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In the field of gene expression analysis, DNA microarray technology is having a major impact on many different areas including toxicology. For instance, a number of studies have shown that transcription profiling can generate the information needed to assign a compound to a mode-of-action class. In this study, we investigated whether compounds inducing similar toxicological endpoints produce similar changes in gene expression. In vitro primary rat hepatocytes were exposed to 11 different hepatotoxicants: acetaminophen, amiodarone, clofibrate, erythromycin estolate, isoniazid, α-naphtylylisothiocyanate, β-naphtoflavone, 4-pentenoic acid, phenobarbital, tetracycline, and zileuton. These molecules were selected on the basis of their variety of hepatocellular effects observed such as necrosis, cholestasis, steatosis, and induction of CYP P450 enzymes. We used a low-density DNA microarray containing 59 genes chosen as relevant toxic and metabolic markers. The in vitro gene expression data generated in this study were generally in good agreement with the literature, which mainly concerns in vivo data. Furthermore, gene expression profiles observed in this study have been confirmed for several genes by real-time PCR assays. All the tested drugs generated a specific gene expression profile. Our results show that even with a relatively limited gene set, gene expression profiling allows a certain degree of classification of compounds with similar hepatocellular toxicities such as cholestasis, necrosis. The clustering analysis revealed that the compounds known to cause steatosis were linked, suggesting that they functionally regulate similar genes and possibly act through the same mechanisms of action. On the other hand, the drugs inducing necrosis and cholestasis were pooled in the same cluster. The drugs arbitrarily classified as the CYP450 inducers formed individual clusters. In conclusion, this study suggests that low-density microarrays could be useful in toxicological studies. [ABSTRACT FROM AUTHOR]

N-Nitrosamine impurities, including nitrosamine drug substance-related impurities (NDSRIs), have challenged pharmaceutical industry and regulators alike and affected the global drug supply over the past 5 years. Nitrosamines are a class of known carcinogens, but NDSRIs have posed additional challenges as many lack empirical data to establish acceptable intake (AI) limits. Read-across analysis from surrogates has been used to identify AI limits in some cases; however, this approach is limited by the availability of robustly-tested surrogates matching the structural features of NDSRIs, which usually contain a diverse array of functional groups. Furthermore, the absence of a surrogate has resulted in conservative AI limits in some cases, posing practical challenges for impurity control. Therefore, a new framework for determining recommended AI limits was urgently needed. Here, the Carcinogenic Potency Categorization Approach (CPCA) and its supporting scientific rationale are presented. The CPCA is a rapidly-applied structure-activity relationship-based method that assigns a nitrosamine to 1 of 5 categories, each with a corresponding AI limit, reflecting predicted carcinogenic potency. The CPCA considers the number and distribution of α-hydrogens at the N-nitroso center and other activating and deactivating structural features of a nitrosamine that affect the α-hydroxylation metabolic activation pathway of carcinogenesis. The CPCA has been adopted internationally by several drug regulatory authorities as a simplified approach and a starting point to determine recommended AI limits for nitrosamines without the need for compound-specific empirical data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

There are more than 3,500 active pharmaceutical ingredients (APIs) on the global market for human and veterinary use. Residues of these APIs eventually reach the aquatic environment. Although an environmental risk assessment (ERA) for marketing authorization applications of medicinal products is mandatory in the European Union since 2006, an ERA is lacking for most medicines approved prior to 2006 (legacy APIs). Since it is unfeasible to perform extensive ERA tests for all these legacy APIs, there is a need for prioritization of testing based on the limited data available. Prioritized APIs can then be further investigated to estimate their environmental risk in more detail. In this study, we prioritized more than 1,000 APIs used in Europe based on their predicted risk for aquatic freshwater ecosystems. We determined their risk by combining an exposure estimate (Measured or Predicted Environmental Concentration; MEC or PEC, respectively) with a Predicted No Effect Concentration (PNEC). We developed several procedures to combine the limited empirical data available with in silico data, resulting in multiple API rankings varying in data needs and level of conservativeness. In comparing empirical with in silico data, our analysis confirmed that the PEC estimated with the default parameters used by the European Medicines Agency often - but not always - represents a worst-case scenario. Comparing the ecotoxicological data for the three main taxonomic groups, we found that fish represents the most sensitive species group for most of the APIs in our list. We furthermore show that the use of in silico tools can result in a substantial underestimation of the ecotoxicity of APIs. After combining the different exposure and effect estimates into four risk rankings, the top-ranking APIs were further screened for availability of ecotoxicity data in data repositories. This ultimately resulted in the prioritization of 15 APIs for further ecotoxicological testing and/or exposure assessment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cristiana Cannata reports financial support was provided by PREMIER (Prioritisation and Risk Evaluation of Medicines in the EnviRonment). Irene Bramke reports a relationship with AstraZeneca that includes: employment., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The unexpected detection of nitrosamine impurities in human medicines has recently seen global regulators act to understand the risks of these contaminations to patients and to limit their presence. Over 300 nitrosamines are known, many of which are highly potent mutagenic carcinogens. Regulators first became aware of the presence of nitrosamines in EU medicines in 2018, with reports of detection of N -nitroso-dimethylamine (NDMA) in valsartan from one manufacturer. A subsequent EU review of all valsartan medicines was triggered by the European Medicines Agency (EMA) and was later extended to other angiotensin receptor blockers/sartans. A separate review was also started for ranitidine medicines. This was followed by an EU-wide examination of the risk of presence of nitrosamines in all human medicines. This article reflects on the investigation of the EU regulatory network into the presence of nitrosamines and the scientific knowledge informing recommendations for developers on how to limit nitrosamines in medicines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ruepp, Frötschl, Bream, Filancia, Girard, Spinei, Weise and Whomsley.)

