Androgen Receptor Binding Affinity Research Articles

Novel Nonsteroidal Ligands with High Binding Affinity and Potent Functional Activity for the Androgen Receptor.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Pharmacology, pharmacokinetics, and metabolism of acetothiolutamide, a novel nonsteroidal agonist for the androgen receptor.

The present study characterized the in vitro androgen receptor (AR) binding affinity, in vitro and in vivo pharmacological activity, and in vivo pharmacokinetics and metabolism of acetothiolutamide, a nonsteroidal AR ligand. AR binding was determined by a competitive binding assay. In vitro AR agonist activity was examined by a cotransfection assay. Acetothiolutamide displayed high AR binding affinity (K(i) = 4.9 +/- 0.2 nM) and full agonist activity in the in vitro studies. Next, the androgenic, anabolic, and antiandrogenic activity of acetothiolutamide was evaluated in a castrated immature rat model. In this animal model, acetothiolutamide exhibited an overall negligible androgenic effect, but a statistically significant anabolic effect at high subcutaneous doses. Also, acetothiolutamide demonstrated a noticeable antiandrogenic effect in castrated rats supplemented with testosterone propionate. To understand the causes for the observed disparity between in vitro and in vivo activities, pharmacokinetics and metabolism of acetothiolutamide were studied in male Sprague-Dawley rats. Acetothiolutamide was rapidly cleared from rat plasma (clearance of about 45 ml/min/kg) in a concentration-independent manner after i.v. dosing. Acetothiolutamide was completely absorbed after subcutaneous administration, but not bioavailable after oral dose. In the metabolism study, the unchanged molecule and its metabolites in urine and fecal samples were detected by high-performance liquid chromatography-mass spectrometry. The structures of major metabolites were elucidated with liquid chromatography-tandem mass spectrometry. After i.v. administration, acetothiolutamide was excreted in urine and feces as unchanged drug and a variety of metabolites. Oxidation, hydrolysis, and sulfate conjugation of phase I metabolites were the major metabolic pathways of acetothiolutamide in rats. Overall, the high plasma clearance of acetothiolutamide, due to its extensive hepatic metabolism, likely contributed to its lack of androgenic activity in vivo.

Pharmacodynamics of selective androgen receptor modulators.

The present study aimed to identify selective androgen receptor modulators (SARMs) with in vivo pharmacological activity. We examined the in vitro and in vivo pharmacological activity of four chiral, nonsteroidal SARMs synthesized in our laboratories. In the in vitro assays, these compounds demonstrated moderate to high androgen receptor (AR) binding affinity, with K(i) values ranging from 4 to 37 nM, and three of the compounds efficaciously stimulated AR-mediated reporter gene expression. The compounds were then administered subcutaneously to castrated rats to appraise their in vivo pharmacological activity. Androgenic activity was evaluated by the ability of these compounds to maintain the weights of prostate and seminal vesicle, whereas levator ani muscle weight was used as a measure of anabolic activity. The maximal response (E(max)) and dose for half-maximal effect (ED(50)) were determined for each compound and compared with that observed for testosterone propionate (TP). Compounds S-1 and S-4 demonstrated in vivo androgenic and anabolic activity, whereas compounds S-2 and S-3 did not. The activities of S-1 and S-4 were tissue-selective in that both compounds stimulated the anabolic organs more than the androgenic organs. These two compounds were less potent and efficacious than TP in androgenic activity, but their anabolic activity was similar to or greater than that of TP. Neither S-1 nor S-4 caused significant luteinizing hormone or follicle stimulating hormone suppression at doses near the ED(50) value. Thus, compounds S-1 and S-4 were identified as SARMs with potent and tissue-selective in vivo pharmacological activity, and represent the first members of a new class of SARMs with selective anabolic effects.

