Interaction of peptides, peptidomimetics and other drug candidates with the DI/tripeptide transport system in intestinal epithelial cells, using the in vitro caco-2 cell culture system

An uptake system was developed using Caco-2 cell monolayers and the dipeptide, glycyl-[3H]L-proline, as a probe compound. Glycyl-[3H]L-proline uptake was via the di-/tripeptide transport system (DTS) and, exhibited concentration-, pH- and temperature-dependency. Dipeptides inhibited uptake of the probe, and the design of the system allowed competitors to be ranked against one another with respect to affinity for the transporter. The structural features required to ensure or increase interaction with the DTS were defined by studying the effect of a series of glycyl-L-proline and angiotensin-converting enzyme (ACE)-inhibitor (SQ-29852) analogues on the uptake of the probe. The SQ-29852 structure was divided into six domains (A-F) and competitors were grouped into series depending on structural variations within specific regions. Domain A was found to prefer a hydrophobic function, such as a phenyl group, and was intolerant to positive charges and H+ -acceptors and donors. SQ-29852 analogues were more tolerant of substitutions in the C domain, compared to glycyl-L-proline analogues, suggesting that interactions along the length of the SQ-29852 molecule may override the effects of substitutions in the C domain. SQ-29852 analogues showed a preference for a positive function, such as an amine group in this region, but dipeptide structures favoured an uncharged substitution. Lipophilic substituents in domain D increased affinity of SQ-29852 analogues with the DTS. A similar effect was observed for ACE-NEP inhibitor analogues. Domain E, corresponding to the carboxyl group was found to be tolerant of esterification for SQ-29852 analogues but not for dipeptides. Structural features which may increase interaction for one series of compounds, may not have the same effect for another series, indicating that the presence of multiple recognition sites on a molecule may override the deleterious effect of anyone change. Modifying current, poorly absorbed peptidomimetic structures to fit the proposed hypothetical model may improve oral bioavailability by increasing affinity for the DTS. The stereochemical preference of the transporter was explored using four series of compounds (SQ-29852, lysylproline, alanylproline and alanylalanine enantiomers). The L, L stereochemistry was the preferred conformation for all four series, agreeing with previous studies. However, D, D enantiomers were shown in some cases to be substrates for the DTS, although exhibiting a lower affinity than their L, L counterparts. All the ACE-inhibitors and β-lactam antibiotics investigated, produced a degree of inhibition of the probe, and thus show some affinity for the DTS. This contrasts with previous reports that found several ACE inhibitors to be absorbed via a passive process, thus suggesting that compounds are capable of binding to the transporter site and inhibiting the probe without being translocated into the cell. This was also shown to be the case for oligodeoxynucleotide conjugated to a lipophilic group (vitamin E), and highlights the possibility that other orally administered drug candidates may exert non-specific effects on the DTS and possibly have a nutritional impact. Molecular modelling of selected ACE-NEP inhibitors revealed that the three carbonyl functions can be oriented in a similar direction, and this conformation was found to exist in a local energy-minimised state, indicating that the carbonyls may possibly be involved in hydrogen-bond formation with the binding site of the DTS.

Uptake and transport of orally-deliverable drugs across caco-2 cell monolayers:the effect of lipid formulations

