A general perspective for the conduct of radiolabelled distribution, metabolism, and excretion studies for antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of monoclonal antibodies (mAbs) with the cytotoxicity of small molecule drugs. 15 ADCs have been approved by regulatory authorities up to now, mainly for indications in oncology, however, this review paper will only focus on the 13 ADCs that have been approved by either the FDA or EMA.ADME (Absorption, Distribution, Metabolism, and Excretion) studies are essential for the development of small molecule drugs to evaluate their disposition properties. These studies help to select drug candidates, determine the optimal dosing regimen and help to identify potential safety concerns for the drug of interest in human. Tissue distribution studies are also important as they facilitate the understanding of the efficacy and safety for parent drug and its metabolites in preclinical and clinical studies.For biologics, ADME studies are usually not required. In this paper, we review the existing approval packages and literature for approved ADCs to determine the extent of ADME studies performed as part of ADC registration packages.We conclude that ADME studies are recommended for the development of ADCs if new linkers and payloads are used that have never been used in humans before as these studies provide valuable information on the pharmacokinetic properties, optimal dosing regimen, and potential safety concerns. However, for the development of ADCs with established linker payload combinations, radiolabelled ADME studies may not be necessary if the distribution, metabolism and excretion properties have been described before. Clinical radiolabelled ADME studies are not recommended where patients are treated for life threating diseases like for indications in oncology.

Preface for special issue: "Emerging strategies, technologies, and approaches for the next generation ADCs".

1. Antibody-drug conjugates (ADCs) represent an advanced category of biotherapeutic agents, typically consisting of an antibody bound to a biologically-active cytotoxic agent. Since the first ADC, MylotargTM, was approved in 2000, there have been fifteen ADCs sanctioned to date, with thirteen receiving approval from the FDA for the treatment of a variety of cancers, including blood malignancies and solid tumors.2. In this Special Issue of Xenobiotica focusing on ADCs, our goal is to compile a collection of papers, featuring both original research and review articles authored by specialists in academia and the pharmaceutical industry, to showcase some of the historical insights gained, current progress, and future prospects to enhance comprehension and tackle obstacles in the field of ADC development for cancer therapy.3. This special issue features articles that evaluate key components of ADC development, including payload design, innovative linker chemistries, and the use of new technologies for site-specific conjugations beyond traditional engineered cysteines. It also spotlights cutting-edge ADC structures like bispecific ADCs, dual-payload ADCs, targeted nanoparticles and antibody oligonucleotide conjugates (AOCs).4. Several other papers discuss bioanalytical and ADME strategies for ADCs as well. In addition, approaches to improve the translation of pharmacokinetics, safety, and therapeutic index (TI) of ADCs are presented.

Potential of measured relative shifts in collision cross section values for biotransformation studies.

Ion mobility spectrometry-mass spectrometry (IMS-MS) separates gas phase ions due to differences in drift time from which reproducible and analyte-specific collision cross section (CCS) values can be derived. Internally conducted in vitro and in vivo metabolism (biotransformation) studies indicated repetitive shifts in measured CCS values (CCSmeas) between parent drugs and their metabolites. Hence, the purpose of the present article was (i) to investigate if such relative shifts in CCSmeas were biotransformation-specific and (ii) to highlight their potential benefits for biotransformation studies. First, mean CCSmeas values of 165 compounds were determined (up to n = 3) using a travelling wave IMS-MS device with nitrogen as drift gas (TWCCSN2, meas). Further comparison with their predicted values (TWCCSN2, pred, Waters CCSonDemand) resulted in a mean absolute error of 5.1%. Second, a reduced data set (n = 139) was utilized to create compound pairs (n = 86) covering eight common types of phase I and II biotransformations. Constant, discriminative, and almost non-overlapping relative shifts in mean TWCCSN2, meas were obtained for demethylation (- 6.5 ± 2.1 Å2), oxygenation (hydroxylation + 3.8 ± 1.4 Å2, N-oxidation + 3.4 ± 3.3 Å2), acetylation (+ 13.5 ± 1.9 Å2), sulfation (+ 17.9 ± 4.4 Å2), glucuronidation (N-linked: + 41.7 ± 7.5 Å2, O-linked: + 38.1 ± 8.9 Å2), and glutathione conjugation (+ 49.2 ± 13.2 Å2). Consequently, we propose to consider such relative shifts in TWCCSN2, meas (rather than absolute values) as well for metabolite assignment/confirmation complementing the conventional approach to associate changes in mass-to-charge (m/z) values between a parent drug and its metabolite(s). Moreover, the comparison of relative shifts in TWCCSN2, meas significantly simplifies the mapping of metabolites into metabolic pathways as demonstrated.

