Application of toxicogenomic tools in the drug research and development process

The cost for the development of new active and safe drugs is higher than ever and continues to increase. At the same time, both the pharmaceutical industry and the Regulatory Authorities are, despite the increasing effort to develop safer drugs, concerned by the risk of unexpected side effects observed in the late steps of the development of new drugs, either in late clinical development or after marketing approval. Then, the early knowledge of any potential toxic effect of a new drug is a key issue to allow adequate decision making. This means that current approaches based on the determination of the No-Adverse-Effect-Level and the Human-Equivalent-Dose are far from being perfect, and fail mainly to detect toxic phenomena of low intensity and/or low frequency. To improve the predictability of the existing experimental models, Toxicogenomics could be included into the in vitro candidate-selection steps and/or during the regulatory preclinical (or clinical) studies.     

Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of a high purity sample of single-wall carbon nanotubes (SWCNTs) was evaluated using a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation test (Ames test), an in vitro chromosomal aberration test, and an in vivo mouse bone marrow micronucleus test. The SWCNTs exerted no genotoxicity in Salmonella typhimurium TA97, TA98, TA100, and TA1535, or in Escherichia coli WP2 uvrA/pKM101, whether in the absence or presence of metabolic activation and at concentrations of 12.5–500 μg/plate. In the chromosomal aberration test, at 300–1000 μg/mL, the SWCNTs did not increase the number of structural or numerical chromosomal aberrations, whether the test was conducted with or without metabolic activation. In the in vivo bone marrow micronucleus test, doses of 60 mg/kg and 200 mg/kg SWCNTs did not affect the proportions of immature and total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of mice. The results of these studies show that the high purity and well-dispersed sample of SWCNTs are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay, chromosomal aberration assay, or in vivo bone marrow micronucleus test, and thus appear not to pose a genotoxic risk to human health in vivo.     

Comparison of the human skin grafted onto nude mouse model with in vivo and in vitro models in the prediction of percutaneous penetration of three lipophilic pesticides

The evaluation of the degree of percutaneous penetration of agrochemicals is a key part of risk assessment for operators. The availability of suitable and predictive experimental models is crucial, in particular in the case of lipophilic compounds which persist in the stratum corneum (SC). Regulatory models (rat in vivo, human and rat in vitro) and the innovative human skin grafted onto nude mice (HuSki) model were compared for their ability to predict the human skin absorption. Radiolabelled malathion, lindane and cypermethrin (4microg/cm(2)) were topically applied to each model. The % of applied dose absorbed and that present in skin and SC were evaluated at 24h. Additionally, the absorption profile of cypermethrin was evaluated in the in vivo rat and HuSki models for up to 11 days. We found that the human in vitro and HuSki models closely predicted the human skin absorption at 24h, while rat models overestimated the human skin absorption. Furthermore, our experiments with cypermethrin indicated that evaluation of % percutaneous absorption over extended periods of time was feasible with the HuSki model. In our studies the HuSki model overcame the limitations of the regulatory models and is promising to realistically refine the dermal absorption assessment of topically applied chemicals.     

Review on in vivo and in vitro methods evaluation of antioxidant activity

A good number of abstracts and research articles (in total 74) published, so far, for evaluating antioxidant activity of various samples of research interest were gone through where 407 methods were come across, which were repeated from 29 different methods. These were classified as in vitro and in vivo methods. And those are described and discussed below in this review article. In the later part of this review article, frequency of in vitro as well as in vivo methods is analyzed with a bar diagram. Solvents are important for extracting antioxidants from natural sources. Frequency of solvents used for extraction is also portrayed and the results are discussed in this article. As per this review there are 19 in vitro methods and 10 in vivo methods that are being used for the evaluation of antioxidant activity of the sample of interest. DPPH method was found to be used mostly for the in vitro antioxidant activity evaluation purpose while LPO was found as mostly used in vivo antioxidant assay. Ethanol was with the highest frequency as solvent for extraction purpose.     

