Intestinal Fluid Samples Research Articles

Characterisation of colloidal structures and their solubilising potential for BCS class II drugs in fasted state simulated intestinal fluid

A suite of fasted state simulated intestinal fluid (SIF), based on variability observed in a range of fasted state human intestinal fluid (HIF) samples was used to study the solubility of eight poorly soluble drugs (three acidic drugs (naproxen, indomethacin and phenytoin), two basic drugs (carvedilol and tadalafil) and three neutral drugs (felodipine, fenofibrate, griseofulvin)). Particle size of the colloidal structures formed in these SIF in the presence and absence of drugs was measured using dynamic light scattering and nanoparticle tracking analysis.Results indicate that drug solubility tends to increase with increasing total amphiphile concentration (TAC) in SIF with acidic drugs proving to be more soluble than basic or neutral drug in the media evaluated. Dynamic light scattering showed that as the amphiphile concentration increased, the hydrodynamic diameters of the structures decreased. The scattering distribution confirmed the polydispersity of the simulated intestinal fluids compared to the monodisperse distribution observed for FaSSIF v1). There was a large difference in the size of the structures found based on the composition of the SIF, for example, the diameter of the structures measured in felodipine in the minimum TAC media was measured to be 170 ± 5 nm which decreased to 5.1 ± 0.2 nm in the maximum TAC media point. The size measured of the colloidal structures of felodipine in the FaSSIF v1 was 86 ± 1 nm. However, there was no simple correlation between solubility and colloidal size.Nanoparticle tracking analysis was used for the first time to characterise colloidal structures within SIF and the results were compared to those obtained by dynamic light scattering. The particle size measured by dynamic light scattering was generally greater in media with a lower concentration of amphiphiles and smaller in media of a higher concentration of amphiphiles, compared to that of the data yielded by nanoparticle tracking analysis.This work shows that the colloidal structures formed vary depending on the composition of SIF which affects the solubility. Work is ongoing to determine the relationship between colloidal structure and solubility.

Herb–drug interactions: Quantitative analysis of levofloxacin absorption and transporter expression in the rat intestine following combined treatment with Persicaria capitata (Buch.-Ham. ex D. Don) H. Gross

Persicaria capitata (Buch.-Ham. ex D. Don) H. Gross, a traditional Chinese medicinal plant, is often used to treat various urologic disorders in China. P. capitata extracts (PCE) have been used in combination with levofloxacin (LVFX) to treat urinary tract infections (UTIs) for a long time. However, little is known about the absorption of LVFX and transporter expression in the intestine after combined treatment with PCE, restricting the development and utilization of PCE. In view of this, a UPLC-MS/MS method was established for the determination of LVFX in intestinal sac fluid samples and in situ intestinal circulation perfusate samples to explore the effect of PCE on the intestinal absorption characteristics of LVFX ex vivo and in vivo. To further evaluate the interaction between LVFX and PCE, western blotting, immunohistochemistry, and RT-qPCR were utilized to determine the expression levels of drug transporters (OATP1A2, P-gp, BCRP, and MRP2) involved in the intestinal absorption of LVFX after combined treatment with PCE. Using the everted intestinal sac model, the absorption rate constant (Ka) and cumulative drug absorption (Q) of LVFX in each intestinal segment were significantly lower in groups treated with PCE than in the control group. Ka at 2 h decreased most in the colon segment (from 0.088 to 0.016 µg/h·cm2), and Q at 2 h decreased most in the duodenum (from 213.29 to 33.92 µg). Using the intestinal circulation perfusion model, the Ka value and percentage absorption rate (A) of LVFX in the small intestine decreased significantly when PCE and LVFX were used in combination. These results showed that PCE had a strong inhibitory effect on the absorption of LVFX in the rat small intestine (ex vivo and in vivo intestinal segments). In addition, PCE increased the protein and mRNA expression levels of efflux transporters (P-gp, BCRP, and MRP2) and decreased the expression of the uptake transporter OATP1A2 significantly. The effects increased as the PCE concentration increased. These findings indicated that PCE changed the absorption characteristics of levofloxacin, possibly by affecting the expression of transporters in the small intestine. In addition to revealing a herb–drug interaction (HDI) between PCE and LVFX, these results provide a basis for further studies of their clinical efficacy and mechanism of action.