Species-specific Bioactivation of Morpholines as a Causative of Drug Induced Liver Injury Observed in Monkeys

BACKGROUND: Everolimus, an allosteric mechanistic target of rapamycin (mTOR) inhibitor, recently demonstrated the therapeutic value of mTOR inhibitors for Central Nervous System (CNS) indications driven by hyperactivation of mTOR. A newer, potent brain-penetrant analog of everolimus, referred to as (1) in this manuscript [(S)-3-methyl-4-(7-((R)-3-methylmorpholino)-2-(thiazol-4-yl)-3H-imidazo[4,5-b]pyridin-5-yl)morpholine,(1)] catalytically inhibits mTOR function in the brain and increases the lifespan of mice with neuronal mTOR hyperactivation. INTRODUCTION: Early evaluation of the safety of 1 was conducted in cynomolgus monkeys in which oral doses were administered to three animals in a rising-dose fashion (from 2 to 30 mg/kg/day). 1 produced severe toxicity including the evidence of hepatic toxicity, along with non-dose proportional increases in drug exposure. Investigations of cross-species hepatic bioactivation of 1 were conducted to assess whether the formation of reactive drug metabolites was associated with the mechanism of liver toxicity. METHOD: 1 contained two morpholine rings known as structural alerts and can potentially form reactive intermediates through oxidative metabolism. Bioactivation of 1 was investigated in rat, human and monkey liver microsomes fortified with trapping agents such as methoxylamine or potassium cyanide. RESULTS: Our results suggest that bioactivation of the morpholine moieties to reactive intermediates may have been involved in the mechanism of liver toxicity observed with 1. Aldehyde intermediates trappable by methoxylamine were identified in rat and monkey liver microsomal studies. In addition, a total of four cyano conjugates arising from the formation of iminium ion intermediates were observed and identified. These findings may potentially explain the observed monkey toxicity. Interestingly, methoxylamine or cyano adducts of 1 were not observed in human liver microsomes. CONCLUSION: The bioactivation of 1 appears to be species-specific. Circumstantial evidence for the toxicity derived from 1 point to the formation of iminium ion intermediates trappable by cyanide in monkey liver microsomes. The cyano conjugates were only observed in monkey liver microsomes, potentially pointing to cause at least the hepatotoxicity observed in monkeys. In contrast, methoxylamine conjugates were detected in both rat and monkey liver microsomes, with only a trace amount in human liver microsomes. Cyano conjugates were not observed in human liver microsomes, challenging the team on the drugability and progressivity of 1 through drug development. The mechanisms for drug-induced liver toxicity are multifactorial. These results are highly suggestive that the iminium ion may be an important component in the mechanism of liver toxicity 1 observed in the monkey.

The application of organ-on-chip models for the prediction of human pharmacokinetic profiles during drug development.

Organ-on-chip (OoC) technology has led to in vitro models with many new possibilities compared to conventional in vitro and in vivo models. In this review, the potential of OoC models to improve the prediction of human oral bioavailability and intrinsic clearance is discussed, with a focus on the functionality of the models and the application in current drug development practice. Multi-OoC models demonstrating the application for pharmacokinetic (PK) studies are summarized and existing challenges are identified. Physiological parameters for a minimal viable platform of a multi-OoC model to study PK are provided, together with PK specific read-outs and recommendations for relevant reference compounds to validate the model. Finally, the translation to in vivo PK profiles is discussed, which will be required to routinely apply OoC models during drug development.

