Introduction

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Following the discovery that the anesthetic steroid alphaxalone potentiated GABA responses (Harrison and Simmonds, 1984), it soon became clear that related steroids, including metabolites of progesterone and deoxycorticosterone, are also positive allosteric modulators of GABA_A receptor complexes (Gee et al., 1987; Harrison et al., 1987; Majewska et al., 1986). By analogy to other known GABA potentiators such as barbiturates and benzodiazepines, this GABAergic mechanism suggested that these novel modulators, now termed neuroactive steroids, could be useful clinically for a number of central nervous system disorders. In addition to the historical (Phillips, 1975) and recent (Anderson et al., 1997) use as intravenous anesthetics, neuroactive steroids have potential uses as antiepileptic agents (Carter et al., 1997; Gasior et al., 1997), sedative-hypnotics (Edgar et al., 1997), anxiolytics (Brot et al., 1997; Carter et al., 1995; Wieland et al., 1995, 1997) and for migraine (Limmroth et al., 1996).

Although specific binding by a radiolabeled steroid has not been convincingly demonstrated, compelling evidence for a unique site on the GABA_A receptor complex for neuroactive steroids has been amassed (Gee et al., 1995). The strongest single argument in favor of a unique binding site is the exquisite SAR, in particular the stereoselectivity of the 3-hydroxy group, which must be in the  configuration for potent modulation of the receptor complex (Gee et al., 1987; Harrison et al., 1987; Hawkinson et al., 1994a; Hogenkamp et al., 1997; Upasani et al., 1997). Presumably, the 3-stereochemistry is required for the correct alignment of the hydroxy group with a hydrogen bond accepting group located in the binding site. In addition, the 20-keto function is thought to contribute to high receptor potency by interacting with a hydrogen bond donating residue in the binding site, although steroids without this group may retain moderate potency (Bolger et al., 1997; Purdy et al., 1990; Hawkinson et al., 1994a).

Substitution of the steroid nucleus at the 3-position was initially explored to increase bioavailability by blocking metabolic oxidation of the critical 3-hydroxy group, preventing conversion to potentially hormonally active steroid metabolites and to slow metabolic conjugation at this position (Hogenkamp et al., 1997). This approach resulted in ganaxolone (Carter et al., 1997), which is currently in phase II clinical trials for epilepsy and migraine. Recently, it was shown that substitution of the 3-position with an ethynyl spacer unit linked to a phenyl group in the 5 steroid series results in highly potent modulators of GABA_A receptors, particularly when the phenyl group is substituted in the para-position with hydrogen bond accepting groups such as acetyl (Upasani et al., 1997). Based on these observations, it was proposed that an auxiliary binding pocket exists adjacent to the site occupied by the steroid A-ring and that this pocket contains a hydrogen bond donating group which interacts with the p-acetyl group of the 3-phenylethynyl substituent (Upasani et al., 1997). The present report examines the role of extended 3-substitution in the 5 steroid series and describes the pharmacology of Co 152791 (fig. 1), the most potent known neuroactive steroid modulator of GABA_A receptors.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Structure of Co 152791 (Compound 16).

	Methods

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Synthesis

The preparation of compounds 1-5 and 18 was described previously (Hogenkamp et al., 1997; Upasani et al., 1997). 3-(Hexyn-1-yl)-3-hydroxy-5-pregnan-20-one (6) was prepared by adding hexyn-1-yl lithium, generated by the reaction of 1-hexyne with n-butyl lithium, to 5-pregnane-3,20-dione 20-ketal. Similarly, addition of phenylmagnesium bromide and benzylmagnesium bromide to the same ketal afforded the 3-phenyl (8) and 3-benzyl (9) derivatives, respectively. 3-Hydroxy-3-phenylethynyl-5-pregnan-20-one derivatives (7, 12-17) were synthesized in ~30% to 50% yields using the previously described (Upasani et al., 1997) coupling reaction of 3-ethynyl-3-hydroxy-5-pregnan-20-one (Hogenkamp et al., 1997) with the corresponding p-substituted iodobenzenes in the presence of catalytic amounts of (PPh₃)₂PdCl₂ and CuI. The 3-phenylethyl derivative (10) was prepared by catalytic hydrogenation of the unsaturated analog (11), which was synthesized from (3R)-spiro[oxirane-2',5-pregnan]-20-one (Hogenkamp et al., 1997) by reaction with methyl phenyl sulfoxide anion and elimination of the -hydroxy sulfoxide formed (Hogenkamp, 1995). All the compounds prepared were purified by column chromatography over silica gel. Purity was ascertained by thin layer chromatography and routine spectral analysis (IR and NMR spectroscopy).