PS-B07-3: ANGIOTENSIN I CONVERTING ENZYME INHIBITORY PEPTIDES DERIVED FROM KATSUOBUSHI

Purpose: Antihypertensive components are typically assessed by screening based on in vitro results; however, since intestinal absorption is closely involved in the antihypertensive mechanism of action, we attempted to analyze the absorption of bonito derived peptides, which have been found to have antihypertensive effects in animal experiments. Methods: The intestinal tracts of rats, 7 week old male SDrats, were excised after performing cervical dislocation. Incisions 5.5 cm were quickly made into the jejunum, one side was ligated and inverted, and after injecting 1 mL reaction solution, the other side was ligated to create an inverted sack. The sack was then quickly placed in 30 mL of the reaction solution, antihypertensive amount: KBP 0.3 mg/per animal and synthetic peptides: each 0.25 mM, and left to react for 30 min at 37°C after which the inverted sack was removed and washed gently with physiological saline before recovering all the internal fluid. Liquid chromatography tandem mass spectrometry LC/MS/MS was used for analysis and the intestinal fluid samples were measured after examining pretreatment and measurement conditions. Results: Pretreatment conditions involved adding 5 μL water, 5 μL IS L-Leu-D-Leu, and 5 μL of 20% sulfosalicylic acid to a 50 μL intestinal fluid sample, stirring the sample, transferring 0.22 μm to a centrifugal filter, performing centrifugation for 3 min at 20000 g and 4°C and then injecting the filtrate into the LC/MS/MS system. LC conditions included a 1.7 μL ACQUITY UPLC BEH C18 column; 2.1, 50 mM, 0%-100% acetonitrile/0.1% TFA linear gradient elution in the mobile phase. Based on the above analysis conditions, the GW, AW, VW, MW, IW, LW, and WL values ng/mL of KBP in the reaction solution were 34.0, 4.64, 7.11, 1.28, 12.7, 4.57, and 0.923, respectively, and 53.2, 3.92, 6.02, 1.10, 8.76, 5.76, and 0.785, respectively. Values for the reaction solution of additive-free peptides serving as a base were estimated at 13.5, 1.09, 2.06,0.350, 5.54, 1.72, and 0.189, respectively. The calibration curve range was set at 0.200–1000ng/ mL. Conclusion: The intestinal membrane permeability of dried bonito-derived dipeptides, which have an antihypertensive effect in vivo, suggests that dipeptides absorbed from the intestinal membrane may have an antihypertensive effect.

Active Multiple-Sampling Capsule for Gut Microbiome

It is well known that the gut microbiome performs necessary physiological functions for the host organism, such as preventing infection from various pathogens, promoting the immune system's maturation, participating in nutrition absorption and metabolism processes, and promoting anticancer functions. To understand the relationship between the gut microbiome and host, multiple microbial samplings and analyses from certain locations in the gastrointestinal (GI) tract are necessary. However, ingestible sampling capsules, previously designed for gut-microbiome sampling, rely on the peristaltic motion of the intestine and can perform sampling only once, when the capsule body is submerged in the gut fluid. In this article, we propose an active multiple-sampling capsule that can be actively moved (by an external magnetic field) to specific locations in the GI tract and collects multiple gut-microbe-containing intestinal fluid samples. In particular, because the proposed capsule includes three microchannels, three inlet ports, and a small permanent magnet (for alignment), it can perform selective sampling with minimal cross-contamination, by allowing the capsule to move to the target location and aligning the inlet port of one of the three microchannels downwards. Through basic performance tests, we evaluate the locomotive and microbial-sampling performances of the proposed capsule (locomotion accuracy: ∼1.39°; inlet-port alignment accuracy: ∼1.48°; maximum suction flow rate: 22.3 <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">μ</i> L/min); furthermore, through phantom and <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">ex vivo</i> tests, we verify the feasibility of the proposed capsule. We confirm that the proposed active multiple-sampling capsule can be moved to the desired target locations in the GI tract and can collect ∼45 <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">μ</i> L gut-microbe-containing intestinal fluid thereat; furthermore, it can perform up to three samplings with minimal cross-contamination. In the future, we expect the proposed capsule to be implemented in medical research, to understand the gut microbiome's role in the human body (via the collection and analysis of these microbiomes) and to diagnose various human diseases.

