Iain Gardner

Design of Ester Prodrugs to Enhance Oral Absorption of Poorly Permeable Compounds: Challenges to the Discovery Scientist

Many drugs are administered at sites that are remote from their site of action. The most common route of drug delivery is the oral route. The optimal physicochemical properties to allow high transcellular absorption following oral administration are well established and include a limit on molecular size, hydrogen bonding potential and adequate lipophilicity. For many drug targets, synthetic strategies can be devised to balance the physicochemical properties required for high transcellular absorption and the SAR for the drug target. However, there are drug targets where the SAR requires properties at odds with good membrane permeability. These include a requirement for significant polarity and groups that exhibit high hydrogen bonding potential such as carboxylic acids and alcohols. In such cases, prodrug strategies have been employed. The rationale behind the prodrug strategy is to introduce lipophilicity and mask hydrogen bonding groups of an active compound by the addition of another moiety, most commonly an ester. An ideal ester prodrug should exhibit the following properties: 1). Weak (or no) activity against any pharmacological target, 2). Chemical stability across a pH range, 3). High aqueous solubility, 4). Good transcellular absorption, 5). Resistance to hydrolysis during the absorption phase, 6). Rapid and quantitative breakdown to yield high circulating concentrations of the active component post absorption. This paper will review the literature around marketed prodrugs and determine the most appropriate prodrug characteristics. In addition, it will examine potential Discovery approaches to optimising prodrug delivery and recommend a strategy for prosecuting an oral prodrug approach.

Modelling and PBPK Simulation in Drug Discovery

Physiologically based pharmacokinetic (PBPK) models are composed of a series of differential equations and have been implemented in a number of commercial software packages. These models require species-specific and compound-specific input parameters and allow for the prediction of plasma and tissue concentration time profiles after intravenous and oral administration of compounds to animals and humans. PBPK models allow the early integration of a wide variety of preclinical data into a mechanistic quantitative framework. Use of PBPK models allows the experimenter to gain insights into the properties of a compound, helps to guide experimental efforts at the early stages of drug discovery, and enables the prediction of human plasma concentration time profiles with minimal (and in some cases no) animal data. In this review, the application and limitations of PBPK techniques in drug discovery are discussed. Specific reference is made to its utility (1) at the lead development stage for the prioritization of compounds for animal PK studies and (2) at the clinical candidate selection and “first in human” stages for the prediction of human PK.

Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae

This study examined the accumulation and metabolism of a number of drugs and commonly used probes for human cytochrome P450s (CYPs) in zebrafish larvae under conditions relevant to pharmacological and toxicological assays. Studies with cisapride, chlorpromazine, verapamil, testosterone, and dextromethorphan showed that the zebrafish larvae catalyze a range of phase 1 (oxidation, N-demethylation, O-de-ethylation, and N-dealkylation) and phase 2 (sulfation and glucuronidation) reactions. Both similarities and differences in the metabolic pathways were observed in zebrafish larvae when compared to mammals. Metabolism of phenacetin to paracetamol and dextromethorphan to dextrorphan (metabolic reactions catalyzed by CYP 1A2 and 2D6 in humans respectively) were observed in the zebrafish larvae. In addition the zebrafish larvae 7 days post fertilization (7 d.p.f.) hydroxylated diclofenac, bupropion, tacrine, and testosterone. Although metabolites of several compounds were detected in zebrafish larvae, in the instances where the metabolite amounts were quantified, the amount of any specific metabolite formed was low, accounting for only a small percentage of the amount of parent compound added. Furthermore, when the concentrations of metabolite present in the zebrafish larvae were compared with the measured level of parent compound, the metabolite concentrations were always much lower than that of parent compound. Overall, for the compounds used in the current study it is unlikely that the quantified metabolites would significantly contribute to the outcome of safety pharmacology or toxicology studies conducted in zebrafish larvae under the paradigms typically used for such investigations.

Simulation of Human Intravenous and Oral Pharmacokinetics of 21 Diverse Compounds Using Physiologically Based Pharmacokinetic Modelling