Development of an in vitro liver toxicity prediction model based on longer term primary rat hepatocyte culture

The assumption that compounds with similar toxic endpoints generate unique gene expression signatures has led to attempts to classify unknown compounds according to their genomic profile. However, studies reported so far have mostly been conducted in vivo. In this proof of concept study, we assessed the use of rat hepatocyte sandwich cultures in combination with a toxicogenomic classification method to generate a predictive in vitro toxicity classification model.After pre-incubation for 3 days, primary rat hepatocytes were treated for up to 9 days with 13 well known model compounds, changes in the global gene expression profile were measured and subsequently used for the establishment of a predictive classification model. A subset of 724 genes was capable of discriminating compounds with a misclassification rate of 7.5%. As a preliminary verification, the resulting classifier was applied to two blinded control compounds. The classification of compounds according to transient changes in global gene expression allowed the correct prediction independently from the knowledge of their underlying toxic mechanisms.The results of this first pilot study demonstrate the possibility of in vitro gene expression models to contribute to candidate selection early in drug discovery by improving the predictivity of toxicological studies and thereby reducing animal usage in toxicology.     

In vitro-in vivo correlation and bioavailability studies of captopril from novel controlled release donut shaped tablet

A controlled release formulation of captopril which was coated and fabricated into a donut shaped tablet formulation, was investigated in rabbit for pharmacokinetic and in vitro-in vivo correlation studies. Coated donut shaped tablets were prepared and in vitro release was studied in simulated gastric fluid at three different RPMs. New Zealand albino male rabbits have been used as animal model for in vivo study. A sensitive and simple HPLC method was developed for the determination of captopril content in rabbit plasma. In vitro release studies showed that release patterns followed zero order for around 4 h. Single oral administration of coated donut shaped tablets in rabbit illustrated retained availability of captopril to the injected drug. Captopril content could pursue the same release pattern over the same time course in in vivo study. The in vivo-in vitro correlation coefficients obtained from point-to-point analysis were greater than 99% between concentrations at certain time points obtained from release study in simulated gastric fluid at different RPMs and HPLC analysis of rabbit's plasma. From the in vitro-in vivo correlation prediction it was evident that the coated donut shaped tablet is a good device for controlled delivery of captopril.     

Design and in vivo evaluation of an indapamide transdermal patch

The aim of the present study was to develop and evaluate a novel drug-in-adhesive transdermal patch system for indapamide. Initial in vitro experiments were conducted to optimize formulation parameters prior to transdermal delivery in rats. The effects of the type of adhesive and the content of permeation enhancers on indapamide transport across excised rat skin were evaluated. The results indicated that DURO-TAK^® adhesive 87-2852 is a suitable and compatible polymer for the development of transdermal drug delivery systems for indapamide. The final formulation contained 4% N-dodecylazepan-2-one, 6% l-menthol and 3% isopropyl myristate. For in vivo studies patch systems were administered transdermally to rats while orally administered indapamide in suspension was used as a control. The PK parameters, such as the maximum blood concentration (C_max), time to reach the peak blood concentration (T_max), mean residence time (MRT), area under the curve (AUC_0–t) and terminal elimination half-life (T_1/2) were significantly (p < 0.05) different following transdermal administration compared with oral administration. In contrast to oral delivery, a sustained activity was observed over a period of 48 h after transdermal administration. This sustained activity was due to the controlled release of drug into the systemic circulation following transdermal administration.     

In vitro andin vivo evaluation of the tissue-to-blood partition coefficient for physiological pharmacokinetic models

An important parameter used in physiologically based pharmacokinetic models is the partition coefficient (Kp), which is defined as the ratio of tissue drug concentration to the concentration of drug in the emergent venous blood of the tissue. Since Kp is governed by reversible binding to protein and other constituents in blood and tissue, an attempt was made here to estimate the Kp values for a model drug ethoxybenzamide (EB) by means of in vitrobinding studies and to compare these Kp values to those obtained from in vivokinetic parameters observed following the administration of EB by two different routes, i.e., i.v. bolus injection and constant rate infusion. The Kp values obtained by using these three different methods were in reasonably good agreement suggesting that binding data obtained in vitrocan successfully be used to estimate in vivodistribution.     