Gene therapy medicinal products have the potential to provide curative treatment for many diseases with current limited therapeutic options. As advanced therapy medicinal products (ATMPs), these therapies undergo a centralised, single European Union authorisation by the European Medicines Agency (EMA), but the risks and potential harm to the environment and population at large are weighted in each application, and different interpretations at national level exist. A streamlined procedure is now in place to facilitate a consistent approach for the assessment of the environmental risks of medicines containing genetically modified organisms for both clinical trial applications and marketing authorisation applications. This article provides an overview of basic requirements across the EU, an overview of the new streamlined process and discusses available guidance for developers with particular emphasis on marketing authorisation applications. All these initiatives are aimed to remove hurdles for ATMP developers and facilitate faster access to patients., (© 2021 British Pharmacological Society.)

Background Transarterial chemoembolization with cytotoxic drugs is standard treatment for unresectable intermediate-stage hepatocellular carcinoma but achieves suboptimal outcomes because of hypoxic stress and the production of detrimental proangiogenic factors. An alternative approach using radiopaque embolization beads loaded with the antiangiogenic drug vandetanib may provide improved anticancer efficacy. Purpose To evaluate the pharmacokinetics, safety, and efficacy of vandetanib-eluting radiopaque bead (VERB) chemoembolization of rabbit liver tumors. Materials and Methods Between April 2015 and March 2016, 60 New Zealand white rabbits with VX2 liver tumors were randomly treated with VERBs at different doses, with nonloaded radiopaque beads (ROBs), or with intra-arterial vandetanib suspension (VS) or were not treated. Vandetanib plasma concentration and tumor growth at US were evaluated. Animals were euthanized after 3 days or 3 weeks. Assessment included bead distribution at x-ray imaging and histologic examination, tumor viability at histologic examination, and vandetanib tissue concentration. Group comparison analysis (Mann-Whitney, Kruskal-Wallis, and χ 2 tests) and predictive factor analysis for tumor growth and viability were performed. Results Vandetanib plasma concentration was lower with VERBs than with VS ( P < .01), while concentration in tumor was higher for VERBs (than for VS) at 3 days (median, 29.2 vs 2.74 ng/mg; P = .48). Tumor growth was lower with VERBs than with ROBs and with VS at both time points, with median values of +114%, +192%, and +466% at 3 weeks, respectively. Tumor viability was lower at 3 days for VERBs than for ROBs and for VS (3%, 18%, and 38%, respectively) but was not significantly different at 3 weeks. The volume of bead in tumor was a significant predictive factor for lower tumor growth in multivariable analysis at 3 days ( P = .03). Drug tumor concentration was a significant predictive factor for lower tumor growth at 3 weeks ( P = .04). Conclusion Vandetanib-eluting radiopaque bead chemoembolization showed a pharmacokinetic advantage over intra-arterial drug administration in a preclinical model of liver cancer. High deposition of beads and high vandetanib concentration in tumor led to stronger antitumor effects. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Kim and Van den Abbeele in this issue.

