibroblasts.
- the cells protein content: 1.7 mg, dish size: 143 cm 2 ) were incubated with medium (15 ml) containing 10% FCS and were harvested at the indicated times. The concentration of cholesterol in FCS was 1.2 mM. All cells were preincubated for 24 h in media containing 10% LDS (see also Table III). For comparison, the production of 27-hydroxycholesterol ( ⁇ ) is also shown.
- the steroids used were obtained in the following way: Diosgenin ((25R)-5-s ⁇ irosten -3 ⁇ -ol) was from Sigma (USA) and was used as the starting material for the synthesis of 27-oxygenated steroids (Arunachalam et al, ( 1981) J. Org. Chem. vol. 46, 2966-2968; Fieser et al., (1967) Reagents for Organic Synthesis, p. 1059, John Wiley and Sons Inc., New York; Shoda et al., (1993) Steroids, vol. 58, 1 19-125).
- 5-cholestene-3 ⁇ ,7 ⁇ ,25-triol was prepared from the 3-acetate,25- trimethylsilyl ether derivative of 25-hydroxycholesterol, and after hydrolysis, this steroid was further oxidized to 7 ⁇ ,25-dihydroxy-4-cholesten-3-one as described for the corresponding 27-hydroxysteroids (Shoda et al., supra). 25-Hydroxycholesterol was oxidized in the same way to 25-hydroxy-4-cholesten-3-one.
- the other steroids were those used in a previous study (Axelson et al (1995) J. Lipid Res vol. 36, 290- 298) and we refer to this article as a disclosure on how to obtain these compounds.
- Line GM 08333 Normal human fibroblasts (line GM 08333) were obtained from NiGMS, Georgia Institute for Medical Research (Camden, NJ) and SV40 virus-transformed human fibroblasts 90-VA IV) were a kind gift from Dr. Stein (University of Colorado, Boulder, CO).
- Human colonic carcinoma (WiDr), breast carcinoma (MDA 231), and malignant melanoma (SK-MEL-2) cell lines were from American Type Culture Collection.
- Cell lines were grown in monolayers in tissue culture flasks maintained in a 95% air, 5% CO 2 atmosphere at 37°C in humidified incubator and were cultured in either Dulbecco's Modified Eagle's Medium (MDA 213) or Minimal Eagle's Medium (the other cells) supplemented with essential and non-essential amino acids and 10% FCS (v/v).
- MDA 213 Dulbecco's Modified Eagle's Medium
- Minimal Eagle's Medium the other cells
- FCS v/v
- normal or transformed fibroblasts (cell number 3-6 x 10 6 in 57-143 cm 2 dishes) were first preincubated for 24 h in medium containing 10% LDS and were then incubated for 3-68 h with 7-10 ml of medium containing 4-10% FCS (with our without 3 H-labeled cholesterol or cholsteryl oleate) or were incubated with the oxysterol in 10% LDS for 24-48 h. Control cells were incubated in the same way, but only for 15 min.
- CsA cyclosporin A
- ketoconazole ketoconazole
- oxysterols were tested on normal and transformed fibroblasts at concentrations of 10-30 ⁇ M, 30 ⁇ M, and 0.12 ⁇ M, respectively, in cell media containing 0-10% FCS and 10-0% LDS.
- the substances were added to the incubation media in freshly prepared ethanol solutions, and the ethanol concentrations of media became 0.1-0.5%.
- Control cells were incubated in the same way, but without CsA, ketoconazole, or oxysterols.
- the dish size and volume of media when HMG-CoA reductase activity was to be determined were 20 cm " and 5 ml, respectively, and the incubations were carried out in duplicate for 3- 24 h. Each oxysterol was tested in 2-5 separate experiments. Determination of
- HMG-CoA reductase activity was then carried out as described previously (Axelson et al (1995) J. Biol. Chem. 270, 15102, Cavenee et al. (1981) J. Biol. Chem. 256, 2675 and Edwards, P.A. et al. (1979) J. Lipids. Res. 20, 40.