Multi-analyte HPLC–DAD Method for Concurrent Analysis of Six Antimicrobials and Three Proton Pump Inhibitors Frequently used in Management of Helicobacter pylori Infection: Application to Simulated Intestinal Fluid Samples

The present work deals with the optimization, validation and application of a versatile HPLC–DAD method for concurrent estimation of nine antimicrobials and proton pump inhibitors, namely amoxicillin (AMX), doxycycline (DOX), furazolidone (FRZ), lansoprazole (LNS), levofloxacin (LVF), metronidazole (MTZ), omeprazole (OMZ), pantoprazole (PNZ) and tinidazole (TNZ). The selected nine drugs are frequently included in various treatment regimens of Helicobacter pylori infection. Successful separation was accomplished using the analytical column Agilent Zorbax Eclipse plus-C18 (250 × 4.6 mm, 5 µm particle size) and a mobile phase prepared from phosphate buffer pH 5 and acetonitrile pumped at a flow rate 1 mL/min using a gradient elution program. The gradient elution started with buffer/acetonitrile ratio 90:10, then it was altered in 15 min to reach 40:60 by volume. Quantification of the analytes was based on measuring peak areas of AMX at 230 nm, LVF, LNS and PNZ at 290 nm, OMZ at 300 nm, MTZ and TNZ at 320 nm, and DOX and FRZ at 360 nm. The separated compounds eluted at retention times 5.68, 6.43, 7.82, 8.84, 9.42, 10.75, 12.82, 13.74 and 14.90 min for AMX, MTZ, LVF, TNZ, DOX, FRZ, OMZ, PNZ and LNS respectively. Validation of the proposed HPLC procedure was carefully studied according to the ICH items: ranges, precision, accuracy, linearity, robustness and limits of detection and quantitation. The linear dynamic ranges were 5–100, 5–50, 2–40, 10–100, 10–100, 5–50, 2.5–30, 3–30 and 2–30 µg/mL for AMX, MTZ, LVF, TNZ, DOX, FRZ, OMZ, PNZ and LNS, respectively with correlation coefficients > 0.9993. Application fields of the validated method included analysis of laboratory-prepared binary dosage forms along with analysis of several ternary mixtures in spiked simulated intestinal fluid.

The differences between fecal microbiota and intestinal fluid microbiota in colon polyps: An observational study.

Generally, intestinal microbiota can be classified into intestinal cavity microbiota and mucosal microbiota, among which, the former is the default type. This study aimed to identify the differences between fecal microbiota and intestinal fluid microbiota in colon polys.This study enrolled patients with colon polys who met the Rome-III criteria to carry out 16s rDNA gene sequencing. Then, both fresh feces as well as intestinal fluid was sampled. Thereafter, α/β diversities, together with the heterogeneities with regard to microbial function and structure were assessed among those intestinal fluid and fresh feces samples collected.According to bioinformatics analysis, difference in α-diversity was not statistically significant between intestinal fluid microbiota and fecal microbiota among patients with colorectal polyps (CPs). Non-metric multidimensional scaling analysis of β-diversity revealed that differences were of statistical significance between both groups. In addition, linear discriminant analysis effect size analysis displayed great heterogeneities in intestinal microbiota of both groups, including Firmicutes, Clostridia, and Phascolarctobacterium. At the phylum level, difference (P = .016) in Spirochaetes was statistically significant between the intestinal fluid group and fecal group. At the family level, differences in Bacteroidaceae, Micrococcaceae, F16, Spirocheatacae, Enterobacteriaceae, Cardiobacteriaceae, Turkish Spirobacteriaceae, Bifidobacteriaceae, and Dethiosulfovibrionaceae were statistically significant between the 2 groups. At the genus level, there were statistical differences between the 2 groups in terms of Bacteroidetes, Rothia, Actinobacillus, F16, Treponema, Oscillospira, Turicibacter, Sharpea, Heamophilus, Veillonella, and Cardiobacterium.There are statistical differences in the composition between intestinal microbiota and fecal microbiota in CP patients, both of which are equally important and indispensable for analyzing the intestinal microbiota in CP patients.

