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Variability of the unbound fraction in plasma (f(u)) between labs, methods and conditions is known to exist. Variability and uncertainty of this parameter influence predictions of the overall pharmacokinetics of drug candidates and might jeopardise safety in early clinical trials. Objectives of this study were to evaluate the variability of human in vitro f(u)-estimates between labs for a range of different drugs, and to develop and validate an in silico f(u)-prediction method and compare the results to the lab variability. A new in silico method with prediction accuracy (Q(2)) of 0.69 for log f(u) was developed. The median and maximum prediction errors were 1.9- and 92-fold, respectively. Corresponding estimates for lab variability (ratio between max and min f(u) for each compound) were 2.0- and 185-fold, respectively. Greater than 10-fold lab variability was found for 14 of 117 selected compounds. Comparisons demonstrate that in silico predictions were about as reliable as lab estimates when these have been generated during different conditions. Results propose that the new validated in silico prediction method is valuable not only for predictions at the drug design stage, but also for reducing uncertainties of f(u)-estimations and improving safety of drug candidates entering the clinical phase.

Pharmacokinetic/toxicokinetic (PK/TK) information for chemicals in humans is generally lacking. Here we applied machine learning, conformal prediction and a new physiologically-based PK/TK model for prediction of the human PK/TK of 65 chemicals from different classes, including carcinogens, food constituents and preservatives, vitamins, sweeteners, dyes and colours, pesticides, alternative medicines, flame retardants, psychoactive drugs, dioxins, poisons, UV-absorbents, surfactants, solvents and cosmetics.About 80% of the main human PK/TK (fraction absorbed, oral bioavailability, half-life, unbound fraction in plasma, clearance, volume of distribution, fraction excreted) for the selected chemicals was missing in the literature. This information was now added (from in silico predictions). Median and mean prediction errors for these parameters were 1.3- to 2.7-fold and 1.4- to 4.8-fold, respectively. In total, 59 and 86% of predictions had errors in silico predictions). Median and mean prediction errors for these parameters were 1.3- to 2.7-fold and 1.4- to 4.8-fold, respectively. In total, 59 and 86% of predictions had errors

Permeability (Pe) and solubility/dissolution are two major determinants of gastrointestinal (GI) drug absorption. Good prediction of these is crucial for predicting doses, exposures and potential interactions, and for selecting appropriate candidate drugs. The main objective was to evaluate screening methods for prediction of GI Pe, solubility/dissolution and fraction absorbed (fa) in humans. The most accurate Pe models for prediction of fa of passively transported and highly soluble compounds appear to be the 2/4/A1 rat small intestinal cell model (in-vitro and in-silico), a newly developed artificial-membrane method, and a semi-empirical approach based on in-vitro membrane affinity to immobilized lipid bilayers, effective molecular weight and physiological GI variables. The predictability of in-vitro Caco-2, in-situ perfusion and other artificial membrane methods seems comparably low. The Pe and fa in humans for compounds that undergo mainly active transport were predicted poorly by all models investigated. However, the rat in-situ perfusion model appears useful for prediction of active uptake potential (complete active uptake is generally well predicted), and Caco-2 cells are useful for studying bidirectional active transport, respectively. Human intestinal in-vitro Pe, which correlates well with fa for passively transported compounds, could possibly also have potential to improve/enable predictions of fa for actively transported substances. Molecular descriptor data could give an indication of the passive absorption potential. The ‘maximum absorbable dose’ and ‘dose number’ approaches, and solubility/dissolution data obtained in aqueous media, appear to underestimate in-vivo dissolution to a considerable extent. Predictions of in-vivo dissolution should preferably be done from in-vitro dissolution data obtained using either real or validated simulated GI fluids.

