Current Drug Metabolism - Volume 7, Issue 2, 2006

Clinical findings indicate that co-administration of the isoxazolyl-penicillin flucloxacillin with cyclosporine may reduce the plasma concentrations of cyclosporine. We have explored in the present study if induction of cytochrome P450 3A4 or P-glycoprotein may offer a mechanistic explanation of the observed effects. Flucloxacillin is neither an inhibitor nor a substrate of drug metabolizing cytochrome P450 isoenzymes (CYP3A4, 1A2, 2C9, 2C19 and 2D6) or Pglycoprotein as shown by an in vitro assay for CYP inhibition, a fluorescent indicator assay for P-glycoprotein inhibition and a functional P-glycoprotein ATPase assay. However, incubation of human LS 180 colorectal adenocarcinoma cells with flucloxacillin led to a dose-dependent induction of MDR1 as well as of CYP3A4 mRNA, which was also confirmed in primary human hepatocytes. At high concentrations, flucloxacillin activated the human Pregnane-X-Receptor, PXR, a ligand-dependent transcription factor that is the target of many drugs that induce CYP3A4, with consequences for the metabolism of other drugs. Liver microsomes from control rats or rats, which received for 3 consecutive days 100 mg/kg of oral flucloxacillin, were used to study the metabolism and metabolite pattern of midazolam, a model substrate of CYP 3A4. There was a trend towards a higher intrinsic microsomal clearance of midazolam using microsomes from flucloxacillin treated rats. In addition, there was a significant increase in the formation of the principal midazolam metabolites 1- hydroxy midazolam, 4-hydroxy midazolam and 1,4-dihydroxy midazolam as compared to controls. These findings indicate that flucloxacillin has the potential to induce expression of both CYP3A4 as well as P-glycoprotein, most likely through activation of the nuclear hormone receptor PXR. This would offer an explanation for the observed clinical drugdrug interactions between the antibiotic and cyclosporine.

A review with 103 references. Fluoxetine is the parent drug of the SSRI (selective serotonin reuptake inhibitor) antidepressant class, and is still one of the most highly used drugs of this class world-wide. Fluoxetine now has largely (albeit not completely) substituted older and less safe drugs such as tricyclic antidepressants. Different cytochrome P450 isoforms are involved in the metabolism of fluoxetine, however, the main active metabolite, norfluoxetine, is produced by the CYP2D6 action in the human liver. In this paper, the main metabolic characteristics of fluoxetine will be reviewed, with particular attention paid to the role of cytochrome isozymes. The pharmacological interactions of the drug will be overviewed, especially those concerning other drugs used in psychiatric clinics, such as antipsychotics and antidepressants and the relationships between pharmacological interactions and cytochrome activity will be discussed. Recently, much attention has been drawn to the therapeutic drug monitoring (TDM) of fluoxetine, and in particular to the analysis of fluoxetine enantiomers for which enantiomeric separations and enantioselective metabolism will also briefly be mentioned.

The purpose of this study was to propose a new method to predict in vivo drug-drug interactions (DDIs) for a high clearance drug from in vitro data. As the high clearance drug, NE-100 (N, N-dipropyl-2-[4-methoxy-3-(2- phenylethoxy)phenyl]ethylamine monohydrochloride) was used. First, approach based on Iu/Ki value was used for the prediction of DDIs between NE-100 and concomitant drugs. When the Ki values (Ki-cal) obtained from the microtiter plate (MTP) assay and the reported Ki values (Ki-rep) for these drugs were used to predict increases at levels of NE-100 AUCoral (AUCoral ratio), the AUCoral ratios from the Iu /Ki-cal correlated with those from the Iu/Ki-rep. This result suggests that the Kical from the MTP assay can be used for prediction of DDIs instead of the Ki-rep value. Second, a new approach combining the inhibition rate (R) calculated from the MTP assay and two physiological models was used to predict DDIs. When the AUCoral ratios of NE-100 by various drugs were predicted using the R value and the well-stirred model, the ratios were similar to those predicted using the Iu/Ki. However, after co-administration of drugs such as quinidine, propafenone and thioridazine (potent inhibitors of CYP2D6), the NE-100 AUCoral ratios predicted from the dispersion model was much greater than those from well-stirred model. This result shows that application of the dispersion model to the prediction method using the R value might sensitively and precisely predict the increased levels of AUCoral by DDIs for high clearance drug, compared with the prediction method using Iu/Ki value.

