Ann Marie Kaukonen

Evaluation of Mesoporous TCPSi, MCM-41, SBA-15, and TUD-1 Materials as API Carriers for Oral Drug Delivery

The feasibility of four mesoporous materials composed of biocompatible Si (TCPSi) or SiO2 (MCM-41, SBA-15, and TUD-1) were evaluated for oral drug delivery applications. The main focus was to study the effect of the materials different pore systems (unidirectional/2D/3D) and their pore diameters, pore size distributions, pore volumes on the maximal drug load capacity, and release profiles of a loaded active pharmaceutical ingredient. Ibuprofen was used as the model drug. The total pore volume of the mesoporous solid was the main factor limiting the maximum drug load capacity, with SBA-15 reaching a very high drug load of 1:1 in weight due to its high pore volume. Dissolution experiments were performed in HBSS buffers of pH 5.5, 6.8, and 7.4 to mimic the conditions in the small intestine. At pH 5.5 the dissolution rate of ibuprofen released from the mesoporous carriers was significantly faster compared with the standard bulk ibuprofen (86–63% versus 25% released at 45 min), with the fastest release observed from the 3D pore network of TUD-1 carrier. The utilization of mesoporous carriers diminished the pH dependency of ibuprofen dissolution (pKa = 4.42), providing an interesting prospect for the formulation of poorly soluble drug compounds.

N-in-One Permeability Studies of Heterogeneous Sets of Compounds across Caco-2 Cell Monolayers

AbstractPurpose. The purpose of the study was to evaluate several n-in-one cocktails of heterogeneous compounds to increase the throughput of permeability studies across Caco-2 monolayers, to investigate the reliability and applicability of the method, and to develop fast and sensitive analysis for the compounds. Compounds with potential interactions in efflux and/or active transport were chosen. Methods Permeability experiments with verapamil, propranolol, midazolam, hydroxyzine, timolol, buspirone, procaine, naproxen, ketoprofen, and antipyrine as single compounds and in cocktails of 5-10 compounds were performed at 50 μM concentration both in the apical-to-basolateral and basolateral-to-apical direction. The compounds were quantified by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). Toxicity tests were performed to determine cellular damage. Results The analytical method was sensitive, accurate, and rapid. The individual permeabilities of compounds in cocktails correlated well with permeabilities as single compounds. No significant interactions between the compounds within the mixtures were observed, except for acidic compounds. The studied mixtures did not show any toxicity. Conclusions The use of n-in-one cocktails is a suitable method to improve the capacity in routine permeability experiments and higher throughput screening of drug candidates, although potential interactions should always be borne in mind. The use of LC-ESI/MS/MS technology provides an excellent tool in fast and accurate analysis of small amounts of heterogeneous compounds.

Water-soluble β-Cyclodextrins in Paediatric Oral Solutions of Spironolactone: Preclinical Evaluation of Spironolactone Bioavailability from Solutions of β-Cyclodextrin Derivatives in Rats

Water‐soluble derivatives of β‐cyclodextrin have been considered for solubilization of spironolactone in the formulation of a safe liquid preparation for premature infants. The oral absorption of spironolactone was studied in rats to evaluate the need to adjust spironolactone dosage in prospective clinical studies. Spironolactone was administered in solutions of sulphobutyl ether β‐cyclodextrin (SBE7) or dimethyl‐β‐cyclodextrin (DM‐β‐CyD) and also as spironolactone‐containing powder papers (reference preparation). Spironolactone in SBE7 solution was administered intravenously to assess the extent of intestinal absorption from the different formulations.

Caco-2 cell monolayers as a tool to study simultaneous phase II metabolism and metabolite efflux of indomethacin, paracetamol and 1-naphthol

The human intestinal cell line, Caco-2, was used to study compounds - indomethacin, paracetamol and 1-naphthol - that undergo intestinal phase II metabolism followed by apical and/or basolateral efflux of the metabolites and/or parent compounds. The interplay was studied during permeability experiments across fully differentiated Caco-2 cell monolayers. The parent compounds and their glucuronide and/or sulfate metabolites were detected by LC-MS/MS. Conjugation of the model compounds and effluxes of their metabolites were observed. The efflux of indomethacin glucuronide was apical, but complementary basolateral efflux was observed at the highest indomethacin concentration (500 microM), probably due to apical saturation. Paracetamol glucuronide was not formed in these experiments, but apical and basolateral effluxes of paracetamol sulfate were observed. A typical bell-shaped inhibition curve was observed for the formation of 1-naphthol glucuronides, indicating substrate or product inhibition of the UGT enzyme(s) at higher 1-naphthol concentrations (200 microM and 500 microM). Based on these results, the fully differentiated Caco-2 cell monolayers can be applied as a platform for qualitative in vitro studies, where phase II metabolism and efflux activities are ongoing simultaneously.