Novel nonsteroidal ligands with high binding affinity and potent functional activity for the androgen receptor

While nonsteroidal androgen receptor (AR) antagonists have been known for many years, and used in the clinic for the treatment of hormone dependent prostate cancer, very little is known about nonsteroidal AR agonists. We designed and synthesized a series of chiral bicalutamide analogs, which bear electron-withdrawing groups (either a cyano or a nitro group at the 4-position and a trifluoromethyl group at the 3-position) in the aromatic A ring, and different substituents at the para position in the aromatic B ring of the parent molecule. We also synthesized a series of racemic bicalutamide analogs, which have a trifluoromethyl group instead of a methyl group at the R 2 position. We examined AR binding affinities of our compounds in a competitive binding assay with a radiolabeled high affinity AR ligand, 3H-mibolerone, and also measured their abilities to stimulate AR-mediated transcriptional activation in a cotransfection assay. These studies demonstrated that (1) nonsteroidal ligands can be structurally modified from known nonsteroidal antiandrogens to generate ligands capable of activating AR-mediated transcriptional activation. (2) R-isomer analogs exhibit higher AR binding affinity and more potent functional activity than their corresponding S-isomers in all cases. (3) All sulphide analogs show higher AR binding affinity and more potent functional activity than their corresponding sulphone analogs, with the exception of ligand R- 8. Those ligands which exhibit high AR binding affinity and potent functional activity for human AR may provide effective clinical uses for male fertility, male contraception, and hormone replacement therapy.

Androgen receptor binding affinity of pesticide "active" formulation ingredients. QSAR evaluation by COREPA method

The COREPA approach for identifying the COmmon REactivity PAttern of biologically similar chemicals was employed to upgrade the recently derived affinity pattern for high androgen receptor (AR) binding affinity. The training set consisted of 28 steroidal and nonsteroidal ligands whose AR binding affinity was determined in competitive binding assays (in terms of p K i ). The interatomic distances between nucleophilic sites and their charges providing distinct and non-overlapping integral patterns for active and inactive chemicals were assumed that it was related with the endpoint, which was under study. These stereoelectronic characteristics were used to predict p K i values of pesticide "active" formulation ingredients in an attempt to identify chemicals with potential AR binding affinity.

Development of radioiodinated nonsteroidal androgen receptor ligands for spect imaging of prostate cancer

The appropriate treatment strategy for prostate cancer is critically dependent on its accurate staging (1). Current screening techniques (CT, MRI, Ultrasound) fail to reliably distinguish clinically-localized disease from metastatic disease. A reliable noninvasive method for detection of prostate cancer metastases would thus be of immense clinical benefit for a) disease staging b) implementing the appropriate treatment strategy c) monitoring the effects of therapy. To address this need, many research groups have focused on the development of radiolabeled androgen receptor (AR) ligands for prostate cancer imaging, based on the observation that most prostate tumors and metastases express AR (2,3). The majority of these studies to date have focused on steroid-based ligands (2,4,5). The antiandrogens RU 59063 and RU 58841 (Table l), are prototypes of a new series of nonsteroidal androgen receptor ligands that display high AR affinity and selectivity (6,7). The ease of synthesis and structural modification of these compounds as compared to steroid-based AR ligands, prompted us to select them as leads for development of a SPECT AR radioligand. We have previously shown that substitution of the trifluoromethyl group of RU 59063 with iodine leads to an improvement in AR binding affinity (8). Accordingly, a series of iodinated analogs of RU 59063 and RU 58841 was synthesized for investigation as in vivo AR radioligands (Table 1). Compounds 1-4 were synthesized from 4-cyano-3-iodoaniline using previously reported methods (8). Radioiodination of compounds 1-4 with iodine-125 were achieved in 50-65% radiochemical yield by an ammonium sulfate catalyzed solid phase exchange procedure (Scheme 1). Radiolabeled products were purified either by elution through an anion exchange resin or

Synthesis of a carbon‐11 labeled nonsteroidal antiandrogen as a potential radioligand for pet imaging of prostate cancer

