Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

Estrogen Enhances Performance of Female Rats during Acquisition of a Radial Arm Maze

Estrogen can influence the expression of behaviors not associated directly with reproduction, including learning and memory. However, the effects of estrogen on learning and memory in mammals are complex, dependent on a variety of factors. The radial arm maze is a traditional experimental task that takes advantage of the natural foraging strategy of rats and provides an appropriate measure for studying the effects of estrogen on working memory in this species. In the experiments reported here, ovariectomized rats were implanted subcutaneously with 5-mm Silastic capsules containing 25% estradiol diluted with cholesterol. Control females received 5-mm Silastic capsules containing 100% cholesterol. Results of three separate experiments demonstrated that estradiol administered by Silastic implants for 30 days prior to eight-arm radial maze training, during the 24 days of maze training, or both significantly improved working memory performance compared to females treated with cholesterol alone, as indicated by improved arm choice accuracy over trials. The positive effect of estradiol exposure prior to training suggests that estrogen may induce neuronal changes that persist beyond the period of exposure with functional consequences for behavior.     

Rate and composition of sweat fluid losses are unaltered by hypohydration during prolonged exercise in horses

Kingston, Janene K., Raymond J. Geor, and Laura JillMcCutcheon. Rate and composition of sweat fluid losses areunaltered by hypohydration during prolonged exercise in horses.J. Appl. Physiol. 83(4):1133-1143, 1997. Rate and ionic composition of sweat fluid losses and partitioning of evaporative heat loss into respiratory and cutaneous components were determined in six horses during three 15-km phases of exercise at ~40% of maximalO₂ uptake. Pattern of change insweat rate (SR) and composition was similar during each phase. SRincreased rapidly for the first 20 min of exercise but remained at~24-28ml · m² · min¹during the remainder of each phase. Similarly, the concentrations of Naand Cl in sweat increased until 30 min of exercise but were unchangedthereafter. Sweat osmolality and concentrations of Na and Cl werepositively correlated with SR. Sweat K concentration decreased duringexercise but was not correlated with SR. Fluid losses were 33.8 ± 1.5 liters, resulting in decreases of ~21% in plasma volume and~11% in total body water. The ~6% hypohydration was notassociated with an alteration in SR, sweat composition, or heatstorage. Respiratory and cutaneous evaporative heat loss represented~23 and 70%, respectively, of the total heat dissipated, and thepartitioning of heat loss was similar in each exercise phase. Weconclude that SR and the relative proportions of respiratory andcutaneous evaporative heat loss are unchanged in horses during prolonged low-intensity exercise despite moderate hypohydration.

     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography     

Genetics of Ia-like alloantigens in chickens and linkage withB major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens (Ia) and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the la antigens are determined by a single codominant locus and that theEa-B and Ia loci are on the same chromosome. Failure to detect a single recombinant between theEa-B and Ia loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The Ia genes are thus included in theB major histocompatibility complex of the chicken.     

Genetics of Ia-like alloantigens in chickens and linkage with B major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens ("Ia") and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the "Ia" antigens are determined by a single codominant locus and that the Ea-B and "Ia" loci are on the same chromosome. Failure to detect a single recombinant between the Ea-B and "Ia" loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The "Ia" genes are thus included in the B major histocompatibility complex of the chicken.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Localization of lysosomal antigens in activated T-lymphocytes

Summary The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel, electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme -glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that -glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density.Fractionation of homogenates from lymphocytes on Percoll gradients revealed that -glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of -glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.