ADME Principles in Small Molecule Drug Discovery and Development: An Industrial Perspective

1

Introduction

As new medicines are being developed to treat patients, it is important to fully characterize the properties of the new molecular entity (NME) to ensure adequate safety and efficacy. As part of the drug development process, much information is gained about the molecule itself including how the drug is absorbed, distributed, metabolized, and excreted. These “ADME” parameters are evaluated both in vitro and in vivo and are studied in both animal models and humans.

In this chapter, important ADME concepts will be discussed in the context of small molecule drug development, further divided into the discovery phase (defined as the time prior to the conduct of human clinical trials) and the development phase (defined as the time during clinical development, prior to submission and approval of new molecular entities). Prior to these phases, during the early discovery period, much work is done to generate hypotheses, identify, and validate targets, as well as develop a myriad of characterization assays to interrogate compound potency, selectivity, and drug properties in the lead optimization phase. However, this early phase is out of scope for this discussion. The general phases of drug development are depicted in Figure 3.1 .

From an ADME-centric view, the goal of the discovery phase is to optimize the structure–activity relationship (SAR) in terms of the balance of pharmacological and toxicological activities. Physical–chemical properties are optimized to support pharmacokinetics (PK)/exposure and metabolism, the presence of reactive intermediate alerts is evaluated, the overall clearance (Cl) pathways are determined at a high level to help predict Cl pathways in humans, and compounds are screened for drug–drug interaction (DDI) risks. At the end of the discovery phase, a single NME is chosen to advance to clinical evaluation in humans. In the development phase, the NME is more fully characterized to support clinical development and ultimately support registration and approval of the new drug by global regulatory agencies. In this chapter, general ADME principles will be reviewed. Additionally, the general types of ADME studies conducted in each of these phases, along with the phase-specific goals of these studies, will also be discussed.

2

General ADME Principles

As previously described, the role of ADME in drug discovery and development is to study the absorption, distribution, metabolism, and excretion of drugs. Both in vitro and in vivo studies are conducted to help guide the early selection of compounds, the appropriate nonclinical toxicology species, and form the basis for clinical studies through dose projections. ADME studies also provide a mechanistic understanding of the link between drug disposition and its effect on pharmacological activity and toxicity.

PK of a molecule describes its plasma concentration versus time profile and can provide a general understanding of its in vivo ADME properties. Two common approaches to understanding the PK of a drug are compartmental analysis and noncompartmental analysis (NCA). Compartmental analysis methods rely on assumptions about interconnected, kinetically homogenous body compartments such as blood and other tissues/organs. This is in comparison to NCA methods, which are less complex, model-independent, and rely almost exclusively on algebraic equations to estimate PK parameters, making this approach an attractive option and the most commonly used approach to determine the exposure of a drug in nonclinical studies. The following list represents some of the most common PK parameters derived from NCA profiles: area under the curve (AUC), maximum plasma concentration (C _max ), the time at which the maximum plasma concentration occurs (T _max ), elimination rate constant (k), elimination half-life (t _1/2 ), volume of distribution (Vd), Cl, and oral bioavailability (%F). Furthermore, these PK parameters can be affected by various factors. For example, age, gender, pregnancy status, genetics, and nutritional state (e.g., fed vs. fasted) are physiological factors that alter the PK of a drug. Different disease states may also affect the PK of a drug, such as hepatic disease, renal impairment, or diseases that cause inflammation to name a few ( ; ).

PK studies are the most useful when the pharmacological/toxicological response is closely related to the drug concentration in the central compartment. As the plasma drug concentration increases, drug levels will correlate with a subtherapeutic range, the optimal range where the benefits of the drug outweigh the risks/side effects, and eventually reach the toxic range where drug concentrations result in toxicity. Ideally, a drug should have a wide efficacious range in which there is large separation between plasma concentrations that are subtherapeutic and those that cause toxicity. Drugs that exhibit a small degree of separation between subtherapeutic and toxic plasma concentrations have a narrow therapeutic range. These concepts are depicted in Figure 3.2 .

From a relatively simple nonclinical in vivo study, discussed in more detail in the sections below, a substantial amount of PK information can be determined for a single molecule. Following a single oral (PO) and intravenous (IV) dose, in which blood samples are typically taken over the course of 24 h after dosing (postdose), a semilog plot of drug concentration versus time can be generated. Multiple PK parameters are derived from this curve including the AUC, C _max (for PO administration), and the concentration at time zero (for IV administration). The elimination rate, k, can be calculated from the slope of the log plot, which in turn can be used to calculate the t _1/2 (= 0.693/k). The t _1/2 is the amount of time it takes for one half of the drug amount to be cleared from the blood or plasma. In general, the assumption is made that it takes five half-lives for compound concentration to either achieve steady state or to be largely eliminated (e.g., 96.875% of the dose) from the body. In Figure 3.3 , from a simple in vivo study design where a single PO dose and a single IV dose were administered, followed by extensive postdose blood sampling in which drug concentrations were determined, a lot of insight about the PK properties of a molecule can be gained.

