In Vivo Absorption Distribution Metabolism and Excretion Studies in Discovery and Development

T Lave and C Funk, F. Hoffmann-La Roche Ltd, Basel, Switzerland © 2007 Elsevier Ltd. All Rights Reserved.

5.03.1 Objectives of Absorption, Distribution, Metabolism, and Excretion (ADME) Studies 32 Support to Drug Discovery 32 Support to Drug Metabolism 32 Support to Pharmacodynamics 32 Support to Toxicology 32 Support to Formulation Testing 32

5.03.2 In Vivo Drug Metabolism and Pharmacokinetics Screening Studies 33

5.03.3 Pharmacokinetic Studies 33

5.03.4 Metabolism Studies 33 The Use of Radiolabeled Compounds 34 Chromatographic Separation and Quantification of Drug Metabolites 34 In Vivo Animal Models Used for Absorption, Distribution, Metabolism, and Excretion Studies 34 Mass Balance and Route of Excretion Studies 34 Metabolite Profiling in Animal Species and Man 35 Systemically available metabolites in plasma 35 Metabolites found in excreta 35 Limitations of animal models for drug metabolism 36 Covalent Binding Studies: Reactive Metabolites 36 Enzyme Induction and Inhibition Studies 37

5.03.5 Mechanistic Pharmacokinetic Studies 38 Intestinal Absorption Studies 38 Liver and Intestinal First Pass Studies 38 Studies to Assess Blood-Brain Barrier Permeation 39 Carotid artery injection technique 39 Intravenous injection technique with brain collection 39 Cerebrospinal fluid sampling technique 39 Intracerebral microdialysis 40

5.03.6 Interspecies Differences in Pharmacokinetics 40 Species Differences in Absorption 40 Physiology of the gastrointestinal tract 40 Interspecies comparison of fraction absorbed 41 Interspecies Differences in Distribution 42 Species Differences in Elimination 44 Interspecies differences in metabolic clearance 44 Interspecies differences in renal clearance 46 Interspecies differences in biliary clearance 46

5.03.7 Prediction to Humans 47 References 48

5.03.1 Objectives of Absorption, Distribution, Metabolism, and Excretion (ADME) Studies

In recent years, there has been an increased awareness about the importance of pharmacokinetics and metabolism data in all stages of the drug discovery and development process. In order to understand better the impact of in vivo drug metabolism and pharmacokinetic studies, it is important to first outline the major objectives of drug metabolism and pharmacokinetics (DMPK) when applied at the discovery and nonclinical development stages. Support to Drug Discovery

DMPK is now a routine part of the lead identification, lead optimization, and clinical candidate selection processes. At these different stages an appraisal of DMPK issues alongside other 'developability' factors is performed. Thus, the deployment of available in vivo and in vitro methods is based on a sound understanding of the relevant DMPK issues and not simply on the capacity to process large numbers of compounds. Support to Drug Metabolism

The main goal of drug metabolism or biotransformation for the body is to eliminate potentially harmful xenobiotics via urine and/or bile. This is typically realized in a stepwise process: the often lipophilic drug molecules are metabolized (biotransformation) to typically inactive, nontoxic, and more hydrophilic products, which can then be readily excreted in urine or bile.1 In some cases, however, drug metabolites might be toxic or might represent activated products (e.g., acylglucuronides), which can potentially lead to organ toxicity or immune-mediated toxicity. Therefore, an extensive knowledge of drug metabolism is required for an overall understanding of the pharmacological and safety properties of new drug molecules.2 Support to Pharmacodynamics

The monitoring of pharmacodynamic studies for exposure and the rigorous design of pharmacokinetic/pharmacody-namic (PK/PD) studies in animals are key to a number of questions that need to be addressed during preclinical development. These questions include: (1) identification of potential pharmacodynamics endpoints for efficacy in animal models; (2) development of mechanism-based models for efficacy; (3) determination of in vivo potency and intrinsic activity and prediction in humans; (4) dosage form and dosage regimen optimization; and (5) supporting dose selection for phase 1 studies.