Receptor Source for Binding Assays

Stable GABA_A 2Lcell line preparation. Human 1, 2, 3 and 2L GABA_A receptor subunits were a gift from Peter Seeburg (University of Heidelberg, Germany). Human 4, 6 and 2 subunits were cloned as described (Yang et al., 1995). Human 5 was cloned from human brain by PCR utilizing oligonucleotide primers corresponding to the proposed ends of the coding region based on the human 5 genomic sequence (Knoll et al., 1993). The amino acid sequence derived from this cDNA was identical to the amino acid sequence previously reported (Wingrove et al., 1991). Human 3 (Wafford et al., 1994) was cloned from human brain by PCR utilizing oligonucleotide primers derived from the published sequences corresponding to the ends of the coding region. All plasmid DNA for transfection was prepared using two cycle cesium chloride gradient centrifugation. The transfection and stable cell line cloning of the HEK293 cells (CRL 1573; American Type Culture Collection) follows the protocol reported previously (Hawkinson et al., 1996).

Membrane preparation. Membranes from stable HEK293 cell lines expressing human recombinant GABA_A receptor subunit combinations and well-washed rat brain cortical homogenates were prepared as described previously (Hawkinson et al., 1996).

Radioligand Binding

[³⁵S]TBPS assay. Steroid inhibition of 2 nM [³⁵S]TBPS (60-100 Ci/mmol; NEN) binding was examined in 200 mM NaCl/50 mM sodium-potassium phosphate buffer (pH 7.4) as previously described (Carter et al., 1997; Hawkinson et al., 1994a, 1996). The GABA concentration was either the approximate IC₅₀ for inhibition of TBPS binding (rat brain) or the concentration producing the peak TBPS binding from the biphasic GABA concentration-effect curve (recombinant receptors) as indicated in table 6. Incubations contained ~350, 100, 100, 120, 140, 200, or 200 µg protein for rat brain, 122L, 222L, 322L, 432L, 522L and 632L membranes, respectively. The incubation and filtration were conducted as previously described (Hawkinson et al., 1996) or in 96-well plates (2.0 ml; Beckman) followed by filtration through GF/B 96-well filter plates (Packard) and rinsed 3 times with ~1.5 ml ice-cold assay buffer. In the latter case, Microscint scintillation cocktail (50 µl; Packard) was added to each well of the dried filter plates, which were then sealed, shaken vigorously for 5 min and counted for 5 min/well on a TopCount 6-detector scintillation counter (Packard).

[³H]Flunitrazepam assay. Steroid enhancement of 1 nM [³H]flunitrazepam (84.5 Ci/mmol; NEN) binding in well-washed rat brain cortical P2 membranes was examined in 200 mM NaCl/50 mM sodium-potassium phosphate buffer (pH 7.4) in the presence of 1 µM GABA as previously described (Carter et al., 1997; Hawkinson et al., 1994a; Hawkinson et al., 1996).

[³H]Muscimol assay. Steroid enhancement of 5 nM [³H]muscimol (10.1 Ci/mmol; NEN) binding in well-washed rat brain cortical P2 membranes was examined in sodium-free buffer (100 mM KCl/40 mM potassium phosphate, pH 7.4) as previously described (Carter et al., 1997; Goodnough and Hawkinson, 1995; Hawkinson et al., 1996).

Data analysis

Nonlinear curve fitting of the overall data for each drug averaged for each concentration was performed using the sigmoidal equation in Prism (GraphPad). The data were fit to a two component instead of a one component model if the sum of squares was significantly lower by F-test. The concentration of test compound producing 50% inhibition (IC₅₀) or enhancement (EC₅₀) of specific binding, the extent of inhibition (% I) or enhancement (% E) corresponding to each component for two component modulators, and the maximal extent of inhibition (I_max) or enhancement (E_max) were determined for the individual experiments with the same model used for the overall data and then the mean ± S.E. of the individual experiments were calculated.

Electrophysiology

Receptor expression and recording in Xenopus oocytes. RNA was prepared as previously described (Hawkinson et al., 1996) and stored at 80°C. Preparation and microinjection of oocytes were performed as reported previously (Woodward et al., 1995). Individual oocytes were injected with ~1 ng each of cRNA encoding the 1, 2 and 2L subunits, and oocytes were stored in Barth's medium containing (in mM): NaCl, 88; KCl, 1; CaCl₂, 0.41; Ca(NO₃)₂, 0.33; MgSO₄, 0.82; NaHCO₃, 2.4; HEPES 5; pH 7.4, with 0.1 mg/ml gentamycin sulfate. Individual oocytes were placed on a mesh in a standard 35-mm culture dish perfused with frog Ringer's solution containing (in mM): NaCl, 115; KCl, 2; CaCl₂, 1.8; HEPES, 5; pH 7.4. Electrical recordings were made using a Dagan TEV-200 voltage clamp. Steroids were initially diluted into DMSO stocks (10 nM to 10 mM) and further diluted into Ringer just prior to experiments. The final DMSO concentration was 0.3%, which had no effect by itself. Drug solutions were applied to oocytes via a triple-barrel linear array as described in detail previously (Hawkinson et al., 1996). Modulatory effects were measured after 1- to 2-min preincubations with steroids, followed by exposure to a mixture of steroid and GABA. Maximal GABA responses were measured before and after steroid modulation experiments, and any change in the maximum current was factored in by calculating fractional currents against a linear sliding scale.

