UNGAP best practice for improving solubility data quality of orally administered drugs.

An important goal of the European Cooperation in Science and Technology (COST) Action UNGAP (UNderstanding Gastrointestinal Absorption-related Processes, www.ungap.eu) is to improve standardization of methods relating to the study of oral drug absorption. Solubility is a general term that refers to the maximum achievable concentration of a compound dissolved in a liquid medium. For orally administered drugs, relevant information on drug properties is crucial during drug (product) development and at the regulatory level. Collection of reliable and reproducible solubility data requires careful application and understanding of the limitations of the selected experimental method. In addition, the purity of a compound and its solid state form, as well as experimental parameters such as temperature of experimentation, media related factors, and sample handling procedures can affect data quality. In this paper, an international consensus developed by the COST UNGAP network on recommendations for collecting high quality solubility data for the development of orally administered drugs is proposed.

Biorelevant media for transport experiments in the Caco-2 model to evaluate drug absorption in the fasted and the fed state and their usefulness.

In this work we developed and characterized transport media that simulate the composition of micellar phase of intestinal fluids in the fasted and, especially, in the fed state and are appropriate for evaluating intestinal drug permeability characteristics using the Caco-2 model (FaSSIF-TM(Caco) and FeSSIF-TM(Caco), respectively). Media composition was based on FaSSIF-V2 and FeSSIF-V2 and recently reported data on total lipid concentrations in the micellar phase of contents of the upper small intestine in the fasted and the fed state and was adapted for cell culture compatibility. Permeation data were evaluated by compartmental kinetic modeling. Permeability coefficients, P, of hydrophilic drugs were not affected by media composition. In contrast, P values of a series of lipophilic compounds measured with FaSSIF-TM(Caco) and FeSSIF-TM(Caco), and reflecting transport by diffusion were smaller than those obtained with a purely aqueous reference transport medium, aq-TM(Caco), following the rank order aq-TM(Caco)>FaSSIF-TM(Caco)>FeSSIF-TM(Caco). The decline of permeability values was stronger as lipophilicity of the compounds increased. Compared with values estimated using aq-TM(Caco), permeability was reduced, depending on the compound, by more than 20- to 100-fold when measured with FeSSIF-TM(Caco) whereas compound ranking in regard to the permeability characteristics was also affected. The impact of reduced P value on flux through the mucosa, hence on drug absorption, in combination with the drug amount loaded on colloidal particles needs to be taken into consideration in PBPK modeling especially when the food effect is evaluated.

Estimation of intragastric drug solubility in the fed state: comparison of various media with data in aspirates.

The suitability of various media to forecast the solubility of ketoconazole and dipyridamole in the fed stomach at various periods after meal administration was evaluated. Solubilities were measured with the shake-flask method in gastric fluids aspirated 30, 60 and 120 min after administration of 500 ml Ensure plus to healthy fasted adults, in three sets of simulated gastric fluids based on milk, and in simple aqueous buffered media. Simple aqueous buffered media vastly underestimated the intragastric solubility of model compounds in the fed state. When using undigested milk-based media, the solubilities of model compounds in aspirates were also underestimated by a factor of 2.5-27. Solubility in milk digested with pepsin was useful for estimating the intragastric solubility of ketoconazole (within 20%) but overestimated the intragastric values of dipyridamole by a factor of 2-19. For both drugs, the solubility in milk digested with pepsin and lipase predicted the solubility in aspirates collected 60 min after meal administration, whereas at other times it overestimated the intragastric solubility (by a factor of <5). Both the use of biorelevant media and simulation of intragastric digestion are necessary for the prediction of drug solubility in the fed stomach. Milk digested with pepsin and lipase enabled the estimation of the intragastric solubility of dipyridamole and ketoconazole at 1 h after meal intake. Simulation of vesicle/micellar structures seems to be key for the prediction of intragastric solubility in the fed stomach.

Simulation of gastric lipolysis and prediction of felodipine release from a matrix tablet in the fed stomach.