Acyl glucuronides are potentially reactive intermediates, which not only undergo hydrolysis and intramolecular acyl migration, but also bind irreversibly to plasma protein in vitro and in vivo. To evaluate the impact of renal failure, liver dysfunction and other disease states on the pharmacokinetics of acyl glucuronides and their parent compounds, a pharmacokinetic model has been established. The model has been successfully utilized to predict the pharmacokinetics of six compounds, diflunisal (DF), valproic acid (VPA), zomepirac (Z), suprofen (S), R-etodolac (R-ET), S-etodolac (S-ET), and their acyl glucuronides in various simulated disease states in experimental animals. Modeling studies revealed that altering the metabolic pathways of these compounds had significant impact on exposure and clearance of acyl glucuoninde. The simulation results also indicated that disease states that affect irreversible metabolic pathways other than glucuronidation may have major impacts on the apparent plasma clearance of the parent compound or exposure to the reactive acyl glucuronide as well. The study concluded that the model is sufficiently robust and applicable for pharmacokinetic studies of acyl glucuronides and their parent compounds in various disease states that may modulate drug clearance. The model is also applicable to understanding the complex disposition of other drugs subject to conjugation, especially those that can be reversible and undergo enterohepatic recycling, such as sulfation and glycine conjugation.

Lack of efficacy and toxicity are considered to be major reasons for drug failures and pharmacokinetics governs them to a large extent. Compound with favorable pharmacokinetics is more likely to be efficacious and safe. Therefore, the preclinical pharmacokinetic evaluation should be comprehensive enough to ensure that compounds do not fail in the clinic. Preclinical ADME screening facilitates early elimination of weak candidates and directs the entire focus of the drug development program towards fewer potential lead candidates. Hence, it is mandatory that the pre-clinical candidates are subjected to as many possible reality checks. Reliance on in-vitro tests should be minimized because they do not represent the real physiological environment but rather slow down the pace of a drug discovery program. Compounds can be straight away subjected to in-vivo high throughput screens such as cassette dosing, cassette analysis or rapid rat screen etc. Candidates with the desired in-vivo pharmacokinetic profile may be further profiled in-vitro, using assays such as metabolic stability, reaction phenotyping, CYP-450 inhibition and induction, plasma protein binding etc. in human microsomes, human recombinant CYP-450 enzymes and human plasma. This also provides an early indication of whether the compound which worked in animals would work in human as well. In-vitro metabolic stability profile is a qualitative as well as quantitative comparison of metabolism of a compound in human and animal models. It helps in identifying the right model for toxicity studies. Extensive metabolism is generally considered a liability as it limits the systemic exposure and shortens the half-life of a compound. Several strategies such as reduction of lipophilicity, modification and / or blocking of metabolically soft spots and use of enzyme inhibitors; have been developed to combat metabolism. In spite of several concerns, the fact that active metabolites of several marketed drugs have been developed as drugs with better efficacy, safety and pharmacokinetics profile; cannot be denied. Therefore, instead of considering metabolic instability a liability it can be exploited as a tool for discovering better drugs. It is equally important to identify the metabolic pathways of the drug candidates by conducting in-vitro CYP450 reaction phenotyping assays. The identification of drug metabolizing enzymes involved in the major metabolic pathways of a compound helps in predicting the probable drug-drug interactions in human. Compounds with more than one metabolic pathway have less likelihood of clinically significant drug interactions. In-vitro CYP450 inhibition and induction screens are used to evaluate the potential of compound towards drug - drug interactions and the most prone candidates may either be discarded or taken ahead with a caution. It is known that only unbound drug is pharmacologically active and therefore the assessment of bound fraction by the estimation of plasma protein binding of a compound is another important parameter to be explored in-vitro. In addition to the process of 'weeding out' weak candidates early in the drug discovery process, it is equally important to identify the probable causes of poor ADME exhibited by some compounds as this information is useful to medicinal chemists for improving upon backbones that exhibit un favorable pharmacokinetic profile. Toxicity study is the foundation of an INDA (Investigational new drug application) and therefore, the final selection of a compound can be performed only after proper toxicological evaluation in animal models. Toxicokinetics forms an integral part of toxicity study and is used to assess the exposure of candidates in toxicity models and correlate the drug levels in blood and various tissues with the toxicological findings. Although in-vivo screening of compounds in animal models and invitro assays in human recombinant CYP-450 enzymes help in drug candidate selection, both approaches have their own limitations. There is no certainty that the selected candidates will exhibit the desired target PK profile in human and real human PK remains suspense until the compound enters Phase-1 clinical trial. The recognition of human micro dosing, (HMD) by medicines and healthcare products regulatory agency (MHRA) and European agency for evaluation of medicinal products [EMEA] is a stepping stone in the direction of obtaining human PK data early in the preclinical stage. This would gradually shift the focus of early drug development away from animal studies directly towards safe and ethical studies in human yielding more relevant and reliable pharmacokinetic data. HMD would provide an answer to the growing public demand for a reduction in the use of animals for pharmaceutical development.