Experimental design and data analysis. GABA concentration-response data were obtained by successive brief exposures to increasing concentrations of GABA, until an apparent maximal current was reached (1-3 mM GABA). These data were fit to the logistic equation (Eq. 1) using Origin (Microcal), where FR = I/GABA_max, n is the slope, EC₅₀ is the concentration that produces a half-maximal response, I is the current at a given concentration of GABA (agonist) and GABA_max is the maximal current in response to GABA.

(1)

Steroid modulation experiments were performed using a concentration of GABA that elicited current ~5% of GABA_max. Oocytes were exposed to steroids for 30-60 seconds prior to coapplication of steroid with the GABA control solution. Fractional response (FR) was calculated by dividing the current obtained in the presence of steroid by the GABA_max. These data were also fit to Eq. 1, where agonist now represents steroid coapplied with the GABA control solution. The mean ± S.E. EC₅₀ values of the individual experiments were then calculated.

In Vivo Pharmacology

Animals. Male NSA mice weighing between 15 and 20 g were obtained from Harlan Sprague-Dawley, Inc. Upon arrival they were housed in standard polycarbonate cages (4 per cage) containing a sterilized bedding material (Sani-Chips, P.J. Murray) in a room of constant temperature (23.0° ± 2.5°C) with a 12 hr (7:00 a.m. to 7:00 p.m.) light/dark cycle. Food (Teklad LM 485; Harlan Sprague-Dawley) and water were freely available. Animals were acclimated a minimum of 4 days prior to experimentation.

PTZ-induced seizures. Seizures were induced by administration of 85 mg/kg, s.c. PTZ (30 min observation period). The dose of PTZ used was previously determined to be the dose producing convulsions in 97% of animals (CD₉₇). A clonic seizure was defined as forelimb clonus of  3 sec duration. Data were treated quantally.

Motor function. The rotorod test used a custom-built apparatus that consisted of an elevated drum of textured surface (diameter: 2.5 cm) that rotated at a constant speed (6 rpm). The height of the drum from the floor of the test apparatus was ~30 cm. Prior to administration of test substance, animals were trained to walk continuously on the drum for a period of 2 min. During testing, animals were given 3 opportunities to remain on the apparatus continuously for 1 min. LRR was also determined in mice. Results were treated quantally.

Pharmacologic procedure. PTZ was obtained from Sigma Chemical Co. and was dissolved in physiologic saline (0.9%). Neuroactive steroids were dissolved in hydroxypropyl--cyclodextrin (Amazio) 50%: distilled water 50% and were placed in solution by warming and sonication for 1-4 hrs. Solutions were prepared on a weight/volume basis on the day of, or evening prior to, use. PTZ was administered s.c.; neuroactive steroids were administered i.v., i.p. or p.o. Drugs were administered in volumes of 100, 100 and 400 µl/20 g for i.v., i.p. and p.o. dosing, respectively.

Data Analysis

Dose-response functions were constructed for graphical presentation by converting the quantal response data to percentages and calculating the mean ± S.E. for each dose of 3 independent experiments. The dose of drug required to produce an anticonvulsant effect (ED₅₀), loss-of-righting reflex (ED₅₀), or motor impairment (TD₅₀) in 50% of animals and its associated 95% confidence limits was calculated on the quantal sum of the data by the method of Litchfield and Wilcoxon (1949) using a commercial computer program (PHARM/PCS v4.2; MicroComputer Specialists). The TI was calculated by dividing the TD₅₀ by the PTZ ED₅₀.

	Results

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Structure-activity of 3-substituted 3-hydroxy-5-pregnan-20-ones defined by [³⁵S]TBPS binding in rat brain membranes. Substitution of the 3 position of 3,5-P (compound 1) with short chain alkyl groups (compounds 2 and 3) reduced potency for inhibition of [³⁵S]TBPS binding, whereas unsaturation of the side chain (compounds 4 and 5) reversed this reduction as previously reported (Hogenkamp et al., 1997) (table 1). Further elongation of the optimal two carbon ethynyl unit (compound 5) with an n-butyl moiety (compound 6) did not alter potency. Extension of the ethynyl unit with a phenyl group resulted in compound (7), which displayed a two component, partial inhibition curve. Considering the high affinity component only, phenyl modification of the ethynyl group further increased potency.

