On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

Bioactivation and reactivity research advances-2023 year in review.

Advances in the field of bioactivation have significantly contributed to our understanding and prediction of drug-induced liver injury (DILI). It has been established that many adverse drug reactions, including DILI, are associated with the formation and reactivity of metabolites. Modern methods allow us to detect and characterize these reactive metabolites in earlier stages of drug development, which helps anticipate and circumvent the potential for DILI. Improved in silico models and experimental techniques that better reflect in vivo environments are enhancing predictive capabilities for DILI risk. Further, studies on the mechanisms of bioactivation, including enzyme interactions and the role of individual genetic differences, have provided valuable insights for drug optimizations. Cumulatively, this progress is continually refining our approaches to drug safety evaluation and personalized medicine.Shuai Wang and Cyrus Khojasteh, on behalf of the authors.

Mass Balance, Metabolic Pathways, Absolute Bioavailability, And Pharmacokinetics of Giredestrant In Healthy Subjects.

Giredestrant is a potent and selective small molecule estrogen receptor degrader. The objectives of this study were to assess the absolute bioavailability (aBA) of giredestrant and to determine the mass balance, routes of elimination and metabolite profile of [14C]giredestrant. In Part 1 (mass balance), a single 30.8 mg oral dose of [14C]giredestrant (105 µCi) was administered to women of non-childbearing potential (WNCBP, n = 6). The mean recovery of total radioactivity (TR) in excreta was 77.0%, with 68.0% of the dose excreted in feces and 9.04% excreted in urine over a 42-day sample collection period. The majority of the circulating radioactivity (56.8%) in plasma was associated with giredestrant. Giredestrant was extensively metabolized with giredestrant representing only 20.0% and 1.90% of the dose in feces and urine, respectively. All metabolites in feces resulted from oxidative metabolism and represented 44.7% of the dose. In Part 2 (absolute bioavailability, aBA), WNCBP (n = 10) received an oral (30 mg capsule) or intravenous (30 mg solution) dose of giredestrant. The aBA of giredestrant after oral administration was 58.7%. Following the intravenous dose, giredestrant had a plasma clearance and volume of distribution of 5.31 L/h and 266 L, respectively. In summary, giredestrant was well tolerated, rapidly absorbed, and showed moderate oral bioavailability with low recovery of the dose as parent drug in excreta. Oxidative metabolism followed by excretion in feces was identified as the major route of elimination of giredestrant. Significance Statement This study provides definitive insight into the absorption, distribution, metabolism, and excretion of giredestrant in humans. The results show that giredestrant exhibits low clearance, high volume of distribution, and moderate oral bioavailability in humans. In addition, the data show that oxidative metabolism followed by excretion in feces is the primary elimination route of giredestrant in humans. These results will be used to further inform the clinical development of giredestrant.

Industry Perspective on the Pharmacokinetic and Absorption, Distribution, Metabolism, and Excretion Characterization of Heterobifunctional Protein Degraders.

