Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.

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Background The prevalence of antibiotic resistance is increasing, and multidrug-resistant Pseudomonas aeruginosa has been identified as a serious threat to human health. The production of β-lactamase is a key mechanism contributing to imipenem resistance in P. aeruginosa . Relebactam is a novel β-lactamase inhibitor, active against class A and C β-lactamases, that has been shown to restore imipenem susceptibility. In a series of studies, we assessed the interaction of relebactam with key mechanisms involved in carbapenem resistance in P. aeruginosa and to what extent relebactam might overcome imipenem non-susceptibility. Results Relebactam demonstrated no intrinsic antibacterial activity against P. aeruginosa , had no inoculum effect, and was not subject to efflux. Enzymology studies showed relebactam is a potent (overall inhibition constant: 27 nM), practically irreversible inhibitor of P. aeruginosa AmpC. Among P. aeruginosa clinical isolates from the SMART global surveillance program (2009, n = 993; 2011, n = 1702; 2015, n = 5953; 2016, n = 6165), imipenem susceptibility rates were 68.4% in 2009, 67.4% in 2011, 70.4% in 2015, and 67.3% in 2016. With the addition of 4 μg/mL relebactam, imipenem susceptibility rates increased to 87.6, 86.0, 91.7, and 89.8%, respectively. When all imipenem–non-susceptible isolates were pooled, the addition of 4 μg/mL relebactam reduced the mode imipenem minimum inhibitory concentration (MIC) 8-fold (from 16 μg/mL to 2 μg/mL) among all imipenem–non-susceptible isolates. Of 3747 imipenem–non-susceptible isolates that underwent molecular profiling, 1200 (32%) remained non-susceptible to the combination imipenem/relebactam (IMI/REL); 42% of these encoded class B metallo-β-lactamases, 11% encoded a class A GES enzyme, and no class D enzymes were detected. No relationship was observed between alleles of the chromosomally-encoded P. aeruginosa AmpC and IMI/REL MIC. Conclusions IMI/REL exhibited potential in the treatment of carbapenem-resistant P. aeruginosa infections, with the exception of isolates encoding class B, some GES alleles, and class D carbapenemases. Electronic supplementary material The online version of this article (10.1186/s12866-019-1522-7) contains supplementary material, which is available to authorized users.

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A four-step synthesis of verrucarin B (3) from verrucarol (2) is described. Coupling of 2 with in situ generated carboxylic acid 5 proceeded with moderate regioselectivity and afforded trichothecene monoester 14 in only 35% yield. It was necessary therefore to block the C(4)-hydroxyl group of verrucarol prior to the coupling reaction with 5. Selective protection of 2 was accomplished in 54% yield (81% corrected for recovered verrucarol after hydrolysis of the other reaction products) by treatment with [ [2-(trimethylsilyl)ethoxy] carbonyl]imidazole ( 16) and DBU in benzene. Esterification of 17 with in situ generated 5 in the presence of BOP-C1, EtgN, and catalytic DMAP as the condensing reagents afforded 18 in 64% yield, deprotection of which with KF in wet MegSO smoothly afforded seco acid 4 (81%). A number of methods for macrolactonization of 4 via mixed anhydride 21 were examined. Best results were obtained when 21 was treated with stoichiometric 4-pyrrolidinopyridine (4-PP) which afforded verrucarin B in 55% yield along with 34% of the (E,E)-muconate isomer 19. Exposure of 19 to I2 in benzene effected rapid isomerization to verrucarin B (60%) and a new isomer 20 (30%). In this manner the yield of 3 from seco acid 4 was 75% and 35% overall from verrucarol.

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