View this table: [in this window] [in a new window]   TABLE 1 Effect of chain length and unsaturation of 3-substituted derivatives of 3-hydroxy-5-pregnan-20-one in the [35S]TBPS binding assay in rat brain membranes IC50 and Imax values for inhibition of 2 nM [35S]TBPS binding to rat brain cortical membranes in the presence of 5 µM GABA. Values are mean ± S.E. of at least three independent experiments. Hill slope values were 1.0 for all one-component compounds.

View this table: [in this window] [in a new window]   TABLE 2 Effect of the spacer group on 3-phenyl substituted derivatives of 3-hydroxy-5-pregnan-20-one in the [35S]TBPS binding assay in rat brain membranes IC50 and Imax values for inhibition of 2 nM [35S]TBPS binding to rat brain cortical membranes in the presence of 5 µM GABA. Values are mean ± S.E. of at least three independent experiments. Hill slope values were 1.0 for all one-component compounds.

View this table: [in this window] [in a new window]   TABLE 3 Effect of para-substitution of 3-phenylethynyl-3-hydroxypregnan-20-one in the [35S]TBPS binding assay in rat brain membranes IC50 and Imax values for inhibition of 2 nM [35S]TBPS binding to rat brain cortical membranes in the presence of 5 µM GABA. Values are mean ± S.E. of at least three independent experiments. Hill slope values were 1.0 for all one-component compounds.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Neuroactive steroid inhibition of [35S]TBPS binding to GABAA receptors in rat brain membranes. Note the high potency of the p-acetyl derivatives 16 (Co 152791) and 18 compared to 3,5-P (1) and the two component modulation by the p-chloro (13) and p-hydroxy (14) compounds. Steroids were incubated with 2 nM [35S]TBPS in the presence of 5 µM GABA for 90 min at room temperature. Symbols represent the mean ± S.E. of at least three independent experiments. IC50, Imax and slope values are found in tables 1 and 3.

Modulatory profile of selected neuroactive steroids in the [³H]flunitrazepam and [³H]muscimol binding assays. In the [³H]flunitrazepam binding assay in rat brain membranes, all of the neuroactive steroids examined exhibited one component enhancement curves (fig. 3). The p-acetyl compounds 16 (Co 152791) and 18 displayed the highest potency of the compounds tested, although the p-chloro compound 13 was also more potent than 3,5-P (1) (table 4). In contrast to the [³⁵S]TBPS result, the p-hydroxy compound 14 displayed only low affinity modulation of [³H]flunitrazepam binding. Although major differences in efficacy of modulation were not observed, the p-acetyl compounds 16 (Co 152791) and 18 had higher E_max values than 3,5-P (1), whereas the p-chloro compound 13 showed lower efficacy.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Neuroactive steroid enhancement of [3H]flunitrazepam binding to GABAA receptors in rat brain membranes. Note the high potency of the p-acetyl derivatives 16 (Co 152791) and 18 and the p-chloro (13) derivatives compared to 3,5-P (1) and the low potency of the p-hydroxy (14) compound. Steroids were incubated with 1 nM [3H]flunitrazepam in the presence of 1 µM GABA for 90 min at room temperature. Symbols represent the mean ± S.E. of at least three independent experiments. EC50, Emax and slope values are found in table 4.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Neuroactive steroid enhancement of [3H]muscimol binding to GABAA receptors in rat brain membranes. Note the two component modulation by 3,5-P (1) and the p-acetyl compound in the 5 series and the high potency of the p-acetyl derivatives in both 5 (compound 16; Co 152791) and 5 series (compound 18). Note also the low efficacy of the p-chloro compound 13 and low potency of the p-hydroxy analog 14. Steroids were incubated with 5 nM [3H]muscimol for 60 min on ice. Symbols represent the mean ± S.E. of at least three independent experiments. EC50, Emax and slope values are found in table 4.

Profile of compounds 13 and 16 (Co 152791) relative to 3,5-P in human recombinant GABA_A receptors. Compounds 13 and 16 (Co 152791) were potent inhibitors of [³⁵S]TBPS binding in membranes prepared from stable HEK cell lines expressing human 122L, 222L, 322L and 522L subunit combinations, with IC₅₀ values ranging from 1.4 to 12 nM (table 5, fig. 5). In these receptor combinations, IC₅₀ values for 3,5-P ranged from 20 to 40 nM (Hawkinson et al., 1996). In the 432L and 632L combinations, 3,5-P inhibited [³⁵S]TBPS binding with much lower potency (IC₅₀ 1700 and 1060 nM, respectively), but retained high efficacy (I_max > 75%). In contrast, the phenylethynyl derivatives 13 and 16 (Co 152791) were considerably more potent at 432L and 632L subunit combinations (IC₅₀ 27-210 nM), although both compounds displayed limited efficacy for inhibition of [³⁵S]TBPS binding at these receptors (table 5, fig. 5).

