Anticonvulsant action of a new analogue of allopregnanolone in immature rats.

Neuroactive steroids represent potential antiepileptic drugs. We tested a newly synthesized analogue of allopregnanolone 3alpha-hydroxy-21xi,22-oxido-21-homo-5alpha-pregnan-20-on (HOHP) against two types of pentylenetetrazol-induced seizures (100 mg/kg s.c.) in 12- and 25-day-old rats. Ganaxolone, a neuroactive steroid in clinical trials, served as a reference drug. Pretreatment with either steroid suppressed generalized tonic-clonic seizures in both age groups, their efficacy was comparable. HOHP as well as ganaxolone were more active in 12- than in 25-day-old rats (effective doses were 40 and 60 mg/kg, respectively). Minimal clonic seizures, which can be elicited only in 25-day-old rats, were not influenced by any drug. Very short duration of anticonvulsant action of HPOP demonstrated in 12-day-old animals indicates that this drug might be used only in acute treatment in epileptology.

Anticonvulsant action of allopregnanolone in immature rats.

Anticonvulsant activity of allopregnanolone, a neurosteroid allosterically modulating GABA(A) receptor was tested in a model of motor seizures elicited by pentetrazol in immature rats. Rats 7, 12, 18, 25 or 90 days old were pretreated with allopregnanolone in doses from 5 to 40 mg/kg i.p. and 15 min later pentetrazol was injected subcutaneously in a dose of 100 mg/kg. Rats were observed in isolation for 30 min. Allopregnanolone dose-dependently suppressed both generalized tonic-clonic and minimal clonic seizures with the highest efficacy in 12-day-old rats. Anticonvulsant action was least expressed in adult animals. Duration of anticonvulsant action tested after a dose of 20 mg/kg in 12- and 90-day-old rats demonstrated markedly longer effects in young rats. Allopregnanolone compromised motor performance of rats but duration of this unwanted effect in 12-day-old rats was shorter than duration of anticonvulsant action. This difference can be important for possible clinical use of neurosteroids.

Immunocytochemistry of p16INK4a in liquid-based cervicovaginal specimens with modified Papanicolaou counterstaining.

AIM: To evaluate the feasibility and value of a modified Papanicolaou counterstain for p16(INK4a) immunostaining in liquid-based cervicovaginal samples. METHODS: Immunocytochemical analyses were carried out with p16(INK4a) and modified Papanicolaou counterstain on 81 liquid-based samples, including 23 of within normal limits (WNL), 6 of low-grade squamous intraepithelial lesion (LSIL), 20 of high-grade squamous intraepithelial lesion (HSIL), 16 of atypical squamous cells of undetermined significance (ASC-US) and 16 of atypical squamous cells, high-grade lesion cannot be excluded (ASC-H). Results were compared with histological or cytological follow-up. For comparison, samples from 29 more cases (10 of LSIL, 10 of ASC-H and 9 of HSIL) were immunostained with p16(INK4a) and conventionally counterstained with haematoxylin. The intensity of immunostaining in cases of squamous intraepithelial lesion (SIL) was assessed using a 0-3 scoring system. Interobserver agreement was calculated by kappa statistics. RESULTS: Expression of p16(INK4a) was detected in 3 of 23 cases of WNL, 4 of 6 cases of LSIL, all cases of HSIL, 5 of 16 cases of ASC-US and 13 of 16 cases of ASC-H. Excluding two cases with no residual dysplastic cells in the immunocytochemistry, all cases of cervical intraepithelial neoplasia (CIN)2 or CIN3 at follow-up expressed p16(INK4a) and none of the p16(INK4a)-negative cases showed a high-grade lesion at follow-up. No evident differences in pattern or intensity of p16(INK4a) expression were observed between the specimens of the study and control groups. Interobserver agreement was significantly better in the study group than in the group with conventional immunostaining (combined kappa 0.773 v 0.549; p<0.05), and still better, albeit statistically not significant, than with conventional immunostaining and cervical smear test together (combined kappa 0.773 v 0.642). CONCLUSION: Immunocytochemistry with p16(INK4a) and modified Papanicolaou counterstain may add to the cervicovaginal cytology the full potentiality of p16(INK4a) without the need of a further slide and the risk of loss of dysplastic cells, yet maintaining the typical morphological features of the smear test.

Action of two neuroactive steroids against motor seizures induced by pentetrazol in rats during ontogeny.

