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PART B: ADME SYSTEMS AND METHODS
9
TECHNICAL CHALLENGES AND RECENT ADVANCES OF IMPLEMENTING COMPREHENSIVE ADMET TOOLS IN DRUG DISCOVERY
Jianling Wang and Leslie Bell
9.1 INTRODUCTION
Discovering and developing new drugs have become extremely challenging and are as risky as ever. Medicinal chemists have been considerate of the chemical space of new chemical entities (NCEs) not only from a ligand affinity and drug efficacy standpoint but also from an absorption, distribution, metabolism, elimination, and toxicity (ADMET) and druggability outlook. Relevant and comprehensive ADMET profiles have seldom been brought into critical evaluation for potential risk and decision-making processes so early in the discovery phase of drug candidates. ADMET has become one of the three indispensable legs of the drug discovery tripod, along with biology (for therapeutic target finding) and chemistry (for candidate optimization). As a result, a panel of in silico, in vitro, and in vivo tools is being developed and integrated into current drug discovery flowcharts from hit finding to lead identification and optimization, all the way to the nomination of development candidates. This enables project teams to identify the most optimal drug candidate(s) for an unmet medical need while, in parallel, mitigating the possible ADMET risks (Figure 9.1). A suite of comprehensive in vitro ADMET assays, as summarized in Table 9.1, is then crucial to assess the ADMET properties and druggability in parallel with preclinical efficacy evaluations. This profiling offers a broad look at the ADMET properties of NCEs and raises flags for those with potential ADMET risks, which can be addressed by medicinal chemists via additional structureāactivity relationships (SARs).
TABLE 9.1. Integrated Tools for In Silico, In Vitro, and In Vivo ADMET Profiling in Drug Discovery
ADMET profiling plays a critical role along with biology and medicinal chemistry. Therefore, it is extremely important that reliable, reproducible, and validated high-quality assays be developed and deployed in support of drug discovery and optimization. However, many technical challenges are present for in vitro ADMET profiling in early drug discovery. This chapter discusses various ADMET assays for support of discovery and the technical challenges involved in developing these assays, and the merits of implementing a tier-based profiling portfolio to meet diverse needs.
9.2 āAā IS THE FIRST PHYSIOLOGICAL BARRIER THAT A DRUG FACES
9.2.1 Solubility and Dissolution
Solubility is a key property affecting both in vitro and in vivo ADMET properties. Multiple solubility expressions (e.g., thermodynamic solubility, equilibrium solubility, kinetic or apparent solubility, and intrinsic solubility) are available but quite distinctive in terms of the data collection and their potential applications. In general, unless otherwise specified, solubility refers to a thermodynamic property (or equilibrium solubility). Thermodynamic solubility, also referred to as āsaturation shake-flaskā method, reflects not only the molecular interactions of NCEs in the solution phase but also the solid packing insights (polymorph character) (Yalkowsky, 1999; Avdeef, 2003; Wang et al., 2007; Zhou et al., 2007). This method, as the āgold standardā for solubility determination by industry and the U.S. Food and Drug Administration (FDA), is widely utilized to project the in vivo gastrointestinal (GI) absorption and bioavailability of NCEs together with in vitro permeability and metabolic clearance data. This approach, however, is time consuming and labor intensive, and thereby applicable for compounds in the late discovery and development phases, where compounds are well characterized for crystalline forms and a sufficient amount of material is available. Thermodynamic solubility is frequently employed to assess the in vivo exposure of test compounds as well as to understand the interplay among ADMET parameters in physiology. In contrast, kinetic or apparent solubility is measured for characterizing and qualifying in vitro biological and ADMET profiling assays (Wang et al., 2007). Intrinsic solubility is applied for assessing solubility of NCEs as neutral species and predicting their impact on permeability, which is believed to correlate better with the availability of neutral molecules in solution.