View this table: [in this window] [in a new window]   TABLE 5 Neuroactive steroid inhibition of [35S]TBPS binding in human recombinant GABAA receptors IC50, Imax and Hill slope values for inhibition of 2 nM [35S]TBPS binding to human recombinant GABAA receptor combinations expressed in HEK 293 cells. Values are mean ± S.E. of at least three independent experiments. Hill slope values were not different from 1.0 (P < .05) except for Co 152791 in 122L (1.7 ± 0.5) and 222L (1.3 ± 0.1) membranes, and for compound 13 in 122L (2.4 ± 0.7), 222L (2.0 ± 0.2), 322L (1.6 ± 0.1) and 522L (constrained to 2.5) membranes.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Inhibition of [35S]TBPS binding to human recombinant GABAA receptors by compound 13 and Co 152791. Note the high potency of the p-chloro (13) (top) and the p-acetyl (16; Co 152791) (bottom) analogs and their limited efficacy at the 432L and the 632L combinations. Steroids were incubated with membranes prepared from stable HEK cell lines expressing human recombinant GABAA receptors and 2 nM [35S]TBPS for 90 min at room temperature in the presence of GABA at the concentrations listed in table 5. Symbols represent the mean ± S.E. of at least three independent experiments. IC50, Imax and slope values are found in table 5.

Electrophysiological characterization of selected neuroactive steroids at 122L receptors expressed in Xenopus oocytes. Oocytes showed robust expression of functional GABA_A receptors 5-17 days after injection with a mixture of 1, 2 and 2L cRNAs. Maximal current response to 10 mM GABA was 2400 ± 100 nA (n = 19) from two batches of oocytes from two separate frogs. The EC₅₀ value for GABA in these oocytes was 24 ± 2 µM, with a slope of 1.3 ± 0.1 (n = 19), consistent with a single population of 122L receptors. Modulatory effects of steroids on 122L receptors were assayed using control GABA-evoked currents that were ~5% of maximal GABA responses in each individual oocyte. Mean concentrations of GABA used to elicit 5% responses were 5.1 ± 0.6 µM (n = 17).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Functional modulation of GABA-activated currents by neuroactive steroids in Xenopus oocytes expressing the human GABAA receptor combination 122L. Steroids and other modulators were co-applied with a concentration of GABA producing a current that was ~5% of the GABAmax. Top, concentration-effect curves. Note the high potency of compound 16 (Co 152791) and the low efficacy of compounds 7 and 13 and, particularly, compound 14. EC50, fractional response compared to the GABAmax (FR) and slope values are found in table 6. Bottom, sample records from a single oocyte illustrating the high potency of compound 16 (Co 152791) relative to 3,5-P, diazepam and pentobarbital. Bars indicate duration and timing of drug applications. The holding potential was 70 mV with steps to 60 mV (upward deflections) to time drug applications and to monitor membrane conductance. Vertical and horizontal scale bars represent 100 nA and 1 min, respectively. Record was taken in sequence, with drug applications separated by a 5-10 min wash.

View this table: [in this window] [in a new window]   TABLE 6 Neuroactive steroid potentiation of GABA-evoked responses in Xenopus oocytes expressing 122L receptors EC50, fractional response (FR) compared with the GABAmax, and slope values for potentiation of currents elicited by a GABA concentration that was 5% of the GABAmax. Values are mean ± S.E. of at least three independent experiments. Due to low solubility, the highest concentration of compound 7 (100 nM) was applied in 1% DMSO; the resultant DMSO-induced current was subtracted from the steroid-induced potentiation current. All other data were obtained in the presence of 0.3% DMSO, which had no effect by itself.