Replacement of in vivo acute oral toxicity studies by in vitro cytotoxicity methods: opportunities, limits and regulatory status

The development of a new medicinal product is a long and costly process in particular due to the regulatory requirements for quality, safety and efficacy. There is a common interest to increase the efficiency of drug development and to provide new, better quality medicinal products much faster to the public. One possible way to economize time and costs, as well as to consider animal protection issues, is to introduce new alternative methods into non-clinical toxicity testing. Currently, animal tests are mandatory for the evaluation of acute toxicity of chemicals and new drugs. The replacement of the in vivo tests by alternative in vitro assays would offer the opportunity to screen and assess numerous compounds at the same time, to predict acute oral toxicity and thus accelerate drug development. Moreover, the substitution of in vivo tests by in vitro methods shows a proactive pursuit of ethical and animal welfare issues. Importantly, the implementation of in vitro assays for acute oral toxicity would require the establishment of common test guidelines across the EU, USA and Japan, i.e., the regions of ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use). Presently, alternative in vitro tests are being investigated internationally. Yet, in order to achieve regulatory acceptance and implementation of in vitro assays, convincing results from validation studies are required. In this review, we discuss the current regulatory status of acute oral toxicity testing and point out achievements of alternative methods. We describe the application of in vitro tests, correlating in vitro with in vivo data. The use of in vitro data to predict in vivo acute oral toxicity is analyzed using the Registry of Cytotoxicity, an official independent database. We have then analyzed opportunities and drawbacks for future implementation of in vitro test methods, with particular focus on industrial use.     

Preparation andin vivo evaluation of huperzine A-loaded PLGA microspheres

Huperzine A-loaded microspheres composed of poly(D,L-lactide-co-glycolide) were prepared by an ONV emulsion solvent evaporation method. The characterization of the microspheres such as drug loading, size, shape and release profile was described. The in vitro release in the initial 7 days was nearly linear with 10% released per day. Thereafter drug release rate became slow gradually and about 90% drug released at day 21. The in vitro release rate determined by dialysis bag method had a good correlation with the in vivo release rate. Huperzine A aqueous solution was intramuscularly injected (i.m.) at 0.4 mg/kg and microspheres were intramuscularly injected at 8.4 mg eq huperzine A/kg in rats. The maxium plasma concentration (Cmax ) after i.m. microspheres was only 32% of that after i.m. solution. Drug in plasma could be detected until day 14 and about 5% of administered dose was residued at the injection site at day 14. The relative bioavailability of huperzine A microspheres over a period of 14 days was 94.7%. Inhibition of acyecholinesterase activity (AchE) in rat's cortex, hippocampus and striatum could sustain for about 14 days. In conclusion, huperzine A-loaded microspheres possessed a prolonged and complete drug release with significant inhibition of AchE for 2 weeks in rats.     

Uptake and efflux of methylmercury in vitro: Comparison of transport mechanisms in C6, B35 and RBE4 cells

