The challenge of predicting drug toxicity in silico

Poor pharmacokinetics, side effects and compound toxicity are frequent causes of late-stage failures in drug development. A safe in silico identification of adverse effects triggered by drugs and chemicals would be highly desirable as it not only bears economical potential but also spawns a variety of ecological benefits: sustainable resource management, reduction of animal models and possibly less risky clinical trials. In computer-aided drug discovery, both existing and hypothetical compounds may be studied; the methods are fast, reproducible, and typically based on human bioregulators, making the question of transferability obsolete. In the recent past, our laboratory contributed towards the development of in silico concepts (--> multi-dimensional QSAR) and validated a series of "virtual test kits" based on the oestrogen, androgen, thyroid, and aryl hydrocarbon receptor (endocrine disruption, receptor-mediated toxicity) as well as on the enzyme cytochrome P450 3A4 (metabolic transformations, drug-drug interactions). The test kits are based on the three-dimensional structure of their target protein (i.e. ER(alphabeta), AR, TR(alphabeta), CYP450) or a surrogate thereof (AhR) and were trained using a representative selection of 362 substances. Subsequent evaluation of 107 compounds different therefrom showed that binding affinities are predicted close to experimental uncertainty. These results suggest that our approach is suited for the in silico identification of adverse effects triggered by drugs and chemicals and encouraged us to compile an Internet Database for the virtual screening of drugs and chemicals for toxic effects.     

Progress in predicting human ADME parameters in silico

Understanding the development of a scientific approach is a valuable exercise in gauging the potential directions the process could take in the future. The relatively short history of applying computational methods to absorption, distribution, metabolism and excretion (ADME) can be split into defined periods. The first began in the 1960s and continued through the 1970s with the work of Corwin Hansch et al. Their models utilized small sets of in vivo ADME data. The second era from the 1980s through 1990s witnessed the widespread incorporation of in vitro approaches as surrogates of in vivo ADME studies. These approaches fostered the initiation and increase in interpretable computational ADME models available in the literature. The third era is the present were there are many literature data sets derived from in vitro data for absorption, drug-drug interactions (DDI), drug transporters and efflux pumps [P-glycoprotein (P-gp), MRP], intrinsic clearance and brain penetration, which can theoretically be used to predict the situation in vivo in humans. Combinatorial synthesis, high throughput screening and computational approaches have emerged as a result of continual pressure on pharmaceutical companies to accelerate drug discovery while decreasing drug development costs. The goal has become to reduce the drop-out rate of drug candidates in the latter, most expensive stages of drug development. This is accomplished by increasing the failure rate of candidate compounds in the preclinical stages and increasing the speed of nomination of likely clinical candidates. The industry now understands the reasons for clinical failure other than efficacy are mainly related to pharmacokinetics and toxicity. The late 1990s saw significant company investment in ADME and drug safety departments to assess properties such as metabolic stability, cytochrome P-450 inhibition, absorption and genotoxicity earlier in the drug discovery paradigm. The next logical step in this process is the evaluation of higher throughput data to determine if computational (in silico) models can be constructed and validated from it. Such models would allow an exponential increase in the number of compounds screened virtually for ADME parameters. A number of researchers have started to utilize in silico, in vitro and in vivo approaches in parallel to address intestinal permeability and cytochrome P-450-mediated DDI. This review will assess how computational approaches for ADME parameters have evolved and how they are likely to progress.     

Application of in silico approaches to predicting drug--drug interactions

In an environment driven to find the next blockbuster drug, failure years into a project should not be an option. Recent studies have shown that poor absorption, distribution, metabolism, and excretion (ADME), and the related properties of toxicity and pharmacokinetics are responsible for a large proportion of failures. One way to understand and potentially predict molecules likely to be successful in humans as drugs from an ADME point of view is to use simulations. Such simulations may include simple rule-based approaches, structure--activity relationships, three-dimensional quantitative structure--activity relationships (3D-QSAR), and pharmacophores. All of these represent useful tools in understanding metabolism by the cytochromes P450, predicting drug--drug interactions (DDIs), and other pharmacokinetic parameters. The present paper briefly reviews the application of some computational tools applied to predicting DDIs and will provide the reader with an idea of their utility.     

In silico approaches for predicting ADME properties of drugs

Combinatorial chemistry and high-throughput screening have increased the possibility of finding new lead compounds at much shorter time periods than conventional medicinal chemistry. However, too much promising drug candidates often fail because of unsatisfactory ADME properties. In silico ADME studies are expected to reduce the risk of late-stage attrition of drug development and to optimize screening and testing by looking at only the promising compounds. To this end, many in silico approaches for predicting ADME properties of compounds from their chemical structure have been developed, ranging from data-based approaches such as quantitative structure-activity relationship (QSAR), similarity searches, and 3-dimensional QSAR, to structure-based methods such as ligand-protein docking and pharmacophore modelling. In addition, several methods of integrating ADME properties to predict pharmacokinetics at the organ or body level have been studied. In this article, we briefly summarize in silico ADME approaches.     