Purpose: To evaluate the plasma and tissue pharmacokinetics, safety and toxicity following intra-arterial hepatic artery administration of Vandetanib (VTB)-eluting Radiopaque Beads (VERB) in healthy swine., Materials and Methods: In a first phase, healthy swine were treated with hepatic intra-arterial administration of VERB at target dose loading strengths of 36 mg/mL (VERB36), 72 mg/mL (VERB72) and 120 mg/mL (VERB120). Blood and tissue samples were taken and analysed for VTB and metabolites to determine pharmacokinetic parameters for the different dose forms over 30 days. In a second phase, animals were treated with unloaded radiopaque beads or high dose VTB loaded beads (VERB100, 100 mg/mL). Tissue samples from embolized and non-embolized areas of the liver were evaluated at necropsy (30 and 90 days) for determination of VTB and metabolite levels and tissue pathology. Imaging was performed prior to sacrifice using multi-detector computed tomography (MDCT) and imaging findings correlated with pathological changes in the tissue and location of the radiopaque beads., Results: The peak plasma levels of VTB (C max ) released from the various doses of VERB ranged between 6.19-17.3 ng/mL indicating a low systemic burst release. The plasma profile of VTB was consistent with a distribution phase up to 6 h after administration followed by elimination with a half-life of 20-23 h. The AUC of VTB and its major metabolite N-desmethyl vandetanib (NDM VTB) was approximately linear with the dose strength of VERB. VTB plasma levels were at or below limits of detection two weeks after administration. In liver samples, VTB and NDM VTB were present in treated sections at 30 days after administration at levels above the in vitro IC 50 for biological effectiveness. At 90 days both analytes were still present in treated liver but were near or below the limit of quantification in untreated liver sections, demonstrating sustained release from the VERB. Comparison of the reduction of the liver lobe size and associated tissue changes suggested a more effective embolization with VERB compared to the beads without drug., Conclusions: Hepatic intra-arterial administration of VERB results in a low systemic exposure and enables sustained delivery of VTB to target tissues following embolization. Changes in the liver tissue are consistent with an effective embolization and this study has demonstrated that VERB100 is well tolerated with no obvious systemic toxicity., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Drugs for allergy are often taken in combination with other drugs, either to treat allergy or other conditions. In common with many pharmaceuticals, most such drugs are subject to metabolism by P450 enzymes and to transmembrane transport. This gives rise to considerable potential for drug-drug interactions, to which must be added consideration of drug-diet interactions. The potential for metabolism-based drug interactions is increasingly being taken into account during drug development, using a variety of in silico and in vitro approaches. Prediction of transporter-based interactions is not as advanced. The clinical importance of a drug interaction will depend upon a number of factors, and it is important to address concerns quantitatively, taking into account the therapeutic index of the compound.

The olfactory neuro-epithelium is highly sensitive to chemicals and its direct microbiological environment. It also plays a role as an interface between the airways and the nervous system, and so it has developed several defence instruments for rapid regeneration or for the detoxification of the immediate environment. This review illustrates three of these defence mechanisms: regeneration of the epithelium, local production of metabolising enzymes and xenobiotic transporters. Toxicants can inflict damage by a direct toxic response. Alternatively, they may require metabolic activation to produce the proximate toxicant. In addition to detoxifying inhaled and systemically derived xenobiotics, the local olfactory metabolism may fulfil multiple functions such as the modification of inhaled odorant, the modulation of endogenous signalling molecules and the protection of other tissues such as the CNS and lungs from inhaled toxicants. Finally, the permeability of nasal and olfactory mucosa is an important efficacy parameter for some anti-allergic drugs delivered by intranasal administration or inhalation. Efflux or update transporters expressed in these tissues may therefore significantly influence the pharmacokinetics of drugs administered topically.

Pharmacokinetics are typically dependent on a variety of physiological variables (e.g., age, ethnicity, or pregnancy) or pathological conditions (e.g., renal and hepatic insufficiency, cardiac dysfunction, obesity, etc.). The influence of some of these conditions has not always been thoroughly assessed in the clinical studies of antiallergic drugs. However, the knowledge of the physiological grounds of the pharmacokinetics can provide some insight for predicting the potential alterations and guiding the initial prescription strategies. It is important to recognize that both pharmacokinetic and pharmacodynamic differences between populations should be considered. The available information on drugs used for the therapy of allergic diseases is reviewed in this chapter.