- Trimethylsilyl ethers of oxysterols and methyl ester trimethylsilyl ether derivatives of steroid acids were prepared (Axelson 1991 supra) and were analyzed by gas chromatography mass spectrometry (GC/MS) as described in Axelson 1995 supra.
- H-labeled cholesterol, cholesteryl oleate, and 25-hydroxycholesterol and/or their radioactive metabolites were analysed by HPLC prior to or after group fractionation and purification as described above.
- H-labeled cholesterol and cholesteryl esters were extracted from small aliquots of the incubation media with mixtures of isopropyl alcohol and hexane and from cells with mixtures of ethanol and water prior to separation by straight-phase HPLC using hexane/isopropyl alcohol, 98:2 (v/v), as the mobile phase (Axelson 1995 supra). Appropriate fractions from the HPLC effluent were collected in vials, and the radioactivity was then determined by scintillation counting.
- Radioactive neutral and acidic metabolites of [ H]cholesterol or [ 3 H]cholesteryl oleate were isolated from media and cells as described above and were then characterized by HPLC:
- three HPLC systems were used in the following order.
- Reversed phase HPLC was carried out on a column of LiChrospher (250 x 4 mm, Hibar, 100RP-18, 5 ⁇ m, Merck, Darmstadt, Germany) using a pump (Constametric III) and a variable wavelength detector (Spectra Monitor D from LDC/Miton Roy, Riviera Beach, FL) set at 220 or 240 nm and a Rheodyne Model 7125 injector with a 100 ⁇ l loop.
- the mobile phase used for neutral metabolites was a mixture of methanol/ethanol/water, 80:20: 10 (by volume, flow rate 1 ml x min ' 1 ), and fractions were collected between 0 and 11 min (fraction 1 ; containing polar metabolites, e.g. 7 ⁇ ,27-dihydroxy-4-cholesten-3-one, retention time about 4.5 min) and between 11 and 14 min (fraction 2; containing 7 ⁇ -hydroxy- 4-cholesten-3-one and 27-hydroxycholesterol having retention times 1 1.5 and 12.5 min, respectively).
- the mobile phase was then changed to 85% aqueous methanol (flow rate 1 ml x min "1 ) for separation of sterols in fraction 1 or for separation of steroid acids (as methyl ester derivatives).
- flow rate 1 ml x min "1 ) for separation of sterols in fraction 1 or for separation of steroid acids (as methyl ester derivatives).
- a fraction of the effluent containing 7 ⁇ ,27-dihydroxy-4-cholesten-3-one (retention time about 8.5 min) was collected between 8.0 and 9.0 min
- a fraction of the effluent containing 7 ⁇ -hydroxy-3-oxo-4-cholestenoic acid (retention time about 12 min) was collected between 1 1.0 and 13.0 min.
- the mobile phase was hexane/isopropyl alcohol, 94:6 (v/v), when fraction 2 was analyzed (retention times of 7 ⁇ -hydroxy-4-cholesten-3-one and 27-hydroxycholesterol were about 8 and 9 min, respectively), and 90: 10 (v/v), when the fractions containing 7 ⁇ ,27-dihydroxy-4-cholesten-3-one or 7 ⁇ -hydroxy-3-oxo-4- cholestenoic acid methyl ester were analyzed.
- the flow rate was 1.0 ml x min " 1 in all cases.
- the HPLC effluent during the latter analyses was collected in scintillation vials with 15-60-s intervals, and after addition of scintillation fluid, the radioactivity was determined.
- Table I shows the oxygenated cholesterol derivatives identified in the neutral and acidic fractions from media (containing 10% FCS) after incubation with normal human fibroblasts and their gas chromatographic/mass spectrometric characteristics as trimethylsilyl ethers and methyl ester trimethylsilyl ether derivatives, respectively.