Lipid-based formulations for intestinal lymphatic delivery

The current state of the art of intestinal lymphatic transport is given by reviewing the more recent publications, which have utilized lipid-based vehicles. The results published often show variable trends depending on, the design of the vehicle, the components used, the physicochemical properties of the drug, the animal model and experimental techniques, these variables often make direct comparisons difficult. Traditionally intestinal lymphatic delivery has been expressed as a percentage of the dose transported in the lymph. Using this parameter results obtained to date, with lipid-based vehicles, are somewhat disappointing maximising at approximately 20–30%, for highly lipophilic compounds including DDT and halofantrine (Hf). Recent data, monitoring Hf, in a fed versus fasted dog study, have shown that a higher degree of lymphatic transport is possible (>50% dose) in the postprandial state, this study should result in stimulating renewed interest in the potential of achieving significant levels of lymphatic targeting. Although some relevant features controlling lymphatic transport have been identified over the years a deeper appreciation of all the mechanisms, which is vital for therapeutic exploitation of lymphatic transport, is still unrealized. This review analyses the success and limitations of a formulation approach using lipid-based vehicles and highlights potential areas for further research.     

Lipid vehicles for intestinal lymphatic drug absorption

The lipoprotein fractions in mesenteric lymph were monitored following intraduodenal administration of arachis oil and oleic, linoleic and linolenic fatty acids to rats. An increase in the chylomicron fraction, but not the VLDL or LDL fraction, was observed with each lipid. The greater the degree of unsaturation of the fatty acid, the more rapid the onset of chylomicron synthesis. The administration of linoleic acid and arachis oil produced the highest concentration of chylomicrons in the lymph. These results reflect differences in the rate of absorption and biochemical metabolism of the lipids and have implications for the selection of vehicles for the delivery of drugs by the lymphatic route.     

In silico methods for predicting ligand binding determinants of cytochromes P450

A large number of computational methodologies have been used to predict, and thus help explain, the metabolism catalysed by the enzymes of the cytochrome P450 superfamily (P450s). A summary of the methodologies and resulting models is presented. This shows that investigations so far have focused on just a few of the many P450s, mainly those that are involved in drug metabolism. The models have evolved from simple comparisons of known substrates to more elaborate models requiring considerable computer power. These help to explain and, more importantly, predict the involvement of P450s in the metabolism of specific compounds.     

Animal models for the study of intestinal lymphatic drug transport

Drug transport via the intestinal lymphatic system has been shown to contribute to the absorption of a number of orally administered highly lipophilic drugs. In order to investigate this phenomenon and assist in the development of improved oral formulations, the use of appropriate animal models is required. This paper reviews the use of various animal models for this purpose, and describes in detail the conscious rat and dog models used in our laboratory. The advantages and disadvantages of both small and large animal models are explored, as well as the factors which have been found to influence the outcome of intestinal lymphatic drug transport studies with these models.     

Bioavailability of lycopene in the rat: the role of intestinal lymphatic transport

Objectives As a natural antioxidant derived from dietary sources, lycopene has attracted considerable attention as a potent chemopreventative agent. Lycopene is an extremely lipophilic compound and absorption from dietary sources is estimated to be low and highly variable. As a result, plasma lycopene concentrations are poorly correlated with dietary intake of lycopene rich food stuffs. The development of an oral formulation remains a challenge that requires a better understanding of the mechanisms involved in the intestinal absorption of this compound. Methods The solubility of lycopene in simulated physiological fluids and bile salt mixed micelle formulations was determined. The extent of intestinal lymphatic transport and the absolute bioavailability of lycopene from a range of biorelevant media was evaluated in a mesenteric lymph duct cannulated anaesthetised rat model. Results The absolute bioavailability of lycopene after 8 h was 1.85 ± 0.39%. The overall extent of the intestinal lymphatic transport was in the range of 0.6–3.4% of the administered dose. A strong positive correlation (r² > 0.9) between intestinal lycopene levels and intestinal triglyceride levels was demonstrated. Conclusions The intestinal lymphatic route is the major uptake mechanism of lycopene from the gastrointestinal tract. Lycopene transport in intestinal lymph was closely associated with triglyceride transport in the lymph. Formulation strategies designed to promote intestinal lymphatic uptake, such as lipid‐based formulations containing long‐chain fatty acids (LCFA) or lecithin, may serve to enhance oral bioavailability of lycopene.     