Meeting report of the 3rd European Biotransformation Workshop.

Challenges, strategies and new technologies in the field of biotransformation were presented and discussed at the 3rd European Biotransformation Workshop which was held in collaboration with the DMDG on 5-6 October 2022 in Amsterdam. In this meeting report we summarise the presentations and discussions from this workshop. The topics covered are listed below:Accelerator mass spectrometry (AMS) for the support of microtracer studiesBiotransformation of the novel myeloperoxidase inhibitor AZD4831 in preclinical species and humansAMS in biotransformation studies: unusual case studiesDiscussion on new FDA draft guidance and AMSMultimodal molecular imaging and ion mobility applications in drug discovery and developmentMetabolites in Safety Testing considerations for large molecules.

A Cross Company Perspective on the Assessment of Therapeutic Protein Biotransformation.

Unlike with new chemical entities, the biotransformation of therapeutic proteins (TPs) has not been routinely investigated or included in regulatory filings. Nevertheless, there is an expanding pool of evidence suggesting that a more in-depth understanding of biotransformation could better aid the discovery and development of increasingly diverse modalities. For instance, such biotransformation analysis of TPs affords important information on molecular stability, which in turn may shed light on any potential impact on binding affinity, potency, pharmacokinetics, efficacy, safety, or bioanalysis. This perspective summarizes the current practices in studying biotransformation of TPs and related findings in the biopharmaceutical industry. Various TP case studies are discussed, and a fit-for-purpose approach is recommended when investigating their biotransformation. In addition, we provide a decision tree to guide the biotransformation characterization for selected modalities. By raising the awareness of this important topic, which remains relatively underexplored in the development of TPs (Bolleddula et al., 2022), we hope that current and developing practices can pave the way for establishing a consensus on the biotransformation assessment of TPs. SIGNIFICANCE STATEMENT: This article provides a comprehensive perspective of the current practices for exploring the biotransformation of therapeutic proteins across the drug development industry. We, the participants of the Innovation and Quality therapeutic protein absorption distribution metabolism excretion working group, recommend and summarize appropriate approaches for conducting biotransformation studies to support internal decision making based on the data generated in discovery and development.

Absorption, Distribution, Metabolism, and Excretion of Therapeutic Proteins: Current Industry Practices and Future Perspectives.

Therapeutic proteins (TPs) comprise a variety of modalities, including antibody-based drugs, coagulation factors, recombinant cytokines, enzymes, growth factors, and hormones. TPs usually cannot traverse cellular barriers and exert their pharmacological activity by interacting with targets on the exterior membrane of cells or with soluble ligands in the tissue interstitial fluid/blood. Due to their large size, lack of cellular permeability, variation in metabolic fate, and distinct physicochemical characteristics, TPs are subject to different absorption, distribution, metabolism, and excretion (ADME) processes as compared with small molecules. Limited regulatory guidance makes it challenging to determine the most relevant ADME data required for regulatory submissions. The TP ADME working group was sponsored by the Translational and ADME Sciences Leadership Group within the Innovation and Quality (IQ) consortium with objectives to: (1) better understand the current practices of ADME data generated for TPs across IQ member companies, (2) learn about their regulatory strategies and interaction experiences, and (3) provide recommendations on best practices for conducting ADME studies for TPs. To understand current ADME practices and regulatory strategies, an industry-wide survey was conducted within IQ member companies. In addition, ADME data submitted to the U.S. Food and Drug Administration was also collated by reviewing regulatory submission packages of TPs approved between 2011 and 2020. This article summarizes the key learnings from the survey and an overview of ADME data presented in biologics license applications along with future perspectives and recommendations for conducting ADME studies for internal decision-making as well as regulatory submissions for TPs. SIGNIFICANCE STATEMENT: This article provides comprehensive assessment of the current practices of absorption, distribution, metabolism, and excretion (ADME) data generated for therapeutic proteins (TPs) across the Innovation and Quality participating companies and the utility of the data in discovery, development, and regulatory submissions. The TP ADME working group also recommends the best practices for condu-cting ADME studies for internal decision-making and regulatory submissions.