In this article, aspects of absorption, distribution, metabolism, and excretion have been described bearing in mind the pathogenesis of allergic diseases and their possible therapeutic opportunities. The importance of the routes of administration of the different therapeutic groups has been emphasized. The classical aspects of drug metabolism and disposition related to oral administration have been reviewed, but special emphasis has been given to intranasal, cutaneous, transdermal, and ocular administration as well as to the absorption and the subsequent bioavailability of drugs. Drug-metabolizing enzymes and transporters present in extrahepatic tissues, such as nasal mucosa and the respiratory tract, have been particularly discussed. As marketed antiallergic drugs include both racemates and enantiomers, aspects of stereoselective absorption, distribution, metabolism, and excretion have been discussed. Finally, a new and promising methodology, microdosing, has been presented, although it has not yet been applied to drugs used in the treatment of allergic diseases.

Competition for uptake and/or efflux transporters can be responsible for drug interactions. Cetirizine is mainly eliminated unchanged in urine through both glomerular filtration and tubular secretion. The aim of this study was to investigate whether the eutomer, levocetirizine, and the distomer, dextrocetirizine, have a similar tubular secretion. The renal clearance associated with tubular secretion was calculated from the renal clearance of levocetirizine and dextrocetirizine obtained in a study in healthy volunteers. The values of the unbound fraction in plasma were obtained in an in vitro study of the binding of (14)C-cetirizine and (14)C-levocetirizine to human plasma proteins using equilibrium dialysis and chiral high-performance liquid chromatography (HPLC) with on-line liquid scintillation counting. The unbound fraction was 0.074 for levocetirizine and 0.141 for dextrocetirizine. The tubular secretion of dextrocetirizine (44.5 mL/min) is higher than that of levocetirizine (23.1 mL/min), which may have consequences for drug interactions at the renal level. The higher tubular secretion for dextrocetirizine may be due to the higher free fraction available for secretion or to a higher affinity for (a) renal transporter(s) mediating the secretion pathway.

In this review, the major enzyme systems involved in vivo in the oxidative metabolism of xenobiotic amines in humans are discussed, i.e. the monooxygenases [cytochrome P450 system (CYPs) and flavin-containing monooxygenases (FMOs)] and the amine oxidases (AOs). Concerning the metabolism of xenobiotic amines (drugs in particular) by monoamine oxidases (MAOs), this aspect has been largely neglected in the past. An exception is the extensive investigation carried out on the inhibition of the metabolism of tyramine, when tyramine-containing food is ingested by subjects taking inhibitors of MAO A or of both MAO A and B. Moreover, investigations in humans on the metabolism of drug amines on the market by AOs, such as semicarbazide-sensitive amine oxidases (SSAOs) and polyamine oxidases (PAOs), are practically nonexistent, with the exception of amlodipine. In contrast to MAOs, monooxygenases (CYP isoenzymes more than FMOs) have been extensively investigated concerning their involvement in the metabolism of xenobiotics. It is possible that the contribution of AOs to the overall metabolism of xenobiotic amines in humans is underestimated or erroneously estimated, as most investigations of drug metabolism are performed using in vitro test systems optimized for CYP activity, such as liver microsomes, and most investigations of drug metabolism in vivo in humans carry out only the identification of the final, stable metabolites. However, for some drugs on the market, the involvement of MAOs in their in vivo metabolism in humans has been demonstrated recently, among these drugs citalopram, sertraline and the triptans are examples that can be mentioned.

This review focuses on one of the key factors accounting for differences in drug/metabolite exposure in paediatric and elderly subjects compared with that of the adult population, that is, differences in drug metabolism (both qualitative and quantitative) and in particular differences due to changes in the activity and/or concentration of drug metabolizing enzymes. Important differences have been found in the paediatric population compared with adults for both phase I (e.g. CYP3A7 versus CYP3A4 and CYP1A2, reductive and hydrolytic enzymes) and phase II (e.g. glucuronosyltransferases) enzymes. In the elderly, some phase I enzymes (e.g. esterases) appear to be impaired. From the information collected thus far, it would appear that phase II reactions, though sometimes decreased, are not extensively affected by old age.

The monooxygenases and the amine oxidases (AOs) are the major enzyme systems involved in vivo in the oxidative metabolism of xenobiotic amines in humans. With the exception of the inhibition of the metabolism of tyramine ingested by subjects taking inhibitors of MAO-A or of both MAO-A and -B, which has been extensively investigated, the involvement of the monoamine oxidases in xenobiotic amine metabolism (drugs in particular) has been largely neglected. Furthermore, with the exception of amlodipine, there have been essentially no studies on the metabolism of drug amines by amine oxidases such as SSAOs and PAOs in humans. In contrast, monooxygenases (CYP isoenzymes, and to a lesser extent, FMOs) have been extensively investigated in terms of their involvement in xenobiotic metabolism. It is possible that the contribution of AOs to the overall metabolism of xenobiotic amines in humans has been underestimated, or erroneously estimated, as most investigations of drug metabolism have been performed using in vitro test systems optimized for CYP activity, such as liver microsomes, and most investigations of drug metabolism in vivo in humans have identified only the final, stable metabolites.