The importance of intragastric lipolysis to felodipine release from a hydrophilic, extended release tablet in the fed stomach was assessed in USP II apparatus with the tablet fixed on a steel wire above the paddle. The release medium, homogenized long-life milk, was gradually digested with shots of acidic solutions of pepsin over the course of the experiment in absence and in presence of biorelevant concentrations of a lipase that was similar to human gastric lipase. Percentage tablet erosion at specific times in the same media was measured in separate experiments. The data were compared to published data for intragastric release in fed healthy adults. In all cases, felodipine release occurred under sink conditions. Lipase facilitated felodipine release from the eroded polymer, bringing the release profile closer to the in vivo data. Likewise, the relationship between tablet erosion and amount of released felodipine reflected the in vivo data only when lipase was added to the medium. It was concluded that modelling intragastric lipolysis is necessary in order to simulate felodipine release from the extended release tablets in the fed stomach.

Estimating drug solubility in the gastrointestinal tract.

Solubilities measured in water are not always indicative of solubilities in the gastrointestinal tract. The use of aqueous solubility to predict oral drug absorption can therefore lead to very pronounced underestimates of the oral bioavailability, particularly for drugs which are poorly soluble and lipophilic. Mechanisms responsible for enhancing the luminal solubility of such drugs are discussed. Various methods for estimating intra-lumenal solubilities are presented, with emphasis on the two most widely implemented methods: determining solubility in fluids aspirated from the human gastrointestinal tract, and determining solubility in so-called biorelevant media, composed to simulate these fluids. The ability of the biorelevant media to predict solubility in human aspirates and to predict plasma profiles is illustrated with case examples.

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of ketoconazole in canine plasma.

An isocratic high-performance liquid chromatographic method with detection at 240 nm was developed, optimized and validated for the determination of ketoconazole in canine plasma. 9-Acetylanthracene was used as internal standard. A Hypersil BDS RP-C18 column (250 mm x 4.6 mm, 5 microm particle size), was equilibrated with a mobile phase composed of methanol, water and diethylamine 74:26:0.1 (v/v/v). Its flow rate was 1 ml/min. The elution time for ketoconazole and 9-acetylanthracene was approximately 9 and 8 min, respectively. Calibration curves of ketoconazole in plasma were linear in the concentration range of 0.015-10 microg/ml. Limits of detection and quantification in plasma were 5 and 15 ng/ml, respectively. Recovery was greater than 95%. Intra- and inter-day relative standard deviation for ketoconazole in plasma was less than 3.1 and 4.7%, respectively. This method was applied to the determination of ketoconazole plasma levels after administration of a commercially available tablet to dogs.

Development and validation of a simple reversed-phase high-performance liquid chromatography method for the determination of testosterone in serum of males.

An isocratic high-performance liquid chromatographic method with detection at 234 nm was developed, optimized and validated for the determination of testosterone in human serum. Propylparaben was used as internal standard. A Hypersil BDS RP-C18 column (150 mmx4.6 mm, 5 microm), was equilibrated with a mobile phase composed of acetonitrile and water (35:65, v/v) and having a flow rate of 1 ml/min. The elution time for testosterone and internal standard was approximately 11.6 and 9.9 min, respectively. Calibration curves of testosterone in serum were linear in the concentration range of 1-20 ng/ml. Limits of detection and quantification in serum were 0.4 and 1.1 ng/ml, respectively. Recovery was greater than 92%. Intra- and inter-day relative standard deviation for testosterone in serum was less than 2.1 and 3.9%, respectively. This method was applied to the determination of testosterone serum levels of 12 healthy males and data were correlated with data obtained using a radioimmunoassay method.

Sensitive and simple liquid chromatographic method with ultraviolet detection for the determination of nifedipine in canine plasma.

