In vitro dissolution absorption system (IDAS2): Use for the prediction of food viscosity effects on drug dissolution and absorption from oral solid dosage forms.

Existing in vitro dissolution or permeation models to predict food effect are mainly based on Pharmacopeias' compendial media, which specify such variables as pH, bile salts, lipolytic enzymes, and phospholipids content. However, the viscosity of food in the gastrointestinal (GI) tract is not taken into account, although it can affect both the dissolution of the oral solid dosage form and absorption of the released drug. Here, a new in vitro dissolution absorption system (IDAS2) is utilized, which comprises a dissolution apparatus USP2 (DISTEK) equipped with specially constructed permeability chambers containing Caco-2 monolayers, thereby allowing dissolution and transepithelial absorption to be ascertained simultaneously. The IDAS2 was used to evaluate the effect of medium viscosity on both the dissolution of oral solid dosage forms and absorption of released drugs. Such information, which is not ordinarily determined in dissolution and permeation studies, will be helpful to the formulators developing robust oral dosage forms. Commercially available solid dosage forms of ten model drugs from across all BCS classifications were used in this evaluation: metoprolol, minoxidil, and propranolol from BCS class 1; carbamazepine, ketoprofen, and simvastatin from BCS class 2; atenolol and ranitidine from BCS class 3; and acetazolamide and saquinavir from BCS class 4. The study revealed the applicability of IDAS2 as a tool for in vitro screening of dissolution and absorption of intact oral solid products to predict food viscosity effect. The most profound viscosity effect on dissolution and absorption was observed of solid dosage forms for the BCS class 2 compounds carbamazepine and simvastatin. A higher medium viscosity significantly slowed down the dissolution rate of tested BSC class 4 compounds acetazolamide and saquinavir, without significant effect on their absorption. The solid dosage forms least affected by the viscosity of the medium tested were the BCS class 1 compounds minoxidil and propranolol.

Simultaneous Analysis of Dissolution and Permeation Profiles of Nanosized and Microsized Formulations of Indomethacin Using the In Vitro Dissolution Absorption System 2.

The in vitro dissolution absorption system 2 (IDAS2), a recent invention comprised a conventional dissolution vessel containing 2 permeation chambers with Caco-2 cell monolayers mounted with their apical side facing the dissolution media, permits simultaneous measurement of dissolution and permeation of drugs from intact clinical dosage forms. The objectives of this study were (1) to assess the utility of IDAS2 in the determination of the effect of particle size on in vitro performance of indomethacin and (2) to find out whether the behavior in IDAS2 of 2 indomethacin products differing in particle size is correlated with their in vivo behavior. Indomethacin dissolution and permeation across Caco-2 cell monolayers were simultaneously measured in IDAS2; the dissolution and permeation profiles were simultaneously modeled using a simple two-compartment model. Compared to microsized indomethacin, the nanosized formulation increased the dissolution rate constant by fivefold, whereas moderately increasing the permeation rate constant and the kinetic solubility. As a result, the drug amount permeated across the Caco-2 cell monolayers doubled in the nanosized versus microsized formulation. The in vitro results showed a good correlation with in vivo human oral pharmacokinetic parameters, thus emphasizing the physiological relevance of IDAS2 data in predicting in vivo absorption.

High energy X-ray pinhole imaging at the Z facility.

A new high photon energy (hν > 15 keV) time-integrated pinhole camera (TIPC) has been developed as a diagnostic instrument at the Z facility. This camera employs five pinholes in a linear array for recording five images at once onto an image plate detector. Each pinhole may be independently filtered to yield five different spectral responses. The pinhole array is fabricated from a 1-cm thick tungsten block and is available with either straight pinholes or conical pinholes. Each pinhole within the array block is 250 µm in diameter. The five pinholes are splayed with respect to each other such that they point to the same location in space, and hence present the same view of the radiation source at the Z facility. The fielding distance from the radiation source is 66 cm and the nominal image magnification is 0.374. Initial experimental results from TIPC are shown to illustrate the performance of the camera.

Rapid isolation of peptidic inhibitors of the solute carrier family transporters OATP1B1 and OATP1B3 by cell-based phage display selections.