Thus, the use of PK/PD modeling in early preclinical development to define the dose-concentration-pharmacological effects and dose-concentration-toxicity relationships, as well as the extrapolation of these results to humans using a combination of in vitro and in vivo data, can be particularly helpful in determining the appropriate dosing regimen for phase 1 studies. Preclinical PK/PD studies may also prompt a series of important mechanistic studies to explore any dissociation between plasma concentration and duration of pharmacological effect (i.e., active metabolites or long half-life in the effect compartment, etc.). For PK/PD models and their use in drug discovery (see 5.38 Mechanism-Based Pharmacokinetic-Pharmacodynamic Modeling for the Prediction of In Vivo Drug Concentration-Effect Relationships - Application in Drug Candidate Selection and Lead Optimization). Support to Toxicology

An important function of animal DMPK studies is in support of preclinical safety evaluation. Thus, determination of pharmacokinetics and exposures during the course of a toxicological experiment is key to interpreting toxicological findings. For example, it is essential to compare exposures in toxicological studies with the exposures expected or achieved in humans to ensure sufficient safety margin. Furthermore, metabolite profiles obtained from the species used in toxicology studies are compared to those in humans. In this case, emphasis is placed on qualitative similarity in metabolite profiles between humans and species used in toxicology studies in order to ensure that both are exposed to the parent drug as well as the same metabolites, any of which may contribute to toxicity. Support to Formulation Testing

During the discovery and development process, the formulation scientist develops formulations to ensure appropriate dosing of the test compound during early DMPK, safety, and pharmacodynamics studies. Various formulations also should be investigated when the in vivo plasma concentration versus time profiles need, for example, to be extended with controlled release dosage forms and when oral bioavailability needs to be improved. In this context, formulations with a desirable pattern in vitro are submitted to in vivo oral absorption studies in animals. Once a favorable plasma concentration versus time profile in animals is achieved, the formulations are developed for testing in humans.

The judicious selection of animal species to support formulation testing is of key importance at this stage. To this end it is important to understand the similarities and differences of the physiology of the gastrointestinal (GI) tract between animals and humans. These issues are discussed in detail later in this chapter. However, it is a fact that the dog has become the most commonly used species for bioavailability studies in the context of formulation testing. Because of some species differences between humans and dogs, the findings in dogs and in other animal species have to be interpreted cautiously for their relevance to humans.

5.03.2 In Vivo Drug Metabolism and Pharmacokinetics Screening Studies

In recent years, several pharmaceutical companies have published reports on the simultaneous administration of several compounds to a single animal (cassette dosing or 'N-in-One' dosing)3-7 as a means to rapidly rank order compounds on the basis of their in vivo pharmacokinetic properties. Compared with conventional pharmacokinetic studies, this method has the advantage of speed, because the slow steps of animal dosing, blood collection, and sample analysis are minimized. Another advantage is that animal usage is greatly reduced. The enabling technology for cassette dosing is liquid chromatography coupled to tandem mass spectrometry (LC/MS), which allows many compounds to be analyzed simultaneously.

Although cassette dosing has been reported to yield useful results when used as a screen, especially to rank order drug candidates, the potential for large errors was shown recently both theoretically and experimentally.4 Consequently, it is recommended that the pharmacokinetic parameters derived from cassette dosing are interpreted very cautiously.

5.03.3 Pharmacokinetic Studies

A pharmacokinetic study involves dosing and sampling of animals or subjects, bioanalysis of the biological samples (e.g., blood, plasma, tissues) and analysis of the resulting blood, plasma, or serum concentration versus time data using for example noncompartmental or compartmental pharmacokinetic methods. Low aqueous solubility of molecules often necessitates significant formulation work prior to dosing. The bioanalytical or assay phase typically involves sample extraction with organic solvents and LC/MS or LC/MS/MS separation and detection of analytes. During drug discovery, pharmacokinetic studies are most commonly conducted in rodents and/or the species used for the assessment of in vivo efficacy. Subsequently, experiments in larger animal species such as dog or monkey are performed to further characterize the compound of interest, to support toxicology studies, and to generate data useful in predicting human pharmacokinetics (see discussion on scaling below). The most common routes of compound administration are oral and intravenous.

5.03.4 Metabolism Studies

Two aspects are of importance in the context of drug metabolism studies. The first aspect is the chemical nature of the metabolites formed in respect to their safety profile. All metabolites that are formed and are systemically available in man should ideally also be formed and reach at least similar systemic exposure in one animal species used in the preclinical toxicity program.8 Second, the enzymes mainly responsible for the primary metabolic clearance steps might exhibit species-specific expression patterns, inducibility, or inhibition potential. Many of the clinically relevant drug-drug interactions are based on an induction or inhibition of enzymes or other active processes involved in drug metabolism and elimination (e.g., transporters).