Bioavailability and first-pass metabolism are also important concepts. First-pass metabolism is defined as the loss of drug between the site of administration and systemic circulation on passing through tissues where elimination occurs. Thus, for PO administration, after the drug is absorbed through the intestinal lumen, a portion of the drug can be lost to metabolism by enzymes in the gut wall and the liver, before the remainder of the drug is ultimately delivered to the systemic circulation. The overall PO bioavailability is the percent of drug that successfully enters the systemic circulation and is compared to the IV exposure, since drug is administered directly into the systemic circulation. The overall bioavailability reflects the fraction of drug absorbed through the intestinal lumen, the fraction of drug that is delivered to the portal system, and the fraction of drug that successfully passes through the liver. This process is represented in Figure 3.4 .

These concepts will be discussed in more detail throughout this chapter. Relevant study designs for several types of ADME studies will also be discussed.

3

Discovery Overview

The goal of the discovery phase of drug development is to identify a series of compounds with pharmacological activity for a given target, characterize these molecules in a myriad of studies across multiple disciplines, and to ultimately decide on the single, best compound to advance into human clinical trials with the most optimal benefit:risk profile based on available data at a particular instance in time. As this can be a daunting task, narrowing the selection from different scaffolds of potentially 1000s of compounds to the ultimate candidate molecule, pharmaceutical companies have created benchmarks using in silico , in vitro , and in vivo databases for decision-making. While the nomenclature may differ across the industry, the general principles are the same. For the sake of discussion within, Candidate Identification (CI) is referred to as the identification of a compound possessing pharmacological activity with optimal drug-like properties to advance to pilot toxicological evaluations and Candidate Selection (CS) is referred to as the ultimate compound chosen to advance to human clinical trials, having demonstrated an acceptable toxicology profile in animals.

ADME properties are thoroughly characterized in compounds that are targeted toward CI or CS using both in vitro and in vivo study data. One of the end goals is to use the available data to translate and predict how the compound is likely to behave in humans while optimizing compounds for demonstration of pharmacology or evaluating toxicology with adequate exposures. Early in discovery, compounds may not advance toward CS due to multiple reasons, including but not limited to the following: lack of potency or lack of selectivity to a given target, inadequate exposure, cardiac toxicity or general toxicity associated with the target itself or with binding to other sites within the body, liabilities such as potential DDI risks by which more than one drug are competing for the same metabolizing enzymes and transporters involved in its clearance, high variability in the ADME parameters and plasma/tissue concentrations, lack of dose response, or an inability to reach target tissues (e.g., brain). Therefore, it is not surprising that there is high and rapid attrition of compounds during this early phase of development. For these reasons, early PK studies in rodents, in conjunction with physicochemical properties, in silico predictions, and in vitro data may be designed to provide a quick answer regarding performance. Multiple CIs may be declared as development teams are unsure of the toxicological outcomes in large animal species or in longer duration rodent toxicology studies, particularly with novel scaffolds that have previously not been studied. After CI, ADME data are collected to more fully characterize the drug to determine whether it is ready for human testing and set the stage for development planning. Thorough in vitro evaluations and PK studies with adequate number of animals, full time course of sampling, and cross-over study designs for such compounds are needed to evaluate clinical candidates.

In recent years, emphasis has also been placed on mechanistic and quantitative understanding of disposition and Cl pathways as they relate to excretion and metabolism/transporter involvement to inform human PK predictions. The data used to understand human ADME properties importantly feed into human PK predictions with uncertainties based on mechanistic understanding of clearance, volume, and absorption properties to inform clinical development. Preclinical evaluations use a combination of physicochemical properties, in vitro, and in vivo metabolism data, as well as PK and excretion data collected in various animal species. Often, scaling methods for metabolic Cl and Vd that are based on in vitro to in vivo extrapolations (IVIVEs) in animals are evaluated to provide confidence for human predicted parameters. In addition, in vivo performance in animals is used as a predictor of that in human to predict extent of renal and biliary excretion. Depending on the properties and performance of the CI molecule, definitive studies to characterize the PK properties of a molecule may include, but are not limited to, bioavailability with the final salt/formulation, dose linearity, and excretion studies in intact and/or bile cannulated animals. Considerations for various in vivo studies include routes of administration: bolus versus infusions, length of infusions to reduce distress to animals, sampling techniques such as dried blood spot (DBS) versus plasma, excreta collection and biomarkers, and tissue distribution into organs of interest ( ).