AbstractBackground: The importance of predicting human pharmacokinetics during compound selection has been recognized in the pharmaceutical industry. To this end there are many different approaches that are applied. Methods: In this study we compared the accuracy of physiologically based pharmacokinetic (PBPK) methodologies implemented in GastroPlus™ with the one-compartment approach routinely used at Pfizer for human pharmacokinetic plasma concentration-time profile prediction. Twenty-one Pfizer compounds were selected based on the availability of relevant preclinical and clinical data. Intravenous and oral human simulations were performed for each compound. To understand any mispredictions, simulations were also performed using the observed clearance (CL) value as input into the model. Results: The simulation results using PBPK were shown to be superior to those obtained via traditional one-compartment analyses. In many cases, this difference was statistically significant. Specifically, the results showed that the PBPK approach was able to accurately predict passive distribution and absorption processes. Some issues and limitations remain with respect to the prediction of CL and active transport processes and these need to be improved to further increase the utility of PBPK modelling. A particular advantage of the PBPK approach is its ability to accurately predict the multiphasic shape of the pharmacokinetic profiles for many of the compounds tested. Conclusion: The results from this evaluation demonstrate the utility of PBPK methodology for the prediction of human pharmacokinetics. This methodology can be applied at different stages to enhance the understanding of the compounds in a particular chemical series, guide experiments, aid candidate selection and inform clinical trial design.

An Introduction to Drug Disposition: The Basic Principles of Absorption, Distribution, Metabolism, and Excretion

A knowledge of the fate of a drug, its disposition (absorption, distribution, metabolism, and excretion, known by the acronym ADME) and pharmacokinetics (the mathematical description of the rates of these processes and of concentration-time relationships), plays a central role throughout pharmaceutical research and development. These studies aid in the discovery and selection of new chemical entities, support safety assessment, and are critical in defining conditions for safe and effective use in patients. ADME studies provide the only basis for critical judgments from situations where the behavior of the drug is understood to those where it is unknown: this is most important in bridging from animal studies to the human situation. This presentation is intended to provide an introductory overview of the life cycle of a drug in the animal body and indicates the significance of such information for a full understanding of mechanisms of action and toxicity.

Application of PBPK modelling in drug discovery and development at Pfizer

Early prediction of human pharmacokinetics (PK) and drug–drug interactions (DDI) in drug discovery and development allows for more informed decision making. Physiologically based pharmacokinetic (PBPK) modelling can be used to answer a number of questions throughout the process of drug discovery and development and is thus becoming a very popular tool. PBPK models provide the opportunity to integrate key input parameters from different sources to not only estimate PK parameters and plasma concentration-time profiles, but also to gain mechanistic insight into compound properties. Using examples from the literature and our own company, we have shown how PBPK techniques can be utilized through the stages of drug discovery and development to increase efficiency, reduce the need for animal studies, replace clinical trials and to increase PK understanding. Given the mechanistic nature of these models, the future use of PBPK modelling in drug discovery and development is promising, however, some limitations need to be addressed to realize its application and utility more broadly.

Pre-clinical pharmacokinetics of UK-453,061, a novel non-nucleoside reverse transcriptase inhibitor (NNRTI), and use of in silico physiologically based prediction tools to predict the oral pharmacokinetics of UK-453,061 in man

1. UK-453,061 is a novel second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI). Following intravenous bolus administration of UK-453,061 in male rat and infusion administration in dog, UK-453,061 had the following mean pharmacokinetic properties: elimination T 1/2 of 1.6 and 2.4 h, CLp of 26 and 10 ml min−1 kg−1 and V ss of 1.6 and 2 l kg−1, respectively. 2. The half-lives of UK-453,061 disappearance in recombinant human CYPs 2C8, 2C9, 2A6, 2E1, 1A2, 2C19, 2D6 and 3A4 were 71, 100, 56, 101, 61, 34, 60 and 8 min, respectively. The disappearance half-life of UK-453,061 in human liver microsomes in the presence of UDPGA was 90 min. 3. Human clearance values were predicted using single-species scaling from in vivo data and from in vitro data using SimCYP. The human distribution of UK-453,061 was estimated using an in silico physiologically based pharmacokinetics (PBPK) methodology and absorption was predicted from measured physicochemical, permeability, and solubility data using GastroPlus and SimCYP. The C max was predicted to be 68, 185, 149% of the actual mean value using rat, dog and in vitro predicted values of human clearance at 30 mg and 53, 150, 29% of actual at 500 mg. The area under the curve (AUC) was predicted to be 73, 285 and 142% of the actual mean value using rat, dog and in vitro predicted values of human clearance at 30 mg and 52, 212 and 35% of actual at 500 mg. 4. This study demonstrates the utility of using in silico PBPK approaches to make predictions of human pharmacokinetics before dosing for the first time in humans.

The metabolism and toxicity of furosemide in the Wistar rat and CD-1 mouse: a chemical and biochemical definition of the toxicophore.