The goal of this study was to determine the most appropriate hydrodynamic model for the Loc-I-Gut in-vivo perfusion system. The general mixing-tank-in-series model, which can approximate single mixing tank and laminar and plug-flow hydrodynamics, was fitted to the observed experimental residence-time distribution curves for the non-absorbable marker [14C]PEG 4000. The residence-time distribution analysis shows that the hydrodynamics of the perfusion solution within the jejunal segment in man is well approximated by a model containing on average between 1–2 mixing tanks in series. The solution is well mixed when using perfusion rates of 20, 30 and 60 mL min−. The average mean residence time estimates from the fitted residence-time distribution were 12 ± 7.6, 15 ± 4.2 and 7.7 ± 4.6 min, respectively, at these three perfusion rates. The mean volumes of the segment (Vs) were 25 ± 15, 45 ± 12 and 46 ± 27 mL, respectively. There were no statistical differences between 20, 30 and 60 mL min− in respect of the number of mixing tanks (n) and mean residence times. This residence-time distribution analysis indicates that the luminal fluid in the Loc-I-Gut perfusion system is well-mixed, and that permeability calculations based on the well-mixed assumption most closely approximate the actual local (average) membrane permeability within the perfused segment.

EXPERIMENTAL ESTIMATION OF THE EFFECTIVE UNSTIRRED WATER LAYER THICKNESS IN THE HUMAN JEJUNUM, AND ITS IMPORTANCE IN ORAL-DRUG ABSORPTION

The main objective was to evaluate and propose methods for predicting biliary clearance (CLbile) and enterohepatic circulation (EHC) of intact drugs in man. Another aim was to evaluate to role of intestinal drug secretion and propose a method for prediction of intestinal secretion CL (CLi). Animal data poorly predict the CL and CLbile of biliary excreted drugs, and the suggested molecular weight threshold for bile excretion as the dominant elimination route does not seem to hold. Active transport, low metabolic intrinsic CL (CLint) and, as an approximation, permeability (Pe) less than that of metoprolol is required for substantial CLbile to occur. The typical EHC plasma concentration vs time profile (multiple peaks) is demonstrated for many low metabolic CLint-compounds with efflux and moderate to high intestinal Pe and fraction absorbed. Physiologically-based in-vitro to in-vivo (PB-IVIV) methodology with in-vitro intrinsic CLbile-data obtained with sandwich-cultured human hepatocytes has generated 2- and 5-fold underpredictions for two compounds with intermediate to high CLbile. This is despite not considering the unbound fraction. Possible explanations include low transporter activity and diffusion limitations in the in-vitro experiments. Intestinal reabsorption and EHC were also neglected in these predictions and in-vivo CLbile estimations. The sandwich model and these reference data are still very useful. Consideration of an empirical scaling factor and a newly developed approach that accounts for intestinal reabsorption and EHC could potentially lead to improved PB-IVIV predictions of CLbile. Apparently, no attempts have been made to predict CLi. Elimination via the intestinal route does not appear to be of great importance for the few compounds with available data, but could be equally as important as bile excretion. Net secretion in-vitro Pe and newly estimated in-vivo intrinsic CLi data for digoxin and rosuvastatin could be useful for approximation of CLi of other compounds.

