Comparison of the CYP3A selective inhibitors CYP3cide, clobetasol, and azamulin for their potential to distinguish CYP3A7 activity in the presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ~50% of the total CYP content in fetal and neonatal livers and is the predominant CYP involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-S (sulfate). The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low, but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We utilized three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and CYP inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that CP was not an MDI of CYP3A5, but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7 and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. Significance Statement These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect probe selection may have on CYP3A P450 inhibition studies.

HCV Antiviral Drugs Have the Potential to Adversely Perturb the Fetal-Maternal Communication Axis through Inhibition of CYP3A7 DHEA-S Oxidation.

The hepatitis C virus (HCV) poses a great risk to pregnant people and their developing fetus, yet no HCV antiviral treatment guidelines have been established. While there has been a substantial increase in the development of HCV antivirals, the effect they have on the developing fetus remains poorly defined. Many of these drugs are metabolized through the cytochrome P450 CYP3A pathway, which is mediated by cytochrome P450 3A7 (CYP3A7) in the fetus and developing infant. In this study, we sought to investigate the effect HCV antivirals have on CYP3A7 metabolism, as this CYP enzyme plays a vital role in proper fetal and neonatal development. Of the 13 HCV antivirals we investigated, 8 (∼62%) inhibited CYP3A7 metabolic activity by 50% or more at a concentration of 20 µM. Furthermore, paritaprevir, asunaprevir, simeprevir, danoprevir, and glecaprevir all had observed half-maximal inhibitory concentrations between the range of 10 and 20 µM, which is physiologically relevant in comparison with the Km of dehydroepiandrosterone-sulfate (DHEA-S) oxidation (reported to be between 5 and 20 µM). We also discovered that paritaprevir is a time-dependent inhibitor of CYP3A7, which shifts the IC50 ∼twofold from 11 µM to 5 µM. Upon further characterization, paritaprevir inactivates DHEA-S metabolism by CYP3A7, with KI and Kinact values of 4.66 µM and 0.00954 minute-1, respectively. Depending on treatment plan and off-label drug use, HCV treatment could adversely affect the fetal-maternal communication axis by blocking fetal CYP3A7 metabolism of important endogenous hormones. SIGNIFICANCE STATEMENT: The prevalence of HCV in pregnant people is estimated at between 1% and 8% of the global population, yet little to no information exists about the risk antiviral treatment poses to the developing fetus. There is a potential risk of drugs adversely affecting mother-fetal communication by inhibiting fetal hepatic CYP3A7, an integral enzyme for estriol production. We discovered that five HCV antivirals inhibited DHEA-S metabolism by CYP3A7, and paritaprevir inactivated the enzyme. Our studies demonstrate the potential threat these drugs pose to proper fetal development.

Per- and polyfluoroalkyl substances (PFAS) inhibit cytochrome P450 CYP3A7 through direct coordination to the heme iron and water displacement.

Per- and polyfluoroalkyl substances (PFAS) are a chemical class of highly stable, fluorinated compounds popular for use in a variety of consumer products. PFAS environmental persistence in drinking water contributes to acute exposure in humans and subsequent bioaccumulation of the compounds in the liver and lung tissue. Prenatal PFAS exposure has been associated with lowered birth weight, premature birth, and developmental defects including cranio-facial abnormalities. The cytochrome P450 enzyme CYP3A7 is responsible for facilitating a variety of reactions essential for proper fetal development in humans. In addition to drug metabolism, CYP3A7 is responsible for metabolizing endogenous ligands in the developing human liver, including the steroid precursor dehydroepiandrosterone 3-sulfate (DHEA-S), essential for estriol synthesis during pregnancy, along with the morphogen all-trans-retinoic acid (atRA). Interference with estriol synthesis during pregnancy, as well as atRA clearance, is known to result in similar effects associated with prenatal PFAS exposure including lowered birth weight, premature birth, and developmental defects. We hypothesized that PFAS compounds bind to the CYP3A7 enzyme resulting in its inhibition. We implemented a series of binding studies using spectral characterization of six PFAS compounds (PFOA, PFOS, GenX, PFNA, PFNS, and PFHxS), and evaluated their interactions with recombinant CYP3A7. In addition, we screened PFAS for their ability to inhibit CYP3A7 oxidative activity using dibenzylfluorescein, a fluorescent probe, and DHEA-S, an endogenous substrate of CYP3A7. Our data demonstrate that of the six PFAS tested, PFOA, PFOS, PFNA, and PFHxS bind to and inhibit CYP3A7.

Optimization of Biocompatibility for a Hydrophilic Biological Molecule Encapsulation System.

Despite considerable advances in recent years, challenges in delivery and storage of biological drugs persist and may delay or prohibit their clinical application. Though nanoparticle-based approaches for small molecule drug encapsulation are mature, encapsulation of proteins remains problematic due to destabilization of the protein. Reverse micelles composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) in low-viscosity alkanes have been shown to preserve the structure and stability of a wide range of biological macromolecules. Here, we present a first step on developing this system as a future platform for storage and delivery of biological drugs by replacing the non-biocompatible alkane solvent with solvents currently used in small molecule delivery systems. Using a novel screening approach, we performed a comprehensive evaluation of the 10MAG/LDAO system using two preparation methods across seven biocompatible solvents with analysis of toxicity and encapsulation efficiency for each solvent. By using an inexpensive hydrophilic small molecule to test a wide range of conditions, we identify optimal solvent properties for further development. We validate the predictions from this screen with preliminary protein encapsulation tests. The insight provided lays the foundation for further development of this system toward long-term room-temperature storage of biologics or toward water-in-oil-in-water biologic delivery systems.

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that is highly prevalent in individuals with cystic fibrosis (CF). A major problem in treating CF patients infected with P. aeruginosa is the development of antibiotic resistance. Therefore, the identification of novel P. aeruginosa antibiotic drug targets is of the utmost urgency. The genome of P. aeruginosa contains four putative cytochrome P450 enzymes (CYPs) of unknown function that have never before been characterized. Analogous to some of the CYPs from Mycobacterium tuberculosis, these P. aeruginosa CYPs may be important for growth and colonization of CF patients' lungs. In this study, we cloned, expressed, and characterized CYP168A1 from P. aeruginosa and identified it as a subterminal fatty acid hydroxylase. Spectral binding data and computational modeling of substrates and inhibitors suggest that CYP168A1 has a large, expansive active site and preferentially binds long chain fatty acids and large hydrophobic inhibitors. Furthermore, metabolic experiments confirm that the enzyme is capable of hydroxylating arachidonic acid, an important inflammatory signaling molecule present in abundance in the CF lung, to 19-hydroxyeicosatetraenoic acid (19-HETE; Km = 41 µM, Vmax = 220 pmol/min/nmol P450), a potent vasodilator, which may play a role in the pathogen's ability to colonize the lung. Additionally, we found that the in vitro metabolism of arachidonic acid is subject to substrate inhibition and is also inhibited by the presence of the antifungal agent ketoconazole. This study identifies a new metabolic pathway in this important human pathogen that may be of utility in treating P. aeruginosa infections.

Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening.

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.