The hormone dependency of prostate cancer is well established and androgen receptor (AR) expression is frequently observed in primary prostate tumors and metastases (1). As a consequence, the development of radioligands that target the AR for prostate tumor imaging is an active area of research (2). The majority of these studies to date have focused on steroid-based ligands, including the naturally occurring androgens (testosterone, dihydrotestosterone) and synthetic steroids (mibolerone, metribolone) (3,4). The recent emergence of AR-selective, high-affinity, nonsteroidal antiandrogens such as RU 59063, (Table l), offers a usehl alternative approach towards AR radioligand development (5). Our goal in this study was to develop a suitable carbon-1 1 labeled nonsteroidal AR radioligand for PET imaging of prostate cancer. We recently showed that replacement of the trifluoromethyl group of RU 59063 with iodine (DTIB, Table 1) leads to a 3-fold enhancement in AR binding affinity (6). This observation led us to synthesize the N-methylated hydantoin and thiohydantoin derivatives (laJb), which were subsequently shown to retain high affinity towards AR (Table 1). Since the synthesis of [C]lJ by direct N-[C]methylation of its normethyl precursor is not feasible (due to preferential methylation on sulfur), the N-methyl derivative was selected for carbon-1 1 labeling. We report here the radiosynthesis of [ C ] b for evaluation as a AR radioligand for PET.

Synthesis and structure–activity studies of side-chain derivatized arylhydantoins for investigation as androgen receptor radioligands

A series of arylhydantoin derivatives modeled after the antiandrogen RU 58841 was generated to identify potential candidates for development as androgen receptor (AR) radioligands. Side-chain modified derivatives of RU 58841, suitable for labeling with either carbon-11 or radiohalogens (fluorine-18, iodine-123), were synthesized and tested for their AR binding affinities. The N-(iodopropenyl) derivative 13 ( K i=13 nM) is a potential candidate for development as a radioiodinated AR ligand.

Chiral nonsteroidal affinity ligands for the androgen receptor. 1. Bicalutamide analogues bearing electrophilic groups in the B aromatic ring.

A series of chiral analogues of bicalutamide bearing electrophilic groups (isothiocyanate, N-chloroacetyl, and N-bromoacetyl) on aromatic ring B of the parent molecule were synthesized. These compounds were designed as affinity ligands for the androgen receptor (AR). We prepared the (R)- and (S)-optical isomers of these compounds as pure enantiomers. The AR binding affinities of these compounds were measured in a competitive binding assay with the radiolabeled high-affinity AR ligand, [(3)H]mibolerone. In accordance with our previous results for the enantiomers of bicalutamide, we found that all (R)-isomers demonstrated much higher binding affinity to the AR as compared to their corresponding (S)-isomers. The para-substituted affinity ligands in ring B bound the AR with higher affinities than the corresponding meta-substituted analogues. Oxidation of thioester affinity ligands to their sulfonyl analogues for the para-substituted compounds decreased AR binding affinities and similar modification increased binding affinities for corresponding meta-analogues. The least potent para-substituted sulfonyl compounds had higher AR binding affinities than the most potent meta-substituted sulfonyl compounds. Overall, the para-substituted unoxidized molecules demonstrated the highest AR binding affinity. Subsequent research using AR exchange assays and Scatchard analyses showed that the isothiocyanate affinity ligands (R)-7, (R)-9, and (R)-10 reported herein are the first specific chemoaffinity ligands for the AR.

Inhibition of skin 5α-reductase by oral contraceptive progestins in vitro

Androgenic disorders of female skin such as hirsutism, acne and alopecia are etiologically caused by androgen excess. Skin 5α-reductase activity is a major factor influencing the manifestation of endogenous androgen excess in women. Oral contraceptives have proven useful for the treatment of androgen disorders of the skin. The mechanisms of action by which oral contraceptives correct skin androgen levels may include inhibition of 5α-reductase and androgen receptor activity. We investigated the inhibitory effect of oral contraceptive progestins and ethinyl estradiol on skin 5α-reductase and their influence on androgen receptor activity and affinity, using three different in vitro assay systems. It was shown that norgestimate blocked 5α-reductase activity with an IC50 value of 10 μM, followed by levonorgestrel (IC50 52 μM), dienogest (IC50 55 μM), cyproterone acetate (IC50 87 μM) and gestodene (IC50 98 μM). To determine the full androgenic potential of the progestins, androgen receptor binding affinities and activation potentials were determined. The progestins norgestimate and dienogest in particular combined 5α-reductase inhibition with minimal androgenic potential. These data demonstrate that the progestins norgestimate and dienogest might help in the treatment of clinical hyperandrogeny in women.