- Table II shows the production of oxysterols in normal and virus transformed human fibroblasts when incubated with lipoproteins.
- the amounts of neutral and acidic oxygenated cholesterol derivatives were determined in the media (10 ml) containing 10% FCS (cholesterol concentration 1.2 mM) after incubation with fibroblasts for 48 hours. Cells incubated for 0.25 h served as controls.
- LDS LDS.
- d n number of incubations.
- e Can also be formed by autooxidation of cholesterol during incubations or during purification of samples.
- Fig. 3 shows a GC/MS analysis of neutral C 27 -steroids isolated from the medium after incubating normal fibroblasts with lipoproteins.
- the identification was based on the GC retention indices of the derivatives and mass spectra, which were compared with those of the authentic steroids. Most of the steroids had additional oxygen groups both at C-7 and in the side chain. No additional steroids were identified in the cell extracts, and, with the exception of the autooxidation products of cholesterol (see above), the amounts of oxysteroids in the cell extracts were low and barely detectable ( ⁇ 10-20% of those in media). Because of this, oxysterols in the cells were usually not analyzed, unless otherwise stated.
- the oxysterols were also studied with regard to the time course of their cellular production, as shown in Table HI which shows time-response for the production of oxysterols in normal fibroblasts when incubated with lipoproteins.
- the amounts of neutral oxygenated cholesterol derivatives were determined in the media (15 ml) containing 10% FCS (cholesterol concentration 1.2 mM) after incubation for 0.25 h with normal human fibroblasts (protein content 1.7 mg, size of dishes 143 cm ) All cells had been preincubated for 24 h in media containing 10% LDS.
- b Can also be formed by autooxidation of cholesterol during the incubations or during purification of samples.
- Example 1 The cell lines, cell culture conditions, analysis of oxysterols and steroid acids and the HPLC procedures were as in Example 1.
- the metabolism of 7 ⁇ -hydroxycholesterol differed from that of 7 ⁇ -hydroxy- cholesterol.
- the major metabolites were 7 ⁇ ,27-dihydroxycholesterol (1%) and 3 ⁇ ,7 ⁇ ,-dihydroxy-5- cholestenoic acid (62%).
- a large portion (about one-third) of 7 ⁇ - hydroxycholesterol was converted to 7-oxocholesterol (20%), 27-hydroxy-7- oxocholesterol (1%), and 3 ⁇ -hydroxy-7-oxo-5-cholestenoic acid (14%). Oxidation of 7 ⁇ -hydroxy group also occurred when 7 ⁇ ,27-dihydroxycholesterol was incubated.
- Table IV shows the formation of radioactive metabolites from LDL [ 3 H ]cholesteryl oleate in normal fibroblasts.
- the amounts of 3 H-labeled 27-hydroxycholesterol, 7 ⁇ ,27-dihydroxy-4- cholesten-3-one and 7 ⁇ -hydroxy-3-oxo-4-cholestenoic acid were determined in media and cells after incubating normal human fibroblasts (protein content, 0.7 mg; size of dishes, 57 cm 2 ) for 68 h with media ( 10 ml) containing LDL (4% FCS; cholesterol concentration, 1.2 mM) labeled with [ 3 H]cholesteryl oleate or [ 3 H]cholesterol.
- Example 1 The cell lines, cell culture conditions, analysis of oxysterols and steroid acids and the HPLC procedures were as in Example 1.
