First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

In vitro disposition profiling of heterocyclic compounds.

Compound libraries that are screened for biological activity commonly contain heterocycles. Besides potency, drug-like properties need to be evaluated to ensure in vivo efficacy of test compounds. In this context, we determined hepatic and intestinal disposition profiles for 17 heterocyclic compounds. All studied compounds showed rapid uptake in suspended rat hepatocytes, whereas metabolism was poor and the rate-limiting step in hepatic elimination. In vitro assays demonstrated a relatively low solubility and high intestinal permeability. Based on these in vitro data, heterocycles were categorized in the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS) to predict disposition characteristics before clinical data are available. Our findings emphasized the importance to use hepatocytes in addition to microsomes to study metabolism, since the latter lack non-microsomal enzymes and cellular context. Moreover, intracellular exposure should be considered to gain insight in the relevant fraction of the compound available at the enzymatic site. Finally, the study reveals discrepancies associated with the classification of heterocycles in BCS versus BDDCS. These probably originate from the binary character of both systems.

Evaluation of fasted state human intestinal fluid as apical solvent system in the Caco-2 absorption model and comparison with FaSSIF.

To date, the Caco-2 model is considered as the gold standard to predict intestinal drug absorption. Often, aqueous phosphate buffers are used as apical medium. The purpose of this study was to use fasted state human intestinal fluid (FaHIF) as apical solvent system to generate biorelevant permeability values for a series of 16 model drugs that can be used as reference data to critically evaluate fasted state simulated intestinal fluid (FaSSIF) as possible substitute medium. Caco-2 compatibility with FaHIF was achieved when 50mg/ml mucus was applied on top of the cells before adding the apical medium. The use of FaHIF as solvent system generated a broad range of apparent permeability values (Papp) for the series of model compounds. When Papp values obtained with FaHIF were compared to those obtained with FaSSIF, a strong correlation was observed (R=0.951). The use of FaSSIF in the absence of mucus did not significantly alter this correlation. For FaHIF, FaSSIF and reference phosphate buffer blank FaSSIF, a strong sigmoidal relationship was found between Papp and fahuman, illustrated by correlation coefficients of 0.961, 0.893 and 0.868, respectively. In terms of inter-subject variability, the use of FaHIF from different volunteers originating from two distinct age groups (18-25 years; 65-72 years) exhibited an average coefficient of variance (CV) of 30%. However, no age dependency in permeability could be observed. In conclusion, the data generated in this article justify the use of FaSSIF as biorelevant apical medium in the Caco-2 assay to accurately predict in vivo drug absorption. Also, the optimized mucus-containing Caco-2 model can be used in combination with intestinal fluid samples aspirated after drug administration to further investigate intraluminal drug and formulation behavior.

Evaluation of fasted and fed state simulated and human intestinal fluids as solvent system in the Ussing chambers model to explore food effects on intestinal permeability.

The Ussing chambers model is almost exclusively used in the presence of plain aqueous phosphate buffers as solvent system. In an attempt to further elucidate the effect of luminal ingredients and postprandial conditions on intestinal permeability, pooled fasted and fed state human intestinal fluids (FaHIFpool, FeHIFpool) were used. In addition, simulated intestinal fluids of both nutritional states (FaSSIF, FeSSIF) were evaluated as possible surrogate media for HIF. The use of FaHIFpool generated a broad range of Papp values for a series of 16 model drugs, ranging from 0.03×10(-6)cm/s (carvedilol) to 33.8×10(-6)cm/s (naproxen). A linear correlation was observed between Papp values using FaSSIF and FaHIFpool as solvent system (R=0.990), justifying the use of FaSSIF as surrogate medium for FaHIF in the Ussing chambers. In exclusion of the outlier carvedilol, a strong sigmoidal relationship was found between Papp and fahuman of 15 model drugs, illustrated by correlation coefficients of 0.961 and 0.936 for FaHIFpool and FaSSIF, respectively. When addressing food effects on intestinal permeability, the use of FeHIFpool resulted in a significantly lower Papp value for nine out of sixteen compounds compared to fasting conditions. FeSSIF as solvent system significantly overestimated Papp values in FeHIFpool. To conclude, the optimized Ussing chambers model using biorelevant media as apical solvent system holds great potential to investigate food effects in a more integrative approach, taking into account drug solubilisation, supersaturation and formulation effects.

Human and simulated intestinal fluids as solvent systems to explore food effects on intestinal solubility and permeability.

The mixed micelles and vesicles present in the intraluminal environment of the postprandial state exhibit suitable solubilizing capacities for lipophilic drugs. This increase in solubility, however, is accompanied by a decrease in the free fraction caused by micellar entrapment of these lipophilic compounds. In this study, both simulated and aspirated human intestinal fluids of fasted and fed state conditions were used to evaluate the influence of food on the intestinal disposition of a series of structurally related ß-blockers, with varying logP values. Using the in situ intestinal perfusion technique with mesenteric blood sampling in rats, it was demonstrated that fed state conditions significantly decreased the absorptive flux of the more lipophilic compounds metoprolol, propranolol and carvedilol, whereas the influence on the flux of the hydrophilic ß-blocker atenolol was limited. The solubility of BCS class II compound carvedilol was found to increase significantly in simulated and aspirated media of the fed state. Intestinal perfusions using intestinal media saturated with carvedilol, revealed a higher flux in the fasted state compared to the fed state, despite the higher solubility in the fed state. This study underscores the importance of addressing the complex nature of the behavior of compounds in the intraluminal environment in fasted and fed state conditions. Moreover, our data point out the value of studying the effect of food on both solubility and permeability using biorelevant experimental conditions.