The anticonvulsant action of two neuroactive steroids, 3alpha-hydroxy-5beta-pregnan-20-one (pregnanolone) and triethylammonium 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-21-yl hydrogensuccinate (THDOC-conjugate), was tested against motor seizures induced by pentetrazol in immature rats. Five age groups (7, 12, 18 and 25 days old and adult rats) were pretreated with the steroids in doses from 2.5 to 40 mg/kg i.p. Twenty minutes later pentetrazol (100 mg/kg s.c.) was administered. Minimal seizures (clonic seizures of head and forelimb muscles with preserved righting ability) could be induced in the three older age groups. They were suppressed by pregnanolone in all these tested groups (this effect was best expressed in 18-day-old rats and decreased with age), whereas significant changes in THDOC-conjugate-pretreated animals appeared only in 18-day-old rats. Generalized tonic-clonic seizures were suppressed by both neuroactive steroids in all age groups, this effect being more marked with pregnanolone and again decreased with age. The 7- and 12-day-old rats exhibited higher sensitivity of the tonic phase so that generalized clonic seizures were observed. Duration of the effect was studied in 12- and 25-day-old animals; it was substantially shorter in the older rats than in 12-day-old animals. Both drugs exhibited an anticonvulsant action in developing rats but, unfortunately, their effect was only shortlasting.

Suppressing aggressive behavior with analogs of allopregnanolone (epalon).

3alpha-Hydroxy-20-oxo-5alpha-pregnan-21-yl hemisuccinate (8) was produced by partial acylation of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (14). 3alpha-Fluoro-5alpha-pregnan-20-one (9) was prepared by treatment of 3beta-hydroxy-5alpha-pregnan-20-one (11) with DAST and by solvolysis of tosylate 12 with tetrabutylammonium fluoride. A behavioral test on mice was performed using 3alpha-hydroxy-5alpha-pregnan-20-one (1) and compounds 8 and 9. Compound 8 was found to be inactive, while the fluoro derivative 9 selectively reduced aggressive behavior in mice more than the corresponding 3alpha-hydroxy compound 1; locomotion and other behavioral features were not affected.

7-Hydroxydehydroepiandrosterone--a natural antiglucocorticoid and a candidate for steroid replacement therapy?

7-Hydroxylated metabolites of dehydroepiandrosterone (DHEA) are believed to be responsible for at least some immunomodulatory and antiglucocorticoid effects of DHEA and hence are considered candidates for hormone replacement therapy. Our experiments in vitro brought the evidence that 3beta, 7beta-dihydroxy-5-androsten-3-one (7beta-OH-DHEA), but not DHEA and its 7alpha-hydroxyisomer, could counteract the immunosuppressive effect of dexamethasone on the formation of plaques in culture of murine spleen lymphocytes. In another experiment, DHEA and after a 3-weeks pause 3beta-hydroxy-5-androstene-7,17-dione (7-oxo-DHEA) were applied transdermally to 6 male volunteers on 5 consecutive days. Blood levels of DHEA, its 7-hydroxylated metabolites, and in the first case also dehydroepiandrosterone sulphate (DHEAS), were measured before, during and one day after the end of treatment. Application of DHEA increased significantly not only DHEA and DHEAS, but also its both 7-hydroxyisomers. Application of 7-oxo-DHEA also led to a significant increase of both 7-hydroxyisomers of DHEA, with 7beta-OH-DHEA being the preferred metabolite the concentration of which was increased more than three times.

A new steroidal alkaloid from the roots of Cynanchum caudatum.

A new steroidal alkaloid, 12-O-nicotinoylsarcostin, gagamine (1), was isolated from the roots of Cynanchum caudatum Max. (Asclepiadaceae), together with a known alkaloid, gagaminine (2). Their structures were established using spectroscopic methods, some 13C-NMR data of 2 have to be revised.

Some reactions of 16 alpha,17 alpha-oxido-5 alpha-cholestane derivatives, synthesis of 17 alpha-hydroxycholest-4-en-3-one.

Careful epoxidation of the delta 16-olefins 3 and 4 yielded 16 alpha,17 alpha-epoxides 5 and 6 which were reduced by lithium aluminium hydride, oxidized, and dehydrated to 17 alpha-hydroxycholest-4-en-3-one 20, i.e., an epitestosterone homolog containing a well tolerated alkyl group at position 17. Under catalysis of acids, epoxide 5 was rearranged to delta 13-16 alpha-alcohol 10. Less careful epoxidation of delta 16-olefin 4 with excess of peroxy acid led to products of double epoxidation (i.e., epoxidation, rearrangement, and another oxidation) 7 and 12. Structures of products of rearrangement were studied mainly by NMR spectroscopy.