Furosemide, a loop diuretic, causes hepatic necrosis in mice. Previous evidence suggested hepatotoxicity arises from metabolic bioactivation to a chemically reactive metabolite that binds to hepatic proteins. To define the nature of the toxic metabolite, we examined the relationship between furosemide metabolism in CD-1 mice and Wistar rats. Furosemide (1.21 mmol/kg) was shown to cause toxicity in mice, but not rats, at 24 h, without resulting in glutathione depletion. In vivo covalent binding to hepatic protein was 6-fold higher in the mouse (1.57 ± 0.98 nmol equivalent bound/mg protein) than rat (0.26 ± 0.13 nmol equivalent bound/mg protein). In vivo covalent binding to mouse hepatic protein was reduced 14-fold by a predose of the cytochrome P450 (P450) inhibitor, 1-aminobenzotriazole (ABT; 0.11 ± 0.04 nmol equivalent bound/mg protein), which also reduced hepatotoxicity. Administration of [14C]furosemide to bile duct-cannulated rats demonstrated turnover to glutathione conjugate (8.8 ± 2.8%), γ-ketocarboxylic acid metabolite (22.1 ± 3.3%), N-dealkylated metabolite (21.1 ± 2.9%), and furosemide glucuronide (12.8 ± 1.8%). Furosemide-glutathione conjugate was not observed in bile from mice dosed with [14C]furosemide. The novel γ-ketocarboxylic acid, identified by nuclear magnetic resonance spectroscopy, indicates bioactivation of the furan ring. Formation of γ-ketocarboxylic acid was P450-dependent. In mouse liver microsomes, a γ-ketoenal furosemide metabolite was trapped, forming an N-acetylcysteine/N-acetyl lysine furosemide adduct. Furosemide (1 mM, 6 h) became irreversibly bound to primary mouse and rat hepatocytes, 0.73 ± 0.1 and 2.44 ± 0.3 nmol equivalent bound/mg protein, respectively, which was significantly reduced in the presence of ABT, 0.11 ± 0.03 and 0.21 ± 0.1 nmol equivalent bound/mg protein, respectively. Furan rings are part of new chemical entities, and mechanisms underlying species differences in toxicity are important to understand to decrease the drug attrition rate.

Are endosomal trafficking parameters better targets for improving mAb pharmacokinetics than FcRn binding affinity

F.W.R. Brambell deduced the existence of a protective receptor for IgG, the neonatal Fc receptor (FcRn), long before its discovery in the early to mid-1990s. With the coincident, explosive development of IgG-based drugs, FcRn became a popular target for tuning the pharmacokinetics of monoclonal antibodies (mAbs). One aspect of Brambells initial observation, however, that is seldom discussed since the discovery of the receptor, is the compliance in the mechanism that Brambell observed (saturating at 10s-100s of μM concentration), vs. the comparative stiffness of the receptor kinetics (saturating in the nM range for most species). Although some studies reported that increasing the already very high Fc-FcRn affinity at pH 6.0 further improved mAb half-life, in fact the results were mixed, with later studies increasingly implicating non-FcRn-dependent mechanisms as determinants of mAb pharmacokinetics. Mathematical modelling of the FcRn system has also indicated that the processes determining the pharmacokinetics of mAbs have more nuances than had at first been hypothesised. We propose, in keeping with the latest modelling and experimental evidence reviewed here, that the dynamics of endosomal sorting and trafficking have important roles in the compliant salvage mechanism that Brambell first observed nearly 50 years ago, and therefore also in the pharmacokinetics of mAbs. These ideas lead to many open questions regarding the endosomal trafficking of both FcRn and mAbs and also to what properties of a mAb can be altered to achieve an improvement in pharmacokinetics.

A Physiologically Based Pharmacokinetic Modeling Approach to Predict Disease–Drug Interactions: Suppression of CYP3A by IL‐6

Elevated cytokine levels are known to downregulate expression and suppress activity of cytochrome P450 enzymes (CYPs). Cytokine‐modulating therapeutic proteins (TPs) used in the treatment of inflammation or infection could reverse suppression, manifesting as TP‐drug–drug interactions (TP‐DDIs). A physiologically based pharmacokinetic model was used to quantitatively predict the impact of interleukin‐6 (IL‐6) on sensitive CYP3A4 substrates. Elevated simvastatin area under the plasma concentration–time curve (AUC) in virtual rheumatoid arthritis (RA) patients, following 100 pg/ml of IL‐6, was comparable to observed clinical data (59 vs. 58%). In virtual bone marrow transplant (BMT) patients, 500 pg/ml of IL‐6 resulted in an increase in cyclosporine AUC, also in good agreement with the observed data (45 vs. 39%). In a different group of BMT patients treated with cyclosporine, the magnitude of interaction with IL‐6 was underpredicted by threefold. The complexity of TP‐DDIs highlights underlying pathophysiological factors to be considered, but these simulations provide valuable first steps toward predicting TP‐DDI risk.