Species-dependent hepatic and intestinal metabolism of selective oestrogen receptor degrader LSZ102 by sulphation and glucuronidation.

LSZ102 is an orally bioavailable selective oestrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass.To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase (SULT) enzymes potentially involved in metabolism of LSZ102 was characterised.Sulphate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolised by several human SULTs expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulphation in human but glucuronidation in rat.As LSZ102 is metabolised by a number of different SULTs, drug-drug interactions resulting from the inhibition of one SULT are unlikely.Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulphate M5, glucuronide M4, and secondary glucuronide/sulphate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Preclinical pharmacokinetics and metabolism of MAK683, a clinical stage selective oral embryonic ectoderm development (EED) inhibitor for cancer treatment.

MAK683 (N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridin-3-yl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine) is a potent and orally bioavailable EED inhibitor for the potential treatment in oncology. Pharmacokinetics (PK) in preclinical species are characterised by low to moderate plasma clearances, high oral exposure, and moderate to high oral bioavailability at the dose of 1-2 mg/kg.A species comparison of the metabolic pathways of MAK683 has been made using [14C]MAK683 incubations with liver microsomes and hepatocytes from rat, dog, cynomolgus monkey, and human. Overall, the in vitro hepatic metabolism pathway of MAK683 in all five species was very complex. A total of 60 metabolites with 19 metabolites >1.5% of the total integrated area in the radiochromatogram of at least one species were identified in five species (rat, mouse, dog, monkey, and human).The primary in vitro hepatic oxidative metabolism pathway identified in humans involved 2-hydroxylation of the dihydrofuran ring to form alcohol (M28), which was in a chemical equilibrium favouring the formation of its aldehyde form. The aldehyde was then oxidised to the carboxylic acid metabolite (M26) or reduced to the O-hydroxyethylphenol (M29). N-dealkylation (M1), 3-hydroxylation of the dihydrofuran ring (M27), N-oxidation of the pyridine moiety (M53), and sulphate conjugation of M28 to form M19 were also important biotransformation pathways in human hepatocytes. The above major human hepatic metabolic pathways were also observed across the animal species (rat, mouse, dog, and monkey) mostly providing precursors for the formation of other metabolites via further oxygenation, glucuronidation, and sulphation pathways.No human-specific metabolites were observed. In addition, in vivo biotransformation was also conducted in bile-duct cannulated (BDC) rat. The metabolism in BDC rat was similar to those observed the in vitro hepatocytes.

Risk-Based Pharmacokinetic and Drug-Drug Interaction Characterization of Antibody-Drug Conjugates in Oncology Clinical Development: An International Consortium for Innovation and Quality in Pharmaceutical Development Perspective.

Antibody-drug conjugates (ADCs) represent a rapidly evolving area of drug development and hold significant promise. To date, nine ADCs have been approved by the US Food and Drug Administration (FDA). These conjugates combine the target specificity of monoclonal antibodies with the anticancer activity of small-molecule therapeutics (also referred to as payload). Due to the complex structure, three analytes, namely ADC conjugate, total antibody, and unconjugated payload, are typically quantified during drug development; however, the benefits of measuring all three analytes at later stages of clinical development are not clear. The cytotoxic payloads, upon release from the ADC, are considered to behave like small molecules. Given the relatively high potency and low systemic exposure of cytotoxic payloads, drug-drug interaction (DDI) considerations for ADCs might be different from traditional small molecule therapeutics. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) convened an ADC working group to create an IQ ADC database that includes 26 ADCs with six unique payloads. The analysis of the ADC data in the IQ database, as well as nine approved ADCs, supports the strategy of pharmacokinetic characterization of all three analytes in early-phase development and progressively minimizing the number of analytes to be measured in the late-phase studies. The systemic concentrations of unconjugated payload are usually too low to serve as a DDI perpetrator; however, the potential for unconjugated payloads as a victim still exists. A data-driven and risk-based decision tree was developed to guide the assessment of a circulating payload as a victim of DDI.