Antihormonal properties of some new A-homo-B, 19-dinor steroids of the androstane series.

On solvolysis of Westphalen-type steroids with a leaving group in the position 6 beta (e.g., 2), products of elimination (followed by rearrangement and fragmentation of the steroid skeleton) were prepared (e.g., 4 and 5). These products were subsequently converted to suitable analogs of the compound, which has been reported to promote hair growth (1). Compounds 11 to 13 exhibited strong antiandrogenic activity in vivo; however, this activity could not be interpreted either in terms of inhibition of 5 alpha-reductase or by strong binding to an androgen receptor.

Progesterone side-chain degradation beside hydroxylation with Rhizopus nigricans depends on the presence of nutrients.

In the absence of nutrients, Rhizopus nigricans transforms progesterone into a mixture of 11 alpha-hydroxy-4-androstene-3,17-dione and 11 alpha-hydroxy-1,4-adrostadiene-3,17-dione. The same mixture is obtained by the transformation of testosterone and its acetate.

Effects of cholesteryl esters on the accessibility of LH/hCG receptors and membrane lipid fluidity in rat testes.

Incubation of rat testicular membranes with cholesteryl hemisuccinate resulted in an increase in both membrane lipid microviscosity and 125I-labelled hCG specific binding. The purpose of this investigation was to establish which functional groups of cholesteryl hemisuccinate are important for the stimulatory effects. The data obtained showed that only esters of cholesterol with dicarboxylic acids, not those of monocarboxylic acids, increase the accessibility of LH/hCG receptors and membrane rigidity. Experiments with cholesteryl sulfates showed that there are polar groups on C3 carbon of cholesterol having no stimulatory effect on receptors, although an increase in membrane rigidity occurred. The side-chain of cholesterol is important for the stimulatory action. Androstenolone hemisuccinate was ineffective in this respect. On the other hand, partially modified side-chains (hemisuccinates of beta-sitosterol and stigmasterol) did not result in a marked reduction of the stimulatory action. The carboxyl group of cholesteryl hemisuccinate must be 'free': its esterification abolishes the stimulatory effect of cholesteryl hemisuccinate on both the LH/hCG receptor and membrane microviscosity. These results suggest that an intact carboxyl group of ester and the side-chain of cholesterol are indispensable for the stimulatory effect of cholesteryl hemisuccinate on the accessibility of LH/hCG receptors.

Binding of [3H]-4,5-secodihydrotestosterone by androgen receptor.

Specific binding of [5,6-3H2]-17 beta-hydroxy-4,5-secoandrostan-3-one (or [3H]-4,5-secodihydrotestosterone, IX) to rat prostate cytosol receptors was observed and confirmed by two independent methods: by adsorption to dextran-charcoal and by polyacrylamide gel electrophoresis. The electrophoresis of [3H]-4,5-secodihydrotestosterone--cytosol mixtures showed a single radio-activity peak at different radioligand concentrations, displaceable by nonradioactive 4,5-secodihydrotestosterone; the mobility of the peak corresponds to a protein with a relative molecular weight of 90 000-130 000. The apparent association constant of the compound with respect to adsorption to dextran-charcoal is 1.48 X 10(9) l X mol-1, similar to the value observed with dihydrotestosterone (4.8 X 10(8) l X mol-1).

Androgen receptor binding and antiandrogenic activity of some 4,5-secoandrostanes and ring B cyclopropanoandrostanes.

Eight androstane derivatives with modified ring A or B (4,5-secoandrostanes and ring B cyclopropanoandrostanes) were assayed in vivo on mice for their antiandrogenic activity and the effect was compared with that of cyproterone acetate. The inhibition of dihydrotestosterone binding to rat prostate cytosol and to human plasmatic sex hormone binding protein was correlated with the in vivo effect. The antiandrogenic activity of 6 alpha,7 alpha-cyclopropano-5 alpha-androstane-3 beta,17 beta-diol was nearly as high as that of cyproterone acetate. The opening of ring A of androgens, such as testosterone or 17 alpha-methyltestosterone, moderately reduced the binding and changed the biological activity to weakly antiandrogenic.