Methylmercury (MeHg) is a neurotoxicant which enters the brain and may cause permanent change. Thus, the properties of MeHg transport across cell membranes are a key factor in designing an appropriate model for MeHg neurotoxicity. This study uses cell cultures to examine the uptake and efflux mechanisms of methylmercury in C6 glioma, B35 neuroblastoma and rat brain endothelial (RBE4) cells. The cellular uptake and efflux of MeHg was investigated using ¹⁴C-labeled MeHg. The uptake of MeHg-chloride was temperature-independent while the uptake of MeHg-l-cysteine was temperature-dependent in all the three cell types. This indicates that uptake of MeHg-chloride is due to passive diffusion and uptake of MeHg-l-cysteine is due to a protein carrier. Substrates of the amino acid transport system L inhibited uptake of MeHg-l-cysteine in C6 and RBE4 cells, but not B35 cells, indicating a role for system L in MeHg-uptake in the former two. Probenecid, Na⁺-free medium, MeHg and several l-amino acids did not alter the efflux of MeHg from C6 and RBE4 cells. The amino acids l-cysteine and cystine however, increased the efflux. Both cysteine and cystine are important in the generation of glutathione (GSH), suggesting the involvement of GSH in MeHg efflux. HgCl₂ at low concentrations (0.5 and 1.0 μM) decreased the MeHg efflux and at high concentrations (5.0 and 10.0 μM) increased the efflux. This inhibiting effect of HgCl₂ at low concentrations is possibly due to binding to GSH while the effect of high HgCl₂ concentrations is attributed to disrupted membrane integrity, as measured by Trypan blue. This study demonstrates differing transport mechanisms of MeHg in the cell lines C6, B35 and RBE4.     

Investigating relationships betweenin vivo andin vitro pharmacological variables for the purpose of prediction

Consistency of effect in vivois an important characteristic of a drug product, and great variation from lot to lot or between manufacturers is not desirable. The variation usually arises when some lots fail to meet minimum standards. It may then be necessary to develop a procedure for detecting and screening out these defective lots. A new, heuristic approach is presented for evaluating procedures in which screening is to be based on in vitromeasurements rather than more costly in vivomeasures. A quantification of risks is defined from which a minimum (in a certain sense) risk procedure may be selected from a set of candidate procedures. This approach is shown to be more appropriate than those based on correlational techniques.The views expressed herein are those of the author and not necessarily those of the Food and Drug Administration. This article was originally presented at the Thirty-first Annual Princeton Conference on Applied Statistics, Princeton, New Jersey, December 1975.     

In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole

AIMS

Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo.

METHODS

A sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs).

RESULTS

Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r = 0.96, P = 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K_m values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r = 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day⁻¹); anastrozole glucuronide was less apparent.

CONCLUSION

Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.     

Smoke chemistry, in vitro and in vivo toxicology evaluations of the electrically heated cigarette smoking system series K

The Electrically Heated Cigarette Smoking System Series K (EHCSS) produces smoke through the controlled electrical heating of tobacco. Evaluation of the EHCSS was accomplished by comparison with commercial and reference cigarettes, using International Organization for Standardization (ISO) and alternative puffing regimens based on nicotine exposures measured in a short-term clinical study. Using the alternative puffing regimen and compared with conventional cigarettes on a per cigarette basis, the EHCSS had 50–60% reductions in tar and nicotine; at least 90% reductions in carbon monoxide, nitrogen oxides, 1,3-butadiene, isoprene, acrylonitrile, polyaromatic hydrocarbons, hydrogen cyanide, aromatic amines, tobacco specific nitrosamines, and phenol; and least a 40% reduction in 2-nitropropane. Other important smoke constituents in EHCSS smoke were reduced as well. The in vitro studies showed similar large reductions in biological activity. Ames mutagenicity of total particulate matter (TPM) from the EHCSS was reduced by 70–90%; cytotoxicity of the TPM was reduced by approximately 82% and 65% for the gas–vapor phase. In vivo testing under ISO smoking conditions in the mouse skin painting assay demonstrated later dermal tumor onset, lower dermal tumor incidence, reduced dermal tumor multiplicity, and a lower proportion of malignant dermal tumors in EHCSS smoke condensate-exposed mice. Thirty-five day and 90-day nose-only inhalation studies in rats showed reductions in pulmonary inflammation and other biological activity, including histopathological endpoints. We conclude that under the conditions of these in vitro and in vivo studies, the EHCSS demonstrated significantly lower biological activity compared to conventional cigarettes, and may suggest the potential for reductions in human smokers.     

