Ekkehard Schillinger

Drospirenone: a Novel Progestogen with Antimineralocorticoid and Antiandrogenic Activity

Drospirenone (ZK 30595; 6 beta, 7 beta, 15 beta, 16 beta-dimethylen-3-oxo-17 alpha-pregn-4-ene-21, 17-carbolactone) is a novel progestogen under clinical development. Drospirenone is characterized by an innovative pharmacodynamic profile which is very closely related to that of progesterone. Potential applications include oral contraception, hormone replacement therapy and treatment of hormonal disorders. The pharmacological properties of drospirenone were investigated in vitro by receptor binding and transactivation experiments and in vivo in appropriate animal models. In qualitative agreement with progesterone, the compound binds strongly to the progesterone and the mineralocorticoid receptor and with lower affinity to androgen and glucocorticoid receptors. There is no detectable binding to the estrogen receptor. Steroid hormone agonistic and antagonistic activities of progesterone and drospirenone were compared in transactivation experiments. Individual steroid hormone receptors were artificially expressed together with a reporter gene in appropriate cell lines. Both hormones were unable to induce any androgen receptor-mediated agonistic activity. Rather, both progesterone and drospirenone distinctly antagonized androgen-stimulated transcriptional activation. Likewise, both compounds only very weakly activated the mineralocorticoid receptor but showed potent aldosterone antagonistic activity. Drospirenone did not induce glucocorticoid receptor-driven transactivation. Progesterone was a weak agonist in this respect. Drospirenone exerts potent progestogenic and antigonadotropic activity which was studied in various animal species. It efficiently promotes the maintenance of pregnancy in ovariectomized rats, inhibits ovulation in rats and mice and stimulates endometrial transformation in the rabbit. Furthermore, drospirenone shows potent antigonadotropic, i.e., testosterone-lowering activity in male cynomolgus monkeys. The progestogenic potency of drospirenone was found to be in the range of that of norethisterone acetate. The majority of clinically used progestogens are androgenic. Drospirenone, like progesterone, has no androgenic but rather an antiandrogenic effect. This property was demonstrated in castrated, testosterone propionate substituted male rats by a dose-dependent inhibition of accessory sex organ growth (seminal vesicles, prostate). In this model, the potency of drospirenone was about a third that of cyproterone acetate. Drospirenone, like progesterone, shows antimineralocorticoid activity, which causes moderately increased sodium and water excretion. This is an outstanding characteristic which has not been described for any other synthetic progestogen before. Drospirenone is eight to ten times more effective in this respect than spironolactone. The natriuretic effect was demonstrable for at least three weeks upon daily treatment of rats with a dose of 10 mg/animal. Drospirenone is devoid of any estrogenic, glucocorticoid or antiglucocorticoid activity. In summary, drospirenone, like progesterone, combines potent progestogenic with antimineralocorticoid and antiandrogenic activity in a similar dose range.(ABSTRACT TRUNCATED AT 400 WORDS)

Drospirenone : a novel progestogen with antimineralocorticoid and antiandrogenic activity : pharmacological characterization in animal models

Drospirenone (ZK 30595; 6 beta, 7 beta, 15 beta, 16 beta-dimethylen-3- oxo-17 alpha-pregn-4-ene-21, 17-carbo-lactone) is a novel progestogen under clinical development. Potential applications include oral contraception, hormone replacement therapy and treatment of hormonal disorders. Drospirenone is characterized by a pharmacodynamic profile very closely related to that of progesterone. The progestogenic activity of drospirenone has been analysed in a variety of animal models. The compound efficiently promotes the maintenance of pregnancy in rats, inhibits ovulation in rats and stimulates endometrial transformation in the rabbit. Furthermore, drospirenone shows potent antigonadotropic, i.e. testosterone-lowering, activity in male cynomolgus monkeys. The progestogenic potency of drospirenone was found to be in the range of that of norethisterone acetate or cyproterone acetate. Like progesterone, drospirenone has been shown to have an antimineralocorticoid effect in rats and humans. It has now been demonstrated that the compound has a long-lasting natriuretic activity in rats on administration of a daily dose of 10 mg s.c. for three weeks. Under identical conditions, spironolactone, a widely-used antimineralocorticoid, becomes ineffective after the initial treatment phase. Drospirenone exhibits antiandrogenic activity in castrated, testosterone-substituted male rats as shown by dose-dependent inhibition of accessory sex organ growth (prostate, seminal vesicles). In this model, the potency of drospirenone was found to be about one-third that of cyproterone acetate. The compound is devoid of androgenic, estrogenic, glucocorticoid and antiglucocorticoid activity. Possible drug interaction between drospirenone and ethinylestradiol (EE) was also investigated. EE did not interfere with either the progestogenic or the antimineralocorticoid activity of drospirenone. In conclusion, drospirenone represents a novel type of synthetic progestogen since it combines potent progestogenic characteristics with antimineralocorticoid and antiandrogenic activity. Thus, the pharmacological profile of drospirenone is more closely related to that of the natural hormone progesterone than is that of any other synthetic progestogen in use today. Therefore, drospirenone is anticipated to give rise to a number of additional health benefits both for users of oral contraceptives and hormone replacement therapy recipients.

