Metabolism of steroidal anti-inflammatory antedrugs in vitro: methyl 3,20-dioxo-11beta,17alpha,21-trihydroxy-1,4-pregnadiene-16alpha-carboxylate; its 9alpha-fluorinated,and their 21-O-acyl derivatives

The in vitro hydrolysis rates of steroidal anti-inflammatory antedrugs, methyl 3,20-dioxo-11beta,17alpha,21-trihydroxy-1,4-pregnadiene-16alpha-carboxylate (P16CM), its 9alpha-fluorinated analogue (FP16CM), and their 21-O-acyl derivatives (P16CM-acetyl, FP16CM-acetyl, FP16CM-propionyl, FP16CM-valeryl, and FP16CM-pivalyl) were investigated in rat plasma. These steroids were synthesized based on the antedrug concept. P16CM and FP16CM were hydrolyzed to inactive steroid-16-carboxylate, with half-lives of 90.0 and 99.4 min, respectively. The metabolite was positively identified by NMR and elemental analysis. To determine the relative hydrolysis rate of the C21-O-acyl versus the C16-methoxycarbonyl group, P16CM- and FP16CM-21-O-acyl derivatives were also studied. The hydrolysis rates of all 21-O-acyl groups were much faster than that of the 16-methoxycarbonyl group. The half-lives of P16CM-acetyl, FP16CM-acetyl, FP16CM-valeryl, and FP16CM-propionyl were 6.3, 16.8, 23.2, and 18.4 min, respectively. On the other hand, FP16CM-pivalyl showed relatively slow hydrolysis rate (T(1/2): 59.7 min). These results clearly indicate that 21-O-acyl group is metabolized first to active compound, P16CM or FP16CM, followed by the hydrolysis of 16-methoxycarbonyl to corresponding inactive steroid-16-carboxylates as the major metabolites. Collectively, the results of the present study support the previous reports where decrease in adverse systemic effects without losing local anti-inflammatory activity was attributed to the hydrolysis of the active agents to inactive acidic metabolites in the systemic circulation. This study thus shows that the incorporation of a 16-methoxycarbonyl coupled with a 21-O-acyl moiety may be a fundamentally sound synthetic strategy in the development of locally active anti-inflammatory steroids having reduced systemic adverse activities.     

Effect of chirality at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives on antiinflammatory activity

In an effort to determine the C-20 chirality effect on the antiinflammatory activity of 17beta-glycolate esters, methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate and its 9alpha-fluoro analog, their acetonide and their carbonate derivatives were synthesized and evaluated. The agents were tested for their binding potency to the macrophage glucocorticoid receptor, and their effect on LPS-induced nitric oxide generation in RAW 264.7 cells. The acetonide derivatives showed the highest binding affinity while the triols and carbonates bound rather poorly to the receptors. With the exception of the triols, the alpha-isomer in each pair of the agents exhibited higher binding affinity to the receptor than its corresponding beta-isomer, clearly indicating that C-20 chirality has a significant effect on antiinflammatory activity. In addition, the alpha-isomers of the acetonides showed substantially higher binding affinity than the parent compound, prednisolone. In contrast to the high binding activity exhibited by some of the acetonides, all of the agents showed weak inhibitory effect on NO generation. Metabolic inactivation during assessment of NO inhibition may play a role in the divergence noted between receptor affinity and the measured biologic activity resulting from the binding.     

Synthesis and pharmacological evaluations of new steroidal anti-inflammatory antedrugs: 9alpha-Fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its derivatives

In continuing efforts to develop potent anti-inflammatory steroids without systemic adverse effects, methyl 9alpha-fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its 16-alkoxycarbonyl derivatives (FP16CE, FP16CP and FP16CB) were synthesized based on the antedrug concept. The steroids were evaluated for their pharmacological activities and adverse systemic effects. All steroidal antedrugs showed both binding affinity to the glucocorticoid receptor in liver cytosol and inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cell. These compounds also inhibited croton-oil-induced ear edema and showed no systemic effects such as thymus atrophy and suppression of corticosterone level after 5-day treatment. Among those compounds tested, FP16CM showed the highest activities in receptor binding, NO inhibition and ear edema, these activities were comparable to those of prednisolone. Hydrolysis study in plasma showed that FP16CB was hydrolyzed rapidly, with the half-live (T1/2) of 3.2 min and the half-lives of other compounds were between 16.9 and 29.4 min. These results support the antedrug concept, of which the decrease in systemic adverse effects is attributed to fast hydrolysis to inactive metabolite in the systemic circulation.     