4

Absorption, Bioavailability, and PK/TK Studies

Early studies in rodents and nonrodents are utilized to understand the PO versus IV exposures (bioavailability), as well as to derive the previously described PK parameters in order to characterize the overall PK profile of the NME. The rate of absorption and bioavailability is compared across multiple species. The profiles are studied at both lower dose levels in the pharmacological range, as well as at higher dose levels to support the toxicology studies, which are generally designed to maximize compound exposure. Study design considerations are discussed below.

When designing nonclinical PK studies, doses should be low to avoid saturation of Cl pathways if possible, but high enough for drug concentrations to be detected in bioanalytical assays with typical sensitivity. It is good practice to ensure similar conditions for the PO and IV dose to ensure similar conditions for the ADME processes driving exposure in each case. For example, if the bioavailability is expected to be approximately 30%, the PO dose should be about threefold higher than the IV dose. If the bioavailability is expected to be close to 100%, then the same PO and IV dose should be used. Typical doses for bioavailability studies are 1 mg/kg IV infusion (administered via tail vein in rodents and femoral vein in larger nonclinical species) and 3 mg/kg, PO (administered via oral gavage). Plasma or blood samples are serially collected for long enough to recover most of the area under the concentration–time curve. To facilitate accurate determination of t _1/2 , and hence most other PK parameters, a trough concentration should be targeted that represents approximately two–three half-lives. Additionally, the use of at least four animals in each “arm” of the study allows for evaluation of outliers and characterization of interanimal variability. Cross-over studies with serial blood collections after PO and IV dosing are typically used, and where blood volumes are prohibitive in mice, microbleeding techniques coupled with DBS bioanalysis have been successfully developed and applied ( ; ). These methods have also been successfully employed in toxicology studies with no substantial impact on animal health. In fact, microbleeding techniques are sometimes preferred because the toxicokinetic (TK) analysis can be done in the same animals as those on study and are not in a separate “satellite” group of animals. Therefore, the relationship between exposure and effect can be assessed in an individual animal.

IV administration via infusion, rather than a bolus injection, tends to give better estimates of Vd and Cl particularly when the initial distribution phase is very fast and/or when the t _1/2 is short. Infusions can be short (e.g., few minutes) and do not need to reach steady state. Each has advantages and disadvantages, and several issues need to be considered as described in Table 3.1 .

IV dosing can be performed in fed or fasted states, unless the compound has a high extraction ratio in the liver, defined as the proportion of drug “extracted” from the blood or plasma via hepatic clearance as it passes through the liver. In this case, the increase in liver blood flow in the fed state can cause an increase in the extraction ratio. Using formulations that cause irritation or pain should be avoided. The IV formulation is preferably delivered as a solution and should have the correct isotonicity and pH. If high nonspecific binding is expected, one should test if the compound will bind to the tubing or the syringe and consider dose formulation bioanalysis to correct for the actual dose administered. For important CI/CS studies, it is highly recommended that dose formulation bioanalysis is utilized for the IV solutions in order to assure accurate calculations of PK parameters.

PO doses can be administered as solutions or suspensions while avoiding formulations that cause irritation or pain. Solutions are homogenous mixtures in which the particle size is adequately small such that the substance is completely dissolved in the matrix. This makes solutions ideal for dosing. While the particle size in suspensions is larger than those found in solutions and will thus eventually settle, the substance can still be evenly distributed by mechanical means such as mixing, also making suspensions amenable to PO dosing. Parenteral (nonoral) routes of administration tend to be more restrictive as a good understanding of compatibility with site of administration (e.g., subcutaneous) or solubility considerations with IV administration can restrict dose volumes. General dose volume recommendations for exposure studies as well as generally accepted and maximum volumes that can be utilized for each species with various routes of administration in exposure studies are listed in Table 3.2 ( ) . These guidelines may differ depending on the specific laboratory conducting the study. It is recommended that these volumes should be modified based on limitations of the final formulation chosen ( ). For example, while PO dose volumes listed in the table below are considered relevant for aqueous formulations, the drug amount may be limited when using solid dispersions or lipid-based formulations. With such approaches, it is important to consider the compatibility of the formulation, the load of excipients that fit into capsules, and the number of capsules that is practical for species selected.