OATP1B1 and OATP1B3 (1B3) are members of organic anion-transporting polypeptides (OATPs), a family of sodium-independent organic anion membrane transporters that contribute to transport of various drugs. To identify peptide inhibitors of OATP1B1, we developed a direct selection system on live cells using phage-displayed peptide libraries. Selections against OATP1B1 overexpressed cell-lines yielded three unique peptides able to inhibit the transport function of OATP1B1 and 1B3. Affinity maturation of one peptide led to identification of two peptides that demonstrated improved inhibition efficacy on drug uptake mediated by OATP1B1 and 1B3. We anticipate that these peptides will assist the identification of novel substrates for OATP1B1 and 1B3. Moreover, our selection system is a practical method for generating inhibitors of other membrane transporters.

Optimization of PEGylated nanoemulsions for improved pharmacokinetics of BCS class II compounds.

The objective of the study was the optimization of nanoemulsion formulations to prevent their rapid systemic clearance after intravenous administration. An amphiphilic PEG derivative DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(polyethylene glycol) with different chain lengths and concentration was used as a nanoemulsion droplet surface modifier. The danazol loading in all nanoemulsions was kept on the same level of ∼2 mg/mL. In the present investigation, PEGylated and non-PEGylated nanoemulsions were compared in vitro phagocytosis by incubating with lung macrophages and in vivo after intravenous administration in rats. Danazol-containing nanoemulsions (NE) modified with various PEG chain lengths (2000-10 000) and concentrations (3-12 mg/mL) were prepared and characterized. Nanoemulsion droplets were reproducibly obtained in the size range of 213-340 nm. The non-PEGylated NE had the surface charge of -25.4 mV. This absolute charge value decreased with increasing chain length and concentration. With increase in chain length and density the macrophage uptake decreased which could be due to decrease in surface charge and hydrophilicity of droplets. The greatest shielding of the NE droplets was reached with DSPE-PEG5000 at the concentration of 6 mg/mL where the surface charge changed to -1.27 mV. Following intravenous administration a maximum danazol exposure (401 ± 68.2 h ng/mL) was observed with the lowest clearance rate (5.06 ± 0.95 L/h/kg) from 6 mg/mL DSPE-PEG5000 nanoemulsion. PEG5000 and PEG10000 altered the pharmacokinetic of danazol by decreasing clearance and volume of distribution which is likely explained by the presence of hydrophilic shields around the droplets that prevent their rapid systemic clearance and tissue partitioning.

Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.

Evaluation of a nanoemulsion formulation strategy for oral bioavailability enhancement of danazol in rats and dogs.

The objective of this study was to determine whether nanoemulsion formulations constitute a viable strategy to improve the oral bioavailability of danazol, a compound whose poor aqueous solubility limits its oral bioavailability. Danazol-containing oil-in-water nanoemulsions (NE) with and without cosurfactants stearylamine (SA) and deoxycholic acid (DCA) were prepared and characterized. Nanoemulsion droplets size ranging from 238 to 344 nm and with surface charges of -24.8 mV (NE), -26.5 mV (NE-DCA), and +27.8 mV (NE-SA) were reproducibly obtained. Oral bioavailability of danazol in nanoemulsions was compared with other vehicles such as PEG400, 1% methylcellulose (MC) in water (1% MC), Labrafil, and a Labrafil/Tween 80 (9:1) mixture, after intragastric administration to rats and after oral administration of NE-SA, a Labrafil solution, or a Danocrine® tablet to dogs. The absolute bioavailability of danazol was 0.6% (PEG400), 1.2% (1% MC), 6.0% (Labrafil), 7.5% (Labrafil/Tween80), 8.1% (NE-DCA), 14.8% (NE), and 17.4% (NE-SA) in rats, and 0.24% (Danocrine), 6.2% (Labrafil), and 58.7% (NE-SA) in dogs. Overall, danazol bioavailability in any nanoemulsion was higher than any other formulation. Danazol bioavailability from NE and NE-SA was 1.8- to 2.2-fold higher than NE-DCA nanoemulsion and could be due to significant difference in droplet size.

Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.

Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes.

Use of transporter knockdown Caco-2 cells to investigate the in vitro efflux of statin drugs.