Rate and extent of ion-exchange process: the effect of physico-chemical characteristics of salicylate anions.

Ten salicylate anions were used as model compounds in order to investigate systematically the impact of compound lipophilicity, valence, aqueous solubility and hydrogen bonding on binding into and release from a strong anion-exchange fiber, Smopex DS-218v. The release of salicylates from the fiber was studied at 1/10, 1/1 or 10/1 molar ratios of the external chloride-ions versus the salicylate bound in the fiber. The Donnan potential between the fiber and external solution (electrostatic interaction) appeared to be the main factor affecting the release of salicylates from the strong base anion-exchange fiber--an increase in the molar amount of the external chloride-ions resulted in a more effective release of all the salicylates from the fiber. The highest chloride-ion concentration (10/1) released the monovalent salicylates practically completely, while the lowest concentration (1/10) released only 10-35% of the loaded salicylates. The nature and strength of salicylate binding to the fiber by non-electrostatic interactions affected also the ion-exchange process, especially in dilute Cl- solutions. Hydrophobic interactions decreased the rate and amount of drug release from the fiber with the most lipophilic salicylates. Hydrogen bonding between the fiber and the compound restricted also the rate and extent of ion-exchange process of the hydrophilic 5-aminosalicylic acid and 5-hydroxysalicylic acid. The amount of divalent 5-carboxylsalicylic acid bound into and released from the fiber was clearly smaller as compared to the monovalent salicylates potentially due to cross-linking of the fiber chains.

High-performance liquid chromatography methods for the separation and quantitation of spironolactone and its degradation products in aqueous formulations and of its metabolites in rat serum

HPLC assays have been developed for the determination of spironolactone and its degradation products 7 alpha-thiospirolactone and canrenone in aqueous solutions of beta-cyclodextrins and for the determination of spironolactone and its metabolites 7 alpha-thiospirolactone, 7 alpha-thiomethylspirolactone, 6 beta-hydroxy-7 alpha-thiomethylspirolactone and canrenone in rat serum samples. Both methods were well suited for their respective applications, i.e., studying the stability of spironolactone in liquid formulations of beta-cyclodextrins and the oral absorption of spironolactone in rats. The HPLC method developed for spironolactone and its metabolites in rat serum requires very small volumes of serum making it possible to take several blood samples over a period of time.

The expression of most UDP-glucuronosyltransferases (UGTs) is increased significantly during Caco-2 cell differentiation, whereas UGT1A6 is highly expressed also in undifferentiated cells.

The human colon carcinoma cell line Caco-2 is often used as a model for intestinal drug absorption. To better understand xenobiotic glucuronidation in Caco-2 cells, we have examined the expression levels of different UDP-glucuronosyltransferases (UGTs) in them. The effects of two main factors were investigated, namely, passage number and cell differentiation. Hence, the mRNA levels of 15 human UGTs of subfamilies 1A and 2B were assessed in both undifferentiated and fully differentiated cells at four passage levels: P31, P37, P43, and P49. Quantitative reverse transcriptase-polymerase chain reaction was used to determine the mRNA levels of individual UGTs, and the values were normalized using β-actin as a reference gene. The results indicate that although passage number in the tested range exerts a mild effect on the expression level of several UGTs, the contribution of cell differentiation is much larger. The expression of nearly all the UGTs that were examined in this study was significantly, sometimes greatly, increased during cell differentiation. UGT1A6 was a distinct exception to this rule, however, because it was already highly expressed in the undifferentiated cells. The mRNA findings were confirmed at the enzyme activity level by measuring the glucuronidation of 1-naphthol, a very good substrate for UGT1A6, as well as estradiol that is not glucuronidated by this enzyme. The results revealed that 1-naphthol glucuronidation activity was high in both the differentiated and undifferentiated cells, whereas estradiol glucuronidation was only detected in the differentiated cells. Thus, Caco-2 cell differentiation plays a major role in UGT expression and ensuing metabolic reactions.