Diversity of effects induced by boron-containing compounds on immune response cells and on antibodies in basal state

BackgroundIt has been reported that boron induces changes in the immune response, including in inflammatory processes. Recently, the effect of boric acid has been documented on the differentiation of lymphocyte clusters in mice and rats. However, the differences among boron-containing compounds (BCC) have been poorly explored. MethodsIn this study, we analyzed the effects after oral administration of boric acid (BOR), methylboronic (MET), 3-thyenylboronic (3TB), 4-hydroxymethyl-phenylboronic (4MP) and 4-methanesulfonyl-phenylboronic (4SP) acids on the populations of lymphocytes from spleen and Peyer’s patch (PP) as well as on antibodies. Groups of six male BALB/c were orally treated with 4.6 mg/kg of body weight with BOR, MET, 3TB, 4MP, and 4SP/daily for 10 days or vehicle (VEH) as a control group. After euthanasia, the spleen and small intestine were dissected. We conducted flow cytometry assays to assess B, CD3+ T, CD4+ T, and CD8+ T cells. Levels of IgG and IgM in serum, and IgA in intestinal fluid samples were analyzed by enzyme immunoassay. ResultsIn particular, we observed the effects of the administration of boronic acids on the number of lymphocytes; these changes were more notable in spleen than in PP. We found different profiles for each boron-containing compound, that is BOR induced an increase in the percentage of CD8+ T and CD19+/IgA+ cells in spleen, but a decrease in CD8+ T and B220+/CD19+ cells in PP. Meanwhile MET induced a decrease of CD4+ T in spleen, but induced an increase of CD4+ T cells and a decrease in the number of CD8+ T cells in PP. Boronic acids with an aromatic ring moiety induced changes in serum immunoglobulins levels, while 3TB acid induced a notable increase in S-IgA. ConclusionsEffects in lymphocyte populations and antibodies are different for each tested compound. These results highlight the establishment of the necessary structure-activity relationship for BCC as immunomodulatory drugs. This is relevant in the biomedical field due to their attractiveness for selecting compounds to develop therapeutic tools.

Alteration of Intestinal Microbiome of Clostridioides difficile-Infected Hamsters during the Treatment with Specific Cow Antibodies.

Clostridioides difficile infection (CDI) often develops after pretreatment with antibiotics, which can lead to damage of the intestinal microbiome. The approach of this study was to use specific polyclonal antibodies isolated from the milk of immunized cows to treat CDI, in contrast to the standard application of nonspecific antibiotics. To gain a deeper understanding of the role of the microbiome in the treatment of CDI with bovine antibodies, stool and intestinal fluid samples of hamsters were collected in large quantities from various treatments (>400 samples). The results show that the regeneration of the microbiome instantly begins with the start of the antibody treatment, in contrast to the Vancomycin-treated group where the diversity decreased significantly during the treatment duration. All antibody-treated hamsters that survived the initial phase also survived the entire study period. The results also show that the regeneration of the microbiome was not an antibody-induced regeneration, but a natural regeneration that occurred because no microbiota-inactivating substances were administered. In conclusion, the treatment with bovine antibodies is a functional therapy for both the acute treatment and the prevention of recurrence in hamsters and could meet the urgent need for CDI treatment alternatives in humans.

In vivo assessment of a delayed release formulation of larazotide acetate indicated for celiac disease using a porcine model.

There is no FDA approved therapy for the treatment of celiac disease (CeD), aside from avoidance of dietary gluten. Larazotide acetate (LA) is a first in class oral peptide developed as a tight junction regulator, which is a lead candidate for management of CeD. A delayed release formulation was tested in vitro and predicted release in the mid duodenum and jejunum, the target site of CeD. The aim of this study was to follow the concentration versus time profile of orally administered LA in the small intestine using a porcine model. A sensitive liquid chromatography/tandem mass spectrometry method was developed to quantify LA concentrations in porcine intestinal fluid samples. Oral dosing of LA (1 mg total) in overnight fasted pigs resulted in time dependent appearance of LA in the distal duodenum and proximal jejunum. Peak LA concentrations (0.32-1.76 μM) occurred at 1 hour in the duodenum and in proximal jejunum following oral dosing, with the continued presence of LA (0.02-0.47 μM) in the distal duodenum and in proximal jejunum (0.00-0.43 μM) from 2 to 4 hours following oral dosing. The data shows that LA is available in detectable concentrations at the site of CeD.