The objective of the present study was to determine the efflux transporters responsible for acid and lactone statin drug efflux using transporter knockdown Caco-2 cells. The bidirectional transport was determined in Caco-2 cell monolayers in which the expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), or multidrug resistance associated protein 2 (MRP2) was knocked down by transduction with lentivirus containing human transporter-targeted small hairpin RNAs (shRNAs). Cells transduced with lentivirus containing nontargeted shRNA served as the vector control. Atorvastatin, lovastatin, and rosuvastatin displayed extremely low apical-to-basolateral (A-to-B) transport, which made the P(app,A-B) values too unreliable to calculate the efflux ratio. Thus, transport comparisons were performed using the B-to-A permeability (P(app,B-A)) values. Presented in the order of vector control, P-gp, BCRP, and MRP2 knockdown Caco-2 cells, the P(app,B-A) values (×10(-6), cm/s) were 28.1 ± 1.3, 8.6 ± 2.9, 20.3 ± 1.8, and 21.5 ± 1.6 for atorvastatin; 96.1 ± 7.1, 25.3 ± 3.5, 57.3 ± 9.8, and 48.2 ± 2.3 for fluvastatin; and 14.1 ± 1.9, 4.6 ± 1.7, 5.8 ± 0.7, and 6.6 ± 1.8 for rosuvastatin, respectively. Lovastatin and simvastatin showed no efflux in the vector control or knockdown cell monolayers in either lactone or acid forms. Results indicate that atorvastatin, fluvastatin, and rosuvastatin were transported by P-gp, BCRP, and MRP2. On the other hand, neither the lactone nor the resulting acid of lovastatin and simvastatin was transported by P-gp, BCRP, or MRP2. The current study demonstrated that the transporter knockdown Caco-2 cells are useful tools for studying drug-transporter interactions and should help eliminate some of the ambiguity associated with the identification of drug-transporter interactions based on chemical inhibitors alone.

Investigation of the involvement of P-glycoprotein and multidrug resistance-associated protein 2 in the efflux of ximelagatran and its metabolites by using short hairpin RNA knockdown in Caco-2 cells.

Liver and bile secretion can be an important first-pass and clearance route for drug compounds and also the site of several drug-drug interactions. In the clinical program for ximelagatran development, an unexpected effect of erythromycin on the pharmacokinetics of the direct thrombin inhibitor ximelagatran and its metabolites was detected. This interaction was believed to be mediated by inhibition of drug transporters, which normally extrude the drug into the bile. Previous Caco-2 cell experiments indicated the involvement of an active efflux mechanism for ximelagatran, hydroxy-melagatran, and melagatran possibly mediated by P-glycoprotein (P-gp). However, the inhibitors used may not have been specific enough and the possibility that transporters other than P-gp were important in the Caco-2 cell assay cannot be excluded. In this study we used RNA interference, a post-transcriptional gene silencing mechanism in which mRNA is degraded in a sequence-specific manner, to specifically knock down P-gp or multidrug resistance-associated protein 2 (MRP2) transporters in Caco-2 cells. The data obtained from bidirectional transport studies in these cells indicate a clear involvement of P-gp but not of MRP2 in the transport of ximelagatran, hydroxy-melagatran, and melagatran across the apical cell membrane. The present study shows that short hairpin RNA Caco-2 cells are a valuable tool to investigate the contribution of specific transporters in the transcellular transport of drug molecules and to predict potential sites of pharmacokinetic interactions. The results also suggest that inhibition of hepatic P-gp is involved in the erythromycin-ximelagatran interaction seen in clinical studies.

Silencing the breast cancer resistance protein expression and function in caco-2 cells using lentiviral vector-based short hairpin RNA.

A series of stable breast cancer resistance protein (BCRP, ABCG2) knockdown cell lines were produced by transduction of Caco-2 cells with lentiviral vector-based short hairpin RNA (shRNA). Caco-2 cell is a human intestinal-derived cell line widely used to study intestinal drug absorption. Caco-2 expresses three apical drug efflux transporters: BCRP, P-glycoprotein (P-gp; ABCB1), and multidrug resistance protein 2 (MRP2, ABCC2). BCRP and P-gp in particular play a significant role in pharmacokinetics because of their expression at several key interfaces. Overexpression of BCRP in cancer cells may also be a mechanism of tumor resistance to chemotherapeutic drugs. The goal of this study was to engineer and characterize Caco-2 cell clones with stable knockdown of BCRP expression. The shRNA/BCRP lentiviral particles were used to infect a stable clone of Caco-2 cells. Expression of BCRP was monitored using quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence microscopy, and bidirectional transport of probe substrates, estrone-3-sulfate (E3S), and pheophorbide A (PhA). Based on qPCR, expression of BCRP mRNA was knocked down in five clones with a maximum of 97% silencing in clone D. Silencing of BCRP gene expression was maintained for at least 25 passages. Expression of BCRP protein was also reduced significantly. Functionally, BCRP knockdown was reflected in significant reduction of the efflux ratio of E3S and PhA. Clone D in particular should be a useful model for identifying and characterizing P-gp substrates and inhibitors without interference from BCRP and/or MRP2. In addition, it can be used in conjunction with wild-type or vector control Caco-2 cells to identify BCRP substrates.