- Table V shows the distribution of 3 H-labeled cholesterol and cholesteryl esters after incubating normal and transformed fibroblasts with lipoproteins labeled with radioactive cholesterol or cholesteryl oleate for 48 h. Distribution of radioactivity after incubating normal and virus-transformed human fibroblasts for 48 hours in media containing lipoproteins (10% FCS) labeled with f 3 HJcholesteryl or [ HJcholesteryl oleate. The total concentration of unlabelled cholesterol in FCS was 1.2 mM. Incubations for 0.25 h served as controls. TABLE V
- Table VI shows the metabolism of 25-[ 3 H]hydroxycholesterol in normal and transformed fibroblasts. In particular it shows the distribution of recovered radioactivity after incubating normal and transformed fibroblasts for 48 h with media containing 3 H-labeled and unlabeled 25-hydroxycholesterol and 10% LDS (cholesterol concentration 0.1 mM) Incubations for 0.25 h served as controls.
- cell lysate were incubated in 200 mM potassium phosphate, 20 mM dithiotheritol, 40 mM glucose-6-phosphate, 5 mM NADPH, and 5 units/ml of glucose-6-phos ⁇ hate dehydrogenase. After a 15-min preincubation at 37°C, 0.9 nmol/L [ 14C]HMG-CoA (57mCi/mmol) and unlabelled HMG-CoA (the final concentration of HMG-CoA was 100 ⁇ M) were added for a 60-min incubation at 37°C. The final reaction volume for each sample was 60 ⁇ L.
- Table VII shows the effects of LDL and oxysterols on HMG-CoA reductase in normal and transformed fibroblasts.
- LDL 0.5- 8% FCS; cholesterol concentration 1.2 mM
- oxysterols (0.12 ⁇ M in media containing 10% LDS)
- the activities of HMG-CoA reductase were determined after incubating the fibroblasts for 24 h. All cells were preincubated for 24 h in media containing 10% LDS.
- HMG-CoA reductase 0 % of control.
- the activities of HMG-CoA reductase in normal and transformed fibroblasts were 58 and 96 pmol/min/mg protein respectively. Difference in degree of suppression of HMG-CoA reductase in normla and transformed fibroblasts induced by LDL or the oxysterol
- LDL cholesterol and a number of oxysterols including) 27- hydroxycholesterols, 3 ⁇ -hydroxy-5 ⁇ -cholest-8(14)-en-15-one, and the autooxidation products of cholesterol 25-hydroxycholesterol, 7-oxo-cholesterol, 7 ⁇ - hydroxycholesterol and 7 ⁇ -hydroxycholesterol), which have been considered to be potent suppressors of HMG-CoA reductase, all appear to have to be metabolized prior to being biologically active.
- an additional aspect of the invention provides the compound 3 ⁇ ,27-dihydroxy-5-cholesten-7-one, preferably in substantially pure form, for instance >75%, preferably > 90% pure and most preferably >95% enantiomerically pure.
- This aspect of the invention further provides the use in medicine of this compound and/or its active metabolites such as the cholestenoate.
- a preferred use is in the manufacture of medicament for suppressing HMG-CoA reductase activity in a cell or mammal, including humans.
- Representative for this aspect of the invention include its administration to a human or mammal to reduce serum cholesterol and/or the biosynthesis of cholesterol. 4.
- Certain sterols with these structures also showed suppression of HMG-
- CoA reductase in other human neoplastic cells including breast carcinoma, colonic carcinoma and malignant melanoma cells, which all displayed a defective regulatory response to LDL. This is shown in Example 5.
- MDA 231 Dulbecco's Modified Eagle's Medium
- Minimal Eagle's Medium the other cells
- FCS fetal calf serum
- cell lysates were incubated in 200 mM potassium phosphate, 20 mM dithiotheritol, 40 mM glucose-6-phosphate, 5 mM NADPH, and 5 units/mL of glucose-6-phosphate dehydrogenase.
- Table VIII shows the effects of LDL and oxysterols on HMG-CoA reductase in human malignant cells.
- LDL 2% FCS; cholesterol concentration 1.2 mM and of selected oxysterols (0.12 ⁇ M in media containing 10% LDS)
- All cells had been pre- incubated for 24 h in media containing 10% LDS.
- the corresponding values on transformed human fibroblasts are also shown.
