**1. Introduction**

The cytochrome P450 (CYP) superfamily is a group of Phase I mono-oxidase enzymes with broad substrate specificity that is responsible for the majority of xenobiotic metabolism [1]. CYP enzymes are also involved in vital endogenous pathways, including prostaglandin metabolism and steroid hormone biosynthesis [2]. Gene names are determined according to a standardized nomenclature. Using CYP3A4 as an example, "CYP" refers to the superfamily of cytochrome P450 genes, "3" refers to the family designation (<40% amino acid identity with other CYPs), "A" refers to the subfamily designation (40–55% amino acid identity with other CYPs in that family), and "4" refers to the specific gene within the subfamily with >55% sequence identity [3]. This enzyme superfamily is ubiquitous in nature—it is present in bacteria, fungi, plants, and animals—with varying expression patterns [4]. In fact, many drug metabolizing isoforms including CYP 1A, 2C, and 3A show significant interspecies differences in enzymatic activity while CYP 2E1 does not [5].

Hepatocytes express an abundance of drug metabolizing CYP enzymes and demonstrate the greatest capacity for Phase I xenobiotic biotransformation, followed by the small intestine and kidneys [6–8]. Of the 57 known human CYP enzymes, five CYP isoenzymes from CYP 1–3 families metabolize the majority of clinically used drugs—CYP3A4/5, CYP2D6, CYP2C9, CYP2C19, and CYP1A2 [9]. Although CYP 1–3 families predominantly aid in xenobiotic detoxification, CYP enzymes are also involved in the bioactivation of xenobiotics, resulting in the formation of toxic intermediates. CYPs 1B1, 1A1, and 2A6 are involved in the bioactivation pathways of cigarette smoke constituents [10,11], while CYP2E1 is involved in the metabolism of alcohol and acetaminophen [12]. These are associated with the generation of hepatotoxic or carcinogenic metabolites and promote reactive oxygen species (ROS) production in vitro and in vivo [13–15], which eventually causes organ damage and cancers.

CYP enzymes are generally upregulated by their own substrates/drugs, resulting in enhanced metabolism and suboptimal plasma concentrations of concurrent drugs [16]. Conversely, CYP inhibition by various drugs contributes to supratherapeutic drug levels and drug-induced toxicity, thus preventing CYPs from performing their protective role in detoxification [16]. In the case of prodrugs that require CYP-mediated conversion to their active form, the reverse is true. Prototypical pharmacologic CYP enzyme inducers and inhibitors that are commonly implicated in drug–drug interactions include rifampin (inducer) and azole antifungals (inhibitors), which may interact with multiple CYP isoenzymes to varying degrees [9]. Further, pharmacogenetic variations in CYP activity may result in reduced, absent or increased metabolic capacity. Drug metabolizing isoforms with functionally relevant polymorphisms include CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 [17,18]. Certain isoforms are more highly polymorphic than others [17] and are associated with clinically significant effects, such as toxicity or lack of therapeutic response [17]. Furthermore, while CYP enzymes are most abundantly expressed in the liver, they are also found in extrahepatic tissues throughout the body. Although the majority of extrahepatic CYPs are involved in endogenous pathways [2], drug metabolism also occurs outside the liver. The small intestine and kidneys are the primary sites of extrahepatic drug metabolizing CYP enzymes [6,7]; however, drug metabolizing CYPs are also expressed in the lungs, blood (monocytes, lymphocytes), brain, and heart [19–22]. Extrahepatic CYP enzymes contribute to cell-specific biotransformation, albeit to a lesser extent than hepatic CYPs. While extrahepatic CYP expression and metabolic capacity are not able to mediate total body clearance of xenobiotics, the enzymes may play a significant role in local tissue exposure and toxicity [19].

Recently, we provided the first evidence that functional CYP isoforms are packaged into extracellular vesicles (EVs) derived from human plasma of healthy volunteers, as well as in EVs derived from hepatic cell lines [23]. Extracellular vesicles (EVs) are nanosized, membrane-bound particles that are secreted from most cell types into biological fluids, namely plasma, and are taken up by other cells [24]. EV cargo includes a heterogeneous array of biomolecules, e.g., lipids, carbohydrates, cytokines, proteins, and nucleic acids—mRNAs, miRNAs, etc. [24–26]. Thus, EVs are thought to be critical in cell-to-cell signaling, protein transfer, and nucleic acid shuttling [24,27,28]. These characteristics suggest that EVs might be potential biomarkers, therapeutic targets, and drug-delivery systems [24].

It is important to note that exosomes are a subgroup of extracellular vesicles with a distinct biogenesis pathway [29]. Although much of the literature prior to 2018 refers to "exosomes", distinguishing exosomes from other EVs has proved challenging due to overlaps in size, composition, and marker proteins [30]. Therefore, in accordance with current ISEV guidelines [30], this review will exclusively refer to "EVs" even when published reports refer to "exosomes".

As drug metabolic capacity is limited in extrahepatic tissues, it is possible that CYP-containing plasma EVs are secreted from the liver, circulate via plasma, and are delivered to distant sites (e.g., brain cells), where they may aid in extrahepatic drug metabolism, detoxification, and may also influence toxicity at these sites (Figure 1). It is also possible that extrahepatic cells in the kidneys, lungs, blood, heart, and brain also secrete EVs that are pooled in the plasma and cerebral spinal fluid (CSF), making an "EV-depot". These EVs may then be delivered to other cells as needed and under specific

conditions, to perform biological functions. This review will provide an overview of the contribution of CYPs to drug metabolism in extrahepatic tissues. Since our primary goal is to discuss relevant drug-metabolizing CYP enzymes and their xenobiotic substrates, discussion pertaining to endogenous pathways is largely outside the scope of this review. Importantly, we will summarize the most recent literature pertaining to CYPs and EVs, the relative abundance of CYPs in human plasma-derived EVs, and potential implications of CYP-containing EVs in xenobiotic biotransformation/bioactivation. Further, we will discuss the potential role of EV CYP enzymes as biomarkers in various pathological conditions and xenobiotic exposure/drug use, as well as suggest novel therapeutic interventions.

**Figure 1.** Cytochrome P450 (CYP)-containing plasma extracellular vesicles (EVs)/exosomes are secreted from the liver and other peripheral organs, circulate via plasma, and are delivered to distant sites (e.g., brain cells), where they may aid in extrahepatic drug metabolism, detoxification, and may also influence toxicity at these sites. Similarly, secretion of EVs from extrahepatic cells, including brain cells are also likely to contain CYPs in addition to other biomolecules, which would also be circulated via plasma and delivered to other distant cells.