New steroidal antiinflammatory antedrugs: Methyl 3,20-dioxo-9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-1,4-pregnadiene-16 alpha-carboxylate and its 21-O-acyl derivatives

In a continuing effort to increase local to systemic activity ratios of potent steroidal antiinflammatory antedrugs, a series of 21-O-acyl derivatives of methyl 3,20-dioxo-9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-1,4-pregnadiene-16 alpha-carboxylate, FP16CM, were synthesized. These derivatives were evaluated for antiinflammatory activity and their adverse effects in an acute and semi-chronic croton oil-induced ear edema bioassay. Following a single topical application in the croton oil-induced ear edema bioassay, treatment with all the compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID(50) values (nmol/ear resulting in a 50% reduction of edema) were calculated: prednisolone (Pred); 454, FP16CM; 255, 21-acetate (FP16CM-acetyl); 402, 21-propionate (FP16CM-propionyl); 474, 21-valerate (FP16CM-valeryl); 446 and 21-pivalate (FP16CM-pivalyl); 219 nmol. In a 5-day semi-chronic study at the equipotent doses, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights or plasma corticosterone levels unlike the parent compound Pred. The compounds were assessed for high-affinity glucocorticoid receptor binding and glucocorticoid-mediated inhibition of nitric oxide (NO) generation in an in vitro RAW 264.7 macrophage cell culture system. Binding affinities for cytosolic glucocorticoid receptors were Pred; 85, FP16CM-acetyl; 86, FP16CM-propionyl; 169, FP16CM-valeryl; 149, FP16CM-pivalyl; 126 nM, respectively. Concomitant potencies for inhibition of NO generation by macrophages stimulated with lipopolysaccharide were Pred; 159, FP16CM-acetyl; 377, FP16CM-propionyl; 405, FP16CM-valeryl; 344, FP16CM-pivalyl; 311 nM, respectively. Collectively, results of these investigations suggest that esterification of 21-OH with various anhydrides did not improve receptor binding, inhibition of NO generation and ear edema inhibition, however, serum corticosterone level and local over systemic activities (L/S) were markedly improved.     

梨头霉11α—羟基化制备16β—甲基—11α,17α21—三烃基孕甾—1, …

选育到一株对１６β－甲基－１７α,２１－二羟基孕甾－１,４＝－二烯－３,２０－二酮（Ⅱａ）１１α－羟基化活性强的梨头霉Ａ２８菌株,并发现底物２１－乙酰化（Ⅱｂ）可明显提高１１α－羟工 能力。在适宜的转化条件下,１１ｂ投料浓度０．５％,产物１６β－基１１α,１７α,２１－三羟基孕甾－１,４－二烯－３,２０－二酮（Ⅲ）收率为７３％,结构经波谱分析确认。     

Use of subcutaneous silastic implants for the study of 11 beta, 17 alpha, 21-trihydroxy-4-pregnene-3,20-dione(cortisol) metabolism

Capsules prepared from polydimethylsiloxane (PDS) tubing and containing tritium labeled 11β, 17α, 21-trihydroxy-4-pregnene-3, 20-dione (cortisol) were implanted subcutaneously in six mongrel dogs. $\text{In}$$\text{vivo}$ diffusion rates were found to decline at a single exponential rate after a period of rapid, initial change. Diffusion of the steroid from the capsule provided an exogenous marker which can be used for the continuous monitoring of metabolic clearance rates and production rates in the normal state and during metabolic insults. Comparison of the temporal aspects of plasma radioactive concentration and excretion of radioactivity suggests the existence of a tightly bound plasma component serving as a reservoir of cortisol.     