The kidneys have the capability to both excrete and metabolise drugs. An understanding of mechanisms that determine these processes is required for the prediction of pharmacokinetics, exposures, doses and interactions of candidate drugs. This is particularly important for compounds predicted to have low or negligible non-renal clearance (CL). Clinically significant interactions in drug transport occur mostly in the kidneys. The main objective was to evaluate methods for prediction of excretion and metabolic renal CL (CLR) in humans. CLR is difficult to predict because of the involvement of bi-directional passive and active tubular transport, differences in uptake capacity, pH and residence time on luminal and blood sides of tubular cells, and limited knowledge about regional tubular residence time, permeability (Pe) and metabolic capacity. Allometry provides poor predictions of excretion CLR because of species differences in unbound fraction, urine pH and active transport. The correlation between fraction excreted unchanged in urine (fe) in humans and animals is also poor, except for compounds with high passive Pe (extensive/complete tubular reabsorption; zero/negligible fe) and/or high non-renal CL. Physiologically based in-vitro/in-vivo methods could potentially be useful for predicting CLR. Filtration could easily be predicted. Prediction of tubular secretion CL requires an in-vitro transport model and establishment of an in-vitro/in-vivo relationship, and does not appear to have been attempted. The relationship between passive Pe and tubular fraction reabsorbed (freabs) for compounds with and without apparent secretion has recently been established and useful equations and limits for prediction were developed. The suggestion that reabsorption has a lipophilicity cut-off does not seem to hold. Instead, compounds with passive Pe that is less than or equal to that of atenolol are expected to have negligible passive freabs. Compounds with passive Pe that is equal to or higher than that of carbamazepine are expected to have complete freabs. For compounds with intermediate Pe the relationship is irregular and freabs is difficult to predict. Tubular cells are comparably impermeable (for passive diffusion), and show regional differences in enzymatic and transporter activities. This limits the usefulness of microsome data and makes microsome-based predictions of metabolic CLR questionable. Renal concentrations and activities of CYP450s are comparably low, suggesting that CYP450 substrates have negligible metabolic CLR. The metabolic CLR of high-Pe UDP-glucuronyltransferase substrates could contribute to the total CL.

Intestinal mucosal cells operate with different metabolic and transport activity, and not all of them are involved in drug absorption and metabolism. The fraction of these cells involved is dependent on the absorption characteristics of compounds and is difficult to predict (it is probably small). The cells also appear comparably impermeable. This shows a limited applicability of microsome intrinsic clearance (CLint)-data for prediction of gut-wall metabolism, and the difficulty to predict the gut-wall CL (CLGW) and extraction ratio (EGW). The objectives of this review were to evaluate determinants and methods for prediction of first-pass and systemic EGW and CLGW in man, and if required and possible, develop new simple prediction methodology. Animal gut-wall metabolism data do not appear reliable for scaling to man. In general, the systemic CLGW is low compared with the hepatic CL. For a moderately extracted CYP3A4-substrate with high permeability, midazolam, the gut-wall/hepatic CL-ratio is only 1/35. This suggests (as a general rule) that systemic CLGW can be neglected when predicting the total CL. First-pass EGW could be of importance, especially for substrates of CYP3A4 and conjugating enzymes. For several reasons, including those presented above and that blood flow based models are not applicable in the absorptive direction, it seems poorly predicted with available methodology. Prediction errors are large (several-fold on average; maximum-15-fold). A new simple first-pass EGW-prediction method that compensates for regional and local differences in absorption and metabolic activity has been developed. It has been based on human cell in-vitro CLint and fractional absorption from the small intestine for reference (including verapamil) and test substances, and in-vivo first-pass EGW-data for reference substances. First-pass EGW-values for CYP3A4-substrates with various degrees of gastrointestinal uptake and CLint and a CYP2D6-substrate were well-predicted (negligible errors). More high quality in-vitro CLint- and in-vivo EGW-data are required for further validation of the method.

Physiologically based methods generally perform poorly in predicting in-vivo hepatic CL (CLH) from intrinsic clearance (CLint) in microsomes in-vitro and unbound fraction in blood (fu,bl). Various strategies to improve the predictability have been developed, and inclusion of an empirical scaling factor (SF) seems to give the best results. This investigation was undertaken to evaluate this methodology and to find ways to improve it further. The work was based on a diverse data set taken from Ito and Houston (2005). Another objective was to evaluate whether rationalization of CLH predictions can be made by replacing blood/plasma-concentration ratio (Cbl/Cpl) measurements with SFs. There were apparently no or weak correlations between prediction errors and lipophilicity, permeability (compounds with low permeability missing in the data set) and main metabolizing CYP450s. The use of CLint class (high/low) and drug class (acid/base/neutral) SFs (the CD-SF method) gives improved and reasonable predictions: 1.3-fold median error (an accurate prediction has a 1-fold error), 76% within 2-fold-error, and a median absolute rank ordering error of 2 for CLH (n = 29). This approach is better than the method with a single SF. Mean (P < 0.05) and median errors, fraction within certain error ranges, higher percentage with most accurate predictions, and ranking were all better, and 76% of predictions were more accurate with this new method. Results are particularly good for bases, which generally have higher CLH and the potential to be incorrectly selected/rejected as candidate drugs. Reasonable predictions of fu,bl can be made from plasma fu (fu,pl) and empirical blood cell binding SFs (B-SFs; 1 for low fu,pl acids; 0.62 for other substances). Mean and median fu,bl prediction errors are negligible. The use of the CD-SF method with predicted fu,bl (the BCD-SF method) also gives improved and reasonable results (1.4-fold median error; 66% within 2-fold-error; median absolute rank ordering error = 1). This new empirical approach seems sufficiently good for use during the early screening; it gives reasonable estimates of CLH and good ranking, which allows replacement of Cbl/Cpl measurements by a simple equation.