In vivo profiles of selected neuroactive steroids. Selected neuroactive steroids were evaluated for in vivo pharmacological activity and compared to reference steroids. Dose-response data for protection against clonic seizures induced by s.c. PTZ administration in mice are summarized in table 7. Consistent with the in vitro data, Co 152791 was a potent anticonvulsant, displaying an ED₅₀ of 0.6 mg/kg, i.p. for inhibition of PTZ-induced clonic seizures (fig. 7). A comparable increase in ataxic potency relative to 3,5-P (1) was observed in the rotorod test after i.p. administration (TD₅₀ 4.8 mg/kg), resulting in a slightly better therapeutic index for Co 152791 (TI 8.0) than 3,5-P (TI 6.7). In contrast to 3,5-P, Co 152791 retained activity after oral administration (ED₅₀ 1.1 mg/kg). A similar shift in ataxic potency (TD₅₀ 7.7 mg/kg) resulted in a TI of 7.0. Conversely, compounds 13 and 18 were less potent than 3,5-P, although compound 18 retained oral activity. Compound 14 was inactive i.p. in the PTZ assay. Anesthetic activity of some of these steroids was examined by their ability to induce loss-of-righting reflex (LRR) in mice. Following i.v. administration, Co 152791 (ED₅₀ 2.4 mg/kg) was 2.3- and 4.5-fold more potent than compound 18 and 3,5-P, respectively, for induction of LRR.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Comparison of the anesthetic, anticonvulsant and ataxic activity of the 5 (compound 16; Co 152791) and 5 (compound 18) 3-(p-acetylphenylethynyl) substituted steroids. Dose-response curves for inhibition of s.c. PTZ-induced seizures or induction of rotorod ataxia after i.p. administration in mice were determined at the time of peak effect for each compound. Each point represents the mean ± S.E. of 3 independent experiments (n = 8). ED50, TD50 and TI values are found in table 7.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Substitution of the naturally occurring progesterone metabolite 3,5-P at the 3-position has lead to the discovery of Co 152791 (3-hydroxy-3-(p-acetylphenylethynyl)-5-pregnan-20-one; compound 16), which is the most potent known neuroactive steroid and may well be most potent GABA_A receptor modulator known. Co 152791 modulated the binding of GABA_A receptor radioligands in rat brain with IC₅₀ or EC₅₀ values of 2-7.5 nM. This neuroactive steroid was 11-, 16- and 49-fold more potent than the endogenous neuroactive steroid 3,5-P (1) in the [³⁵S]TBPS, [³H]flunitrazepam and [³H]muscimol assays, respectively. Similarly, Co 152791 inhibited [³⁵S]TBPS binding with IC₅₀ values of 1.4-5.7 nM in the human recombinant receptor combinations 122L, 222L, 322L and 522L, being 3- to 20-fold more potent than 3,5-P. This compound was remarkably potent in potentiating GABA-evoked currents in Xenopus oocytes expressing 122L receptors (EC₅₀ 0.87 nM), being 184-fold more potent than 3,5-P.

The high potency of Co 152791 in vitro was also observed in vivo. Thus, Co 152791 exhibited exceptional anesthetic and anticonvulsant potency, inducing loss-of-righting reflex and protecting against clonic seizures induced by PTZ in mice with an ED₅₀ of 2.4 mg/kg, i.v. and 0.6 mg/kg, i.p., respectively. In both instances, Co 152791 was ~5 times more potent than 3,5-P in vivo. Although an increase in ataxic potency determined by impairment of rotorod performance was also observed after i.p. administration (TD₅₀ 4.8 mg/kg), Co 152791 exhibited a wide separation between anticonvulsant and ataxic activities, reflected in a therapeutic index of 8. Although slightly less active after oral administration, Co 152791 displayed potent anticonvulsant activity (PTZ ED₅₀ 1.1 mg/kg) with a TI of 7, superior to that for any previously reported orally active neuroactive steroid (Carter et al., 1997; Gasior et al., 1997; Kokate et al., 1994; Wieland et al., 1995).

The structure-activity relationship (SAR) for 3-substituted derivatives of 5-pregnane steroid modulators of the GABA_A receptor indicates the presence of an auxiliary pocket in the neuroactive steroid binding site near the region occupied by the steroid A-ring (Upasani et al., 1997). The SAR in the 5 series reported here confirms this interpretation. As in the 5 series, 3 substitution of 3,5-P (1) with small alkyl groups reduced potency in the [³⁵S]TBPS binding assay, although unsaturation of the side chain reversed this decrement so that the 3-ethynyl derivative 5 had similar potency to 3,5-P. Although the effect of unsaturation could be due to reduction in the effective size of the substituent, this is unlikely since extension of the ethynyl group with phenyl actually increased potency. Indeed, a spacer group is required to extend the phenyl group from the steroid A-ring and this spacer must be unsaturated, with ethynyl being optimal. Compounds with spacers of 0, 1, or 2 carbon atoms were essentially inactive (IC₅₀ > 10 µM). The key features of the ethynyl spacer are its length and rigidity, which places the phenyl group in a constrained volume in the binding pocket. These requirements are more critical in the 5 series since saturated spacers or direct attachment of the phenyl group to the 3 position in the 5 series results in compounds which retain moderate activity (IC₅₀ 100-400 nM) (Upasani et al., 1997). para-Substitution of the phenyl ring with the hydrogen bond accepting acetyl group as in Co 152791 confers optimal potency.

