**3.2 CYP450 inhibition studies**

Once the metabolic pathways are identified, *in vitro* P450 inhibition and induction studies are conducted in order to predict clinically significant DDIs. Per the FDA's January 2020 guidance, "The sponsor should evaluate an investigational drug's potential to inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A in both a reversible manner (i.e., reversible inhibition) and time-dependent manner (i.e., time-dependent inhibition (TDI))" [14].

In general, each microsomal assay includes the preparation of both test compound and positive control samples in order to assess the metabolite formation specific to each CYP450 isoform. Experiments are typically prepared with both the test compound at increasing concentrations and a selective P450 probe substrate prepared at a predetermined concentration that will produce first-order kinetics. Following an incubation under physiologic conditions (pH and temperature), the experiment is quenched and resulting incubations are prepared for analysis employing LC–MS/MS. The data is then analyzed by comparing the metabolite formation of the test samples relative to the control samples and dose–response plots are created to determine the specific endpoints of the assay.

### **3.3 Screening DDI cocktail assay**

Concurrent with the early optimization stage P450 assessments, a cocktail of the test compound and substrates that exclusively bind to specific P450 isoforms is incubated with human liver microsomes across multiple concentrations of the test compound. Following incubation, P450 isoform specific metabolites are measured in each sample and compared to a control sample. Based on the metabolite abundance of the test samples relative to the control sample, as measured by liquid chromatography and tandem mass spectrometry (LC–MS/MS), a potential DDI liability can be identified early to inform future *in vitro* and potentially future *in vivo* investigations. Based on the results of this screening assay and the P450 inhibition and induction assays, definitive *in vitro* DDI screening may be warranted during the Drug Candidate Selection stage.

### **3.4 CYP450 induction studies**

CYP induction is typically measured *in vitro* using three separate lots of human hepatocytes. A metabolically active human hepatocyte cell line (e.g. HepaRG) can sometimes be substituted for one of the human hepatocyte lots. Three P450s are commonly measured for induction – CYP1A2, 2B6 and 3A4. If CYP3A4 induction is observed, it is recommended to assess CYP2C8, 2C9 and 2C19 induction in a separate experiment. For the induction experiment, human hepatocytes are incubated in a sandwich culture format prior to the experiment. The media is changed to serum-free prior to the start of the experiment in order to lower the basal expression of CYPs. On day one, test compounds are added and incubated with the cells for 48-72 hours, with daily replenishment of media and test compound. Induction can be assessed by either measuring mRNA levels or activity levels. For the mRNA method, the cells are harvested after 48 hours, mRNA is extracted and the expression level of CYPs is measured by qPCR. For the activity assay, specific substrates for each of the CYPs are added following 72 hours of incubation and the rate of metabolism is measured over a defined time point, e.g. 2 hours. A positive result is considered to be ≥2-fold increase over the vehicle control.

#### **3.5 Membrane drug transporters**

Membrane drug transporters play an important role in the uptake, distribution and elimination of both endogenous substances and drugs in the body. Because they help regulate the flux of many substances across cell membranes they are often implicated in detoxification mechanisms, multidrug resistance and clinical DDIs [15]. Drug transporters control the concentration of drug substrates available for P450 reactions by regulating drug disposition within the cell for both the parent drug and metabolites. Drug metabolizing enzymes are often coupled with transporters to efficiently modify the level of drug present in a specific tissue. A well-known example of this is the efficient removal of bilirubin from plasma by OATP1B1-mediated uptake into the liver, UGT1A1-mediated formation of monoand diglucuronide metabolites and subsequent elimination into the bile via the MRP2 transporter. In addition, the expression levels of drug metabolism enzymes are closely tied to transporters. For example, the nuclear receptor PXR regulates the expression of both drug metabolism enzymes such as CYP3A4 and CYP2C9 as well as several efflux drug transporters including P-gp and MRP2.

Membrane transporters relevant to drug development include two major superfamilies – ATB-binding cassette (ABC) and solute carrier (SLC). Members of the ABC superfamily utilize ATP hydrolysis to actively transport a solute across a cell membrane against a concentration gradient. The most relevant ABC transporters include P-glycoprotein (P-gp, MDR1), breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2). One of the primary functions of these transporters is to efflux small molecule substrates out of the cell in order to reduce cellular exposure and protect cells and organs against potentially harmful drugs or toxins. They are widely expressed in the epithelia of the intestine, liver and kidney, and in the endothelium of the blood–brain barrier and other blood-tissue barriers where they are localized on apical membranes.

In contrast, members of the SLC superfamily utilize solute exchange mechanisms to drive drug transport, including endogenous anions/cations or electrogenic mechanisms. SLC transporters include the major uptake transporters such as organic anion (OAT, OATP), organic cation (OCT) and the multidrug and toxin extrusion transporters (MATE1, MATE2-K). OATP1B1 and OATP1B3 are located

#### *Drug Metabolism in Drug Discovery and Preclinical Development DOI: http://dx.doi.org/10.5772/intechopen.97768*

primarily on the hepatocyte plasma-facing apical membrane, while OAT1, OAT3 and OCT2 are located on the basolateral membrane of the kidney proximal tubule. MATE1 and MATE2-K are located both in the proximal tubule and in the liver (canalicular membrane).