Intestinal absorption characteristics of Polygonum orientale extract in normal and isoproterenol-induced myocardial ischemia model rats via everted intestinal sac models

The present study is to investigate the absorption characteristics of the main components in Polygonum orientale extract in normal and isoproterenol-induced myocardial ischemia model rats with everted intestinal sac models. Intestinal sac fluid samples were collected in different part of intestine(duodenum, jejunum, ileum, colon) at different time after administration of different concentration of P. orientale extract(5.0,10.0, 20.0 mg·mL~(-1)). An UPLC-TQD method was employed for the determination of six components including orientin, isoorientin, vitexin, protocatechuic acid, kaempferol-3-O-β-D-glucoside and quercitrin in the intestinal sac samples. The absorption rate and cumulative absorption were calculated to analyze the intestinal absorption characteristics of six components in normal and myocardial ischemia model rats. The P-glycoprotein(P-gp) inhibitor was applied to investigate influence of intestinal absorption of six components in P. orientale extract. The results showed that the main absorption sites were concentrated on the duodenum at low concentration, while they were the colon at the medium concentration and the ileum at high concentration in control groups. In the condition of myocardial ischemia model, the main absorption sites focus on the ileum and jejunum at low concentration; the main absorption sites were in the ileum at the medium concentration and main absorption sites were the duodenum and ileum at high concentration. Compared with the normal group, the absorption rate and cumulative absorption of the six components significantly decreased in the model group. P-gp inhibitor markedly increased the absorption rate and cumulative absorption of six components in the model group, inferring that the 6 components may be the substrates of P-gp, and the mechanism needs further study. In this study, it is revealed that the six components of P. orientale extract can be absorbed into the intestinal sac, and it is an effective method to assess the intestinal absorption characteristics of P. orientale extract through everted intestinal sac model, providing data support for the clinical application and further development of P. orientale.

Characterisation of fasted state gastric and intestinal fluids collected from children

Fundamental knowledge about the composition of intestinal fluids in paediatric populations is currently unavailable. This study aimed to characterise gastric and intestinal fluid from paediatric populations.Gastric and intestinal fluid samples were obtained during routine clinical endoscopy from paediatric patients at a large teaching hospital. These fluids were characterised to measure the pH; buffer capacity; osmolality; bile acid concentration and composition.A total of 55 children were recruited to the study aged from 11 months to 15 years of age where 53 gastric fluid samples and 40 intestinal fluid samples were obtained. pH values recorded ranged from pH 0.57 to 11.05 (median: 2.50) in gastric fluids and from 0.89 to 8.97 (median: 3.27) in intestinal fluids. The buffer capacity did not change significantly between gastric and intestinal fluids with median values of 12 mM/L/ΔpH for both fluids. Gastric fluid osmolality values ranged from 1 to 615 mOsm/kg, while intestinal fluid values ranged from 35 to 631 mOsm/kg.Gastric fluid bile acid concentrations ranged from 0.002 to 2.3 mM with a median value of 0.017 mM whilst intestinal fluid bile acid concentrations ranged from 0.0008 to 3.3 mM with a median value of 0.178 mM. Glycocholate; taurocholic acid; glycochenodeoxycholate and taurochenodeoxycholate were the most commonly identified bile acids within paediatric intestinal fluids.All compositional components were associated with large inter-individual variability. Further work is required to develop simulated paediatric media and to explore the impact of these media on drug solubility and dissolution.

Immunological effects of Kefir produced from Kefir grains versus starter cultures when fed to mice