A Computationally Based Identification Algorithm for Estrogen Receptor Ligands: Part 1. Predicting hERalpha Binding Affinity

The common reactivity pattern (COREPA) approach is a 3-dimensional, quantitative structure activity relationship (3-D QSAR) technique that permits identification and quantification of specific global and local stereoelectronic characteristics associated with a chemical's biological activity. It goes beyond conventional 3-D QSAR approaches by incorporating dynamic chemical conformational flexibility in ligand-receptor interactions. The approach provides flexibility in screening chemical data sets in that it helps establish criteria for identifying false positives and false negatives, and is not dependent upon a predetermined and specified toxicophore or an alignment of conformers to a lead compound. The algorithm was recently used to screen chemical data sets for rat androgen receptor binding affinity. To further explore the potential application of the algorithm in establishing reactivity patterns for human estrogen receptor alpha (hERalpha) binding affinity, the stereoelectronic requirements associated with the binding affinity of 45 steroidal and nonsteroidal ligands to the receptor were defined. Reactivity patterns for relative hERalpha binding affinity (RBA; 17ss-estradiol = 100%) were established based on global nucleophilicity, interatomic distances between electronegative heteroatoms, and electron donor capability of heteroatoms. These reactivity patterns were used to establish descriptor profiles for identifying and ranking compounds with RBA of > 150%, 100-10%, 10-1%, and 1-0.1%. Increasing specificity of reactivity patterns was detected for ligand data sets with RBAs above 10%. Using the results of this analysis, an exploratory expert system was developed for use in ranking relative ER binding affinity potential for large chemical data sets.

TWO MUTATIONS IDENTIFIED IN THE ANDROGEN RECEPTOR OF THE NEW HUMAN PROSTATE CANCER CELL LINE MDA PCA 2A

We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth. Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations. These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP. Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.

Affinity labeling of the androgen receptor with nonsteroidal chemoaffinity ligands

We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [ 3H]mibolerone ([ 3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [ 3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites ( B max), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [ 3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate ( R-4), the p-thio-isothiocyanate ( R-6), and the m-isothiocyanate ( R-3), reduced exchangeable specific binding of [ 3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate ( S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [ 3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in B max at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.

Season and physiological parameters modulate salmonid leucocyte androgen receptor affinity and abundance

Immune responses of ectothermic animals are known to vary seasonally. A loss of sensitivity to cortisol and testosterone (both normally immunosuppressive) in leucocytes from juvenile salmonids during winter has been previously documented. In an attempt to understand how the salmonid immune system changes in the winter, the present study investigated changes in androgen receptor affinity and abundance in salmonid leucocytes brought on by season, and physiological states such as stress and sexual maturation. In sexually immature fish, leucocyte androgen receptor affinity dropped more than four-fold during winter. TheKdrose from less than 1nMin July to 4nMin January. The number of androgen receptors present also rose dramatically in January (from 50fmol mg−1protein to 400fmol mg−1protein). An 18h confinement stress caused a significant drop in leucocyte androgen receptor binding affinity in sexually immature fish (Kdrose to 6nMfrom 2·7nM) but had no effect on the number of receptors present. Sexual maturation of rainbow trout led to an increased number of leucocyte androgen receptors, similar to the number seen in immature fish sampled during the spawning season (300fmol mg−1protein), and leucocyte androgen receptor affinity decreased significantly after ovulation (Kd14nM, up from 4nMbefore ovulation).