Double Trap Interface: A novel gas interface for high throughput analysis of biomedical samples by AMS.

Although Accelerator Mass Spectrometry (AMS) offers unparalleled sensitivity by investigating the fate of 14C-labeled compounds within the organism, its widespread use in ADME (absorption, distribution, metabolism, excretion) studies is limited. Conventional approaches based on Liquid Scintillation Counting (LSC) are still preferred, in particular because of complexity and costs associated with AMS measurements. Progress made over the last decade towards more compact AMS systems increased the interest in a combustion-based AMS approach allowing the analysis of samples in gaseous form. Thus, a novel gas Double Trap Interface (DTI) was designed, providing high sample throughput for the analysis of biomedical samples. DTI allows the coupling of an Elemental Analyzer (EA) for sample combustion to the hybrid ion source of a MICADAS (MIni CArbon DAting System) AMS system. The performance was evaluated in two studies through the analysis of more than 1000 samples from 14C-labeled biomatrices and fractions collected after liquid chromatography (LC). The covered activity ranged from 1 to 1000 mBq/g for labeled biomatrices and from 1 to 10000 mBq/g(C) for LC fractions. The implemented routine allows automated measurements requiring less than 5 min per sample (12-13 analyses per hour) without the need for sample conversion to graphite.

Evaluation of the Absorption, Metabolism, and Excretion of a Single Oral 1-mg Dose of Tropifexor in Healthy Male Subjects and the Concentration Dependence of Tropifexor Metabolism.

Tropifexor (NVP-LJN452) is a highly potent, selective, nonsteroidal, non-bile acid farnesoid X receptor agonist for the treatment of nonalcoholic steatohepatitis. Its absorption, metabolism, and excretion were studied after a 1-mg oral dose of [14C]tropifexor was given to four healthy male subjects. Mass balance was achieved with ∼94% of the administered dose recovered in excreta through a 312-hour collection period. Fecal excretion of tropifexor-related radioactivity played a major role (∼65% of the total dose). Tropifexor reached a maximum blood concentration (Cmax) of 33.5 ng/ml with a median time to reach Cmax of 4 hours and was eliminated with a plasma elimination half-life of 13.5 hours. Unchanged tropifexor was the principal drug-related component found in plasma (∼92% of total radioactivity). Two minor oxidative metabolites, M11.6 and M22.4, were observed in circulation. Tropifexor was eliminated predominantly via metabolism with >68% of the dose recovered as metabolites in excreta. Oxidative metabolism appeared to be the major clearance pathway of tropifexor. Metabolites containing multiple oxidative modifications and combined oxidation and glucuronidation were also observed in human excreta. The involvement of direct glucuronidation could not be ruled out based on previous in vitro and nonclinical in vivo studies indicating its contribution to tropifexor clearance. The relative contribution of the oxidation and glucuronidation pathways appeared to be dose-dependent upon further in vitro investigation. Because of these complexities and the instability of glucuronide metabolites in the gastrointestinal tract, the contribution of glucuronidation remained undefined in this study. SIGNIFICANCE STATEMENT: Tropifexor was found to be primarily cleared from the human body via oxidative metabolism. In vitro metabolism experiments revealed that the relative contribution of oxidation and glucuronidation was concentration-dependent, with glucuronidation as the predominant pathway at higher concentrations and the oxidative process becoming more important at lower concentrations near clinical exposure range. The body of work demonstrated the importance of carefully designed in vivo and in vitro experiments for better understanding of disposition processes during drug development.

Pharmacokinetics, metabolism, and excretion of licogliflozin, a dual inhibitor of SGLT1/2, in rats, dogs, and humans.