Background: Natural kefir grains have a unique microbiota. The structure contains lactic acid bacteria (LAB), acetic acid bacteria and yeast in specific ratios in a polysaccharide matrix. Authentic kefir is produced by a traditional method using kefir grains cultured in milk. In contrast, starter cultures are used instead of kefir grains in the industry. The commercial kefir starter cultures used are limited and often very different from the kefir grain microbiota. The resultant commercial “kefir” is just a fermented drink containing some probiotic microorganisms and does not possess the same microbial population or chemical and physical characteristics of authentic kefir. The aim of this project was to determine and compare the effects on the mouse immune system of kefir produced using natural kefir grain versus commercial kefir produced by starter culture.Methods: Kefir produced with different cultures was fed to Balb/c mice (6-8 weeks, 20-25 grams, male) by gavage for two weeks at 300 μl/day. Intestinal tissues were collected from sacrificed mice at the end of the trial. The control group of mice (CNI group) were fed with phosphate buffered saline (PBS). The experimental treatments were mice fed mice fed authentic kefir produced using kefir grains (KGI group) and mice fed kefir produced using starter culture (STI group). Immunoglobulin (Ig) A, Immunoglubulin G, Interleukin (IL)-4, Interleukin-10, Interleukin-12, Toll Like Receptor (TLR)-4 were analyzed immunologically in intestinal fluid samples. Results: Results indicated that IgA values were 60.87, 72.78 and 55.31 ng/mL; IgG values were 26.59, 38.90 and 29.44 ng/mL; IL-4 values were 84, 40.28 and 53.28 pg/mL; IL-10 values were 110.98, 175.91 and 134.77 pg/mL; IL-12 values were 53.90, 22.93 and 24.75 pg/mL; TLR-4 values were 0.53, 0.43 and 1.37 ng/mL, for the CNI, KGI and STI groups, respectively.Conclusion: The high probiotic content of grain kefir had the ability to modulate many immunological mechanisms.Keywords: immune system, in vivo, kefir grain, probiotic, starter kefir culture

HPLC Estimation, Ex vivo Everted Sac Permeability and In Vivo Pharmacokinetic Studies of Darunavir.

Darunavir ethanolate (DRV) is an efficient protease inhibitor (PI) used in the treatment of human immunodeficiency virus (HIV) type-1 patients. An isocratic reversed-phase HPLC method was developed to monitor concentration of darunavir in in vitro intestinal fluid samples in everted sac absorption model in the presence of bioenhancers, viz., piperine, quercetin, naringenin. The method was validated and successfully applied to everted sac and pharmacokinetic studies in rats. The absorption profiles of DRV and apparent permeability coefficients were determined. The proposed method was found to be simple, rapid, robust and selective and was applied for continuous ex vivo monitoring of DRV in everted sac absorption studies. Of the three bioenhancers screened at different concentrations, piperine caused highest and significant 1.5-fold increase in apparent permeability of DRV across everted sac tissue. Further, co-administration of piperine significantly increased the maximum plasma concentration of DRV by 1.18-fold confirming the enhancement in its absorption.

Exploring drug solubility in fasted human intestinal fluid aspirates: Impact of inter-individual variability, sampling site and dilution

One of the main factors defining intestinal drug absorption is the solubility of the compound in the gastrointestinal environment. This study reports the solubility of a series of 27 commonly used acidic, neutral and basic drugs in human intestinal fluid samples collected from the duodenum or jejunum of healthy volunteers under fasted state conditions. The interindividual variability as well as the impact of factors such as pH, sampling site and bile salts on the solubility in human intestinal fluids was investigated. The solubility measurements were evaluated using a statistical experimental design. Variability in solubility across volunteers and sampling sites was highly compound-specific and appeared to be substantial for weak acids and bases and for lipophilic drugs. Both pH of the samples and the abundance of amphiphilic components were responsible for the variability observed in the solubility values obtained. The results confirm strong interindividual differences in intraluminal solubility, especially for compounds with high lipophilicity and/or compounds with a pKa value within the physiological pH range. It is important to recognize this variability in intestinal drug solubility as it may considerably influence the therapeutic outcome among patients.

Effect of infectious bursal disease (IBD) vaccine on Salmonella Enteritidis infected chickens

BackgroundChickens infected with both infectious bursal disease virus (IBDV) and Salmonella had higher mortality. In this work, we investigated the effect of IBDV vaccine (modified live-virus bursal disease vaccine, Nobilis strain 228E®) on experimentally infected chickens with Salmonella Enteritidis (SE). MethodsFour experimental groups were included in this study, negative control group, 228E®group, 228E®+SE infected group, and SE infected group. Chickens were ocularly administrated 228E® at 12days of age and orally infected with S. Enteritidis at 13days of age. Sera, intestinal fluid, blood, cloacal swabs and tissue samples were collected at 1, 2 and 3weeks post vaccination (PV). ResultsThe recorded mortalities were higher in the 228E®+SE infected group, compared to the SE infected group. The anti-S. Enteritidis serum antibody titer and the intestinal mucosal IgA level were higher in the SE infected group at 2 and 3weeks PV, compared to 228E®+SE infected group. S. Enteritidis fecal shedding and organ colonization were significantly higher in the 228E®+SE infected group than the SE infected group at 2 and 3weeks PV. The 228E®+SE group had significantly lower bursa to body weight ratios at 2 and 3weeks PV, as well as had higher bursal lesion scores than the SE infected group. IBDV vaccine depressed the specific-SE systemic and mucosal antibody responses, but did not affect the specific-SE cellular immune responses. ConclusionChickens administrated IBDV vaccine, followed by S. Enteritidis infection, could cause a significant effect on the bursa of Fabricius, resulting in failure of systemic and mucosal antibody responses to the S. Enteritidis and reduce the elimination and the clearance of S. Enteritidis.