9.2.1.1 Thermodynamic or Equilibrium Solubility
The āshake-flaskā thermodynamic solubility assay typically starts with dry powders of the test compound, with a shaking at room temperature for 24 h. This is generally acceptable as most of discovery NCEs are prepared in amorphous format and typically present relatively fast dissolution curve to reach >90% of its maximal solubility within 24 h (Zhou et al., 2007). The obtained test solution or solution/precipitation mixture is then separated either by a filtration or centrifugation procedure. A calibration curve (in 0.1ā500 ĀµM) is derived (using high-performance liquid chromatography (HPLC)/ultraviolet (UV) at multiple wavelengths such as 210, 254, 280, and 360 nm) from standard solution of the compound in an organic solvent such as dimethyl sulfoxide (DMSO), acetonitrile, or methanol (100 ĀµM). The equilibrium solubility of the test compound is determined by quantifying the concentration of the filtrate (via the filtration procedure) or supernatant (via the centrifugation protocol) of the test sample against the calibration curve. Lately industry is shifting toward utility of liquid chromatography-mass spectrometry (LC-MS) for quantification, which is beneficial for low soluble compounds and also for those lacking an appropriate UV chromophore.
9.2.1.2 High-Throughput (HT) Kinetic Solubility
In contrast to thermodynamic solubility measured under the equilibrium condition between drug solid and saturated solution, kinetic solubility typically offers a snapshot of drugās dissolution at a specific time point (e.g., with 15ā30 min incubation) approaching their equilibrium. The typical turbidity-based HT kinetic solubility, initiated by Lipinski et al. (1997), involves nephelometric titration using either optical (Thermo [Thermo Scientific, Hudson, NH, USA]) or laser nephelometry (BMG [BMG LABTECH, Inc., Cary, NC, USA]) in 96-well microplates (384-well plates for laser nephelometry) to monitor the concentration where NCEs start precipitating out of solution (see Figure 9.2). While HT kinetic solubility provides a fast and cost-effective surrogate solubility filter for in vitro ADMET assays, its application to project the solubility impact on formulation and in vivo exposure is significantly limited by its poor correlation to the conventional solubility data derived under thermodynamically equilibrated condition (Zhou et al., 2007).
9.2.1.3 HT Equilibrium Solubility
For HT equilibrium solubility measurement (Figure 9.3), samples reconstituted (on a GeneVac [Genevac, Inc., Gardiner, NY, USA] HT-4X evaporator) from the DMSO stock solution of test compounds (e.g., 10 mM) in 96-well microplates, instead of dry powder, are used to start the assay, thereby evading the burden to dispatch solid powder and drastically reducing sample consumption. The reconstituted sample plates are filled with designated buffers and sealed by aluminum foil before loading onto shakers for agitated incubation (24ā72 h). After equilibrium, a centrifugation procedure is run and the supernatant liquid is transferred into an analytical plate for analysis by HPLC or other HT mass spectrometry (MS) analysis (Brown et al., 2010). The HT equilibrium solubility data collected using the microtiter plate format show reasonable predictivity to conventional thermodynamic solubility using the shake-flask approach using a set of Novartis (Novartis Institutes for Biomedical Research, Cambridge, MA, USA) discovery NCEs (Figure 9.3).
9.2.1.4 Intrinsic Solubility and Solubility pH Profile
Intrinsic solubility (SO) refers to the equilibrium solubility of NCEs in the neutral form. Conventionally, intrinsic solubility may be estimated by identifying the lowest value from the multiple equilibrium solubility data collected under a broad pH range. Nowadays, intrinsic solubility for ionizable NCEs may be determined using the potentiometric titration approach named pSOL introduced by pION Inc. (Woburn, MA) and Sirius Analytical Inc. (Beverly, MA), respectively (Avdeef 2003; Box et al., 2009).
The pSOL protocol, requiring ionization constant (pKa) data, is initiated by a partition coefficient (LogP)-based solubility estimate (Eq. 9.1) of solubility (Ran et al., 2001):
The titration preferably starts from the low soluble to the high soluble species of the test compound (i.e., from acidic to alkaline pH for an acid) in order to avoid supersaturation. Typically, the compound solid is first dissolved using strong acid (for a base) or strong base (for an acid) and then quickly reprecipitated in the titration solution, allowing for equilibrium to establish before the data collection starts. The intrinsic solubility and the solubility pH profile are calculated from the difference between the titration curves in the presence (causing precipitation) and absence (calculated using pKa) of an excess of the test compound (Wang et al., 2007).
9.2.1.5 Solubility and Dissolution in Biorelevant Media
It should be noted that thermodynamic or equilibrium solubility in aqueous buffers offers simplified determinants to flag solubility risks to oral absorption of NCEs. Practically, the composition of the intestine is far more complex than aqueous buffers. Therefore, the latest industry trend is to project solubility impact on...
