Ryungsoon Song Kim

Opiate receptor binding activity of 17-Alpha estrogenic steroids☆

A large number of steroids of the estrane, androstane, pregnane and, etianic acid series, including their sulfate and glucuronide conjugates, were inactive in an opiate radioreceptor assay (> 10% displacement of 3H-naloxone at 10−4M). Only certain estrogens were active, the most potent being 17α-hydroxy-estrogens. The displacement curves for the active steroids were parallel to those of known opiate drugs. SC 22,000, a semisynthetic steroid with opiate effects, was a potent agonist in the assay. The sodium effect indicated the active estrane steroids to be of mixed agonist-antogonist character. IC50 for 17α-dihydroequilin, 17α-dihydroequilenin, and 17α-estradiol was 1.0, 1.8, and 3.0 μm, respectively. Sulfation or acetylation of the 3-OH or 17-OH abolished binding activity.

Ouabain receptor binding of hydroxyprogesterone derivatives.

1 A specific and sensitive radioreceptor assay has been devised which is based on high affinity, saturable binding of 9 nm [3H]‐ouabain to the total particulate fraction isolated from dog heart. Ouabain and other cardiac glycosides, including the aglycones, were about equipotent in their ability to displace [3H]‐ouabain from its receptor, the IC50s ranging from 10 to 30 nm. 2 The only other substances found to compete significantly in the assay were derivatives of hydroxy‐progesterone having a 17α‐acetate substituent: chlormadinone acetate, megestrol acetate, cyproterone acetate and medroxyprogesterone acetate, with IC50s of 2, 7.4, 9 and 21 μm, respectively. Prednisolone‐3,20‐bisguanyl‐hydrazone, reported to have inotropic activity, gave an IC50 of 6.4 μm. Cyproterone‐17α‐OH was less active (IC50 90 μm) than cyproterone‐17α‐acetate. 3 A large number of peptide and protein hormones, steroid hormones and their metabolites, amines, and drugs were inactive.

Antiproliferative properties of aminosteroid antioxidants on cultured cancer cells

The antiproliferative properties of three 21-aminosteroid antioxidants (lazaroids), 21-[4-[5,6-bis(diethylamino)-2-pyridinyl]-1- piperazinyl]-16 alpha-methylpregna-1,4,9(11)triene-3,20-dione, hydrochloride (U74500A), 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piper- azinyl]-pregna-1,4,9(11)-triene-3,20-dione, monomethanesulfonate (U74389F), 2H-1-benzopyran-6-ol, 2-[[4-[3-(ethylamino)-2-pyridinyl]-1-piperzinyl]methyl]-3,4- dihydro-2,5,7,8-tetramethyl-, (Z)-2-buten dioate (U78518F), on human breast cancer cells are described. U74500A inhibited cell proliferation in a dose-dependent manner with IC50 values of approximately 1.7 and 1.2 microM when cells were exposed to the drug for 3 and 5 days, respectively. The non-steroid antioxidants, alpha-tocopherol and nordihydroguaiaretic acid, showed weak or no activity at the same dose range. All three lazaroids inhibited, dose dependently, the proliferation of mouse lymphocytes but only at IC50 values ranging between 20-30 microM. The specificity of action was studied by including other steroids: progesterone, testosterone and hydrocortisone. Both sex hormones stimulated cell proliferation at low (less than 10(-5) M) concentrations but inhibited at higher doses with IC50 values of 26 (progesterone) and 80 (testosterone) microM. Hydrocortisone (IC50 0.17 microM), on the other hand, inhibited cell proliferation by 70% over a wide range of doses. Human breast cancer cells appear to have a greater sensitivity than the mouse lymphocytes to lazaroids. The antiproliferative effects of lazaroids in cancer cells may be, at least in part, due to an interaction with glucocorticoid receptors.

The effect of linoleic and arachidonic acid derivatives on calcium transport in vesicles from cardiac sarcoplasmic reticulum

The effect of linoleic and arachidonic acid derivatives on ATP-dependent calcium transport was studied in the isolated vesicles from cardiac sarcoplasmic reticulum of guinea-pigs. Oxidation products of linoleic and arachidonic acids, obtained either by autoxidation or incubation with soybean lipoxygenase, effectively blocked in a dose-dependent manner, the net influx of calcium in the absence or presence of 5 mM of oxalate. Unoxidized fatty acids were much weaker at lower concentrations as compared to their oxidized counterparts, except the lipoxygenase-generated product of arachidonic acid which had only a marginal effect even at high concentrations. Autoxidation products of arachidonic acid were the most potent inhibitors of calcium transport. Likewise, autoxidation products of linoleic and arachidonic acids and lipoxygenase-generated products of linoleic acid induced a dose-dependent release of calcium from vesicles previously loaded with 45Ca, and release was further enhanced in the presence of 0.5 mM of EGTA. In contrast, lipoxygenase metabolites of arachidonic acid caused a transient increase in net calcium content. The effect of the fatty acid derivatives on calcium transport did not appear to be due either to the inhibition of Ca2+-ATPase activity or to a non-specific detergent-like action. The effects of oxidized fatty acids, on ATP-dependent calcium accumulation into and release from cardiac microsomal fraction were similar but less potent than those of classical calcium ionophores, X537A or A23187.