Olfactory sensitivity of the marine flatfish Solea senegalensis to conspecific body fluids.

Chemical communication is better understood in freshwater fish than marine fish. The Senegalese sole (Solea senegalensis) is a marine flatfish wherein one of the problems in aquaculture is the poor reproductive performance of hatchery-bred males. Is chemical communication involved in the reproduction of this species? Urine, intestinal fluid and mucus samples were taken from adult fish (wild-caught and hatchery-bred) over the spawning season (March-May), and assessed for olfactory potency using the electro-olfactogram (EOG). The effect of stimulation of the olfactory system with adult female urine on circulating luteinizing hormone (LH) levels was also tested in males. Intestinal fluid and urine were potent olfactory stimuli for both juvenile and adult conspecifics, evoking large-amplitude, concentration-dependent EOG responses, with thresholds of detection at approximately 1:106 However, the amplitude of the response to urine depended on the sex and state of maturity of both the donor and the receiver. Most olfactory activity could be extracted by C18 solid-phase cartridges. Urine from mature females evoked a slight, but significant, increase in circulating LH levels in mature males 30 min after exposure. Furthermore, the olfactory potency of urine differed between wild-caught and hatchery-bred fish; however, contrary to expectations, urine from wild-caught females was less potent than that from hatchery-bred females. Taken together, these results strongly suggest that faeces- and urine-released odorants are involved in reproduction in the Senegalese sole, and establish a basis for further investigation into pheromonal communication in marine teleosts.

Statistical investigation of simulated fed intestinal media composition on the equilibrium solubility of oral drugs

Gastrointestinal fluid is a complex milieu and it is recognised that gut drug solubility is different to that observed in simple aqueous buffers. Simulated gastrointestinal media have been developed covering fasted and fed states to facilitate in vitro prediction of gut solubility and product dissolution. However, the combination of bile salts, phospholipids, fatty acids and proteins in an aqueous buffered system creates multiple phases and drug solubility is therefore a complex interaction between these components, which may create unique environments for each API. The impact on solubility can be assessed through a statistical design of experiment (DoE) approach, to determine the influence and relationships between factors. In this paper DoE has been applied to fed simulated gastrointestinal media consisting of eight components (pH, bile salt, lecithin, sodium oleate, monoglyceride, buffer, salt and pancreatin) using a two level D-optimal design with forty-four duplicate measurements and four centre points. The equilibrium solubility of a range of poorly soluble acidic (indomethacin, ibuprofen, phenytoin, valsartan, zafirlukast), basic (aprepitant, carvedilol, tadalafil, bromocriptine) and neutral (fenofibrate, felodipine, probucol, itraconazole) drugs was investigated. Results indicate that the DoE provides equilibrium solubility values that are comparable to literature results for other simulated fed gastrointestinal media systems or human intestinal fluid samples. For acidic drugs the influence of pH predominates but other significant factors related to oleate and bile salt or interactions between them are present. For basic drugs pH, oleate and bile salt have equal significance along with interactions between pH and oleate and lecithin and oleate. Neutral drugs show diverse effects of the media components particularly with regard to oleate, bile salt, pH and lecithin but the presence of monoglyceride, pancreatin and buffer have significant but smaller effects on solubility. There are fourteen significant interactions between factors mainly related to the surfactant components and pH, indicating that the solubility of neutral drugs in fed simulated media is complex. The results also indicate that the equilibrium solubility of each drug can exhibit individualistic behaviour associated with the drug's chemical structure, physicochemical properties and interaction with media components. The utility of DoE for fed simulated media has been demonstrated providing equilibrium solubility values comparable with similar in vitro systems whilst also providing greater information on the influence of media factors and their interactions. The determination of a drug's gastrointestinal solubility envelope provides useful limits that can potentially be applied to in silico modelling and in vivo experiments.