- the compounds of this type are potent suppressors of cholesterol production in many different cells, including those having low activities of sterol-metabolizing enzymes. This is in contrast to most other oxysterols that have been used as HMG-CoA reductase suppressors. Like tumor cells, normal cells such as macrophages, apparently lack the 7 ⁇ -hydroxylating enzyme, which is required for the formation of HMG-CoA reductase suppressors from cholesterol or side-chain hydroxylated 3 ⁇ -hydroxy-5 sterols.
- the described group of sterols also reduces the cellular uptake of LDL-cholesterol by suppressing the number of LDL receptors on the cell surface. This is shown in Example 6.
- the cells were washed twice in phosphate buffered saline and were incubated with 5 ml of medium containing 10% lipoprotein deficient serum (LDS) prepared by treating FCS with Cab-O-Sil (Weinstein supra). (Cell growth was not negatively affected when this serum was used).
- LDS lipoprotein deficient serum
- the sterols 24-hydroxy-4-cholesten-3-one, 25-hydroxy-4-cholesten-3-one and 27-hydroxy-4-cholesten-3-one were then added to the incubation media (concentration 1.1 ⁇ M) in freshly prepared ethanol solutions and the ethanol concentrations of the media were then 0.2%.
- the degradation of 1251- labeled LDL in the control cells were found to be 105-109 ng/h x mg cell protein.
- the corresponding values for cells treated with 24-hydroxy-4-cholesten-3-one, 25- hydroxy-4-cholesten-3-one and 27-hydroxy-4-cholesten-3-one were 40-41 , 58-59 and 62-69 ng/h x mg cell protein, respectively. This shows that the sterols reduced the LDL-receptor activity of the cells by approximately 50% under the conditions used.
- Normal human fibroblasts (line GM 08333) and SV-40 virus transformed human fibroblasts (90- VA VI) were grown in monolayers in tissue cultered flasks maintained in a 95% air/5% CO 2 atmosphere at 37°C in a humidified incubator and were cultured in Dulbecco's Minimal Eagle's Medium supplemented with essential and non-essential amino acids and 10% fetal calf serum (FCS).
- FCS fetal calf serum
- Tumor-transformed fibroblasts were selectively affected by the oxysterols when compared with normal cells, resulting in not only ceased growth but also in cell death of the former cells. The effect on the normal cells was relatively small.
- 7-hydroxylated sterols are more potent than sterols lacking a hydroxyl group or having an oxo group in this position.
- the latter group included the potent HMG-CoA reductase suppressors 25-hydroxy-4-cholesten-3-one, 27- hydroxy-4-cholesten-3-onc and 3 ⁇ ,27-dihydroxy-5-cholesten-7-one, suggesting that the induction of cell death was not mainly due to a suppression of HMG-CoA reductase.
- Sterols with an hydroxy group in the side chain were more potent than the corresponding sterols without such a group.
- Example 8 Effects of 7 ⁇ ,27-dihydroxy-4-cholesten-3-one on proliferation and viability of human malignant cells
- FCS Dulbecco's Minimal Eagle's Medium supplemented with essential and non-essential amino acids and 10% fetal calf serum
- Control cells were incubated with the same volumes of ethanol but without sterols.
- the proliferation of cells was registered by counting in a light microscope the cell number in marked areas in the dishes after incubations for 24 h and 48 h, and cell death was recognized microscopically as detachment or lysis of cells in the monolayer cultures.
- the acetate is prepared from 2 mg dried commercially available 25- hydroxycholesterol (Sigma) to which 0.5 ml pyridine is added prior to ultrasound. 0.5 ml acetic anhydride is added and the mixture resubjected to ultrasound. The reaction mixture is allowed to stand at room temperature for 2.5 hours and the reaction is quenched with 5 ml H 2 O and allowed to stand for 15 minutes. 3 ml ethyl acetate is added and the mixture agitated and subjected to ultrasound and then centrifuged for several minutes. The aqueous phase (the bottom phase) is transferred to a fresh vessel and saved.