An isocratic high-performance liquid chromatographic method with detection at 240 nm was developed, optimized and validated for the determination of nifedipine in canine plasma. Liquid-liquid extraction was used as the sample preparation technique. Carbamazepine was used as internal standard. A Hypersil BDS RP-C18 column (250 mm x 4.6 mm, 5 microm) was equilibrated with a mobile phase composed of water and methanol, 45:55 (v/v). Its flow rate was 1 ml min(-1). The elution time for nifedipine and carbamazepine was approximately 12 and 8 min, respectively. Calibration curves of nifedipine in plasma were linear in the concentration range of 1-200 ng ml(-1). Limits of detection and quantification in plasma were 0.5 and 1.5 ng ml(-1), respectively. Recovery was greater than 98%. Intra- and inter-day relative standard deviation for nifedipine in plasma was less than 8.5 and 10%, respectively. This method was applied to the determination of nifedipine plasma levels after administration of commercially available soft gelatine capsules to dogs.

Optimized determination of lycopene in canine plasma using reversed-phase high-performance liquid chromatography.

An isocratic high-performance liquid chromatographic method with detection at 472 nm was developed, optimized and validated for the determination of lycopene in canine plasma. Ethyl-beta-apo-8'-carotenoate was used as internal standard. A Hypersil BDS RP-C18 column (150 mm x 4.6 mm), 5 microm particle size, was equilibrated with a mobile phase composed of acetonitrile and methanol (50:50, v/v). Its flow rate was 1.5 ml/min. The elution time for lycopene and ethyl-beta-apo-8'-carotenoate was approximately 11 and 5 min, respectively. Calibration curves of lycopene were linear in the concentration range of 3-200 ng/ml in plasma. Limits of detection and quantification in plasma were 1 and 4 ng/ml, respectively. Recovery was greater than 97%. Intra- and inter-day relative standard deviation for lycopene in plasma was less than 1.8 and 3.1%, respectively. This method was applied to the determination of lycopene plasma levels after single dose administration to dogs.

Development and optimization of a reversed-phase high-performance liquid chromatographic method for the determination of acetaminophen and its major metabolites in rabbit plasma and urine after a toxic dose.

A reversed-phase high-performance liquid chromatographic method with detection at 242 nm was developed, optimized and validated for the determination of acetaminophen (A) and its major metabolites glucuronide (AG) and sulfate (AS) conjugates in rabbit plasma and urine after a toxic dose. m-Aminophenol was used as internal standard (IS). A Hypersil BDS RP-C18 column (250 x 4.6 mm), 5 microm particle size, was equilibrated with a mobile phase composed of aqueous buffer solution of KH2PO4 0.05 M containing 1% CH3COOH (pH 6.5) and methanol (95:5, v/v). Its flow rate was 1.5 ml/min. Calibration curves of A, AG and AS were linear in the concentration ranges of 0.5-250, 1-200, 0.5-100 microg/ml in plasma and 1-200, 0.5-150, 0.5-100 microg/ml in urine matrix, respectively. Limits of detection and quantitation were calculated in all cases and extensive recovery studies were also performed. Intra-day relative standard deviation (R.S.D.) for A, AG and AS in plasma was less than 5, 4, 2% and in urine less than 4, 7, 4%, respectively, while the corresponding inter-day values were 7, 6, 4% and 5, 8, 6%, respectively.

Study on the inclusion complexes of bromazepam with beta- and beta-hydroxypropyl-cyclodextrins.

Solubility enhancement of the water insoluble bromazepam was studied during the formation of its inclusion complexes with beta-cyclodextrin (beta-CD) and beta-hydroxypropyl-cyclodextrin (beta-HP-CD). The phase solubility technique established by Higuchi and Connors and UV-spectrophotometric methods (zero- and second-order derivative approaches) were used to measure the changes introduced in this chemical system. The amount of time, which was necessary to reach equilibrium between inclusion complexes and their free components, was estimated and found equal to 24 h. The study was carried out at (i) pH 7.0 and 25 degrees C and (ii) pH 7.4 and 37 degrees C. The solubility of bromazepam increased linearly as a function of concentration for both beta-and beta-hydroxypropyl-cyclodextrins. Thus, the phase solubility diagrams were classified as of A(L) type in all cases. Under the above-mentioned conditions, the formation constants of the inclusion complexes were calculated and their stoichiometry was evaluated, found in the range of 69-85 M(-1) and 1:1, respectively.