In vitro anti-inflammatory activities of new steroidal antedrugs: [16alpha,17alpha-d] Isoxazoline and [16alpha,17alpha-d]-3'-hydroxy-iminoformyl isoxazoline derivatives of prednisolone and 9alpha-fluoroprednisolone

A series of new anti-inflammatory steroidal antedrugs with C-16,17-isoxazoline ring system were synthesized and their pharmacological activities were evaluated. We reported earlier that these compounds are promising antedrugs based on the results of 5-day rat croton oil ear edema assay. In the present study, most of these compounds showed high binding affinities to the glucocorticoid receptor of liver cytosol. 21-acetyloxy-9alpha-fluoro-11beta-hydroxy-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d] isoxazoline (FP-ISO-21AC) and 11beta,21-dihydroxy-9alpha-fluoro-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d] isoxazoline (FP-ISO-21OH) were found 5.0-, 5.3-fold more potent than prednisolone, respectively. Inhibitory effects of the antedrugs on the nitric oxide (NO) production were assessed using LPS-stimulated RAW 264.7 murine macrophage cells. All these steroidal antedrugs exhibited concentration-dependent inhibition of NO production, but their relative potencies were lower than prednisolone. In vitro metabolism study in rat plasma showed that FP-ISO-21AC and 21-acetyloxy-9alpha-fluoro-11beta-hydroxy-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d]-3'-hydroxyiminoformyl isoxazoline (FP-OXIM-21AC) were hydrolyzed rapidly, with the half-lives of 2.1 and 4.2 min, respectively. The half-lives of FP-ISO-21OH and 11beta,21-dihydroxy-9alpha-fluoro-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d]-3'-hydroxyiminoformyl isoxazoline (FP-OXIM-21OH) were 92.2 and 110.2 min, respectively.     

Identification of 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation

This study describes the identification of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) as a major steroid product of the ovary of Atlantic croaker (Micropogonias undulatus) incubated in vitro. This is the first report which describes 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as a major product of teleost steroidogenic tissue. The steroid was identified by a variety of methods, including HPLC, TLC, GC-MS, UV absorbance, and reactions with specific enzymes. Full grown oocytes, prior to final oocyte maturation (FOM), accumulated only small amounts of 20 beta-S. However, a substantial increase in 20 beta-S production occurred at the time of FOM. These results suggest that 20 beta-S is a maturational steroid in this species.     

Induction of maturation of Atlantic croaker oocytes by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one in vitro: consideration of some biological and experimental variables

We characterized the in vitro control of germinal vesicle breakdown (GVBD) by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) in intact ovarian follicles of gonadotropin-primed Atlantic croaker. 20 beta-S-induced GVBD was determined in relation to ovarian (oocyte) morphology, duration of incubation, steroid metabolism, and interaction with other steroids. The rate of GVBD in vitro in the absence of exogenous steroid was positively correlated with initial stage of ovarian morphological development. Maximal responsiveness to 20 beta-S was seen in ovaries with oocytes showing the first signs of morphological maturation. Dose-response experiments with 20 beta-S and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) over a range of incubation times yielded similar results for both steroids, suggesting that conversion of 17 alpha,20 beta-P to 20 beta-S is not required for 17 alpha,20 beta-P-induced GVBD. The ED50 of these steroids markedly decreased with increasing incubation times. Comparisons between patterns of follicular transformation of various radiolabelled steroids to 20 beta-S and their respective activities (using unlabelled steroids) in the GVBD bioassay suggested that, in addition to 17 alpha,20 beta-P, progesterone has some intrinsic maturational activity. However, the maturational effects of 11-deoxycortisol and pregnenolone may be explained by their conversion to 20 beta-S. For the first time in any vertebrate, we showed that the proposed maturation-inducing steroid (20 beta-S) is not significantly transformed to any extractable, potentially active metabolite by intact, maturing ovarian follicles. These findings strongly suggest that 20 beta-S is the terminal product of the MIS biosynthetic pathway in Atlantic croaker ovaries. Estradiol had no acute effects on 20 beta-S-induced GVBD. However, testosterone decreased and cortisol augmented the maturational activity of 20 beta-S. Excess progesterone reduced the activity of a maximally effective dose of 20 beta-S, but pregnenolone was without effect. The effects of these steroids on 20 beta-S-induced GVBD are discussed in relation to their possible interactions with 20 beta-S at the MIS receptor level.