Although the majority of oxidative metabolic reactions are mediated by the CYP superfamily of enzymes, non-CYP-mediated oxidative reactions can play an important role in the metabolism of xenobiotics. The (major) oxidative enzymes, other than CYPs, involved in the metabolism of drugs and other xenobiotics are: the flavin-containing monooxygenases, the molybdenum hydroxylases (aldehyde oxidase and xanthine oxidase), the prostaglandin H synthase, the lipoxygenases, the amine oxidases (monoamine, polyamine, diamine and semicarbazide-sensitive amine oxidases) and the alcohol and aldehyde dehydrogenases. In a similar manner to CYPs, these oxidative enzymes can also produce therapeutically active metabolites and reactive/toxic metabolites, modulate the efficacy of therapeutically active drugs or contribute to detoxification. Many of them have been shown to be important in endobiotic metabolism, and, consequently, interactions between drugs and endogenous compounds might occur when they are involved in drug metabolism. In general, most non-CYP oxidative enzymes appear to be noninducible or much less inducible than the CYP system, although some of them may be as inducible as some CYPs. Some of these oxidative enzymes exhibit polymorphic expression, as do some CYPs. It is possible that the contribution of non-CYP oxidative enzymes to the overall metabolism of xenobiotics is underestimated, as most investigations of drug metabolism in discovery and lead optimisation are performed using in vitro test systems optimised for CYP activity.

Rationale: The aim of this review article is to analyse which antiepileptic drugs (AEDs) alter thyroid hormone homeostasis in humans and when this can be explained, at least partially, by the induction of the glucuronoconjugation pathways., Methods: Electronic databases were searched which have provided more than 300 articles. These have been integrated with fundamental books and personal information by experts in the different areas examined., Results: Alteration of thyroid hormone homeostasis by phenobarbital/primidone, phenytoin, and carbamazepine clearly occurs in humans. However, it is not associated with thyroid-stimulating hormone (TSH) increase and the clinical significance of altered serum concentrations of thyroid hormones by these antiepileptic drugs has remained unclear. The published information on the effect of the other antiepileptic drugs examined in this review article on thyroid hormones is lacking (felbamate, pregabalin, zonisamide) or limited. Oxcarbazepine appears to have some effects. Topiramate would need further investigations as well as gabapentin. Levetiracetam, tiagabine, vigabatrine, and lamotrigine do not alter at all, or only minimally, thyroid hormone homeostasis., Conclusion: Concerning the antiepileptic drugs which alter thyroid hormone homeostasis, it is highly probable that the mechanism of induction of uridine diphosphate glucuronosyltransferases (UGT) is involved, at least partially, in such an alteration. However, it is not possible to estimate the relative contribution of the UGT induction by these drugs on the total alteration observed in thyroid hormone levels, as other mechanisms not investigated, or not examined in the present article, could contribute.

In children, the therapeutic benefits and potential risks associated with drug treatment may be different from those in adults and will depend on the exposure, receptor sensitivity and relationship between effect and exposure. In this paper, key factors undergoing maturational changes accounting for differences in drug metabolism and disposition in the paediatric population compared with adults are reviewed. Gastric and duodenal pH, gastric emptying time, intestinal transit time, secretion and activity of bile and pancreatic fluid, bacterial colonisation and transporters, such as P-glycoprotein (P-gp), are important factors for drug absorption, whereas key factors explaining differences in drug distribution between the paediatric population and adults are organ size, membrane permeability, plasma protein concentration and characteristics, endogenous substances in plasma, total body and extracellular water, fat content, regional blood flow and transporters such as P-gp, which is present not only in the gut, but also in liver, kidney, brain and other tissues. As far as drug metabolism is concerned, important differences have been found in the paediatric population compared with adults both for phase I enzymes (oxidative [e.g., cytochrome P450 (CYP)1A2, and CYP3A7 versus -3A4], reductive and hydrolytic enzymes) and phase II enzymes (e.g., N-methyltransferases and glucuronosyltransferases). Generally, the major enzyme differences observed in comparison with the adult age are in newborn infants, although for some enzymes (e.g., glucuronosyltransferases and other phase II enzymes) important differences still exist between infants and toddlers and adults. Finally, key factors undergoing maturational changes accounting for differences in renal excretion in the paediatric population compared with adults are glomerular filtration and tubular secretion. The ranking of the key factors varies according to the chemical structure and physicochemical properties of the drug examined, as well as to the characteristics of its formulation. It would be important to generate additional information on the developmental aspects of renal P-gp and of other renal transporters, as has been done and is still being done with the different -isozymes involved in drug metabolism.