Targeted protein degraders (TPDs), specifically the bifunctional protein degraders discussed in this manuscript, consist of two linked ligands for a protein of interest and an E3 ligase, resulting in molecules that largely violate accepted physicochemical limits (e.g., Lipinski's Rule of Five) for oral bioavailability. In 2021, the IQ Consortium Degrader DMPK/ADME Working Group undertook a survey of 18 IQ member and nonmember companies working on degraders to understand whether the characterization and optimization of these molecules were different from any other beyond the Rule of Five (bRo5) compounds. Additionally, the working group sought to identify pharmacokinetic (PK)/absorption, distribution, metabolism, and excretion (ADME) areas in need of further evaluation and where additional tools could aid in more rapid advancement of TPDs to patients. The survey revealed that although TPDs reside in a challenging bRo5 physicochemical space, most respondents focus their efforts on oral delivery. Physicochemical properties required for oral bioavailability were generally consistent across the companies surveyed. Many of the member companies used modified assays to address challenging degrader properties (e.g., solubility, nonspecific binding), but only half indicated that they modified their drug discovery workflows. The survey also suggested the need for further scientific investigation in the areas of central nervous system penetration, active transport, renal elimination, lymphatic absorption, in silico/machine learning, and human pharmacokinetic prediction. Based on the survey results, the Degrader DMPK/ADME Working Group concluded that TPD evaluation does not fundamentally differ from other bRo5 compounds but requires some modification compared with traditional small molecules and proposes a generic workflow for PK/ADME evaluation of bifunctional TPDs. SIGNIFICANCE STATEMENT: Based on an industry survey, this article provides an understanding of the current state of absorption, distribution, metabolism, and excretion science pertaining to characterizing and optimizing targeted protein degraders, specifically bifunctional protein degraders, based upon responses by 18 IQ consortium members and non-members developing targeted protein degraders. Additionally, this article puts into context the differences / similarities in methods and strategies utilized for heterobifunctional protein degraders compared to other beyond Rule of Five molecules and conventional small molecule drugs.

Identification of a Discrete Diglucuronide of GDC-0810 in Human Plasma after Oral Administration.

GDC-0810 is a small molecule therapeutic agent having potential to treat breast cancer. In plasma of the first-in-human study, metabolite M2, accounting for 20.7% of total drug-related materials, was identified as a discrete diglucuronide that was absent in rats. Acyl glucuronide M6 and N-glucuronide M4 were also identified as prominent metabolites in human plasma. Several in vitro studies were conducted in incubations of [14C]GDC-0810, synthetic M6 and M4 with liver microsomes, intestinal microsomes, and hepatocytes of different species as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes to further understand the formation of M2. The results suggested that 1) M2 was more efficiently formed from M6 than from M4, and 2) acyl glucuronidation was mainly catalyzed by UGT1A8/7/1 that is highly expressed in the intestines whereas N-glucuronidation was mainly catalyzed by UGT1A4 that is expressed in the human liver. This complicated mechanism presented challenges in predicting M2 formation using human in vitro systems. The absence of M2 and M4 in rats can be explained by low to no expression of UGT1A4 in rodents. M2 could be the first discrete diglucuronide that was formed from both acyl- and N-glucuronidation on a molecule identified in human plasma. SIGNIFICANCE STATEMENT: A discrete diglucuronidation metabolite of GDC-0810, a breast cancer drug candidate, was characterized as a unique circulating metabolite in humans that was not observed in rats or little formed in human in vitro system.

Smarca2 genetic ablation is phenotypically benign in a safety assessment of tamoxifen-inducible conditional knockout rats.

SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.

An Integrated Strategy for Assessing the Metabolic Stability and Biotransformation of Macrocyclic Peptides in Drug Discovery toward Oral Delivery.

Macrocyclic peptides (MCPs) are an emerging class of promising drug modalities that can be used to interrogate hard-to-drug ("undruggable") targets. However, their poor intestinal stability is one of the major liabilities or obstacles for oral drug delivery. We therefore investigated the metabolic stability and biotransformation of MCPs via a systematic approach and established an integrated in vitro assay strategy to facilitate MCP drug discovery, with a focus on oral delivery liabilities. A group of diverse MCPs were incubated with representative matrices, including simulated intestinal fluid with pancreatin (SIFP), human enterocytes, liver S9 fractions, liver lysosomes, plasma, and recombinant enzymes. The results revealed that the stability and biotransformation of MCPs varied, with the major metabolic pathways identified in different matrices. Under the given conditions, the selected MCPs generally showed better stability in plasma compared to that in SIFP. Our data suggest that pancreatic enzymes act as the primary metabolic barrier for the oral delivery of MCPs, mainly through hydrolysis of their backbone amide bonds. Whereas in enterocytes, multiple metabolic pathways appeared to be involved and resulted in metabolic reactions such as oxidation and reduction in addition to hydrolysis. Further studies suggested that lysosomal peptidase cathepsin B could be a major enzyme responsible for the cleavage of side-chain amide bonds in lysosomes. Collectively, we developed and implemented an integrated assay for assessing the metabolic stability and biotransformation of MCPs for compound screening in the discovery stage toward oral delivery. The proposed question-driven assay cascade can provide biotransformation insights that help to guide and facilitate lead candidate selection and optimization.

