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.     

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.     

Chirality effect of C-20 on metabolism of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives

Epimers at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oates, their 9alpha-fluoro analogs, their carbonate derivatives, and their acetonide derivatives were subjected to metabolism study in rat plasma and rat liver homogenate. These steroids were synthesized based on the antedrug concept. In rat plasma, the carboxy ester bonds of 20beta-triols and their acetonides were hydrolyzed with half-lives (T(1/2)) of between 5.7 and 7.7 min, while their corresponding alpha-epimers had longer half-lives of more than 2.5 h. A more profound difference was observed between the alpha- and beta-epimers of the carbonates, with the latter showing a T(1/2) less than 1 min (0.3 and 0.43 min for P20beta- and PF20beta-carbonate, respectively), while that of the former about 3 h (165 min for P20alpha-carbonate and 191 min for PF20alpha-carbonate). In rat liver homogenate, the triol and acetonide derivatives showed greater stability than they did in rat plasma, with T(1/2) for the beta-group in the range of 54-108 min, and T(1/2) for the alpha-group over 7 h. A significant difference in hydrolysis of the carbonate derivatives was also observed in rat liver homogenate. The half-lives of P20beta- and PF20beta-carbonate were 0.67 and 0.66 min, respectively, and the alpha-isomers showed the similar metabolic rate with other alpha-isomers. An esterase inhibitor effectively blocked the hydrolysis of the ester bond, indicating that this metabolism is an enzymatic reaction. Molecular modeling studies show that steric hindrance around the ester group of the alpha-epimers is much greater than that of their beta-counterparts, affording one explanation for the large difference in the metabolic hydrolysis rate; i.e. the carboxy ester bond of beta-isomer which is less hindered sterically than their counter alpha-isomers is hydrolyzed faster than that of alpha-isomers. In conclusion, this study confirms that chirality at C-20 had profound effects on metabolism and pharmacological profile of the steroid acid ester derivatives.     

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.     

New steroidal anti-inflammatory antedrugs: methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-3,20-dioxo-1, 4-pregnadiene-16alpha-carboxylate, methyl 21-desoxy-21-chloro-11beta-hydroxy-3,20-dioxo-1, 4-pregnadiene-16alpha-carboxylate, and their 9alpha-fluoro derivatives*

To a series of 21-desoxy-21-chloro-corticosteroids, a metabolically labile methoxycarbonyl group at C-16 has been incorporated. The approach is to synthesize locally active compounds that are hydrolyzed to inactive and readily excretable acid metabolites upon entry into the systemic circulation. Novel antedrugs were evaluated for anti-inflammatory activity and their adverse effects in an acute and semichronic croton oil-induced ear edema bioassay. Binding affinity to glucocorticoid receptors and induction of L-tyrosine-2-oxoglutarate aminotransferase were studied in hepatoma tissue culture cells. After 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: 540, 618, 454, and 346 nmol for prednisolone (P), methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-3,20-dioxo-1, 4-pregnadien-16alpha-carboxylate (PClCM), methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-9alpha-fl uoro-3, 20-dioxo-1,4-pregnadien-16alpha-carboxylate (FPClCM), and methyl 21-desoxy-21-chloro-9alpha-fluoro-11beta-hydroxy-3,20-dioxo- 1, 4-pregnadien-16alpha-carboxylate (FDPClCM), respectively. Results of the 5-day rat croton oil ear edema bioassay indicated that, in contrast with the parent compound P, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, or plasma corticosterone levels. The binding affinities for cytosolic hepatoma tissue culture glucocorticoid receptors were 33, 201, 471, 5304, and 3765 nM for P, PClCM, FPClCM, methyl 21-desoxy-21-chloro-11beta-hydroxy-3,20-dioxo-1, 4-pregnadien-16alpha-carboxylate (DPClCM), and FDPClCM, respectively. Collectively, results of these investigations suggest that modifications of P, which included replacement of 21-hydroxyl group with chlorine and addition of 16-methoxycarbonyl group with or without 17-hydroxyl moiety, retained the topical anti-inflammatory activity of the parent compound P without significant adverse systemic effects.