While numerous in vitro tools are available to study the main processes involved in drug metabolism and the metabolites formed, quantitative correlations between in vitro and in vivo metabolism are often difficult to make.9 In the early lead optimization process, the metabolic stability, biotransformations, and metabolite structures of new chemical entities (NCEs) are typically studied first using appropriate in vitro tools such as expressed human enzymes or cellular systems (e.g., hepatocytes).9 Often a ranking of compounds in terms of metabolic stability and metabolites formed is sufficient as output from these in vitro studies.9 Later on during drug development, differences in the systemic exposure to metabolites and the overall excretion pathways have to be addressed. As compounds are increasingly optimized for metabolic stability, extrahepatic metabolic routes and direct excretion of unchanged drug molecules are often seen, requiring in vivo studies in animals and man. The completeness of excretion, the excretory pathways, and the metabolite patterns in plasma and excreta are studied sequentially in the animal species involved in toxicity studies and finally in man using radiolabeled drugs. This chapter will focus on in vivo metabolism studies; in vitro studies are discussed in detail in Chapter 5.10. The Use of Radiolabeled Compounds

Radiolabeled drug molecules are useful for most in vitro and in vivo drug metabolism studies. The total sum of all drug-related molecules can be easily quantified in different biological matrices and the recoveries of sample work-up procedures can be determined. Also, chromatographic separation of the individual metabolites with easy quantification and detection of unknown metabolites for which no chemical standard is available is greatly facilitated by radiolabeled isotopes.10 Therefore, radiolabeled drugs are used in most in vivo ADME studies; 14C-labeled compounds are preferred for theses studies due to the higher metabolic stability of this isotope in the molecule as compared to the 3H label. The isotope is typically placed on the metabolically stable core group of the molecules. However, for more specific questions or depending on the metabolic steps involved, the label might be placed both on stable and labile moieties. Furthermore, double-labeled compounds with different isotopes (13C/14C or 3H/14C) might be synthesized to aid in metabolite identification and quantification of the individual moieties.10 Chromatographic Separation and Quantification of Drug Metabolites

High-performance liquid chromatography (HPLC) is the method of choice to analyze and quantify the parent drug and any metabolites, both from in vitro drug metabolism assays or in vivo animal studies using radiolabeled compounds. A new technique with a much higher sensitivity for the detection of 3H and 14C isotopes is accelerated mass spectroscopy (AMS), which has potential especially in human ADME studies. ADME studies in man can be performed with radioactive doses of very low specific activity, the total amount of radioactivity being in the range of nanocuries. Information on the completeness of excretion (mass balance), the excretion pathways, and also limited information on metabolites formed can be obtained very early on in drug development. In Vivo Animal Models Used for Absorption, Distribution, Metabolism, and Excretion Studies

A number of different animal models are used for in vivo drug metabolism studies. Nonoperated, naive animals are used for excretion balance studies and to assess the metabolite profiles in plasma, urine, and feces. For compounds with a high proportion of biliary elimination of total drug-related material, bile duct-cannulated animals are often used in addition. The total gastrointestinal drug absorption, the importance of first pass metabolism, and also secretion of drug and metabolites into the gastrointestinal tract (after intravenous administration) can be evaluated in this animal model.10 However, operated rats often show inflammatory reactions leading to induced or repressed phase I and phase II enzyme systems (cytochrome P450 and glucuronyltransferases) thereby significantly altering metabolic capacity. For specific questions, knock-out animals or strains with deficiencies in certain metabolic enzymes or transporters can be used to complement in vitro findings. P-glycoprotein knock-out mice are used to study the contribution of this transporter for limitations in brain uptake and to a minor degree also drug absorption in the GI tract. Humanized mice can be used to study the impact of the respective cytochrome P450 isoenzymes on the metabolism and the drug-drug interaction potential in an in vivo environment.13 Mass Balance and Route of Excretion Studies

In animals the full urinary and fecal balance is typically assessed at different developmental stages in the main species involved in safety studies, e.g., rats and dog or monkey.8 The nonrodent species used for teratology studies, typically the rabbit, and the second species used for carcinogenicity studies, typically the mouse, are not routinely investigated to this extent.14 The results are used for the preparation of the ADME study using a radiolabeled dose in man, which is typically initiated during clinical development phase I.