Biotransformation novel advances - 2021 year in review.

Biotransformation field is constantly evolving with new molecular structures and discoveries of metabolic pathways that impact efficacy and safety. Recent review by Kramlinger et al. (2022) nicely captures the future (and the past) of highly impactful science of biotransformation (see the first article). Based on the selected articles, this review was categorized into three sections: (1) new modalities biotransformation, (2) drug discovery biotransformation, and (3) drug development biotransformation (Table 1).

Bioactivation and reactivity research advances - 2021 year in review.

This year's review on bioactivation and reactivity began as a part of the annual review on biotransformation and bioactivation led by Cyrus Khojasteh (see references). Increased contributions from experts in the field led to the development of a stand alone edition for the first time this year focused specifically on bioactivation and reactivity. Our objective for this review is to highlight and share articles which we deem influential and significant regarding the development of covalent inhibitors, mechanisms of reactive metabolite formation, enzyme inactivation, and drug safety. Based on the selected articles, we created two sections: (1) reactivity and enzyme inactivation, and (2) bioactivation mechanisms and safety (Table 1). Several biotransformation experts have contributed to this effort from academic and industry settings.[Table: see text].

Comparative assessment for rat strain differences in metabolic profiles of 14 drugs in Wistar Han and Sprague Dawley hepatocytes.

Knowledge of inter-strain and inter-gender differences in drug metabolism studies is important for animal selection in pharmacokinetic and toxicological studies. The effects of rat strain and gender in in vitro metabolism were investigated in Sprague Dawley (SD) and Wister Han (WH) rats based on the hepatocyte metabolic profiles of 14 small molecule drugs. Similarities were found between the hepatocyte metabolic clearances of SD and WH strains, suggesting that only one strain can be confidently used for the evaluation of hepatic clearance. Neither strain of rat was preferable over the other to cover human metabolites. Higher similarities in metabolic pathways were found between the same gender than the same strain. Differences in metabolite identities, metabolite formation rates and potential biotransformation pathways were observed between SD and WH rat strains. Eleven metabolites from six drugs were "disproportionally" formed between SD and WH rats. The use of a specific rat strain model and gender for ADME and toxicity testing should, therefore, be carefully considered as metabolic profiles may differ, even though metabolic clearance was similar between SD and WH rats.

Novel advances in biotransformation and bioactivation research-2019 year in review.

Biotransformation is one of the main mechanisms used by the body to eliminate drugs. As drug molecules become more complicated, the involvement of drug metabolizing enzymes increases beyond those that are typically studied, such as the cytochrome P450 enzymes. In this review, we try to capture the many outstanding articles that were published in the past year in the field of biotransformation (see Table 1). We have divided the articles into two categories of (1) metabolites and drug metabolizing enzymes, and (2) bioactivation and safety. This annual review is the fifth of its kind since 2016 (Baillie et al. 2016; Khojasteh et al. 2017, 2018, 2019). This effort in itself also continues to evolve. We have followed the same format we used in previous years in terms of the selection of articles and the authoring of each section. I am pleased of the continued support of Rietjens, Miller, Zhang, Driscoll and Mitra to this review. We would like to welcome Klarissa D. Jackson as a new author for this year's issue. We strive to maintain a balance of authors from academic and industry settings. We would be pleased to hear your opinions of our commentary, and we extend an invitation to anyone who would like to contribute to a future edition of this review. Cyrus Khojasteh, on behalf of the authors.

NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD: Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition.

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.

Novel Homodimer Metabolites of GDC-0994 via Cytochrome P450-Catalyzed Radical Coupling.