The aim of this thesis is to investigate the physicochemical parameters which can influence drug loading within liposomes and to characterise the effect such formulations have on drug uptake and transport across in vitro epithelial barrier models. Liposomes composed of phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) and cholesterol (0, 4, 8, 16 µM) were prepared and optimised in terms of drug loading using the hand-shaking method (Bangham et al., 1965). Subsequently, liposomes composed of 16 µM PC or DSPC and cholesterol (4 µM) were used to monitor hydroxybenzoate release and transport from Iiposomes. The MIT (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and crystal violet assays were employed to determine toxicity of the Iiposome. formulations towards the Caco-2 cell line, employed to model the epithelial barrier in vitro. Uptake and transport of mannitol, propranolol, glutamine and digoxin was measured in the presence and absence of Iiposome formulations to establish changes in absorption resulting from the presence of lipid formulations. Incorporation of the four hydroxybenzoates was shown to be influenced by a number of factors, including liposome composition and drug conformation. Methyl hydroxybenzo.ate (MP) was incorporated into the bilayer most effectively with percentage incorporation of 68% compared to 45% for butyl hydroxybenzoate (BP), despite its increased Iipophilicity. This was attributed to the decreased packing ability of BP within the hydrocarbon core of the lipid bilayer compared to MP. Release studies also suggested that the smaller MP was more strongly incorporated within the lipid bilayer with only 8% of the incorporated solute being released after 48-hours compared to 17% in the case of BP. Model transport studies were seen to reflect drug release profiles from the liposome bilayers with significantly (p < 0.01) higher amounts of BP partitioning from the liposome compared to MP, Caco-2 cell viability was maintained above 86% in the presence of all Iiposome formulations tested indicating the liposome formulations are non-toxic towards Caco-2 cells. Paracellular (apical-to-basolateral) transport of mannitol was significantly increased in the presence of DSPC, PC / DSPC:Cholesterol (16:4 µM; 1000 µg). Glutamine uptake and transport via the carrier-mediated route was Significantly (p < 0.01) increased in the presence of PC I DSPC:Cholesterol (16:0; 16:4 µM). Digoxin apical-to-basolateral transport was significantly increased (p < 0,01) in the presence of PC / DSPC:Cholesterol (16:0; 16:4 µM); thus reducing digoxin efflux via P-glycoprotein. In contrast, PC:ChoJesterol (16:0; 16:4 µM) significantly (p < 0.01) decreased propranolol uptake via the passive transcellular route. Bi-directional transport of propranolol was significantly (p < 0,01) decreased in the presence of PC/DSPC:Cholesterol (16:0; 16:4 µM). The structure of a solute is an important determinant for the incorporation and release of a solute from liposome formulations. PC, DSPC and cholesterol liposome formulations are nontoxic towards Caco-2 cell monolayers and improved uptake and transport of mannitol, glutamine. and digoxin across Caco-2 cell monolayers; thus providing a potential alternative delivery vehicle.

Interaction of quinolone antibiotics with the human intestinal CACO-2 cell model

The transport of a group of quinolone antibiotics across the human intestinal model, Caco-2 cells, was investigated. It was found that the transport of the quinolones generally correlated with the lipophilicity of the compounds, indicating the passive diffusional transcellular processes were involved. However, it was observed that the transport in both directions apical-to-basolateral and basolateral-to-apical was not equivalent, and polarised transport occurred. For all the quinolones studied except, BMS-284756-01, it was found that the basolateral-to-apical transport was significantly greater than the apical-to-basolateral transport. This finding suggested that the quinolones underwent a process of active secretion. The pKas and logPs for the quinolones were determined using potentiometric titrations. The measured logP values were compared with those determined using theoretical methods. The theoretical methods for calculating logP including the Moriguchi method correlated poorly with the measured logP values. Further investigations revealed that there may be an active transporter involved in the apical-to-basolateral transport of quinolones as well. This mechanism was sensitive to competing quinolones, but, it was unaffected by the metabolic inhibitor combination of sodium azide (15mM) with 2-deoxy-D-glucose (50mM). The basolateral-to-apical transport of quinolones was found to be sensitive to inhibition by a number of different inhibitors. The metabolic inhibitors, sodium azide (15mM) with 2-deoxy-D-glucose (50mM) and 2,4-dinitrophenol (1mM), were able to reduce the basolateral-to-apical transport of quinolones. A reduction in temperature from 37°C to 2°C caused an 80-fold decrease in the transport of gatifloxacin in both directions, however, this effect was not sufficient to abolish the greater basolateral-to-apical secretion. As with apical-to-basolateral transport, it was found that quinolones competed with gatifloxacin for basolateral-to-apical transport, both ofloxacin (100μM) and norfloxacin (100μM) significantly (P<0.003) decreased the basolateral-to-apical transport of gatifloxacin; however, ciprofloxacin (100μM and 300μM) had no effect. A number of inhibitors of various transport systems were also investigated. It was found that the anion transport inhibitor, probenecid (100 μM) had a significant inhibitory effect on the basolateral-to-apical transport of ciprofloxacin (P=0.039), while the cation transport inhibitor cimetidine (100μM and 500μM) had no effect. The organic anion exchange inhibitor 4,4'diisothiocyanostilbene-2-2' -disulphonic acid DIDS (400μM) also had a significant inhibitory effect (P=O.O 13). The PgP inhibitor and anion exchange inhibitor verapamil (400Mμ) was able to completely abolish the basolateral-to-apical secretion of gatifloxacin and bring it into line with the apical-to-basolateral flux. In conclusion, the apical-to-basolateral and basolateral-toapical transport of quinolones involved an active component. The basolateral-to-apical secretion was abolished by a verapamil (400μM), a bisubstrate for PgP and the anion transporter.

Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 μM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 μM 1α,25-dihydroxyvitamin D3 to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213±54.35%) compared to that of parent cells (100±6.05%). Furthermore, they retained significantly less [3H]vincristine sulphate (p<0.05), a P-gp substrate, after efflux (272.89±11.86 fmol/mg protein) than the parent cells (381.39±61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1α,25-dihydroxyvitamin D3 treatment was quantified to be 76.2±7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0±20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0±20.0 to 1057.0±46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Investigation of the absorption pathways of some poorly absorbed drugs using human intestinal (CAC0-2) cell monolayers

Absorption across the gastro-intestinal epithelium is via two pathways; the transcellular and paracellular pathway. Caco-2 cells, when cultured on polycarbonate filters, formed a confluent monolayer with many properties of differentiated intestinal epithelial cells, As a model of human gastro-intestinaJ tract epithelia they were used to elucidate and characterise the transepithelial transport of two protein kinase C inhibitors, N-(3-chlorophenyl)-4-[2-(3-hydroxypropylamino)-4-pyridyl]-2-pyrimidinamin (CHPP) and N-benzoyl-staurosporine (NBS), and the polypeptide, human calcitonin. Lanthanum ions are proposed as a paracellular pathway inhibitor and tested with D-mannitol permeability and transepithelial electrical resistance measurements. The effect La3+ has on the carrier-mediated transport of D-glucose and Sodium taurocholate as well as the vesicularly transcytosed horseradish peroxidase was also investigated. As expected, 2 mM apical La3+ increases transepithelial electrical resistance 1.S-fold and decreases mannitol permeability by 63.0 % ± 1.37 %. This inhibition was not repeated by other cations. Apical 2 mM La3+ was found to decrease carrier-mediated D-glucose and taurocholate permeability by only 8.7 % ± 1.6 %, 26.3 % ± 5.0 %. There was no inhibitory effect on testosterone or PEG 4000 permeability observed with La3+. However, for horseradish peroxidase and human calcitonin permeability was decreased by 98.7 % ± 11.7%, and 96.2 % ± 0.8 % respectively by 2 mM La3+. Indicating that human calcitonin could also be transported by vesicular transcytosis. The addition of 2 mM La3+ to the apical surface of Caco-2 monolayers produces a paracellular pathway inhibition. Therefore, La3+ could be a useful additional tool in delineating the transepithelial pathway of passive drug absorption.

Systems biology approach to study permeability of paracetamol and its solid dispersion

Physiological changes that take place at cellular level are usually reflective of their level of gene expression. Different formulation excipients have an impact on physiological behavior of the exposed cells and in turn affect transporter genes, enterocyte-mediated metabolism and toxicity biomarkers. The aim of this study was to prepare solid dispersion of paracetamol and evaluate genetic changes that occur in Caco-2 cell lines during the permeability of paracetamol alone and paracetamol solid dispersion formulations. Paracetamol-PEG 8000 solid dispersion was prepared by melt fusion method and the formulation was characterised using differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Formulation of solid dispersion resulted in the conversion of crystalline drug into an amorphous form. Permeability studies showed that paracetamol absorption was higher from the solid dispersion formulation. DNA microarrays analysis was carried out in order to investigate the involvement of any efflux/uptake transporters in paracetamol or its solid dispersion permeability. Neither transporter carriers nor efflux proteins were found to be involved in the absorption of paracetamol or its PEG solid dispersion. Gene expression analysis established that paracetamol toxicity was potentially reduced upon formulation into solid dispersion when ATP binding cassette (ABC) and solute carrier transporter (SLC) genes were analyzed.