PNU 157706, a novel dual type I and II5 α-reductase inhibitor

PNU 157706 is a novel dual inhibitor of 5 α-reductase (5 α-R), the enzyme responsible for the conversion of testosterone (T) to 5 α-dihydrotestosterone (DHT). Tested on a crude preparation of human or rat prostatic 5 α-R, PNU 157706 caused enzyme inhibition with ic 50 values of 20 and 34 nM, respectively, compared to the values of 32 and 58 nM shown by finasteride. Furthermore, PNU 157706 was highly potent in inhibiting human recombinant 5 α-R type I and II isozymes, showing ic 50 values of 3.9 and 1.8 nM and, therefore, it was several folds more potent than finasteride ( ic 50 values of 313 and 11.3 nM), particularly on the type I isozyme. PNU 157706 was shown to have no binding affinity for the rat prostate androgen receptor (RBA 0.009% that of DHT). In adult male rats, a single oral dose of 10 mg/kg of PNU 157706 caused a marked and longer lasting reduction of prostatic DHT than did finasteride (at 24 h inhibition by 89 and 47%, respectively). In prepubertal, T- or DHT-implanted castrated rats, PNU 157706, given orally for 7 days at the dose of 10 mg/kg/day, markedly reduced ventral prostate weight in T- but not in DHT-implanted animals, thus showing to be devoid of any anti-androgen activity. In adult rats treated orally for 28 days, PNU 157706 resulted markedly more potent (16-fold) than finasteride in reducing prostate weight, the ed 50 values being 0.12 and 1.9 mg/kg/day, respectively. These results indicate that PNU 157706 is a promising, potent inhibitor of both type II and I human 5 α-R with a very marked antiprostatic effect in the rat.

The role of ligand flexibility in predicting biological activity: Structure–activity relationships for aryl hydrocarbon, estrogen, and androgen receptor binding affinity

AbstractRecent studies indicate that the potency and agonist or antagonist activity of steroid hormone ligands are dependent, in part, on ligand–receptor binding affinity as well as the conformation of the ligand–receptor complex. The binding of ligands to hormone receptors is thought to involve interactions by which shapes of both the receptor and ligand are modified in the formation of the ligand–receptor complex. As a consequence, it is essential to explore the significance of ligand flexibility in the development of screening‐level structure–activity relationships. In this review, examples are provided of techniques used to generate and screen ligand conformers in the development of quantitative structure–activity relationships and active analogue search algorithms. The biological endpoint modeled was binding affinity of natural ligands and xenobiotics to the aryl hydrocarbon, estrogen, and androgen receptors. These approaches may be useful in future studies to evaluate relationships between ligand structure, receptor binding affinity, and, ultimately, transactivational events associated with receptor interactions with DNA response elements and associated proteins. An improved understanding of ligand–receptor interactions in the context of well‐defined effector systems will enhance the development of credible predictive models that can be used to screen large sets of chemicals for potential agonist or antagonistic activity.

Reduced expression and normal nucleotide sequence of androgen receptor gene coding and promoter regions in a family with partial androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is an X-linked disorder of XY males characterized by varying degrees of impaired masculinization. In many AIS cases, mutations have been identified in the coding sequence of the human androgen receptor (AR) gene which impair receptor function. Cases have also been reported in which reduced AR mRNA expression may contribute to AIS in association with AR gene mutations. The purpose of this study was to define the molecular basis of AIS in members of a family with clinical and laboratory features of partial androgen insensitivity (PAIS). Genital skin fibroblast (GSF) cultures were established from foreskin tissue for androgen receptor binding analysis. Genomic DNA was obtained from blood leucocytes for AR gene nucleotide sequence analysis. AR mRNA levels were determined in total RNA extracted from GSF cultures. Three related subjects with perineo-scrotal hypospadias, bifid scrotum and microphallus were studied. The family pedigree of these subjects suggested an X-linked pattern of inheritance. Hormone assay results were consistent with AIS. AR binding capacity and affinity were determined in three subjects and compared with unaffected male controls. The coding sequence and 1.4 kb of promoter region of the AR gene were amplified in overlapping fragments by polymerase chain reaction from genomic DNA and sequenced. GSF AR mRNA was measured by a competitive PCR technique. In the PAIS subjects, AR affinity in cultured GSF was normal (Kd = 0.24, 0.30, 0.48 vs 0.27 +/- 0.07 (SD) nmol/l) but binding capacity was reduced (Bmax = 0.31, 0.36, 0.27 vs 1.26 +/- 0.37 (SD) fmol/microgram DNA). Sequence analysis of the CAG repeat polymorphism within exon 1 of the AR gene showed that both mothers were heterozygous at this locus, and that the three subjects had inherited the same allele. GSF AR mRNA levels were reduced in all three patients compared with controls (0.25, 0.74 and 0.74 vs 3.8 +/- 0.9 (SEM), range 1.8-7.3 amol/microgram total RNA). The nucleotide sequences of the entire AR coding region and of a 1.4 kb segment containing the promoter region were normal. Members of this family with clinical and biochemical evidence of X-linked partial androgen insensitivity syndrome demonstrated normal androgen receptor binding affinity and androgen receptor gene nucleotide sequence but reduced androgen receptor binding capacity and reduced androgen receptor mRNA. These results suggest that partial androgen insensitivity syndrome in this family may be caused by reduced expression of a normal androgen receptor gene.