- the resulting ethyl acetate phase is rinsed with a small amount of ethyl acetate into a fresh vessel and blown dry.
- the trimethylsilyl ether (TMS) of the 25 hydroxy group is prepared by subjecting the product to 0.5 ml pyridine/hexamethyldisilane/ trimethylchlorosilazane, 3:2: 1 which is allowed to react at 60° for 30 minutes. The product is blown dry and dissolved in hexane, ultrasounded and transferred to a 25 ml Florence flask with a little hexane.
- the resulting product is subject to TBB-oxidation as follows: The hexane phase is blown dry, dissolved in 1.5 ml concentrated acetic acid and ultrasounded. 1.5 mg copper II bromide, 10 ⁇ l TBB (tert-butylperbenzoate) and an agitating bead is added and the reaction proceeds under N 2 at 100°C for 5 minutes. The product is allowed to cool and is transferred to a separating funnel. The flask is rinsed out into the funnel with 5 ml H 2 O, then 40 ml hexane. The mixture is agitated and the aqueous phase discarded. 5 ml of 5% NaHCO ⁇ is added and the product agitated, while allowing for gas evolution. The bicarbonate phase is discarded. The latter step is repeated. The product is washed twice with 5 ml H 2 O. Th pH should be neutral. The hexane phase is transferred to a Florence flask and blown dry.
- the resulting product is hydrolysed as follows: 5 ml of 5% KOH in methanol is added, ultrsounded and the mixture is incubated for 1 hour at 50°C in a water bath with gentle agitation. The reaction is quenched with 5 ml H 2 O which is mixed and the product is neutralised to ca. pH 7 with concentrated acetic acid. The product is subject to a Cl 8 column (ODS silica) (1.5 x 0.8 cm) rinsed with 10 ml H 2 O. The product is eluted with 8 ml methanol, evaporated and dissolved in 1 ml methanol. lO ⁇ l is retained for GLC.
- the resulting 7 ⁇ ,25-diydroxy-4-cholesten-3-one and 7 ⁇ ,25-diydroxy-4-cholesten-3- one are then purified with reversed phase HPLC (column: LiChrosper, 250 x 4 mm, Hibar, 100 RP-18, 5 ⁇ , Merck) in 85% methanol, UV detector at 240 nm, retention time 7.3 minutes for the 7 ⁇ anomcr and 7.8 minutes for the 7oc anomer.
- the effluent is collected over a Florence flask, evaporated, transferred to a test tube with methanol, evaporated and dissolved in 0.5 ml ethanol. 20 ⁇ l is retained forGLC and GC/MS.
- a Clemmensen reduction of diosgenin is carried out with fresh zink amalgam prepared from 60 g zink filings, 4.5 g mercury II chloride, 3.0 ml cone. HCI and 75 ml H 2 0. The mixture is agitated in a multinecked round flask for 5 minutes and decanted. 200 ml ethanol and 1.2 g diosgenin (Sigma) is added and refluxed. Over 45 minutes 60 ml cone. HCI is added dropwise, followed by 15 min continued reflux. The mixture is allowed to cool to room temperature. 1.5 1 ice-cold water is added dropwise and the reaction allowed to stand under refrigeration for around an hour. The water is then filtered away and the dry mass transferred to a Florence flask.
- the product is oxidised in 4 batches as follows. 20 mg chrome trioxide in 0.1 ml H 2 O and 0.2 ml acetic acid is added dropwise to a stirred mixture of 125 mg tetrahydrodiosgenin and 0.62 g NaAc in 22.5 ml glacial acetate. The reaction continues for 18 hours at 25 °C. A few drops of methanol are then added to destroy excess reagent. The mixture is diluted with 25 ml cold water and extracted with 40 ml MeCl 2 . The methylene chloride phase is rinsed with around 10 ml H 2 O and then with 5% sodium bicarbonate and H 2 O until neutral by pH paper.