1: The pharmacokinetics and metabolism of 14C-levetiracetam, a new anti-epileptic agent, in mouse, rat, rabbit and dog after a single oral dose were investigated. Moreover, the in vitro hydrolysis of levetiracetam to its major carboxylic metabolite by rat tissue homogenates was investigated to identify tissues involved in the production of the metabolite. Data are also presented on the induction of the enzyme(s) involved in levetiracetam hydrolysis in the rat. 2: Levetiracetam was rapidly and almost completely absorbed. The unchanged drug accounted for a very high percentage of plasma radioactivity. Levetiracetam did not bind to plasma proteins. Although brain radioactivity concentrations were lower than those of whole blood at early time points, brain-to-blood ratios increased over time. The predominant route of elimination of total 14C was excretion via urine, accounting for about 81, 93, 87 and 89% of the dose in the mouse, rat, rabbit and dog, respectively. Consequently, levetiracetam was poorly metabolized. It was submitted in vivo to hydrolysis and/or oxidation. Hydrolysis of the amide function of levetiracetam produced the corresponding acid. However, levetiracetam could also be oxidized at positions 3 and 4 of the 2-oxopyrrolidine ring. Finally, the compound and the corresponding acid metabolite could be oxidized at position 5 of the 2-oxopyrrolidine ring and then hydrolysed with the opening of the ring. 3: All the investigated rat tissues (liver, kidney, lung, brain, small intestine mucosa) had the potential to produce the acid metabolite. By contrast, the acid was undetectable following incubation of levetiracetam with buffer alone or heat-denaturated liver fractions. 4: No marked species or sex differences were observed in the absorption, disposition and metabolism of levetiracetam. 5:The hydrolysis of levetiracetam is carried out by an enzymatic process characterized by a broad tissue distribution. In the rat, the enzyme system hydrolysing levetiracetam is not induced by phenobarbital, at least under the experimental conditions used herein, whereas the enzyme system(s) involved in the other metabolic pathways is induced., (Copyright 2004 Taylor and Francis Ltd.)

The absorption, disposition and metabolism of levetiracetam, a new antiepileptic drug, have been investigated after a single oral dose of the (14)C-labelled molecule administered to male healthy volunteers. As chiral inversion can occur during drug metabolism, the chiral inversion of levetiracetam and/or of its major metabolite produced by hydrolysis (the corresponding acid) was also investigated. Finally, the in vitro hydrolysis of levetiracetam to its major metabolite and the inhibition of this reaction in human blood have been studied. Levetiracetam was very rapidly absorbed in man, with the peak plasma concentration of the unchanged drug occurring at 0.25-0.50 h. The unchanged drug accounted for a very high percentage of plasma radioactivity (97-82%) at all the times measured, i.e. until 48 h after administration. The apparent volume of distribution of the compound was close (0.55-0.62 l/kg) to the volume of total body water. Total body clearance (0.80-0.97 ml/min/kg) was much lower than the nominal hepatic blood flow. The plasma elimination half-life of the unchanged drug varied between 7.4 h and 7.9 h. Plasma to blood ratio of total radioactivity concentrations was 1.1-1.3, showing that radioactivity concentrations were similar in blood cells and plasma. The balance of excretion was very high in all four volunteers. The predominant route of excretion was via urine, accounting for a mean of 95% of the administered dose after 4 days. Two major radioactive components were present in urine, the unchanged drug and the acid obtained by hydrolysis, accounting for 66% and 24% of the dose after 48 h, respectively. Hydrolysis of levetiracetam in human blood followed Michaelis-Menten kinetics with Km and V(max) values of 435 microM and 129 pmol/min/ml blood, respectively. Among the inhibitory agents investigated in this study, only paraoxon inhibited levetiracetam hydrolysis (92% inhibition at 100 microM). Oxidative metabolism occurred in man, although it accounted for no more than 2.5% of the dose. There was no evidence of chiral inversion.