Water-soluble β-cyclodextrins in paediatric oral solutions of spironolactone: solubilization and stability of spironolactone in solutions of β-cyclodextrin derivatives

Abstract Water-soluble β -cyclodextrins, hydroxypropyl- β -cyclodextrin (HP β CD), dimethyl- β -cyclodextrin (DM β CD) and sulphobutyl ether β -cyclodextrin (SBE7), were evaluated as potential solubilizers of spironolactone (SP) in paediatric enteral formulations. 1 H-NMR was used to verify the formation of inclusion complexes and to detect possible degradation of spironolactone through deacetylation. The effect of temperature on spironolactone stability was studied in solutions of HP β CD and SBE7 to estimate shelf-lives of possible liquid preparations. HP β CD, DM β CD and SBE7 formed true inclusion complexes with spironolactone with a stoichiometry of 1:2 (SP: β CD). No degradation of spironolactone could be detected in the presence of DM β CD, whereas spironolactone degraded through deacetylation according to pseudo-first order kinetics in the presence of HP β CD and SBE7. Spironolactone degradation was slower in solutions of SBE7 than in HP β CD, with the slowest degradation at 6°C in SBE7 solution. Estimated shelf-lives ( t 90% ) for solutions containing 3 mg/ml of spironolactone were, even at 6°C, below 2 h in the presence of HP β CD. The t 90% -values in the presence of SBE7 were 4.1, 8.3 and 24.5 h at 22, 13 and 6°C, respectively. According to these results, SBE7 could be considered for the solubilization of spironolactone in paediatric enteral solutions, if the solution were to be prepared and stored at 6°C or below.

Evolution of paediatric off-label use after new significant medicines become available for adults: a study on triptans in Finnish children 1994–2007

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Off-label use in children is widespread. New medicines lack marketing authorization for paediatric use, even when they represent significant therapeutic advantages and are intended for treatment of conditions common in children. • Until now no information exists on how off-label use in children develops over time after a significant new medicine is approved for adults and what happens when it is later labelled for one paediatric age group. WHAT THIS STUDY ADDS • Off-label use of a new significant medicine begins in adolescents and extends to younger children with delay. First marketing authorization to adolescents, providing a more child-friendly formulation, results in increase of off-label use in younger children, and has limited effect on total off-label use. AIM To investigate the evolution of paediatric off-label use after a therapeutically new group of medicines for a common condition becomes available for adults but is labelled for children with a delay of several years. METHODS Triptans were used as a model, because migraine is common in children, and is the only indication for triptans. Data on all triptan prescriptions 1994-2007 were extracted from the nationwide Finnish Prescription Register. Prescriptions for children were compared over time. RESULTS Paediatric patients with triptan prescriptions increased from 204 in 1994 to 2618 in 2007. Sumatriptan accounted for 64% of all paediatric triptan prescriptions. When sumatriptan in a nasal formulation was labelled for children ≥ 12 years in 2003, off-label prescribing to younger children (6-11 years) doubled in 2003-2004. Sumatriptan on-label prescriptions increased to 728 adolescents (45% of sumatriptan in the age group) in 2007, but its off-label use continued also to increase to 1119 (61% of paediatric sumatriptan prescriptions) in 2007. In that year 72% of paediatric triptan use was off-label, 28% on-label. CONCLUSIONS When a new significant medicine becomes available in adults, off-label use in children starts slowly but continues to extend to younger children reaching a market size which is little influenced by late appearance of a labelled product. Paediatric treatment remains dominated by off-label use despite labelling of a product in an age appropriate formulation to the most relevant age group.

A comparison of the effect of medium- vs. long-chain triglycerides on the in vitro solubilization of cholesterol and/or phytosterol into mixed micelles.

Despite clinical evidence of the cholesterol-lowering effects of phytosterols, the exact mechanisms involved are still unclear. Displacement of cholesterol by phytosterols from mixed micelles, which is due to their greater hydrophobicity, is one of the hypotheses for the lumenal effects contributing to the reduction of intestinal cholesterol absorption. In this study a dynamic in vitro lipolysis method was used to examine the solubilization behavior of cholesterol and/or phytosterols during lipolysis to probe the efficacy of cholesterol displacement from mixed micelles by phytosterols. The effects of lipid chain length on sterol solubilization were studied by using microcrystalline suspensions containing 17% phytosterol or cholesterol, formulated in long-chain TG (LCT) and medium-chain TG (MCT). When digesting cholesterol suspended in LCT, the entire cholesterol dose was incorporated into the micellar phase. For the cholesterol formulation suspended in MCT, 50.3% of the initial dose was recovered in the micelles. Under the respective conditions, we observed lower solubilization of phytosterols than of cholesterol (roughly fourfold). Only 25% of the initial phytosterol dose was solubilized from suspensions formulated with LCT, and 13% was solubilized from MCT formulations. Co-administration of phytosterol and cholesterol suspensions showed a significant reduction of cholesterol solubilization, particularly when dosed in MCT, with ≈25% of the cholesterol dose solubilized. Insignificant amounts of cholesterol were displaced by phytosterols when cholesterol was presolubilized in the mixed micelles. The results show that, compared with LCT, mixed micelles containing MCT lipolysis products have a reduced solubilizing capacity for cholesterol, which adds to the effectiveness of the phytosterols in displacing cholesterol. This suggests potential benefits of using medium chain length lipids in cholesterol-lowering phytosterol products.