Several of the ATP and SLC transporters have been implicated in clinical DDIs and the FDA has focused on the following list as relevant for *in vitro* screening: P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2-K. *In vitro* evaluation of specific transporter interactions can employ a variety of tools including human and animal monolayer systems, transfected cell lines with single or multiple transporters over-expressed and membrane vesicles, along with a panel of substrates and inhibitors as control probes (**Table 1**).

#### *3.5.1 Monolayer systems*

The standard assay format for transporter function involves measuring the permeability of a test article through a confluent monolayer of cells grown on a permeable membrane. The cells and membrane are part of a transwell insert which fits into a normal 24-well plate, thus creating two media chambers. The upper reservoir is referred to as the apical (or A) chamber while the lower is the basolateral (or B) chamber. Addition of test article to each reservoir in separate wells, allows measurement of the apparent permeability in both directions (A to B, and B to A). The cell line used, therefore, must have the ability to form tight junctions between cells to prevent leakage of test article through the monolayer, and must express the transporters in a polarized fashion, enabling measurement of transport of substrates in two directions. Two cell lines are commonly used in this format – Caco-2 and MDCKII. The Transwell format is used not only to measure the permeability of the test article in a single direction, but also determine the efflux ratio, calculated as the ratio of permeability in both directions (B to A/A to B). If the efflux ratio is greater than 2, an interaction with a transporter is probable and needs further study.

Caco-2 cells were originally derived from a human colorectal adenocarcinoma. Although the cells originated from the human colon, they are widely used as a model of intestinal absorption and transporter activity [16]. When placed in culture, these cells undergo differentiation to an intestinal phenotype. The cells are characterized by a well-defined apical brush border, formation of tight junctions, and the endogenous expression of the majority of uptake and efflux transporters normally present in intestinal enterocytes [17]. Caco-2 cells are regarded as the most sophisticated *in vitro* tool for medium to high throughput modeling of drug transport across human plasma/tissue barriers.

A non-human alternative cell line that is also widely used in monolayer studies is MDCKII (Madin-Darby canine kidney strain II). A number of MDCKII cell lines


**Table 1.**

*Examples of* in vitro *systems to investigate transporter-mediated drug interactions.*

have been generated with single or double human transporters transfected into the cells. These modified animal cell lines enable the study of individual human transporters in the absence of competition from other human transporters. One caveat with MDCK cells is that there is an active form of canine P-gp present that is functionally similar to the human form and needs to be accounted for when transporter data is analyzed.

Other animal cell lines used for transporter studies include LLC-PK1 (porcine) and Chinese hamster ovary (CHO) cells.

#### *3.5.2 Membrane vesicle assays*

An alternative to cell-based transporter assays involves membrane vesicles, in which the assay is conducted with membrane preparations from baculovirusinfected insect cells or mammalian cells that have been transfected with the transporter of interest. When isolated, a small portion of the membrane vesicles end up in an inverted configuration ("inside-out") and are particularly useful for studying efflux transporters. Transporter activity (activation or inhibition) can be assessed by either measuring uptake of a substrate into the vesicles over time, or by measuring ATPase activity.

#### *3.5.3 Genetically modified cell lines*

Transporters recognize and interact with of a broad range of compounds, and each transporter has been characterized for their physicochemical preferences in substrates [18]; however, there remains a large overlapping area of substrate recognition between transporters. Some of the current ambiguity in assigning specificity toward a single transporter is due to the use of different cellular systems to define the interaction. Small molecule inhibitors are commonly used to define transporter interactions, but inhibitors often show overlapping interactions between transporters [19]. Novel test systems that avoid the use of small molecule inhibitors may be able to improve our ability to unambiguously identify specific substrate interactions Two approaches have recently been used to address this situation. Knockdown of transporter gene expression can be accomplished using small inhibitory RNA (siRNA). Alternatively, complete gene knockout can be accomplished using zinc finger nucleases (ZFNs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). For example, several individual or dual knockout cell lines were recently developed in the C2BBe1 cell line (a subclone of Caco-2) and are commercially available [20]. In addition, the canine P-gp transporter in MDCK cells has recently been knocked out.

### **4. Drug candidate selection**

Following the investigations of the Lead Optimization stage, the pharmaceutical organization should have weaned their list of promising drug candidates to less than five entities. From a drug metabolism perspective, there are three major investigations that occur: (1) ADME profiling in rodents and nonrodent species; (2) definitive *in vitro* DDI screening and potentially *in vivo* confirmatory investigations; and (3) metabolite profiling in the identified toxicology species. By the end of the Drug Candidate Selection stage, the pharmaceutical organization should have all the information they need to support their Investigational New Drug application (IND) or Exploratory IND application.