Pharmacological profile of a novel carbacyclin derivative with high metabolic stability and oral activity in the rat.

A novel carbacyclin derivative (16S)-13,14-dehydro-16,20-dimethyl-3-oxa-18,18,19,19-tetradehydro- 6a- carbaprostaglandin-I2 (3-oxa-analogue) has been synthesized in order to find chemically and metabolically stable prostacyclin-mimetics with a potency equal or even superior to PGI2. The 3-oxa-analogue was found to be stabilized against beta-oxidation, a main metabolic degradation step also for chemically stable PGI2-analogues. The compound is orally available and displays a long duration of 4.5-48 h of antiaggregatory and hypotensive action. The 3-oxa-analogue inhibits ADP-induced platelet aggregation with an IC50 of 3.0 nM. Following intravenous application the 3-oxa-analogue lowers diastolic blood pressure in a dose dependent manner, the ED20 being 0.1-0.2 micrograms/kg after injection and less than or equal to 0.05 micrograms/kg/min after infusion respectively. In vivo platelet aggregation is inhibited after i.v. infusion of the 3-oxa-analogue with an IC50 of 0.037 micrograms/kg/min. As compared to Iloprost, the 3-oxa-analogue is 5-12 fold more potent with respect to in vivo hypotensive and anti-aggregatory effects. The results of the present studies indicate that the 3-oxa-analogue has a pharmacological profile comparable to prostacyclin (PGI2) and Iloprost. Due to the fact that the 3-oxa-analogue is chemically and metabolically stable, long term oral treatment can be achieved in clinical conditions in which PGI2 and Iloprost have already been shown to be therapeutically useful principles.

Pharmacological and cardiovascular properties of a hydantoin derivative, BW 245 C, with high affinity and selectivity for PGD2 receptors

Initial experiments demonstrated that the hydantoin prostaglandin derivative, BW 245 C, has potent anti-aggregatory activity on human platelets which may result from its structural similarity with one of the natural prostaglandins. The aim of the present study was to extend this preliminary pharmacological characterization and to determine which, if any, prostaglandin receptor-type is responsible for mediating the biological activity of BW 245 C. A marked species variation was observed in the anti-aggregatory potency of BW 245 C such that in the human (0.36 X PGE1) it was about one hundred times more effective than in the rat (0.003 X PGE1). The relative potencies of PGI2 (ca. 10 X PGE1) and PGE1 were, however, similar in both species. An intravenous bolus injection of 250 micrograms/kg BW 245 C lowered systolic (-23%) and diastolic (-34%) blood pressure in spontaneously hypertensive rats. In radioligand binding studies it showed a high affinity and selectivity for PGD2 platelet receptors, binding to PGI2 or PGE2 receptors was not detectable. Therefore it is concluded that the platelet and cardiovascular actions of BW 245 C are mediated by PGD2 receptors and this accounts for the observed species variation which is a characteristic of this prostaglandin.

Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: Inhibition of cat expression by anti-androgens

A hormone-inducible transcriptional system has been established, based on the stable transfection of the rat androgen receptor (rAR) and a reporter plasmid containing the mouse mammary tumour virus promoter linked to the chloramphenicol acetyltransferase gene (pMMTV-CAT) into steroid receptor-negative CV-1 cells. First, the rAR was stably introduced into CV-1 cells. Single clones were tested for stable expression of functionally active AR by analysing the effect of dihydrotestosterone on induction of transiently transfected pMMTV-CAT. Stable transfection and the expression of AR was confirmed by steroid-binding assays. In a second step, a clone expressing physiological amounts of AR protein (30 fmol/mg protein) was stably transfected with pMMTV-CAT to yield a permanent cell line that stably expresses functional AR and MMTV-CAT sequences. This cell line provides a powerful tool for the efficient and accurate determination and quantification of the effects of androgens and anti-androgens on reporter gene transcription. This was demonstrated by investigating the action of the three anti-androgens hydroxyflutamide, casodex and cyproterone acetate. The three compounds were shown to reverse the effects of the androgen R1881 on gene expression but were themselves devoid of agonistic activity.