The blood-brain barrier (BBB) remains one of the greatest challenges for the discovery and development of treatments for CNS disorders, which to this day remains one of the riskiest disease areas in terms of clinical success rates. Although the BBB is currently seen predominantly as a permeability obstacle for CNS drug delivery, it is becoming increasingly clear that the BBB has many more implications for the pharmaceutical industry impacting on CNS pharmacology and pathology, CNS pharmacokinetics and pharmacodynamics, and adverse CNS effects, to name but a few areas. The present review does not intend to summarize the activities in the field of BBB research per se, which has been accomplished by a number of excellent recent reviews, but instead to provide an overview of the role of BBB studies from a pharmaceutical industry perspective. This review will elaborate on the specific needs in terms of BBB-related issues across the different drug discovery and development phases, i.e. target identification and validation, lead generation and optimization, candidate selection and profiling, preclinical development and clinical studies. The specific approaches taken will be discussed in terms of specific requirements, questions to be asked, feasibility, interpretability, and impact. It becomes clear that few of the existing BBB models fully meet the requirements of the industrialized drug discovery process, highlighting the need for an array of new or modified tools and approaches that are more effective in helping make decisions which are more specifically tailored to the various stages of the lengthy process from target to the clinic. In looking at the numerous ongoing activities in the area of BBB research from the drug discovery and development point of view, an attempt has been made to place a stronger emphasis on the applicability of particular techniques and approaches, to identify gaps and areas for future activities. In order to materialize the considerable knowledge gained in recent years, the review is intended to foster an increased awareness of the need to better integrate basic academic research with the specific requirements of the pharmaceutical industry for the search of effective and safe new CNS medicines.

Phospho-glycoprotein (P-gp) is an efflux transporter expressed in many organs (ex: kidney, lung, liver and spleen) and in hormone producing or responsive tissues (ex: adrenal cortex, testis and placenta). It is involved in many important physiological functions. Among them the major one is extrusion of xenobiotics in order to detoxify the cells. This property of P-gp is associated with multidrug resistance (MDR) for many pathological conditions. While the experimental determination of three-dimensional structure is not yet successful, the transmembrane (TM) 5, 6, 11 and 12 are sensitive to mutations and contain substrate binding sites. Designing of potential and selective inhibitors of P-gp is still hampered by a lack of information upon the three dimensional structure of P-gp. The design of P-gp inhibitors was traditionally driven by quantitative structure activity relationship studies, which is complicated by factors such as different types of assays, multiple drug binding sites and diverse chemical structures. Clearly a conclusive and predictive SAR does not seem to be practical, despite progress in the last few years towards more specific SAR suggesting well defined structural features responsible for activity. Advances made recently in solving the crystal structure of prokaryotic ATP binding cassette proteins (ABC) transporters, Ec-MsbA, Vc-MsbA and BtuCD yielded suitable templates for construction of homology models of P-gp. Few molecular dynamics (MD) simulations aimed at elucidating the functional dynamics of ABC transporters have provided useful insights to their mechanism and structure. The present review aims at the general overview of importance, expression, structure, organization and drug binding sites of P-gp. This review also highlights recent developments in the homology modeling, molecular dynamics simulations of P-gp and progress in QSAR, pharmacophore modeling of P-gp modulators.