Two novel homodimer metabolites were identified in rat samples collected during the in vivo study of GDC-0994. In this study, we investigated the mechanism of the formation of these metabolites. We generated and isolated the dimer metabolites using a biomimetic oxidation system for NMR structure elucidation to identify a symmetric dimer formed via carbon-carbon bond between two pyrazoles and an asymmetric dimer formed via an aminopyrazole-nitrogen to pyrazole-carbon bond. In vitro experiments demonstrated formation of these dimers was catalyzed by cytochrome P450 enzymes (P450s) with CYP3A4/5 being the most efficient. Using density functional theory, we determined these metabolites share a mechanism of formation, initiated by an N-H hydrogen atom abstraction by the catalytically active iron-oxo of P450s. Molecular modeling studies also show these dimer metabolites fit in the CYP3A4 binding site in low energy conformations with minimal protein rearrangement. Collectively, the results of these experiments suggest that formation of these two homodimer metabolites is mediated by CYP3A, likely involving activation of two GDC-0994 molecules by a single P450 enzyme and proceeding through a radical coupling mechanism. SIGNIFICANCE STATEMENT: These studies identified structures and enzymology for two distinct homodimer metabolites and indicate a novel biotransformation reaction mediated by CYP3A. In it, two molecules may bind within the active site and combine through radical coupling. The mechanism of dimerization was elucidated using density functional theory computations and supported by molecular modeling.

Bioactivation of α,ß-Unsaturated Carboxylic Acids Through Acyl Glucuronidation.

After oral administration to monkeys of [14C]GDC-0810, an α,ß-unsaturated carboxylic acid, unchanged parent and its acyl glucuronide metabolite, M6, were the major circulating drug-related components. In addition, greater than 50% of circulating radioactivity in plasma was found to be nonextractable 12 hours post-dose, suggesting possible covalent binding to plasma proteins. In the same study, one of the minor metabolites was a cysteine conjugate of M6 (M11) that was detected in plasma and excreta (urine and bile). The potential mechanism for the covalent binding to proteins was further investigated using in vitro methods. In incubations with glutathione (GSH) or cysteine (5 mM), GSH and cysteine conjugates of M6 were identified, respectively. The cysteine reaction was efficient with a half-life of 58.6 minutes (k react = 0.04 1/M per second). Loss of 176 Da (glucuronic acid) followed by 129 Da (glutamate) in mass fragmentation analysis of the GSH adduct of M6 (M13) suggested the glucuronic acid moiety was not modified. The conjugation of N-glucuronide M4 with cysteine in buffer was >1000-fold slower than with M6. Incubations of GDC-0810, M4, or M6 with monkey or human liver microsomes in the presence of NADPH and GSH did not produce any oxidative GSH adducts, and the respective substrates were qualitatively recovered. In silico analysis quantified the inherent reactivity differences between the glucuronide and its acid precursor. Collectively, these results show that acyl glucuronidation of α,ß-unsaturated carboxylic acids can activate the compound toward reactivity with GSH, cysteine, or other biologically occurring thiols and should be considered during the course of drug discovery. SIGNIFICANCE STATEMENT: Acyl glucuronidation of the α,ß-unsaturated carboxylic acid in GDC-0810 activates the conjugated alkene toward nucleophilic addition by glutathione or other reactive thiols. This is the first example that a bioactivation mechanism could lead to protein covalent binding to α,ß-unsaturated carboxylic acid compounds.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Biotransformation and bioactivation reactions - 2018 literature highlights.

