An overall comparison of small molecules and large biologics in ADME testing

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Biologics mainly monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as new therapeutics are becoming increasingly important biotherapeutics. This review is intended to provide an overall comparison between small molecules (SMs) and biologics or large molecules (LMs) concerning drug metabolism and pharmacokinetic (DMPK) or associated with absorption, distribution, metabolism and elimination ADME testing from pharmaceutical industry drug discovery and development points of view, which will help design and conduct relevant ADME testing for biologics such as mAbs and ADCs. Recent advancements in the ADME for testing biologics and related bioanalytical methods are discussed with an emphasis on ADC drug development as an example to understand its complexity and challenges from extensive in vitro characterization to in vivo animal PK studies. General non-clinical safety evaluations of biologics in particular for ADC drugs are outlined including drug-drug interaction (DDI) and metabolite/catabolite assessments. Regulatory guidance on the ADME testing and safety evaluations including immunogenicity as well as bioanalytical considerations are addressed for LMs. In addition, the preclinical and human PK data of two marked ADC drugs as examples are briefly discussed with regard to PK considerations and PK/PD perspectives.

1. Introduction Biologics or large molecules (LMs) primarily monoclonal antibodies (mAbs) and antibody-drug conjugate (ADC) currently represent main stream therapeutics and continue to grow in number of new approvals and targets recently. A majority of these biotherapeutics are mAbs or mAb-derived proteins, which are subject to transformation mechanisms such as deamidation, oxidation, and isomerization. These processes usually result in relatively small structural changes in the parent drugs. Such small structural changes may be difficult for a conventional immunoassay to differentiate, but they can still affect biological activity, PK and immunogenicity of a therapeutic protein. Whereas ADCs are an emerging class of biotherapeutics that combines the target specificity of an antibody with the potent small-molecule drugs or cytotoxins, which can selectively deliver a potent cytotoxic drug to tumor cells via tumor-specific and/or over-expressed antigens with more favorable therapeutic window. This new type of antibody-drug conjugate or antibody-linker-drug currently shows its great promising therapeutic options, which led to the recent FDA approvals of ADCETRIS (brentuximab vedotin, SGN-35) and KADCYLA (ado-trastuzumab emtansine, T-DM1) for the treatment of Hodgkin’s lymphoma (HL) and anaplastic large-cell lymphomas (ALCL), and HER2-positive metastatic breast cancer, respectively, as well as a rich clinical pipeline of potential new cancer therapies. Throughout several decades of advancements and evolutions, ADMET profiling of small molecule (SM) has becomes a standardized paradigm in drug discovery and development in terms of in vitro screening, in vivo animal studies, LC-MS/MS based bioanalysis as well as regulatory considerations including DDI and drug metabolite safety testing etc. However, ADME testing of LMs lags behind that of SMs due to the complex nature of the biological molecules and also lack of appropriate tools to study drug exposure, biotransformation and target engagement in the vascular and tissue spaces. ADME of LMs or biologics are still based on the lessons learned from the SMs and the tools that have applied to SM drugs. In general, there is a similar high level PK/PD relation concept between the SMs and LMs, although they have different ADME mechanisms and underlying ADMET determinants at different stages. Characterizing the absorption, distribution, metabolism, and excretion of these LM drugs (mAbs and ADCs) in preclinical animal models can better predict their efficacy and tolerability in clinic. Accordingly, it is necessary to understand general characteristics and the difference between SMs and LMs in order to apply relevant approaches for ADME testing and safety evaluation of LMs as reviewed in a number of recent publications . In a previous review, what ADME tests should be conducted for small molecule drugs for preclinical studies was highlighted. This review will provide an overall comparison between SM and LM properties with a particular focus on ADC’s characteristics to gain a better understanding of in vitro and in vivo ADME testing as well as toxicity evaluations. The in-depth information of ADME will also be valuable for the designing of novel mAb constructs and next generation of ADCs with desirable PK profile and safety window.