In the field of gene expression analysis, DNA microarray technology is having a major impact on many different areas including toxicology. For instance, a number of studies have shown that transcription profiling can generate the information needed to assign a compound to a mode-of-action class. In this study, we investigated whether compounds inducing similar toxicological endpoints produce similar changes in gene expression. In vitro primary rat hepatocytes were exposed to 11 different hepatotoxicants: acetaminophen, amiodarone, clofibrate, erythromycin estolate, isoniazid, alpha-naphtylylisothiocyanate, beta-naphtoflavone, 4-pentenoic acid, phenobarbital, tetracycline, and zileuton. These molecules were selected on the basis of their variety of hepatocellular effects observed such as necrosis, cholestasis, steatosis, and induction of CYP P450 enzymes. We used a low-density DNA microarray containing 59 genes chosen as relevant toxic and metabolic markers. The in vitro gene expression data generated in this study were generally in good agreement with the literature, which mainly concerns in vivo data. Furthermore, gene expression profiles observed in this study have been confirmed for several genes by real-time PCR assays. All the tested drugs generated a specific gene expression profile. Our results show that even with a relatively limited gene set, gene expression profiling allows a certain degree of classification of compounds with similar hepatocellular toxicities such as cholestasis, necrosis. The clustering analysis revealed that the compounds known to cause steatosis were linked, suggesting that they functionally regulate similar genes and possibly act through the same mechanisms of action. On the other hand, the drugs inducing necrosis and cholestasis were pooled in the same cluster. The drugs arbitrarily classified as the CYP450 inducers formed individual clusters. In conclusion, this study suggests that low-density microarrays could be useful in toxicological studies.

Cetirizine, terfenadine, loratadine, astemizole and mizolastine were compared for their ability to inhibit marker activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and for some glucuronidation isoenzymes in human liver microsomes. The most pronounced effects were observed with terfenadine, astemizole and loratadine which inhibited CYP3A4-mediated testosterone 6beta-hydroxylation (IC50 of 23, 21 and 32 microM, respectively) and CYP2D6-mediated dextromethorphan O-demethylation (IC50 of 18, 36 and 15 microM, respectively). In addition, loratadine markedly inhibited the CYP2C19 marker activity, (S)-mephenytoin 4-hydroxylation (Ki of 0.17 microM). Furthermore, loratadine activated the CYP2C9-catalyzed tolbutamide hydroxylation (ca. 3-fold increase at 30 microM) and inhibited some glucuronidation enzymes. Mizolastine appeared to be a relatively weak and unspecific inhibitor of CYP2E1, CYP2C9, CYP2D6 and CYP3A4 (IC50Ss in the 100 micromolar range). Cetirizine demonstrated no effect on the investigated activities. A comparison of the inhibitory potencies of cetirizine, terfenadine, loratidine, astemizole and mizolastine with their corresponding plasma concentrations in humans suggests that these antihistamines are not likely to interfere with the metabolic clearance of coadministered drugs, with the exception of loratidine, which appears to inhibit CYP2C19 with sufficient potency to warrant additional investigation.

Studies of a series of 1-(benzofuran-2-ylmethyl)imidazoles, 1-5, previously proposed as potential agents for prostatic cancer by their inhibition of 17beta-hydroxylase:17,20-lyase (P450 17), have been extended to their selectivity against placental microsomal aromatase (P450(Arom)) in man. The compounds were 3-7-fold more potent than aminoglutethimide and had some selectivity for P450 17 as expressed by the ratio (IC50 P450(Arom))/(IC50 P450) 17)/17.0 (2), 10.3 (3), 34.6 (4) and 42.0 (5), where IC50 is the concentration resulting in 50% inhibition. The lower potency of 1-5 towards P450(Arom) compared with the racemic alpha-phenyl-substituted compounds (6, 80-1000 x aminoglutethimide) and some racemic alpha-methyl (8.5 and 12.2 x aminoglutethimide) and alpha-ethyl (12.1 and 32.9 x aminoglutethimide) analogues has been rationalized. This work selectively extends studies of the P450 17 inhibitor 5, a potential prostatic cancer agent, towards other cytochrome P450 enzymes in the steroidogenic pathway and provides a general method for determining the relative influence of chemical manipulation of a parent inhibitor towards two enzymes in the pathway using additional literature data.

