Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

In vitro inhibition of carboxylesterase 1 by Kava (Piper methysticum) Kavalactones.

Kava refers to the extracts from the rhizome of the plant Piper methysticum which is of particular significance to various indigenous cultures in the South Pacific region. Kavalactones are the active constituents of kava products and are associated with sedative and anxiolytic effects. Kavalactones have been evaluated in vitro for their potential to alter the activity of various CYP450 enzymes but have undergone little systematic investigation as to their potential influence on esterases. This study investigated the inhibition effects of kava and its kavalactones on carboxylesterase 1 (CES1) in an in vitro system and established associated kinetic parameters. Kava and its kavalactones were found to produce reversible inhibition of CES1 to varying degrees. Kavain, dihydrokavain, and desmethoxyyangonin displayed competitive type inhibition, while methysticin, dihydromethysticin, and yangonin displayed a mixed competitive-noncompetitive type inhibition. The inhibition constants (Ki) values for each of the kavalactones were as follows: methysticin (35.2 µM), dihydromethysticin (68.2 µM), kavain (81.6 µM), dihydrokavain (105.3 µM), yangonin (24.9 µM), and desmethoxyyangonin (25.2 µM). With consideration to the in vitro Ki for each evaluated kavalactone as well as available clinical kavalactone concentrations in blood circulation, co-administration of CES1 substrate medications and kava products at the recommended daily dose is generally free of drug interaction concerns. However, uncertainty around kavalactone exposure in humans has been noted and a clinically relevant CES1 inhibition by kavain, dihydrokavain, and dihydromethysticin is indeed possible if the kavalactone consumption is higher than 1000 mg in the context of over-the-counter usage. Further clinical studies would be required to assess the possibility of clinically significant kava drug-drug interactions with CES1 substrate medications.

Natural Products as Modulators of CES1 Activity.

Carboxylesterase (CES) 1 is the predominant esterase expressed in the human liver and is capable of catalyzing the hydrolysis of a wide range of therapeutic agents, toxins, and endogenous compounds. Accumulating studies have demonstrated associations between the expression and activity of CES1 and the pharmacokinetics and/or pharmacodynamics of CES1 substrate medications (e.g., methylphenidate, clopidogrel, oseltamivir). Therefore, any perturbation of CES1 by coingested xenobiotics could potentially compromise treatment. Natural products are known to alter drug disposition by modulating cytochrome P450 and UDP-glucuronosyltransferase enzymes, but this issue is less thoroughly explored with CES1. We report the results of a systematic literature search and discuss natural products as potential modulators of CES1 activity. The majority of research reports reviewed were in vitro investigations that require further confirmation through clinical study. Cannabis products (Δ 9-tetrahydrocannabinol, cannabidiol, cannabinol); supplements from various plant sources containing naringenin, quercetin, luteolin, oleanolic acid, and asiatic acid; and certain traditional medicines (danshen and zhizhuwan) appear to pose the highest inhibition potential. In addition, ursolic acid, gambogic acid, and glycyrrhetic acid, if delivered intravenously, may attain high enough systemic concentrations to significantly inhibit CES1. The provision of a translational interpretation of in vitro assessments of natural product actions and interactions is limited by the dearth of basic pharmacokinetic data of the natural compounds exhibiting potent in vitro influences on CES1 activity. This is a major impediment to assigning even potential clinical significance. The modulatory effects on CES1 expression after chronic exposure to natural products warrants further investigation. SIGNIFICANCE STATEMENT: Modulation of CES1 activity by natural products may alter the course of treatment and clinical outcome. In this review, we have summarized the natural products that can potentially interact with CES1 substrate medications. We have also noted the limitations of existing reports and outlined challenges and future directions in this field.