Substitution of the para position of the phenyl ring with groups that do not hydrogen bond or are weaker hydrogen bond acceptors than acetyl results in compounds having lower potency. Thus, the para-unsubstituted (7) and the para-methyl (12), -chloro (13), -methoxy (15) and -carbethoxy (17) compounds were 2.1- to 4.5-fold less potent as inhibitors of [³⁵S]TBPS binding in rat brain (high affinity components). Compounds 7, 12, 13 and 15 were also 2.2- to 13-fold less potent as potentiators of GABA-evoked currents in oocytes expressing 122L receptors. Similar effects of para-substitution have been observed in the 5 series (Upasani et al., 1997). The corresponding 5 analog 18 of the highly potent 5 steroid Co 152791 was also consistently less potent in all assays.

Although Co 152791 (compound 16) was the most potent compound in vitro and in vivo, the correlation between in vitro and in vivo potency did not extend to all compounds examined. For example, compound 18, the 5-epimer of 16, was ~3-fold less potent than 3,5-P as an anticonvulsant, but was more potent than 3,5-P in vitro by a factor of 5- to 24-fold. Similarly, compound 13 was ~2-fold less potent than 3,5-P as an anticonvulsant, but was consistently more potent than 3,5-P in vitro, particularly in electrophysiological assays where it was > 80-fold more potent than 3,5-P. Presumably, 3,5-P has better bioavailability than compounds 13 and 18 after i.p. administration. The situation is reversed after oral administration in that compound 18 retains activity whereas 3,5-P is inactive, consistent with previous reports (Carter et al., 1997). Although lack of 3 substitution probably contributes to the lack of oral activity of 3,5-P, 3 substitution per se does not automatically confer oral activity as compound 13 was inactive orally.

In addition to potency differences, para-substitution of the 3-phenylethynyl group in the 5 series also affects the efficacy of modulation. In the [³⁵S]TBPS binding assay in rat brain membranes, the unsubstituted (7), p-methyl (12), p-chloro (13) and p-hydroxy (14) compounds displayed two component modulation with high and low affinity components corresponding to 27-40% and 36-57% of maximal inhibition, respectively. In contrast, para-substitution with hydrogen bond acceptors, such as methoxy (compound 15), acetyl (compound 16; Co 152791) and carbethoxy (compound 17), resulted in compounds that exhibited only high affinity binding. Thus, para-substitution with strong hydrogen bond accepting groups increases the proportion of the high affinity component relative to para-substitution with groups that are hydrogen bond donors, do not hydrogen bond, or are weak hydrogen bond acceptors. These effects of hydrogen bonding groups on [³⁵S]TBPS binding in the 5-pregnane series are similar, but not identical, to that observed in the 5 series, where hydrogen bonding groups affect the potency but not the efficacy of modulation (Upasani et al., 1997).

Two component modulation of radioligand binding to the GABA_A receptors present in brain membranes by neuroactive steroids has been noted in several cases (Goodnough and Hawkinson, 1995, 1994b; Zhong and Simmonds et al., 1996; Upasani et al., 1997). This phenomenon is suggestive of subtypes of GABA_A receptors with differential affinities for certain neuroactive steroids, but also could be due to negative cooperativity between multiple binding sites per receptor complex, differential GABA sensitivities, partial agonism and/or complex combinations of these actions.

In an attempt to address the issue of potential neuroactive steroid subtype selectivity, the inhibition of [³⁵S]TBPS binding by the high affinity, one component modulator 16 (Co 152791) and the two component modulator 13 was determined in membranes prepared from stable cell lines expressing six different  subunit combinations. In these six human recombinant receptors, the potency and efficacy profile for compound 13 was similar to that for Co 152791, suggesting that subtype selectivity does not explain the two component modulation of [³⁵S]TBPS binding observed in rat brain membranes. On the other hand, two component modulation was not observed in any recombinant receptor combination examined suggesting that the two component modulation by these compounds occurs only in native receptors.

Interestingly, the profiles of the 3-phenylethynyl substituted steroids 13 and 16 (Co 52791) differ somewhat from that for 3,5-P in these recombinant receptors. In 1, 2, 3 and 5-containing receptors, compounds 13 and 16 (Co 152791) have higher potency (IC₅₀ 1-12 nM) and generally lower efficacy (I_max 77-93%) than 3,5-P (IC₅₀ 20-41 nM; I_max ~100%) as predicted from rat brain membranes. In 4 and 6-containing receptor complexes, 3,5-P has low potency (IC₅₀ 1-2 µM) but high efficacy (I_max 76-85%), whereas compounds 13 and 16 (Co 152791) have higher potency (IC₅₀ 27-210 nM), but lower efficacy (I_max 16-40%). The low potency or efficacy of modulation of [³⁵S]TBPS binding at 4/632L GABA_A receptor complexes suggests that these neuroactive steroids display selectivity for 1, 2, 3 and 5-containing complexes. Alternatively, these modulators may have low activity at 3 relative to 2-containing complexes. Unfortunately, direct comparisons could not be made because membranes from 422L cells did not bind [³⁵S]TBPS and cells expressing 622L were not sufficiently viable.