PK studies using subcutaneous, topical, or inhalation routes are perhaps less frequently conducted as these intended routes of administration are less common than PO routes of administration, particularly for small molecule drug development. Nevertheless, study design considerations are worthy of discussion. For example, for topical/dermal administration, consideration of skin physiology in animals relative to humans is important for translation of PK to human; minipigs may be the best model for skin absorption ( ). The rate of absorption from subcutaneous administration may be slower than with other parenteral routes. Subcutaneous infusions can be administered with the use of an oily depot or osmotic mini pumps. Topically applying dosing material to skin that is unbroken and free of hair and avoiding application to sites that animals can reach during grooming is recommended. Transdermal dosing is typically accomplished by application of a patch impregnated with the drug of interest. The patch is applied in such a way as to avoid inadvertent ingestion or removal by the animal.

As compounds advance toward CI, toxicology assessment at high doses necessitates an understanding of dose–exposure relationships. These are in turn used to inform the design of future rodent and nonrodent toxicology studies. Ideally, exposure (AUC and/or C _max ) should increase in a linear manner with an increase in dose. However, limitations in absorption and potential saturation of Cl may result in lack of dose proportionality in exposure. Examples of dose-proportional, dose-limited, and superproportional increases in exposure are depicted in Figure 3.5 .

In addition to dose response, evaluation of the TK allows further understanding of species and sex differences in exposure, as well as the effect of repeated and multiple dosing. Comparing exposures following single and multiple doses gives insight into the extent of accumulation, as well as possible enzyme autoinduction. Autoinduction, where the drug itself increases the abundance or activity of the enzymes/metabolic pathways necessary for its own clearance, can have significant impact on multiple dose exposures and jeopardize the evaluation of the toxicity profile of a compound. In the example depicted in Figure 3.6 , following a single dose of 15, 50, or 150 mg/kg in rats, the AUC exposures increase with an increase in dose, as expected. However, after only 4 days of daily dosing, the exposures at each dose level are substantially reduced such that it is impossible to achieve exposures high enough to adequately assess the toxicity profile of the NME. Since autoinduction can be profound, understanding the effects as early as possible in drug discovery is beneficial and may result in the need for a different NME devoid of this liability to be evaluated. Ultimately, the exposure data obtained from the toxicology studies are utilized to determine the margin of safety, which compares the animal exposure data with predicted or observed human exposure data.

Occasionally, the delivery of compound to the systemic circulation for toxicology studies is challenging, due to considerable dose exposure subproportionality in the absence of target organ toxicity. If the limitation in absorption is due to poor permeability, options for enhancing exposures are limited. However, if dissolution rate limited absorption is driving the poor exposure, strategies for overcoming dose-limited absorption such as twice-daily dosing or investigation of methods for enhancing exposures through salt selection or formulation optimization are considered. The dose formulation, as well as the active pharmaceutical ingredient, is often not optimized and fully characterized in early discovery PK studies. Consequently, free bases and carboxylic acids are often administered in their native state, as an in situ salt or as a simple salt form. Prior to CI, salt screening is performed to identify form(s) with optimized solubility that meet criteria for GLP toxicology studies, as well as first-in-man studies, as the NME advances through development. It is not uncommon for the dose form of an NME to evolve from free base to an in situ salt to a salt that is close to the form of the drug that will ultimately be commercially available. In this case, bridging studies to assess the impact of formulation and any formulation changes will be conducted to evaluate their effect on drug exposure. For example, PK studies with salt forms of a drug are necessary to understand the impact of salt form on exposure. In early discovery, formulations used for PO dosing are often standard suspensions. The selection of alternative formulations may be informed by the physicochemical properties of the NME and routine PK studies with standard vehicles. As optimization of both the dose form and formulation occurs to ultimately support the more stringent and regulatory acceptable “good laboratory practice” toxicology studies in development, it may be necessary to conduct bridging studies to compare the PK/TK using prior formulations. The bridging study is conducted in the appropriate species at a dose level relevant to the subsequent study between “arms” that include the prior and subsequent salt form/formulation. The bridging study may be conducted in a parallel or cross-over design with due consideration to adequate washout between study arms. If multiple formulations need to be assessed, a Latin square study design can be utilized to control the variation from different formulations and different experimental runs. If three formulations are tested, with adequate washout periods between runs, each animal receives a different formulation in run one and then rotates to a different formulation in run two, and another rotation in run three.

Finally, in the lead up to CS, PK/TK studies and in silico simulations using commercial software such as Simcyp or GastroPlus can be used to optimize preclinical dose formulations and to model preclinical to clinical translation of absorption. While not the focus of this chapter, these can be powerful tools for predicting the absorption behavior of NMEs in humans.

5

Distribution