Physicochemical characterisation, drug polymer dissolution and in vitro evaluation of phenacetin and phenylbutazone solid dispersions with polyethylene glycol 8000

Poor water solubility leads to low dissolution rate and consequently, it can limit bioavailability. Solid dispersions, where the drug is dispersed into an inert, hydrophilic polymer matrix can enhance drug dissolution. Solid dispersions were prepared using phenacetin and phenylbutazone as model drugs with polyethylene glycol (PEG) 8000 (carrier), by melt fusion method. Phenacetin and phenylbutazone displayed an increase in the dissolution rate when formulated as solid dispersions as compared with their physical mixture and drug alone counterparts. Characterisation of the solid dispersions was performed using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). DSC studies revealed that drugs were present in the amorphous form within the solid dispersions. FTIR spectra for the solid dispersions of drugs suggested that there was a lack of interaction between PEG 8000 and the drug. However, the physical mixture of phenacetin with PEG 8000 indicated the formation of hydrogen bond between phenacetin and the carrier. Permeability of phenacetin and phenylbutazone was higher for solid dispersions as compared with that of drug alone across Caco-2 cell monolayers. Permeability studies have shown that both phenacetin and phenylbutazone, and their solid dispersions can be categorised as well-absorbed compounds.

Solid dispersions: formulation, characterisation, permeability and genomic evaluation

Poor water solubility is characterised by low dissolution rate and consequently reduced bioavailability. Formulation of solid dispersion of the drug has attracted considerable interest as a means of improving dissolution process of a range of poorly water soluble drugs. This current study investigates the formulation of solid dispersion for a range of poorly water soluble drugs with varying physicochemical properties including paracetamol, sulphamethoxazole, phenacetin, indomethacin, chloramphenicol, phenylbutazone and succinylsulphathiazole. Solid dispersions were prepared using various drugs to polymer ratios. PEG 8000 was selected as a carrier in the solid dispersions. The study revealed that inclusion of drug within the polymeric matrix, ratio of drug to polymer and physicochemical properties of the drug molecules enhance the dissolution rate. Characterisations of the solid dispersions were performed using DSC, FTIR and SEM. These studies revealed that all seven drugs were present in the amorphous form within the solid dispersions and there was a lack of interaction between the PEG 8000 and drug. Stability studies for solid dispersions showed that all seven drugs studied were unstable at accelerated conditions (40°C±2°C/75%RH±5%RH) whereas, they were found to be stable for 12 months at room conditions. Permeability of indomethacin, phenacetin, phenylbutazone and paracetamol were higher for solid dispersions as compared to drug alone across Caco-2 cell monolayers. From the cell uptake studies it was shown that PEG 8000 enhanced rhodamine123 uptake which suggested that PEG 8000 may increase the permeability of these drugs in solid dispersions. Gene expression profiles analyzing the expression changes in the ABC and solute carrier transporter during permeability studies.ABCA10, ABCB4, ABCC12, SLC12A6, MCT13, SLC22A12 and SLC6A6 gene expression were increased by indomethacin alone whereas solid dispersion of indomethacin resulted in a slight increase in expression. ABCC12 and SAMC gene expression was increased in case of paracetamol alone but slightly increased when exposed to solid dispersion of paracetamol.