Lipoxygenase-induced lipid peroxidation of isolated cardiac microsomes modulates their calcium-transporting function

We demonstrated previously that products of linoleic and arachidonic acids, arising from enzymatic or non-enzymatic oxidation, inhibit ATP-dependent calcium accumulation into and promote release of calcium from vesicles derived from sarcoplasmic reticulum of guinea-pig heart. In the present study, direct enzymatic peroxidation of cardiac membrane lipids was performed and the effect on calcium transport was examined. Vesicles were preincubated at 37 degrees C with soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) for up to 1 h prior to the initiation of calcium accumulation. The extent of membrane peroxidation was assessed by monitoring the production of malondialdehyde. Pretreatment of vesicles with lipoxygenase for 40 and 60 min markedly depressed calcium accumulation. The lipoxygenase-induced suppression of calcium transport was completely antagonized by nordihydroguaiaretic acid (1 microM), not at all by indomethacin (1 microM), and only partially by 5,8,11,14-eicosatetraynoic acid (0.3 microM). Low concentrations of calcium (10(-5)-5 X 10(-5) M) enhanced, and a high concentration (10(-3) M) inhibited lipoxygenase-induced peroxidation of membrane lipids. The calcium-accumulating ability of the vesicles was inversely related to the extent of membrane peroxidation. The vesicles which showed the highest degree of peroxidation in the presence of 5 X 10(-5) M calcium, accumulated the lowest amount of calcium. In contrast, calcium at 10(-3) M suppressed lipid peroxidation, resulting in higher calcium uptake than in vesicles peroxidized in the absence of calcium. Thus, calcium transport is depressed in microsomes undergoing lipoxygenase-induced peroxidation, a process which in turn is modulated by calcium.

Calcium translocation by fatty acid derivatives in a two-phase partition model. Structure-activity relationships.

The relationship between structures of fatty acid derivatives, long-chain fatty alcohols, phospholipids and their calcium-transporting activity was investigated using the two-phase model system in which 45Ca is transported from an aqueous to an immiscible organic phase. Calcium translocation by all saturated and unsaturated fatty acids was significant only at 10 mM concentrations, but minimal or negligible below 1 mM; the corresponding methyl esters and alcohols were inactive at 10 mM. Polyunsaturated fatty acid derivatives, prepared by incubation with lipoxygenase (linoleate: oxygen oxidoreductase; EC 1.13.11.12) or by autoxidation in air, showed a markedly increased potency over the parent compounds. The oxidation products of linoleic and arachidonic acids were most potent. For example, the equieffective concentrations were 10 mM for linoleic acid, 0.4 mM for its lipoxygenase metabolites and 0.094 mM for its autoxidation products. Similarly, for arachidonic acid and its derivatives, equieffective concentrations were 10, 0.104 and 0.112 mM, respectively. The potency of the autoxidized fatty acid derivatives varied with both duration of autoxidation and the specific structure. Methyloleate and oleyl alcohol remained inactive even after a prolonged oxidation, whereas methyllinoleate and linoleyl alcohol were very potent only after 4 weeks but not after 1 week autoxidation. The potency of esters and alcohols with three or more double bonds increased significantly even after a short-term autoxidation, reflecting the differences in both the rate of formation and the contribution to calcium-transporting properties of the primary and secondary oxidation products. All phospholipids tested, with the exception of phosphatidylcholine and lysophosphatidylcholine, showed considerable calcium-transporting activities at 0.01 mM or greater concentrations; some members were of similar or greater potencies than the classical calcium ionophores, X537A and A23187.

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

Abstract The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4≫C-6>C-2.

Lipid fractions from liver inhibit specific binding of [3H]ouabain to dog heart ☆

Ethyl acetate extracts of bovine liver contain organic material which inhibits specific binding of [3H]ouabain in a radioreceptor binding assay. Filtration of Sephadex LH-60 resolved active material in a cholesterol-rich fraction which was further purified on a silica gel column and by TLC and found by GLC/MS to be composed mainly of long-chain fatty acids and their methyl esters. Certain authentic saturated and unsaturated fatty acids were active in the binding assay: lauric, myristic and myristoleic acids being most active. Since the amount of active saturated acids in the extracts is too small to account for the total observed activity, the presence of active unsaturated fatty acids is indicated. Furthermore, another active unidentified substance was associated with the neutral lipid fraction which also contained cholesterol and methyl esters of palmitic and stearic acids as major identifiable components, and gave a concentration-displacement curve parallel to that of ouabain. Authentic cholesterol and methyl esters of both saturated and unsaturated fatty acids were inactive in the binding assay. Thus, inhibition of specific binding of [3H]ouabain by lipid extracts of liver appears to be due, in part, to certain fatty acids and, in addition, to a potent unidentified substance associated with neutral lipids.

Synthesis of ring-A and -B substituted 17α-acetoxypregnan-20-one derivatives with potential activity on the digitalis receptor in cardiac muscle

The synthesis of C-6 substituted (methyl, hydroxy, acetoxy, methoxy, ethoxy, methoxycarbonyloxy, ethoxycarbonyloxy, fluoro, chloro, bromo) 5α,5β,C-4 unsaturated and C-4,6 unsaturated derivatives of 17α-acetoxypregn-4-ene-3,20-dione (17α-acetoxyprogesterone) are reported. A convenient synthesis of 6α-hydroxy derivatives by selective reduction of the 4-ene-3,6-dione system is described as is the formation of 6α-alkoxycarbonyl derivatives. Synthesis of a 5α-hydroxy-6-ketone via the 5β,6β-epoxide with N-bromosuccinimide is reported. A new preparation of 3α-hydroxy-5α-derivatives from the 4-en-3-one group has been carried out. 1H n.m.r. spectra are recorded. These compounds were synthesized to test for ouabain displacement activity on the digitalis receptor in cardiac muscle.

Metabolism of 17β-hydroxy-2α-methyl-5α-androstan-3-one in the rabbit

Abstract The neutral urinary excretion products of 17β-hydroxy-2α-methyl-5α-androstan-3-one from the rabbit dosed orally were investigated. Together with oxidation-reduction of the oxygen functions at C-3 and C-17 hydroxylation occurred at C-15, C-16, and at the 2α-methyl positions of the steroid nucleus.