In the past three decades, ADME sciences have become an integral component of the drug discovery and development process. At the same time, the field has continued to evolve, thus, requiring ADME scientists to be knowledgeable of and engage with diverse aspects of drug assessment: from pharmacology to toxicology, and from in silico modeling to in vitro models and finally in vivo models. Progress in this field requires deliberate exposure to different aspects of ADME; however, this task can seem daunting in the current age of mass information. We hope this review provides a focused and brief summary of a wide array of critical advances over the past year and explains the relevance of this research ( Table 1 ). We divided the articles into categories of (1) drug optimization, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation. This annual review is the fourth of its kind (Baillie et al. 2016 ; Khojasteh et al. 2017 , 2018 ). We have followed the same format we used in previous years in terms of the selection of articles and the authoring of each section. This effort in itself also continues to evolve. I am pleased that Rietjens, Miller, and Mitra have again contributed to this annual review. We would like to welcome Namandjé N. Bumpus, James P. Driscoll, and Donglu Zhang as authors for this year's issue. We strive to maintain a balance of authors from academic and industry settings. We would be pleased to hear your opinions of our commentary, and we extend an invitation to anyone who would like to contribute to a future edition of this review. Cyrus Khojasteh, on behalf of the authors.

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

Carfilzomib Is Not an Appropriate Payload of Antibody-Drug Conjugates Due to Rapid Inactivation by Lysosomal Enzymes.

Carfilzomib (CFZ) is a proteasome inhibitor used for oncology indications including treating multiple myeloma. CFZ is a potent cytotoxic agent with an IC50 value in the nanomolar range in various cancer cell lines and was considered as a potential payload for antibody drug conjugates (ADCs); however, the conjugated CFZ to anti-CD22 or anti-HER2 antibody totally abolishes the in vitro potency. This was a surprise since with other payloads such as monomethyl auristatin E (MMAE), where potent antiproliferation efficacy was retained as MMAE alone or as a payload in an ADC. Further investigations were conducted using CFZ alone, CFZ with a linker, and CFZ-ADC with tissue matrices including lysosomal enzymes. With CFZ linked to the ADC, cathepsin B (a lysosomal enzyme) was efficient in liberating CFZ from the ADC by cleavage of the valine-citrulline linker. At the same time, the liberated CFZ in the lysosome was inactivated due to further metabolism by lysosomal enzymes. The products from epoxide and amide hydrolysis were identified from these incubations. These results suggested that the CFZ-ADC upon uptake and internalization specifically delivers CFZ payload to the lysosomes, where CFZ was inactivated. On the other hand, CFZ by itself is not as vulnerable and could reach its target. Therefore, lysosomal stability is an important criterion in the selection of a payload for making the next generation of potent ADC therapeutics.

Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues.

Antibody-drug conjugates (ADCs) contain a disease-receptor antibody and a payload drug connected via a linker. The payload delivery depends on both tumor properties and ADC characteristics. In this study, we used different linkers, attachment sites, and doses to modulate payload delivery of several ADCs bearing maytansinoids (e.g., DM1), auristatins (e.g., MMAE), and DNA alkylating agents [e.g., pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD)] as payloads in HER2- or CD22-expressing xenograft models. The tumor growth inhibition and ADC stability and exposure data were collected and analyzed from these dosed animals. The trend analysis suggests that intratumoral payload exposures that directly related the combination of conjugate linker and dose correlate with the corresponding efficacies of three payload types in two antigen-expressing xenograft models. These preliminary correlations also suggest that a minimal threshold concentration of intratumoral payload is required to support sustained efficacy. In addition, an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor payload concentrations, the assessments of systemic exposures of total antibody (Tab) as well as the linker, dose, site of attachment, plasma stability, and drug-to-antibody ratio changes of these ADCs did not consistently rationalize the observed ADC efficacies. The requirement of a threshold payload concentration for efficacy is further supported by dose fractionation studies with DM1-, MMAE-, and PBD-containing ADCs, which demonstrated that single-dose regimens showed better efficacies than fractionated dosing. Overall, this study demonstrates that 1) the linker and dose together determine the tissue payload concentration that correlates with the antitumor efficacy of ADCs and 2) an ADC can deliver an unnecessary level of payload to tumors in xenograft models.

A Novel Depurination Methodology to Assess DNA Alkylation of Chloro-Bis-Seco-Cyclopropylbenzoindoles Allowed for Comparison of Minor-Groove Reactivity.

Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.