Salmonella in companion animals

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides

BACKGROUND & AIMS: The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunolglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. METHODS: We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgACD71 complexes and intestinal permeability to the gliadin 3H-p3149 peptide were analyzed in polarized monolayers of Caco-2 cells. RESULTS: Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgACD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p3149, transport of intact 3H-p3149 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. CONCLUSIONS: Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apicalbasal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

Are transglutaminase 2 inhibitors able to reduce gliadin-induced toxicity related to celiac disease? A proof-of-concept study

Purpose Celiac disease is an autoimmune-mediated enteropathy characterized by adaptive and innate immune responses to dietary gluten in wheat, rye and barley in genetically susceptible individuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2), leading to an immune response in the small-intestinal mucosa. TG2 inhibitors have therefore been suggested as putative drugs for celiac disease. In this proof-of-concept study we investigated whether two TG2 inhibitors, cell-impermeable R281 and cell-permeable R283, can prevent the toxic effects of gliadin in vitro and ex vivo. Methods Intestinal epithelial Caco-2 cells were treated with peptic-tryptic-digested gliadin (PT-gliadin) with or without TG2 inhibitors and thereafter direct toxic effects (transepithelial resistance, cytoskeletal rearrangement, junction protein expression and phoshorylation of extracellular-signal-regulated kinase 1/2) were determined. In an organ culture of celiacpatient- derived small-intestinal biopsies we measured secretion of TG2-autoantibodies into the culture medium and the densities of CD25- and interleukin (IL) 15-positive cells, forkhead box P3 (FOXP3)-positive regulatory Tcells (Tregs) and Ki-67- positive proliferating crypt cells. Results Both TG2 inhibitors evinced protective effects against gliadin-induced detrimental effects in Caco-2 cells but the cellimpermeableR281seemedslightlymorepotent. Inaddition,TG2 inhibitor R281 modified the gluten-induced increase in CD25- and IL15-positive cells,Tregs and crypt cell proliferation, but had no effect on antibody secretion in celiac-patient-derived biopsies. Conclusions Our results suggest that TG2 inhibitors are able to reduce certain gliadin-induced effects related to responses in vitro and ex vivo. © Springer Science+Business Media, LLC 2012.

Phenotypic and genotypic analysis of intestinal spirochacies

Pulsed field gel electrophoresis of 82 intestinal spirochaete isolates showed specific differentiation of Serpulina pilosicoli and Serpulina hyodysenteriae although considerable heterogeneity was observed, especially amongst S. pilosicoli isolates. In several cases genotypically similar isolates originated from different animals suggesting that cross-species transmission may have occurred. The Caco-2 and Caco-21HT29 cell models have been proposed as potentially realistic models of intestinal infection. Quantitation of adhesion to the cells showed isolate 3 82/91 (from a bacteraemia) to adhere at significantly greater numbers than any other isolate tested. This isolate produced a PFGE profile which differed from other S. pilosicoli isolates and so would be of interest for further study. Comparison of bacteraemic and other S. pilosicoli isolates suggested that bacteraemic isolates were not more specifically adapted for adhesion to, or invasion of the epithelial cell layer than other S. pilosicoli isolates. Genotypically similar isolates from differing animal origins adhered to the Caco-2 model at similar levels. Generation of a random genomic library of S. pilosicoli and screening with species specific monoclonal antibody has enabled the identification of a gene sequence encoding a protein which showed significant homology with an ancestral form of the enzyme pyruvate oxidoreductase. Immunoscreening with polyclonal serum identified the sequences of two gene clusters and a probable arylsulphatase. One gene cluster represented a ribosomal gene cluster which has a similar molecular arrangement to Borrelia burgdorjeri, Treponema pallidum and Thermatoga maritima. The other gene cluster contained an ABC transporter protein, sorbitol dehydrogenase and phosphomannose isomerase. An ELISA type assay was used to demonstrate that isolates of S. pilosicoli could adhere to components of the extracellular matrix such as collagen (type 1), fibronectin, laminin, and porcine gastric mucin.