- the MeCl 2 is dried with a spoon of waterfree Na 2 SO 4 and the MeCl 2 phase filtered down into a Florence flask and evaporated. A GLC sample retained. The remaining product is subjected to a silicon column 8 x 0.8 cm in 30% ethyl acetate/TMP (trimethylpentane) in one fifth aliquots.
- the fractionation comprises:
- step b) The resulting product from step b) (170 mg) is transferred to a small Florence flask and 0.11 g KOH in 3.5 ml triethylene glycol, 0.1 ml hydrazine and a couple of agitation beads are added. The mixture is refluxed for 15 minutes and then refrigerated for 1 hour. The mixture is dropwise added to 15 ml 0.5 M HCI, filtered and rinsed with ice cold water. The dry material is transferred to a small evaporating flask and recrystallised with ethyl acetate. Purity is assayed by GLC & GC/MS. Yield: 75 mg 27-hydroxycholesterol, 45 mg as crystals.
- step A The acetate of the product of step A is prepared in the same manner as
- Example 9 step a with the exception that the reaction was quenched with 5 ml H 2 O.
- the TMS step b) of Example 9 is omitted.
- step d) Fraction 6 from step d) is oxidised by cholesterol oxidase as described in example 9, step e) and the resultant 7 ⁇ ,27-dihydroxy-4-cholesten-3-one purified by HPLC as described in example 9. Retention time 8.7 minutes.
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- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
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Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97926333A EP0906331A1 (en) | 1996-05-30 | 1997-05-29 | Cytostatic sterols |
CA002255677A CA2255677A1 (en) | 1996-05-30 | 1997-05-29 | Cytostatic sterols |
AU31122/97A AU716389B2 (en) | 1996-05-30 | 1997-05-29 | Cytostatic sterols |
JP09542238A JP2000512626A (en) | 1996-05-30 | 1997-05-29 | Antimitotic sterol |
NZ332776A NZ332776A (en) | 1996-05-30 | 1997-05-29 | Cytostatic cholesterol derivatives with an oxo group and a conjugated double bond in the steroid nucleus |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE9602100A SE9602100D0 (en) | 1996-05-30 | 1996-05-30 | New pharmaceuticals |
SE9602100-1 | 1996-05-30 |
Publications (1)
Publication Number | Publication Date |
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WO1997045440A1 true WO1997045440A1 (en) | 1997-12-04 |
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ID=20402780
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/SE1997/000936 WO1997045440A1 (en) | 1996-05-30 | 1997-05-29 | Cytostatic sterols |
Country Status (8)
Country | Link |
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EP (1) | EP0906331A1 (en) |
JP (1) | JP2000512626A (en) |
AU (1) | AU716389B2 (en) |
CA (1) | CA2255677A1 (en) |
IL (1) | IL127039A0 (en) |
NZ (1) | NZ332776A (en) |
SE (1) | SE9602100D0 (en) |
WO (1) | WO1997045440A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20150013254A (en) * | 2012-05-10 | 2015-02-04 | 베타 인노브 | Sterol derivatives and use thereof for treating diseases involving transformed astrocyte cells or for treating malignant haemopathies |
CN110551166A (en) * | 2018-05-31 | 2019-12-10 | 华东师范大学 | Cholic acid derivative and preparation method and application thereof |
-
1996
- 1996-05-30 SE SE9602100A patent/SE9602100D0/en unknown
-
1997
- 1997-05-29 EP EP97926333A patent/EP0906331A1/en not_active Withdrawn
- 1997-05-29 WO PCT/SE1997/000936 patent/WO1997045440A1/en not_active Application Discontinuation