Application and limitation of inhibitors in drug-transporter interactions studies.

The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug-transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER=17) and MDR-MDCK (ER=120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER=15 and 88, respectively) but not in MDR-MDCK cells (ER=1.1 and 1.3, respectively). CsA at 10 microM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug-drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug-drug interactions.

Treatment effects and short-term relapse of maxillomandibular expansion during the early to mid mixed dentition.

INTRODUCTION: The treatment effects and the short-term (0.9 +/- 0.45 years) relapse potential of phase I slow maxillary expansion, with a bonded palatal expander or a quad-helix appliance combined with a mandibular banded Crozat/lip bumper and followed by 12 to 15 months of retention, were examined. METHODS: Pretreatment (8.8 +/- 1.7 years) and posttreatment (11.1 +/- 1.7 years) models of 54 patients were used to evaluate treatment effects. Posttreatment (11.0 +/- 1.3 years) and follow-up (11.9 +/- 1.4 years) models of 23 patients who returned for phase II treatment were used to evaluate relapse over the 11 months, during which no retention was used. The models were digitized, and 15 measures were computed. RESULTS: Significant treatment increases were observed for all measurements in both arches. Treatment gains in arch perimeter (6%-8%) were due more to increases in intermolar width (11%-15%) than to increases in arch depth (5%). Posttreatment relapse was significant (P <.05) for all measures except mandibular intercanine width and maxillary molar arch depth. After accounting for normal growth, net changes (pretreatment to follow-up) indicated significant increases for all measures except maxillary molar arch depth. In addition to maintaining leeway space, the maxilla and the mandible showed net perimeter gains of 2.9 and 1.0 mm, respectively. CONCLUSIONS: Slow maxillary expansion combined with a mandibular banded Crozat/lip bumper during the early mixed dentition produced clinically useful increases in arch dimensions that subsequently underwent mild-to-moderate amounts of relapse after removal of all retention appliances.

Inter-species comparison of 7-hydroxycoumarin glucuronidation and sulfation in liver S9 fractions.

UDP glycosyltransferases (UGTs) and sulfotransferases (SULTs) are phase II enzymes that interact with a number of xenobiotics in humans and animals. Species differences in enzymatic characteristics have seldom been investigated. Liver S9 fractions are commonly used for studying phase II metabolism in vitro. The objective of this study was to characterize the UGT and SULT activities in liver S9 fractions from various species including humans, monkeys, dogs, and rats. A single substrate, 7-hydroxycoumarin (7-HC), at several concentrations was incubated at 37 degrees C with the S9 reaction matrices along with necessary cofactors. The rate of formation of two metabolites, 7-HC-glucuronide (7-HC-G) and 7-HC-sulfate (7-HC-S), was determined with Liquid Chromatography/Tanderm Mass Spectrometry (LC/MS/MS). Apparent Km and Vmax values were calculated for each species. For the UGTs, the apparent Km and Vmax for 7-HC-G formation varied greatly among different species, with dog UGTs having both the highest Km and Vmax values. In contrast to UGTs, the Km for 7-HC-S formation showed no significant difference among humans, monkeys, and rats (approximately 3 microM). However, the Km in dog was 8.7 microM. Species differences with respect to phase II metabolism must be carefully considered when selecting an in vitro model system to study various aspects of drug metabolism.

A secondary structural model of the 28S rRNA expansion segments D2 and D3 for Chalcidoid wasps (Hymenoptera: Chalcidoidea).

We analyze the secondary structure of two expansion segments (D2, D3) of the 28S ribosomal (rRNA)-encoding gene region from 527 chalcidoid wasp taxa (Hymenoptera: Chalcidoidea) representing 18 of the 19 extant families. The sequences are compared in a multiple sequence alignment, with secondary structure inferred primarily from the evidence of compensatory base changes in conserved helices of the rRNA molecules. This covariation analysis yielded 36 helices that are composed of base pairs exhibiting positional covariation. Several additional regions are also involved in hydrogen bonding, and they form highly variable base-pairing patterns across the alignment. These are identified as regions of expansion and contraction or regions of slipped-strand compensation. Additionally, 31 single-stranded locales are characterized as regions of ambiguous alignment based on the difficulty in assigning positional homology in the presence of multiple adjacent indels. Based on comparative analysis of these sequences, the largest genetic study on any hymenopteran group to date, we report an annotated secondary structural model for the D2, D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related apocritan taxa.

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome p450 induction studies.

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.