2. General differences between SMs and LMs (mAb and ADC) in ADME testing Table 1 summarizes general differences between SMs and LMs with an overall comparison of mAb and ADC. As highlighted in Table 1, due to the nature of various characteristics between SMs and LMs, the focus on ADME studies of LMs is thus different from SMs in particular for drug metabolite safety testing and DDI evaluations. In these aspects, in vitro models and in vivo studies and related bioanalysis including transporter studies and safety evaluation and high-throughput screening approaches for SMs have well been established across pharmaceutical industry. Typical in vitro ADME tests for SMs are metabolic stability by liver microsmoes or/and hepatocytes and passive permeability on cell-line models based on Caco-2 or MDCK assays, which are commonly utilized to predict in vivo clearance and absorption or bioavailability as well as potential drug-drug interaction (DDI) evaluations and metabolism pathway studies. In particular, a common consensus has been reached on drug metabolite testing across various species from in vitro models to in vivo studies following regulatory guidance, which clearly suggests critical criteria for decision-tree making in assessing key drug metabolite safety in human. Similarly, cytochrome P450 (CYP) enzymes based DDI as well as transporter-mediated DDI of SMs are also defined and guided on the basis of many years industrious practice of drug discovery and development. On the contrary, ADME testing of biologics (mAbs and ADCs) can be rather diverse as highlighted in Table 1 and Figure 1. Biodistribution of mAbs and ADCs are usually similar , but both have much lower Vd than that of a typical SM drug as the structure of mAbs or ADCs is dominated by the antibody backbone with initial distribution limited to the vascular space or plasma, not organ tissues. While the drug metabolism of mAbs is more complex due to receptor binding target-mediated drug disposition (TMDD), FcRn binding and lysosomal degradation, tissue protease, immune response antibody mediated metabolism etc in addition to other metabolic or metabolic-like biotransformation such modifications as deamidation, oxidation, isomerization, disulfide bond reduction or shuffling and proteolytic or glycolytic hydrolysis (ref. therein). The metabolism /catabolism of ADCs can be more complicated than mAbs due to a cytotoxic drug linked to the antibody via a linker. Typically, the circulating unconjugated drug after the ADC administration has metabolic properties of small-molecule compounds. Hence, drug metabolite and DDI should be concerned for ADC drugs, but maybe not for mAbs wherein the DDI risk is presumably low or not as prominent as small molecules. Once small-molecule drugs released from the ADCs, they may be metabolized by CYP enzymes and thus subject to potential DDI from CYP enzyme inhibitors or inducers due to payload/small molecule component. Furthermore, one or more active small-molecule drugs may be released from an ADC in vivo by additional catabolism mechanism. Accordingly, both unconjugated smallmolecule drug and released catabolites small molecules may be considered for metabolite safety and DDI potential evaluations for ADCs. This is considered to be a major differentiation of ADCs from mAbs regarding ADME testing as the degraded small molecules of mAbs are often amino acids, small peptides or small carbohydrates that are readily eliminated by renal excretion or return to the nutrient pool without biological effects or safety consideration. However, it should be noted that examples of therapeutic protein (TP) and small-molecule drug (D) interactions in clinical studies were observed although the changes in exposures (AUC, Cmax) have not been as remarkable as with small-molecule drugs, and the types of study designs used to assess TP and D interactions were thereby outlined in CDER’s special subject on therapeutic protein–drug interactions and implications for drug development. On the other hand, for LMs at early stage, more extensive in vitro characterizations are required as an example of screening cascade depicted in Figure 1. These typical studies include antibody primary structure by chemical sequencing (Edman degradation) as well as peptide mapping by means of ESI-MS and MALDIMS ; higher order structure by RP-HPLC–ESI–MS, Ellman’s assay CD, FTIR, hydrogen deuterium exchange (HDX)-MS and X-ray; post-translational modifications (PTM) and charge variants by MSion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), capillary electrophoresis (CE) and isoelectric focusing (IEF); glycan profile and variants and size heterogeneity by HPLC-fluorescence, size-exclusion chromatography (SEC), native gel, capillary electrophoresis, RP-HPLC-MS and native intact MS, as well as solubility measurement by ultrafiltration or PEG-induced precipitation methods. Moreover, the immunogenicity should be evaluated for both antibody and ADCs, but unnecessary for SMs. Unlike the antibody, additional in vitro studies of ADC include drug antibody ratio (DAR) characterizations, conjugate site analysis, linker stability and toxin evaluations as highlighted in Figure 1 may be required. Furthermore, absorption needs to be understood as well if LMs are targeting for an SC administration. Despite distinct characteristics and differences of in vitro testing between antibody and ADCs and SMs as shown in Table 1 and Figure 1, similar principle of in vivo PK studies including mass balance and elimination as well as safety evaluations for SMs are generally applicable to both antibody and ADCs. However, different bioanalytical methods have to be applied for antibody and ADCs, which will be discussed in more details in bioanalytical section.