Intestinal absorption barriers as modelled by p-glycoprotein and cytochrome p450 a34 in caco2 cells

The passage number and origin of two populations of Caco-2 cells influence their enterocyte-like characteristics. Caco-2 cells of passage number >90 from Novartis pharmaceutical company possess higher levels of expression of alkaline phosphatase and P-glycoprotein and a greater cellular uptake of Gly-1.-Pro than those of passage number <40 from the American Type Tissue Culture collection. High P-gp expressing Caco-2 cells have been developed through stepwise selection of the cells with doxonibicin. This newly-developed cell line (hereafter referred to as Type I) possesses approximately twice as much P-gp protein than non-exposed cells, restricts the transepithelial transport of vincristine in the apical-to-basolateral direction whilst facilitating its transport in the reverse direction and accumulates less vincristine than non-exposed cells. There is no apparent evidence of the co-existence of the multidrug resistance protein (MIT) in Type I cells to account for the above-listed observations. Stopping the exposure for more than 28 days decreases the P-gp protein expression in previously doxorubicin-exposed Type I Caco-2 cells and reduces the magnitude of vincristine transepithelial fluxes in both directions to the levels that are almost similar to those of non-exposed cells. Exposing Caco-2 cells to 0.25 JAM la, 25-dihydroxyvitamin D3 induces their expression of cytochrome P450 3A4 protein to the level that is equivalent to that from isolated human jejunal cells. Under the same treatment, doxorubiein-exposed (Type I) cells metabolise naidazolam poorly and less extensively compared to non-exposed cells, suggesting that there is no such co-regulation of P-gp and CYP3A4 in Caco-2 cells. However, there is evidence which suggests CYP3A metabolises mida_zolam into 1- and 4-hydroxymidazolam, the latter may possibly be a P-gp substrate and is transported extracellularly by P-gp, supporting the hypothesis of P-gp-CYP3A4 synergistic roles in keeping xenobiotics out of the body. Doxoru.bicin-exposed (Type I) cells are less effective in translocating L-proline and glycyl-L-proline across the cell mono layers.

Investigating the modulation of oral drug absorption using in vitro models

Dipeptides can be absorbed into cells via the dipeptide transporter (which also transported tripeptides and dipeptide derivatives). The optimum conditions for measuring the inhibition of Gly-Pro uptake in Caco-2 cells were identified. A number of structure-activity relationships were identified. These included the effects of increasing the amino-acid chain-length, and the presence of a thiol or hydroxyl group in the side-chain increased IC50 while the presence of a hydroxyl group did not. The benzyl esters had lower or equal IC50 values compared to the parent dipeptides while the methyl esters had higher values. These results indicated that while molecular properties did affect IC50, the size, charge and composition of three particular groups caused the most significant effects, supporting the structure-activity relationship identified. An assay was developed using calcein-AM to show the inhibition of p-glycoprotein activity. There was no significant change due to the presence of mannitol but there was in the presence of clyclosporin A (p<0.01). Incubating the cells with the test solution for 30 minutes before the addition of the ester resulted in a significant (p<0.001) difference. The assay was specific for p-glycoprotein, as the presence MRP inhibitors had no effect (p>0.05). The modified protocol allowed the identification of p-glycoprotein inhibitors quickly and simply using a cell suspension of unmodified cells. The clinically relevant buffering of grapefruit juice to pH 7 led to a four-fold increase in intracellular calcein and hence significant inhibition of p-glycoprotein. Buffered orange and lemon juices had no effect on the assay. Flavone derivatives had previously been found to be inhibitors of CYP3A4 yet neither naringin nor naringenin had any significant effect at concentrations found in grapefruit juice. Of the other (non-grapefruit) flavone derivatives tested, hesperidin, found in orange juice, had no significant effect, kaempferol and rutin also had no effect while genistein significantly inhibited p-glycoprotein (results that support previous studies). Hydroxycinnamic acids had no effect on p-glycoprotein. Studies on other compounds found that the balance between inhibiting p-glycoprotein and disrupting cell membranes depends on the compound containing an oxygen atom and the size of the negative charge on it, as well as three-dimensional arrangement of the atoms.