- 1997-05-29 JP JP09542238A patent/JP2000512626A/en active Pending
- 1997-05-29 CA CA002255677A patent/CA2255677A1/en not_active Abandoned
- 1997-05-29 IL IL12703997A patent/IL127039A0/en unknown
- 1997-05-29 AU AU31122/97A patent/AU716389B2/en not_active Ceased
- 1997-05-29 NZ NZ332776A patent/NZ332776A/en unknown
Non-Patent Citations (7)
Title |
---|
BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1344, no. 3, 1997, pages 241 - 249 * |
CHEMICAL ABSTRACTS, vol. 122, no. 13, 27 March 1995, Columbus, Ohio, US; abstract no. 157013, M. AXELSON ET AL: "Structural Specificity in The Suppression of HMG-CoA Reductase in Human Fibroblasts by Intermediates in Bile Acid Synthesis" page 646; column 1; XP002043825 * |
CHEMICAL ABSTRACTS, vol. 126, no. 13, 31 March 1997, Columbus, Ohio, US; abstract no. 169459, J. ZHANG ET AL: "Studies on The Relationship between 7.alpha.-Hydroxylation and the Ability of 25- and 27-Hydroxylcholesterol to Suppress the Activity of HMG-CoA Reductase" page 396; column 2; XP002043826 * |
D. PAYNE ET AL: "A Novel Nonhepatic Hydroxycholesterol 7.alpha.-Hydroxylase That is Markedly Stimulated by Interleukin-1.beta.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 32, 11 August 1995 (1995-08-11), MD US, pages 18888 - 18896, XP002043823 * |
JOURNAL OF LIPID RESEARCH, vol. 36, no. 2, 1995, pages 290 - 298 * |
L. SWELL ET AL: "An in vivo evaluation of the quantitative significance of several potential pathways to cholic and chenodeoxycholic acids from cholesterol in man", JOURNAL OF LIPID RESEARCH, vol. 21, no. 4, 1980, pages 455 - 466, XP002043822 * |
M. AXELSON ET AL: "27-Hydroxylated Low Density Lipoprotein (LDL) Cholesterol Can Be Converted to 7.alpha.,27-Dihydroxy-4-cholesten-3-one (Cytosterone) before Suppressing Cholesterol Production in Normal Fibroblasts", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 271, no. 22, 31 May 1996 (1996-05-31), MD US, pages 12724 - 12736, XP002043824 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20150013254A (en) * | 2012-05-10 | 2015-02-04 | 베타 인노브 | Sterol derivatives and use thereof for treating diseases involving transformed astrocyte cells or for treating malignant haemopathies |
US20150086615A1 (en) * | 2012-05-10 | 2015-03-26 | Beta Innov | Sterol derivatives and use thereof for treating diseases involving transformed astrocyte cells or for treating malignant haemopathies |
KR102160316B1 (en) | 2012-05-10 | 2020-09-25 | 베타 인노브 | Sterol derivatives and use thereof for treating diseases involving transformed astrocyte cells or for treating malignant haemopathies |
US10800806B2 (en) * | 2012-05-10 | 2020-10-13 | Beta Innov | Sterol derivatives and use thereof for treating diseases involving transformed astrocyte cells or for treating malignant haemopathies |
CN110551166A (en) * | 2018-05-31 | 2019-12-10 | 华东师范大学 | Cholic acid derivative and preparation method and application thereof |
CN110551166B (en) * | 2018-05-31 | 2022-06-21 | 华东师范大学 | Cholic acid derivative and preparation method and application thereof |
Also Published As
Publication number | Publication date |
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EP0906331A1 (en) | 1999-04-07 |
AU716389B2 (en) | 2000-02-24 |
CA2255677A1 (en) | 1997-12-04 |
AU3112297A (en) | 1998-01-05 |
JP2000512626A (en) | 2000-09-26 |
SE9602100D0 (en) | 1996-05-30 |
IL127039A0 (en) | 1999-09-22 |
NZ332776A (en) | 2000-02-28 |
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