Three-Dimensional Quantitative Structure–Activity Relationships for Androgen Receptor Ligands

Comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure–activity relationship (3D-QSAR) paradigm, was used to examine androgen receptor-binding affinities of a series of structurally diverse natural, synthetic, and environmental chemicals of interest. The CoMFA/3D-QSAR model successfully illustrates that the overall steric and electrostatic properties of structurally diverse ligands for the androgen receptor are necessary and sufficient to describe the binding affinity. The practical utility of models of this type is demonstrated using parent compounds in the training set and known as well as putative biological metabolites as test set molecules. The ability of the model to accurately predict binding affinity of test set molecules suggests that structure-based 3D-QSAR models may be used to supplement the process of hazard identification. The application of 3D-QSAR models within a toxicological framework is, at present, limited by the quantity and quality of biological data for relevant biomarkers of toxicity.

Enantioselective binding of Casodex to the androgen receptor.

1. The biologic activity of androgens is mediated through the formation of a non-covalent androgen receptor (AR)-steroid complex. Casodex and other antiandrogens inhibit formation of this complex and thus negate the role of endogenous steroids in androgen-dependent growth of prostate. 2. Casodex is currently available as a racemic mixture. The goal of this investigation was to determine the in vitro AR binding affinities of the individual isomers of Casodex. 3. The (R) or (S) isomers of Casodex were synthesized according to the general synthetic scheme proposed by Tucker et al. for (S)-Casodex, using (R) or (S)-proline as the chiral matrix respectively. 4. ARs were isolated from rat ventral prostate tissue by homogenization and differential centrifugation, and used as the receptor source. 5. AR binding studies were conducted by incubation of the cytosol with 1 nM 3H-mibolerone (a synthetic androgen) and increasing concentrations of each isomer (10(-12) - 10(-5) M). Bound radioligand was quantitated by liquid scintillation counting. 6. Ki for (R)-Casodex (11.0 +/- 1.5 nM) was about 30 times lower than that of (S)-Casodex (364 +/- 10 nM). Ki for the racemate was 20.2 +/- 2.0 nM. 7. This study demonstrated that (R)-Casodex has a higher binding affinity than its stereoisomer and suggests that the antiandrogenic activity of racemic Casodex is almost completely due to the (R)-isomer.

Abnormal androgen receptor binding affinity in subjects with Kennedy's disease (spinal and bulbar muscular atrophy).

We have investigated androgen-binding properties of the androgen receptor (AR) in cultured suprapubic skin fibroblasts from six subjects with Kennedy's disease (X-linked spinal and bulbar muscular atrophy). Binding of the synthetic androgen methyltrienolone (R1881) was measured in a monolayer assay, and Scatchard analysis was performed to determine the total number of binding sites and the apparent binding affinity (Kd) of the AR for androgen. Five of the six subjects investigated had an abnormal apparent binding affinity, with Kd values ranging from 0.34-11.7 nmol/L, more than 2 SD from the mean of the normal range (0.19 +/- 0.06 nmol/L). In this group of six patients, there was a significant correlation between the AR Kd and the severity of testicular atrophy and gynecomastia. The number of CAG repeats in the expanded region of exon A of the AR gene was determined in all subjects from whom suprapubic skin fibroblasts were cultured and an additional 12 subjects with Kennedy's disease. In the total group of 18 subjects investigated, there was a trend for an increasing number of CAG repeats associated with decreasing age at onset of different symptoms; however, this correlation was not statistically significant. Thus, we report for the first time a quantitative abnormality of the AR apparent binding affinity in subjects with Kennedy's disease, which appears to be related to the severity of the symptoms of androgen insensitivity.