This review has evaluated the Biopharmaceutics Classification System (BCS) and improvements have been proposed. The BCS has a very strict solubility/dissolution limit, a generous Pe-limit (≥ 14-times higher rate constant limit for dissolution than for permeation), and is stricter for drugs with a long half-life (t1/2). Available human in-vivo, in-vitro, and in-silico Pe-methods cannot classify Pe for moderately to highly permeable substances sufficiently well, and in-vitro data often underpredict the in-vivo dissolution potential and rate. Good in-vivo dissolution and absorption can be expected for most high Pe drug products. It has not been possible to find a highly permeable product with a Dose number (Do) < 385 (< 2400 in the fed state) that is clearly incompletely absorbed, and near complete uptake has been shown for a drug product with a Do of 660000. The potential implication of these findings is that many true BCS Class I drug products are incorrectly classified. This could be a reason for the limited use of this system. On this basis, it has been suggested that: the limit for high for solubility/dissolution is decreased (to >40 and >95% dissolved within 30min and 3 h, respectively); the limit for high Pe is increased (to >Pe of metoprolol); accurate Pe-models or in-vivo fraction absorbed data are used; solubility/dissolution tests are performed using real or validated simulated gastrointestinal fluids; in-vitro/in-vivo dissolution relationships are established; the t½ is considered; and the rate-limiting step for in-vivo absorption is determined. A major change could be to reduce the BCS into two classes: permeation-rate (Class I) or dissolution-rate (Class II) limited absorption. It is believed that this could give a better balance and increase the number of biowaivers.

Cyclooxygenase inhibiting nitric oxide donators (CINODs) are a new class of anti-inflammatory and analgesic drugs that may minimise gastrointestinal toxicity compared with standard non-steroidal anti-inflammatory drugs (NSAIDs) by virtue of nitric oxide donation.A proof of concept study of the gastrointestinal safety of AZD3582, the first CINOD available for human testing, was conducted. Thirty one subjects were randomised to receive placebo, naproxen 500 mg twice daily, or its nitroxybutyl derivative AZD3582 in an equimolar dose (750 mg twice daily) for 12 days in a double blind three period crossover volunteer study. At the start and end of each dosing period, gastroduodenal injury was assessed by endoscopy and small bowel permeability by differential urinary excretion of lactulose and L-rhamnose. Pharmacokinetic profiles were assessed at steady state.On naproxen, the mean total number of gastroduodenal erosions was 11.5 (and one subject developed an acute ulcer) versus 4.1 on AZD3582 (p0.0001). More than half of the subjects had no erosions on AZD3582. Differences were seen for both the stomach and duodenum. Naproxen increased intestinal permeability (lactulose:L-rhamnose ratio 0.030 before v 0.040 after treatment) whereas AZD3582 (0.029 before, 0.029 after; p=0.006 v naproxen) and placebo (0.030 before, 0.028 after; p0.001 v naproxen) did not. The steady state bioavailability of naproxen metabolised from AZD3582 was 95% (95% confidence interval 87-101%) of that after naproxen administration.This human study supports animal data showing reduced gastrointestinal toxicity with the CINOD AZD3582. The potential combination of effective pain relief and gastrointestinal protection offered by AZD3582 warrants further evaluation in human clinical studies.