An update on oral drug delivery via intestinal lymphatic transport

Orally administered drug entities have to survive the harsh gastrointestinal environment, penetrate the enteric epithelia and circumvent hepatic metabolism before reaching the systemic circulation. Whereas the gastrointestinal stability can be well maintained by taking proper measures, hepatic metabolism presents as a formidable barrier to drugs suffering from first-pass metabolism. The pharmaceutical academia and industries are seeking alternative pathways for drug transport to circumvent problems associated with the portal pathway. Intestinal lymphatic transport is emerging as a promising pathway to this end. In this review, we intend to provide an updated overview on the rationale, strategies, factors and applications involved in intestinal lymphatic transport. There are mainly two pathways for peroral lymphatic transport—the chylomicron and the microfold cell pathways. The underlying mechanisms are being unraveled gradually and nowadays witness increasing research input and applications.     

In silico approaches to predicting cancer potency for risk assessment of genotoxic impurities in drug substances

The current risk assessment approach for addressing the safety of very small concentrations of genotoxic impurities (GTIs) in drug substances is the threshold of toxicological concern (TTC). The TTC is based on several conservative assumptions because of the uncertainty associated with deriving an excess cancer risk when no carcinogenicity data are available for the impurity. It is a default approach derived from a distribution of carcinogens and does not take into account the properties of a specific chemical. The purpose of the study was to use in silico tools to predict the cancer potency (TD₅₀) of a compound based on its structure. Structure activity relationship (SAR) models (classification/regression) were developed from the carcinogenicity potency database using MultiCASE and VISDOM. The MultiCASE classification models allowed the prediction of carcinogenic potency class, while the VISDOM regression models predicted a numerical TD₅₀. A step-wise approach is proposed to calculate predicted numerical TD₅₀ values for compounds categorized as not potent. This approach for non-potent compounds can be used to establish safe levels greater than the TTC for GTIs in a drug substance.     

An examination of the factors affecting intestinal lymphatic transport of dietary lipids

Lipophilic drugs are carried by chylomicrons secreted by the small intestine and transported in lymph. The intent of this review is to update the reader on the digestion, uptake, and transport of dietary lipids and how these processes impact the absorption of lipophilic drugs by the gut. The digestion of lipids in the gastric and intestinal lumen is discussed as well as the role of bile salts in the solubilization of lipid digestion products for uptake by the gut. Both passive and active uptake of lipid digestion products is reviewed. Also examined is how intestinal lipid transporters located at the brush border membrane may play a role in the uptake of lipids by the enterocytes. The intracellular trafficking and the resynthesis of complex lipids from lipid digestion products are explored. Finally, the formation and secretion of chylomicrons and their potential clinical disorders are described.     

Effect of lipid vehicle on the intestinal lymphatic transport of isotretinoin in the rat

Many lipophilic compounds are absorbed to some degree via the lymphatics, however, the mechanisms and factors controlling this absorption process are unclear. In order to provide some information on this area we have studied the effect of lipid vehicle on the lymphatic transport of isotretinoin following oral dosing to the rat. Oils containing higher percentages of the linoleate triglyceride ester appeared to promote both enhanced lymph flow and chylomicron concentration. Long chain triglyceride oils proved to be the most effective vehicles for increasing lymphatic transport - especially cottonseed oil and peanut oil. The solubility of the drug in the oil was also shown to be a key factor in lymphatic transport     

Enhanced intestinal lymphatic absorption of saquinavir through supersaturated self-microemulsifying drug delivery systems

The therapeutic potential of saquinavir, a specific inhibitor of human immunodeficiency virus (HIV)-1 and HIV-2 protease enzymes, has been largely limited because of a low solubility and consequnt low bioavailability. Thus, we aimed to design a supersaturated self-microemulsifying drug delivery system (S-SMEDDS) that can maintain a high concentration of saquinavir in gastro-intestinal fluid thorugh inhibiting the drug precipitation to enhance the lymphatic transport of saquinavir and to increase the bioavailability of saquinavir considerably. Solubilizing capacity of different oils, surfactants, and cosurfactants for saquinavir was evaluated to select optimal ingredients for preparation of SMEDDS. Through the construction of pseudo-ternary phase diagram, SMEDDS formulations were established. A polymer as a precipitation inhibitor was selected based on its viscosity and drug precipitation inhibiting capacity. The S-SMEDDS and SMEDDS designed were administered at an equal dose to rats. At predetermined time points, levels of saquinavir in lymph collected from the rats were assessed. SMEDDS prepared presented a proper self-microemulsification efficiency and dispersion stability. The S-SMEDDS fabricated using the SMEDDS and hydroxypropyl methyl cellulose 2910 as a precipitation inhibitor exhibited a signficantly enhanced solubilizing capacity for saquinavir. The drug concentration in a simulated intestinal fluid evaluated with the S-SMEDDS was also maintained at higher levels for prolonged time than that examined with the SMEDDS. The S-SMEDDS showed a considerably enhanced lymphatic absoprtion of saquinavir in rats compared to the SMEDDS. Therefore, the S-SMEDDS would be usefully exploited to enhance the lymphatic absorption of hydrophobic drugs that need to be targeted to the lymphatic system.