Absorption, metabolism, and excretion (AME) of licogliflozin, a sodium-glucose co-transporters (SGLTs) 1 and 2 inhibitor, were studied in male rats, dogs, and healthy male volunteers and reported.Oral absorption of licogliflozin was rapid (tmax < 1 h) with absorption estimated at 87%, 100% and 77% in rats, dogs and humans, respectively.Excretion of licogliflozin-related radioactivity was rapid and nearly complete following oral administration with total radioactivity recovery ranging from 73% in dogs, 92.5% in humans, to 100% in rats. Dose-related radioactivity was excreted in both urine and faeces with urinary excretion playing a slightly more important role in humans (∼56%) than in animal species (∼19-41%).Elimination of licogliflozin was predominantly via metabolism with the majority of the radioactivity dose (∼54-74%) excreted as metabolites across species.The principal biotransformation pathways involved direct glucuronidation and oxidation across all species. In humans, direct glucuronidation to M17 and M27 was the major pathway observed, accounting for ∼38% of the dose in excreta while oxidative metabolism also contributed to >29% of the dose in excreta. Oxidative pathways were predominant in animal species.

New Perspectives on Drug-Induced Liver Injury Risk Assessment of Acyl Glucuronides.

Drug-induced liver injury (DILI) remains one of the key challenges in drug development due to the mechanisms of action being multifactorial in nature. This is particularly the case for idiosyncratic DILI which occurs in a very low frequency in humans (e.g., 1:10,000). Despite perceptions that acyl glucuronide metabolites are defacto risks for DILI, scientific evidence suggests that acyl glucuronide formation alone does not pose an increased risk compared to other drug metabolites. This applies in particular to those acyl glucuronides which are not reactive and do not form covalent adducts with proteins. The goal of this paper is to provide guidance on preclinical and clinical strategies to evaluate the potential for acyl glucuronide formation to contribute to DILI. A key element of our proposed safety assessment is to investigate whether a particular acyl glucuronide is reactive or not and whether systemic exposure in humans can be demonstrated in animal toxicology studies following administration of the parent drug. While standard animal toxicology studies can identify overtly hepatotoxic compounds, these studies are not predictive for drugs that produce idiosyncratic forms of DILI. In addition, we do not recommend conducting toxicology studies of administered individual acyl glucuronides due to differences in pharmacokinetic and dispositional properties from the endogenously produced metabolites. Once a drug candidate has entered clinical trials, the focus should be on clinical safety data and emerging risk-benefit analysis.

Functional assessment of rat pulmonary flavin-containing monooxygenase activity.

The expression of flavin-containing monooxygenase (FMO) varies extensively between human and commonly used preclinical species such as rat and mouse. The aim of this study was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the contribution of rat lung to the clearance of benzydamine was investigated using an in vivo pulmonary extraction model. Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is predominantly FMO mediated while any contribution from CYPs is negligible. The predicted lung clearance (CLlung) estimated from microsomes and slices was 16 ± 0.6 and 2.1 ± 0.3 mL/min/kg, respectively. The results from in vivo pulmonary extraction indicated no pulmonary extraction following intravenous and intra-arterial dosing to rats. Interestingly, the predicted CLlung using rat lung microsomes corresponded to approximately 35% of rat CLliver suggesting that the lung makes a smaller contribution to the whole body clearance of benzydamine. Although benzydamine clearance in rat appears to be predominantly mediated by hepatic metabolism, the data suggest that the lung may also make a smaller contribution to its whole body clearance.

19F-NMR-based determination of the absorption, metabolism and excretion of the oral phosphatidylinositol-3-kinase (PI3K) delta inhibitor leniolisib (CDZ173) in healthy volunteers.

1. Leniolisib is a novel oral phosphatidylinositol-3-kinase (PI3K) delta inhibitor, currently in clinical development for the treatment of inflammatory and autoimmune diseases. 2. We investigated the absorption, metabolism, and excretion of leniolisib in healthy subjects after a single oral 400 mg dose as part of a first-in-human clinical study. The parent drug and metabolites were quantified by 19F-NMR in plasma, urine and faeces after liquid chromatography separation, and structures were determined by liquid chromatography coupled to tandem mass spectrometry. 3. Drug-related material was mainly excreted as oxidative metabolites in urine and faeces, providing evidence that elimination occurs mainly by metabolism. No metabolites were abundant in plasma relative to the parent drug. An average mass balance of 66% was obtained, demonstrating that relatively extensive elimination/excretion data can be obtained by 19F-NMR in a first in human clinical study without the use of a radiolabeled drug.