The purpose of this study was to characterize the rat jejunal passive transport and the possible active efflux of three local anaesthetics, ropivacaine, lidocaine and bupivacaine using two different absorption models, the in situ single-pass intestinal perfusion and the in vitro Ussing chamber model, as well as P-glycoprotein (Pgp)-mediated calcein transport inhibition in Caco-2 cells. Concentration and pH dependence, efflux inhibition by verapamil and digoxin and bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption of the local anaesthetics. In the jejunal perfusion the permeability of these agents appeared to be high, predicting complete intestinal absorption (>90%). There was no effect of the Pgp inhibitors on net absorption for any of the local anaesthetics in the two absorption models. However, in the Ussing chamber at an equal pH of 7.4 at mucosal and serosal sides, the observed jejunal permeability ratios (S-M)/(M-S), of 2.3, 1.8 and 3.0 for ropivacaine, lidocaine and bupivacaine, respectively, indicated at least some involvement of carrier-mediated intestinal secretion. This idea was supported in the calcein AM Pgp transport assay in which two of the tested local anaesthetic agents affected cellular calcein retention. As anticipated for these agents, the mucosal pH conditions were shown to largely affect the gut permeability. The jejunal permeabilities of the local anaesthetics as measured in the two absorption models fitted well in a model comparison that incorporated the permeabilities of six other structurally unrelated drugs. In conclusion, the jejunal permeability of ropivacaine, lidocaine and bupivacaine was high and although evidence was obtained for carrier-mediated intestinal efflux this process appeared not to have a significant influence on the rate and extent of in vivo intestinal absorption. Rather, passive diffusion of these agents seems to be the major mechanism for the intestinal absorption.

The aim of this study was to investigate if the effective in-situ permeability (Peff) of a new growth hormone-releasing peptide, hexarelin, along rat intestine was enhanced by a lipid matrix drug-delivery system comprising a mixture of soybean phosphatidyl choline and medium-chain monoacylglycerol (PC-MG). The study was performed with and without a protease inhibitor, Pefabloc SC. To enable better understanding of the mechanism of action of this delivery system we also studied the uptake of a small hydrophilic molecule, atenolol. PC-MG at a concentration of 15 mmol L−1 increased the jejunal Peff of hexarelin approximately 20-fold, both in the presence and absence of Pefabloc SC, whereas Peff was not increased in the ileum and colon. PC-MG had no effect on the jejunal, ileal and colonic Peff of atenolol. Complete recovery of the non-absorbable molecule PEG 4000 showed that functional intestinal viability was maintained in all experiments. Although the results obtained in this study are promising, pharmacokinetic and toxicological studies are required to investigate if this delivery system is a suitable and safe candidate for improving the oral bioavailability of hexarelin.

Because the absorption of orally administered drugs depends on intestinal permeability, we have investigated how absorptive capacity varies from the proximal to distal intestine in rats. The effective permeabilities of compounds with a range of physicochemical properties and different absorption mechanisms were estimated by use of a previously validated in-situ, single-pass perfusion model. The low colonic permeabilities of d-glucose and l-dopa indicate the absence or low capacity of the glucose-and amino-acid-transporters in this region. With the exception of the small and moderately lipophilic nonsteroidal anti-inflammatory drug, naproxen, for which permeability was maintained throughout the intestine, the passive intestinal permeabilities for hydrophilic and lipophilic drugs were approximately twice as high in the jejunum and ileum as in the colon. These observations are in accord with those made in recent studies. However, the reasons for the high colonic permeability of non-steroidal anti-inflammatory drugs, and results obtained in previous animal experiments demonstrating that the colon is the region of the intestine with the highest absorptive capacity were not fully clarified. These data show that the permeability to hydrophilic and lipophilic drugs decreases along the intestine, whereas it is maintained throughout the intestine for the small and moderately lipophilic naproxen. Further investigations are required to clarify the interplay between membrane composition, fluidity and permeability under various conditions in different absorption models.