Modulatory Effects of Oral Bovine Lactoferrin on the IgA Response at Inductor and Effector Sites of Distal Small Intestine from BALB/c Mice.

Bovine lactoferrin (bLf) up-modulates intestinal IgA that is essential for homeostasis and which might confer protection to the distal small intestine that is vulnerable to inflammation. This study analyzed the effects of bLf administered orally on the IgA response at inductive (Peyer's patches) and effector (lamina propria) sites of the distal small intestine in mice. Groups of five healthy male BALB/c mice were orally treated with 5mg of bLf for 7, 14, 21, or 28days. Then, mice were killed and the distal small intestine was dissected. Intestinal fluid samples were analyzed to determine IgA and IgM levels by enzyme-immuno assay. Peyer's patches and lamina propria were analyzed for IgA(+) or IgM(+) plasma cells, B, CD4(+) T and CD8(+) T cells as well as CD4(+) T cells positive for either pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon-γ and interleukin (IL)-12] or for IgA-producing ILs (IL-4, -5, -10 and -6) by cytofluorometry. Antibodies, antibody-secreting cells, and B and T responses in both Peyer's patches and lamina propria were higher in bLf-treated than bLf-untreated mice. The generation of IL-10 and IL-6 CD4(+) T cells in Peyer's patches or TNF-α and IL-12 CD4(+) T cells in lamina propria showed similar response patterns. On days 14 and 28, cytokine/IL CD4(+) T cell responses were increased in Peyer's patches or decreased in lamina propria. The effect of bLf on the elicitation of IgA indicates a potential application of bLf as a nutraceutical to control inflammation in the distal small intestine.

Development and validation of HPLC-DAD method for the simultaneous determination of amoxicillin, metronidazole and rabeprazole sodium. Application to spiked simulated intestinal fluid samples

This work deals with the development, validation and application of an HPLC-DAD method for the determination of a ternary mixture containing amoxicillin (AX), metronidazole (MZ) and the proton pump inhibitor rabeprazole sodium (RB). This triple therapy is used for treatment of Helicobacter pylori infection. Effective chromatographic separation between the three drugs was achieved using Thermo Hypersil BDS-C8 (4.6×250mm, 5μm particle size) column and a mobile phase composed of phosphate buffer pH 7 and acetonitrile (70: 30, by volume). The mobile phase was pumped isocratically at a flow rate of 1mL/min. Quantification of the analytes was based on measuring their peak areas at 230nm for both AX and RB, and at 319nm for MZ. AX, MZ and RB eluted at retention times 2.36, 3.55 and 8.72min respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection and quantification limits. The linear dynamic ranges were 25-250, 25-250 and 5-50μg/mL for AX, MZ and RB respectively with correlation coefficients>0.9998. The validated method was successfully applied to the analysis of several laboratory-prepared mixtures as well as simulated intestinal fluid samples spiked with the three drugs.

Intestinal behavior of the ester prodrug tenofovir DF in humans

Tenofovir-disoproxil-fumarate (TDF) is a double ester prodrug which enables intestinal uptake of tenofovir (TFV) after oral administration in humans. In this study, prodrug stability was monitored in situ in the human intestine and in vitro using biorelevant media. In fasted state human intestinal fluids, the prodrug was completely degraded within 90min, resulting in the formation of the mono-ester intermediate and TFV; in fed state intestinal fluids, the degradation rate of TDF was slightly reduced and no TFV was formed. Intestinal fluid samples aspirated after administration of TDF confirmed extensive intraluminal degradation of TDF in fasted state conditions; a relatively fast absorption of TDF partly compensated for the degradation. Although food intake reduced intestinal degradation, the systemic exposure was not proportionally increased. The lower degradation in fed state conditions may be attributed to competing esterase substrates present in food, lower chemical degradation in the slightly more acidic environment and micellar entrapment, delaying exposure to the “degrading” intestinal environment. The results of this study demonstrate premature intestinal degradation of TDF and suggest that TFV may benefit from a more stable prodrug approach; however, fast absorption may compensate for fast degradation, indicating that prodrug selection should not be limited to stability assays.