Comparison of Rat and Human Pulmonary Metabolism Using Precision-cut Lung Slices (PCLS).

BACKGROUND: Although the liver is the primary organ of drug metabolism, the lungs also contain drug-metabolizing enzymes and may, therefore, contribute to the elimination of drugs. In this investigation, the Precision-cut Lung Slice (PCLS) technique was standardized with the aims of characterizing and comparing rat and human pulmonary drug metabolizing activity. METHOD: Due to the limited availability of human lung tissue, standardization of the PCLS method was performed with rat lung tissue. Pulmonary enzymatic activity was found to vary significantly with rat age and rat strain. The Dynamic Organ Culture (DOC) system was superior to well-plates for tissue incubations, while oxygen supply appeared to have a limited impact within the 4h incubation period used here. RESULTS: The metabolism of a range of phase I and phase II probe substrates was assessed in rat and human lung preparations. Cytochrome P450 (CYP) activity was relatively low in both species, whereas phase II activity appeared to be more significant. CONCLUSION: PCLS is a promising tool for the investigation of pulmonary drug metabolism. The data indicates that pulmonary CYP activity is relatively low and that there are significant differences in enzyme activity between rat and human lung.

Models and Approaches Describing the Metabolism, Transport, and Toxicity of Drugs Administered by the Ocular Route.

The eye is a complex organ with a series of anatomic barriers that provide protection from physical and chemical injury while maintaining homeostasis and function. The physiology of the eye is multifaceted, with dynamic flows and clearance mechanisms. This review highlights that in vitro ocular transport and metabolism models are confined by the availability of clinically relevant absorption, distribution, metabolism, and excretion (ADME) data. In vitro ocular transport models used for pharmacology and toxicity poorly predict ocular exposure. Although ocular cell lines cannot replicate in vivo conditions, these models can help rank-order new chemical entities in discovery. Historic ocular metabolism of small molecules was assumed to be inconsequential or assessed using authentic standards. While various in vitro models have been cited, no single system is perfect, and many must be used in combination. Several studies document the use of laboratory animals for the prediction of ocular pharmacokinetics in humans. This review focuses on the use of human-relevant and human-derived models which can be utilized in discovery and development to understand ocular disposition of new chemical entities. The benefits and caveats of each model are discussed. Furthermore, ADME case studies are summarized retrospectively and capture the ADME data collected for health authorities in the absence of definitive guidelines. Finally, we discuss the novel technologies and a hypothesis-driven ocular drug classification system to provide a holistic perspective on the ADME properties of drugs administered by the ocular route.

Evaluation of cAMS for 14C microtracer ADME studies: opportunities to change the current drug development paradigm.

AIM: Although regulatory guidances require human metabolism information of drug candidates early in the development process, the human mass balance study (or hADME study), is performed relatively late. hADME studies typically involve the administration of a 14C-radiolabelled drug where biological samples are measured by conventional scintillation counting analysis. Another approach is the administration of therapeutic doses containing a 14C-microtracer followed by accelerator mass spectrometry (AMS) analysis, enabling hADME studies completion much earlier. Consequently, there is an opportunity to change the current drug development paradigm. MATERIALS & METHODS: To evaluate the applicability of the MICADAS-cAMS method, we successfully performed: the validation of MICADAS-cAMS for radioactivity quantification in biomatrices and, a rat ADME study, where the conventional methodology was assessed against a microtracer MICADAS-cAMS approach. RESULTS & DISCUSSION: Combustion AMS (cAMS) technology is applicable to microtracer studies. A favorable opinion from EMA to complete the hADME in a Phase I setting was received, opening the possibilities to change drug development.