Straightforward entry to pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones and their ADME properties.

A straightforward synthesis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones was developed starting from 2-chloropyridine-3-carboxylic acid by esterification, nucleophilic aromatic substitution and amide formation in one step, and ring closure allowing their synthesis with two identical or two different group attached to nitrogen. The structural diversity of these [2,3-d]pyrimidine-2,4(1H,3H)-diones resulted in significant variation in the biopharmaceutical properties. This was reflected by the broad range in fasted state simulated intestinal fluid solubility values (12.6 µM to 13.8 mM), Caco-2 permeability coefficients (1.2 × 10(-6)cm/s to 90.7 × 10(-6)cm/s) and in vitro-predicted human in vivo intrinsic clearance values (0 to 159 ml/min/kg).

A review of drug solubility in human intestinal fluids: implications for the prediction of oral absorption.

The purpose of this paper is to collate all recently published solubility data of orally administered drugs in human intestinal fluids (HIF) that were aspirated from the upper small intestine (duodenum and jejunum). The data set comprises in total 102 solubility values in fasted state HIF and 37 solubility values in fed state HIF, covering 59 different drugs. Despite differences in the protocol for HIF sampling and subsequent handling, this summary of HIF solubilities provides a critical reference data set to judge the value of simulated media for intestinal solubility estimation. In this regard, the review includes correlations between the reported solubilizing capacity of HIF and fasted or fed state simulated intestinal fluid (FaSSIF/FeSSIF). Correlating with HIF solubilities enables the optimal use of solubility measurements in simulated biorelevant media to obtain accurate estimates of intestinal solubility during drug development. Considering the fraction of poorly soluble new molecular entities in contemporary drug discovery, adequate prediction of intestinal solubility is critical for efficient lead optimization, early candidate profiling, and further development.

Biopharmaceutical profiling of a pyrido[4,3-d] pyrimidine compound library.

The pyrido-pyrimidine structure is associated with different biological activities, including kinase inhibition and antibacterial activity. However, drug-like properties of this scaffold have not been explored thoroughly. Therefore, the biopharmaceutical profile of ten pyrido[4,3-d]pyrimidines with different substitution pattern was determined at the intestinal and hepatic level. The compounds' biopharmaceutical properties were significantly affected by the substitution pattern, resulting in a broad range in fasted state simulated intestinal fluid solubility values (1.9 µM-4.2 mM) and Caco-2 permeability coefficients (0.17×10(-6) cm/s to 52×10(-6) cm/s). A phenylhydrazido group was responsible for the low FaSSIF solubility. Caco-2 permeability was impaired by a dimethoxyphenyl substituent. All analogs were metabolically stable in human intestinal microsomes. The hepatic metabolism, reflected by the extraction ratio, was intermediate to high (ER>0.3). Aliphatic chains, methoxy groups on a phenyl substituent, ketone and amine substituents were predicted as most susceptible sites for hepatic metabolism. Correlations were found between polar surface area of the compound and Caco-2 permeability (R=0.86) and metabolic stability (R=0.76). No toxicity was seen for the pyrido[4,3-d]pyrimidines with Caco-2 cells and sandwich-cultured rat hepatocytes. In conclusion, the large diversity of substituents on the pyrido[4,3-d]pyrimidine core highly influenced the compounds' drug-like properties.

Solubility profiling of HIV protease inhibitors in human intestinal fluids.

The present study pursued to profile the intestinal solubility of nine HIV protease inhibitors (PIs) in fasted- and fed-state human intestinal fluids (FaHIF, FeHIF) aspirated from four volunteers. In addition, the ability of fasted- and fed-state simulated intestinal fluids (FaSSIF, FeSSIF) to predict the intestinal solubility was evaluated. All PIs were poorly soluble in FaHIF (from 7 µM for ritonavir to 327 µM for darunavir) and FeHIF (from 15 µM for atazanavir to 409µM for darunavir). For four of nine PIs, food intake significantly enhanced the solubilizing capacity of intestinal fluids (up to 18.4-fold increase for ritonavir). The intersubject variability (average coefficient of variance CVfed = 60.6%, CVfasted = 40.4%) was higher as compared with the intrasubject variability (CVfed = 41.3%, CVfasted = 20.5%). PI solubilities correlated reasonably well between FaSSIF and FaHIF (R = 0.817), but not between FeSSIF and FeHIF (R = 0.617). To conclude, postprandial conditions increased the inter- and intrasubject variability of the PIs. The inability of FeSSIF to accurately predict the FeHIF solubility emphasizes the need for a multivariate approach to determine solubility profiles, taking into account solid-state characteristics, pH, mixed bile acid/phospholipid micelles, and digestive products.