Several phenyl ethyl and phenyl propyl imidazole based compounds have been synthesised and their biological activity evaluated against human placental Aromatase (AR), rat testicular 17 alpha-hydroxylase/ 17,20-lyase (P450(17) alpha) and bovine cholesterol side chain cleavage (CSCC). The compounds showed good selectivity towards AR with N-[2-(4'-Nitrophenyl) ethyl] imidazole (2) (IC50 = 0.16 +/- 0.01 microM, Ki = 0.09 +/- 0.01 microM), the most potent AR inhibitor, showing some 130 times greater potency over Aminoglutethimide (AG) (IC50 = 20.0 +/- 2.6 microM, Ki = 11.0 +/- 2.0 microM). N-[3-(4'-Fluorophenyl) propyl] imidazole (10) (IC50 = 0.31 +/- 0.01 microM, Ki = 0.34 +/- 0.05 microM), N-(2-(4'-Fluorophenyl ethyl) imidazole (5) (IC50 = 0.74 +/- 0.01 microM, Ki = 0.40 +/- 0.02 microM), N-(3-(4'-Chlorophenyl propyl) imidazole (9) (IC50 = 0.82 +/- 0.02 microM) and N-[3-(4'-Nitrophenyl) propyl] imidazole (7) (IC50 = 0.84 +/- 0.02 microM, Ki = 0.10 microM) were also more potent than AG. Of the compounds tested for P450(17) alpha activity, 7 (IC50 = 25.0 +/- 2.0 microM), N-[2-(4'-Aminophenyl) ethyl] imidazole (3) (IC50 = 27.6 +/- 0.10 microM), 9 (IC50 = 29.0 +/- 4.0 microM) and 2 (IC50 = 30.2 +/- 2.0 microM) showed the highest activity, possessing approximately half the activity of Ketoconazole (IC50 = 12.1 +/- 2.9 microM). Compounds 1, 2, 3, and 7 showed 0% inhibitory activity towards CSCC at 200 microM whilst AG showed 83% inhibition under the same conditions. The compounds proved themselves to be excellent lead compounds and supported the novel models developed by Ahmed for AR and P450(17) alpha.

Several novel pyrrolidine-2,5-dione based compounds have been synthesised and evaluated for their biological activity against human placental aromatase (AR), rat testicular 17 alpha-hydroxylase/17,20-lyase (P450(17) alpha) and bovine cholesterol side chain cleavage (CSCC). The compounds showed good inhibition towards AR with 1-cyclohexyl-3-[2'(4"-aminophenyl) ethyl] pyrrolidine-2,5-dione (3) (IC50 = 23.8 +/- 4.6 microM) and 1-octyl-3-[2'(4"-aminophenyl) ethyl] pyrrolidine-2,5-dione (4) (IC50 = 24.6 +/- 1.8 microM) showing equipotent activity with Aminoglutethimide (AG) (IC50 = 20.0 +/- 2.6 microM, Ki = 11.0 +/- 2.0 microM). Of the compounds tested for P450(17) alpha activity, 3 (IC50 = 18.5 +/- 1.9 microM) again showed the highest activity, being equipotent to Ketoconazole (IC50 = 12.1 +/- 2.9 microM). 3 was a poor inhibitor of CSCC with some 22% inhibitory activity at an inhibitor concentration of 200 microM, as compared to AG with 72% inhibitory activity under the same conditions. The compounds proved themselves to be excellent lead compounds and supported the novel models developed by Ahmed for AR and P450(17) alpha.

1-Alkyl-3-(4'aminophenyl)pyrrolidine-2,5-diones (7,R = C3H7-C7H15) are potent inhibitors of aromatase in vitro, the 1-hexyl (Ki = 62nM) being about 100-fold more potent than aminoglutethimide (AG), and more selective in their ratio of aromatase:CSCC inhibitory potency. The 1-pentyl, 1-hexyl and 1-heptyl derivatives are more stable to liver microsomal metabolism in vitro than AG possibly due to inhibition of the liver cytochrome P450s. 1,3-Dialkyl-3(4'-aminophenyl)pyrrolidine-2,5-diones (9) have been synthesised by a novel method. Although the higher homologues (di-pentyl and di-hexyl) are more potent in vitro as inhibitors of aromatase than AG, they are less active than their 1-alkyl counterparts with the same alkyl substituent.

The effect of dose on the bioavailability of oral etoposide was investigated in ten patients with malignant mesothelioma who received single-agent etoposide as part of a phase II study. Etoposide pharmacokinetics were studied in each patient at oral dose levels of 100, 200, 300, 400 and 600 mg. At doses above 200 mg, the AUC and peak concentrations of etoposide were substantially lower than predictions based on the 100-mg dose. This study confirms previous observations that etoposide absorption is dose-dependent and that a mean bioavailability of approximately 50% cannot be assumed at total oral doses greater than 200 mg.