The mass balance study in rats is most often performed prior to initiation of clinical phase I studies14 in order to evaluate the kinetics and completeness of excretion and the excretion routes in this species. Both oral and intravenous administration are studied for most compounds and three to five animals are typically used per administration mode. The doses selected are within the pharmacologically relevant range and typically about 20 mCi of radiolabel are used per animal enabling enough sensitivity for quantification. A 1:1 mixture of cold and radiolabeled drug is ideal for later metabolite identification by LC/MS/MS. If the excretion is incomplete within 5 to 7 days (<90-95% recovery), the remaining carcasses are analyzed after dissolution for any remaining radiolabel. For the second species used (dogs or monkeys) the excretion balance study is often performed later, in parallel to the phase I studies, in preparation for the human mass balance study. Urine and feces samples from both rat and nonrodent studies can be further used for the analysis of metabolite patterns. Plasma samples are typically not taken from rats, so as not to interfere with the main objective of the study, the completeness of mass balance; for larger animals, however, samples can be taken for additional analyses. Metabolite Profiling in Animal Species and Man

One of the main goals of in vivo ADME studies is the evaluation of metabolite profiles in plasma and excreta and to investigate any species differences. Systemically available metabolites in plasma

The plasma exposure of parent drug and major metabolites along with any related interspecies differences are of special interest for interpretation of toxicology or carcinogenicity studies. However, it is only data from the radiolabeled human ADME study that allows safety margins for all the relevant metabolites to be finally established, as only then the systemic availability of the metabolites formed in man is known. It is possible to detect known or postulated metabolites early on in plasma samples from the first clinical studies in man with newer, more sensitive, and selective technologies such as LC/MS/MS. This technology, however, does not allow the quantification of all those metabolites for which no reference material is available. Furthermore, it is extremely difficult to detect all unknown metabolites.

Special attention should be drawn to major human metabolites, which account for a considerable amount of the AUC (area under the plasma concentration-time curve) relative to the parent drug, and metabolites, that are only seen in man.15 For both types of metabolites, a quantitative analytical assay should be set up in order to establish their kinetics and exposure in animals and man. For the human-specific metabolites and metabolites that do not reach comparable systemic exposure in at least one animal species used in the different toxicity studies, separate toxicity studies should be considered.15

Major metabolites in plasma are not necessarily major metabolites of the overall metabolism, as both the rates of formation and elimination as well as other kinetic parameters are important factors. An example of species-specific differences in the exposure of a major metabolite is given in Figure 1. Most of the radiolabeled test drug was eliminated in the feces in both man and cynomolgus monkey. The oxidative metabolite D was formed in addition to other oxidative metabolites in both the monkey and man to a similar extent (~ 15% of the dose) based on analysis of pooled feces samples. However, this metabolite was only a minor peak in cynomolgus monkey plasma, while in man it reached systemic plasma exposures comparable to, or even exceeding, that of the parent drug. Species-specific differences in the rates of elimination of this metabolite might be one reason for this observation, which was only apparent once human plasma samples were analyzed quantitatively for these metabolites. Metabolites found in excreta

The main aim of analyzing metabolites in excreta is to quantitatively evaluate the contribution of the different excretion routes and metabolic pathways to the overall elimination and metabolism of a test drug. This information is used to estimate the fraction metabolized by various pathways. Furthermore, metabolites can be purified from urine for elucidation of their structures. Purification from bile and feces, although more difficult, can also be successful.

For mostly renally cleared drug-related material, typically smaller molecular weight entities or some phase II metabolites, analysis and quantification is in most cases easy to accomplish. For compounds that are mainly eliminated via bile and feces, the metabolite patterns should be studied in both bile and feces for an overall evaluation of the nature and route of drug metabolites excreted. A feasible approach in the rat is to analyze urine and feces samples from nonoperated animals. This can easily be done with samples from the excretion balance study, allowing an overall quantitative assessment of the individual metabolites in excreta. In parallel studies bile and feces samples from bile duct-cannulated animals after intravenous and oral drug administration should be analyzed. This helps to understand the nature of biliary eliminated metabolites, their stability in feces, the fraction absorbed, the effect of first pass metabolism and bioavailability, and the potential for direct secretion of drug/metabolites into feces.10 A similar approach can be taken for dogs and monkeys, while for man the analysis of excreta is typically limited to urine and feces. It must be emphasized, however, that bile duct-cannulated animals often show altered metabolic activities or incomplete excretion within the study period. Therefore, it is very important to compare these observations with those in naive animals.


Cynomolgus monkey plasma, 4 h

-i—|—i—|—i—|—i—|—i—|—i—|—i—(—i—(—i—|—r-| 0 5 10 15 20 25 30 35 40 45 50

Human plasma, 4 h


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