Non-steroidal anti-inflammatories—XII: Mode of action of anti-inflammatory methane sulfonanilides☆

The influence of anti-inflammatory methane sulfonanilides on the arachidonic acid metabolism in ram seminal vesicle homogenate depends on the pK3 of the individual substance: more acidic compounds suppress, less acidic compounds stimulate PG synthesis and enhance the PGH2/PGG2 ratio. As oxygen radicals are liberated during the conversion of PGG2 to PGH2 it is suggested that the less acidic methane sulfonanilides are scavengers of the (pro-inflammatory) oxygen radicals.

Stable 9ß- or 11α-halogen-15-cyclohexyl-prostaglandins with high affinity to the PGD2-receptor

Various chemically stable prostaglandin analogues were studied for their affinity towards the PGD2-receptor in human platelet membranes in order to define the requirements for specific ligand binding to this receptor. On replacing the 11- or 9-hydroxyl groups of PGF2 alpha by an 11 alpha- or 9 beta-chloro- or fluoro atom, stable prostaglandin analogues were obtained, which showed high affinity towards the PGD2-receptor. The lower side chain consisted of a 15-cyclohexyl group or of the natural 15-n-pentyl group, other substitutents decreased the affinity substantially. The highest PGD2-mimetic activity with a relative affinity of 0.5 to the PGD2-receptor was found in 9-deoxy-9 beta-chloro-16,17,18,19,20-pentanor-15-cyclohexyl-PGF2 alpha (ZK 110 841, compound 16 in Table 1). ZK 110 841 is a chemically stable crystalline substance, which is orally active and which might thus turn out to be an interesting tool for the study of PGD2-receptor interactions. Some other prostaglandin as well as prostacyclin analogues with a 15-cyclohexyl or 15-n-pentyl group exhibited in addition to their known high affinity to the PGE2-receptor of human uterine membranes or the PGI2-receptor of human platelets also affinities to the PGD2-receptor. Generally, the receptor affinities correlate with the activities as stimulators of adenylate cyclase and inhibitors of thrombin induced elevation of cytoplasmic free calcium as well as their ability to inhibit ADP-induced platelet aggregation. The PGI2-character regarding the effector systems prevails in compounds with affinity to both the PGI2- and PGD2-receptor. Compounds which bind to the PGE2- and PGD2-receptor show a flat dose response curve regarding platelet activation suggesting a mixture of pro- and antiaggregatory properties within these molecules.

Identification and characterisation of a prostacyclin-like receptor in bovine coronary arteries using a specific and stable prostacyclin analogue, ciloprost, as radioactive ligand.

Abstract It has been proposed that blood vessel walls contain specific receptors which are responsible for mediating the vasodilating effect of prostacyclin. However, since PGI2 is inactivated in physiological saline and is rapidly metabolized in the vasculature it has not been possible to directly demonstrate binding of the prostaglandin to such receptors. Therefore a stable prostacyclin analogue, Ciloprost, was chosen as radioactive ligand in the present study. Tritium labelled Ciloprost was incubated in an ice bath with a particulate fraction of bovine coronary arteries in the presence of various concentrations of unlabelled compound or natural prostaglandins. After 30 minutes incubation the free and particle bound ligand were separated on a Sephadex G50 column. Binding was rapid (half time ca. 5 min), saturable and readily reversed by addition of excess (10−4M) non-radioactive ligand. A Scatchard plot of the equilibrium binding data was curvilinear and could be resolved into two binding sites with dissociation constants of 2.1 × 10−8M and 7.5 × 10−7M, the corresponding binding site concentrations were 40 and 1140 fmol/mg protein respectively. PGE1, PGE2 and PGD2 also displaced [3H]-Ciloprost binding, their relative potencies were (Ciloprost = 1) 0.14, 0.01 and 0.02 respectively. PGF2α was ineffective. PGI2 did ot retain its biological activity in the vascular preparation under the conditions of the receptor assay. It is concluded that Ciloprost binds with high affinity and specificity to receptors in bovine coronary arteries which may be responsible for the mediation of the vascular effects of PGI2.

Synthesis and pharmacological evaluation of 8α-estradiol derivatives

Abstract A number of 2- and 16-alkyl 8α-estradiol derivatives were synthesized and pharmacologically characterized with respect to estrogenic/antiestrogenic activities.

Prostaglandin I2 receptors in a particulate fraction of platelets of various species

Abstract The binding of [ 3 H]PGI 2 to a particulate fraction of human, bovine, canine and rat platelets was measured. Saturation of the binding sites was reached within 10–15 min at 0°. The dissociation constant K D was in the range of 3.5 to 6.3 × 10 −8 moles/l with only little interspecies variation. A structure-binding relationship was obtained from the competition curves after a logit-log transformation. [ 3 H]PGI 2 was displaced by a five-fold higher concentration of PGE 1 , PGE 2 and PGF 2α were much less effective. The high specificity of the platelet receptor should allow the characterization of stable PGI 2 -derivatives.