Electrophysiological studies, although somewhat incomplete and inconsistent, do not support the finding that neuroactive steroids have low potency and/or very low efficacy at 4 and 6-containing complexes as indicated by [³⁵S]TBPS binding. Whereas 5THDOC had lower efficacy for potentiation of GABA-evoked currents in 632S than in 132S complexes expressed in HEK 293 cells (Zhu et al., 1996), 3,5-P produced higher maximal potentiation in oocytes expressing 612L complexes relative to complexes containing 1, 2, or 3 subunits (Lambert et al., 1996). In both expression systems, these neuroactive steroids had similar modulatory potency at the receptor combinations examined (Lambert et al., 1996; Zhu et al., 1996). In the case of alpha-4-containing complexes, 3,5-P (30 nM) produced similar levels of potentiation at 422L as that observed for 122L receptors expressed in oocytes (Whittemore et al., 1996).

In addition to differences observed between native and recombinant receptors, the apparent efficacy of the neuroactive steroids examined was assay-dependent, further complicating determination of their true modulatory efficacy. In the [³H]muscimol assay in brain membranes, the p-acetyl compound in the 5 series (18) was a two-component modulator, as has previously been shown for 3,5-P (1) (Carter et al., 1997; Goodnough and Hawkinson, 1995). Thus, compounds displaying two component enhancement in the [³H]muscimol assay are different from those exhibiting two component inhibition in the [³⁵S]TBPS assay. Large efficacy differences were observed in the [³H]muscimol assay, with the highest overall enhancement observed for the two component modulators 18 and 1 (overall E_max 81 and 53%, respectively). The one component [³H]muscimol modulators 13 and 16 (Co 152791) displayed reduced enhancement (E_max 23% and 42%, respectively). In contrast, all of the steroids examined displayed one component modulation of [³H]flunitrazepam binding with relatively small efficacy differences, although the p-chloro compound 13 had a relatively low maximal enhancement (E_max 54%) relative to the p-acetyl compounds 16 (Co 152791) and 18 (E_max 73%). These different profiles may be due in part to the differential influence of GABA on steroid modulation of the binding of these three radioligands.

Electrophysiological assays appear to be better in quantifying the relative efficacy of neuroactive steroids compared to allosteric binding assays. In Xenopus oocytes expressing 122L receptors, steroid potentiators of GABA-evoked currents can be grouped into high efficacy (compounds 1 and 18; FR 0.91-0.94), intermediate efficacy (compounds 12, 15 and 16; FR 0.65-0.77), low efficacy (compounds 7 and 13; FR 0.39-.46) and very low efficacy (compound 14; FR 0.14). Although these low efficacy compounds may be partial agonists, this possibility was not explored in antagonism experiments. In this regard, 3-hydroxy-3-trifluoromethyl-5-pregnan-20-one (Co 2-1970; Hawkinson et al., 1996) and 3,21-dihydroxy-5-pregnan-20-one (5THDOC; Xue et al., 1997) have previously been shown to be partial agonists for the neuroactive steroid site. The two component modulators in the [³⁵S]TBPS assay showed limited efficacy for potentiation of GABA-evoked currents and the potencies of the high affinity components in the [³⁵S]TBPS assay appear to correspond to their potencies in the electrophysiological assay. Binding assays are useful in predicting neuroactive steroid potency (Hawkinson et al., 1994b; Hogenkamp et al., 1997; Upasani et al., 1997), whereas electrophysiological measurements may be required to establish compound efficacy.

The p-hydroxy derivative 14 retained reasonable activity in both [³⁵S]TBPS and electrophysiological assays (IC₅₀ and EC₅₀ 110 nM), although had only micromolar activity in the [³H]flunitrazepam and [³H]muscimol assays and was inactive in vivo. Apparently, these latter assays did not detect the potent, low efficacy modulation observed in the [³⁵S]TBPS and electrophysiological assays. Although the lack of in vivo activity may be due to the low efficacy of this compound, other possibilities include poor bioavailability and/or metabolic lability of the p-hydroxy group.

A simplified pharmacophore model is presented that describes the key features of the interactions between neuroactive steroids and their binding site on GABA_A receptors (fig. 8). In this model, the steroid backbone occupies a hydrophobic region in the binding site and acts as a scaffold to maintain the requisite 3-hydroxy and facilitory 20-keto groups in appropriate positions to make hydrogen bonding interactions with a