Hypotensive and spasmolytic activities of crude extract ofCyperus scariosus

Intravenous administration of hydro-methanolic extract ofCyperus scarious (3–10 mg/kg) produced hypotensive and bradycardiac effects. These effects remained unaltered in atropinized animals indicating that cardiovascular effects of the plant extract are not mediated through activation of muscarinic receptors. In thein vitro studies, it suppressed the spontaneous contractions of guinea-pig paired atria, rat uterus and rabbit jejunum in a concentration-dependent (0.1–1 mg/ml) manner. It also inhibited histamine or acetylcholine-induced contractions of guinea-pig ileum indicating non-specific spasmolytic action. In rabbit aorta, it inhibited norepinephrine (10 μM) as well as K⁺ (80 mM)-induced contractions at similar concentrations (0.1–1mg/ml). These data indicate thatCyperus scariosus contains Ca²⁺ channel blocker-like constituent(s) which may explain hypotensive effect observedin vivo and the general spasmolytic activity of plant may explain its folkloric use in diarrhoea.     

The absence of genotoxicity of sucralose

Sucralose is a non-nutritive sweetener that is approximately 600 times sweeter than table sugar. It is currently approved for use in over 80 countries. Evidence from chronic studies demonstrates that this compound is not carcinogenic. This report summarizes the results of genotoxicity studies that were part of the original safety assessment of sucralose–conducted early in the safety investigation and shared with regulatory agencies around the world. Studies included the Ames (Salmonella typhimurium) reverse mutation test, the Escherichia coli pol A+/A− test, an in vitro chromosome damage assay in human lymphocytes, mutation in TK +/− mouse lymphoma cells, an in vivo chromosome aberration test in rats and two separate micronucleus tests in mice. All results were evaluated as negative. These results support the overall conclusion by regulatory and heath agencies that sucralose is safe for its intended use.     

Development and in vitro/in vivo Evaluation of a Silastic Intravaginal Ring for Mifepristone Delivery

Preparation and in vitro/in vivo evaluation of mifepristone intravaginal ring formulations were investigated. In the present study, it is reported that a mifepristone intravaginal ring of reservoir design comprising of a mifepristone silicone elastomer core enclosed in a silicone layer. During the preparation of intravaginal ring solid dispersion method was employed which improved the release rate of drug from the intravaginal ring. In vitro release studies performed under sink conditions and the released drug amounts were estimated using UV spectrometry at 310 nm. In addition, the in vivo release profile of in-house devices was evaluated in female New Zealand white rabbits. The rabbit plasma samples were processed and analyzed using a validated HPLC-MS method. Norgestrel was used as internal standard, and plasma samples contained mifepristone and internal standard were deproteinized, and then subjected to HPLC-MS analysis under condition of electrospray ionization in the selected ion monitoring mode. The drug release from intravaginal ring made in house was constant for 21 days in rabbits, which suggested the mifepristone intravaginal ring release system would be useful in clinical practice in the future. The result indicated the in vitro/in vivo correlation is perfect, which explained in vitro release analysis method developed was feasible.     

Respiratory sensitization: Advances in assessing the risk of respiratory inflammation and irritation

Respiratory sensitization provides a case study for a new approach to chemical safety evaluation, as the prevalence of respiratory sensitization has increased considerably over the last decades, but animal and/or human experimental/predictive models are not currently available. Therefore, the goal of a working group was to design a road map to develop an ASAT approach for respiratory sensitisers. This approach should aim at (i) creating a database on respiratory functional biology and toxicology, (ii) applying data analyses to understand the multi-dimensional sensitization response, and how this predisposes to respiratory inflammation and irritation, and (iii) building a systems model out of these analyses, adding pharmacokinetic–pharmacodynamic modeling to predict respiratory responses to low levels of sensitisers. To this end, the best way forward would be to follow an integrated testing approach. Experimental research should be targeted to (i) QSAR-type approaches to relate potential as a respiratory sensitizer to its chemical structure, (ii) in vitro models and (iii) in vitro–in vivo extrapolation/validation.