Detection of live M. bovis BCG in tissues and IFN-γ responses in European badgers (Meles meles) vaccinated by oropharyngeal instillation or directly in the ileum.

BACKGROUND: Oral vaccination with Mycobacterium bovis Bacille of Calmette and Guerin (BCG) has provided protection against M. bovis to badgers both experimentally and in the field. There is also evidence suggesting that the persistence of live BCG within the host is important for maintaining protection against TB. Here we investigated the capacity of badger inductive mucosal sites to absorb and maintain live BCG. The targeted mucosae were the oropharyngeal cavity (tonsils and sublingual area) and the small intestine (ileum). RESULTS: We showed that significant quantities of live BCG persisted within badger in tissues of vaccinated badgers for at least 8 weeks following oral vaccination with only very mild pathological features and induced the circulation of IFNγ-producing mononuclear cells. The uptake of live BCG by tonsils and drainage to retro-pharyngeal lymph nodes was repeatable in the animal group vaccinated by oropharyngeal instillation whereas those vaccinated directly in the ileum displayed a lower frequency of BCG detection in the enteric wall or draining mesenteric lymph nodes. No faecal excretion of live BCG was observed, including when BCG was delivered directly in the ileum. CONCLUSIONS: The apparent local loss of BCG viability suggests an unfavorable gastro-enteric environment for BCG in badgers, which should be taken in consideration when developing an oral vaccine for use in this species.

Validation of the IntelliCap® system as a tool to evaluate extended release profiles in human GI tract using metoprolol as model drug.

A clinical study was conducted to validate the in vivo drug release performance of IntelliCap® CR capsules. 12 healthy, male volunteers were administered IntelliCap® CR capsules, filled with metoprolol as a BCS 1 model drug, and programmed to release the drug with 3 different release profiles (2 linear profiles extending over 6h and 14h, respectively, and a pulsed profile with two equal pulses separated by 5h) using a cross-over design. An oral metoprolol solution was included as a reference. Standard bioavailability variables were determined. In vivo drug release-time profiles for the IntelliCap® CR capsules were calculated from the plasma drug concentrations by deconvolution, and they were subsequently compared with the in vitro drug release profiles including assessment of level A in vitro/in vivo correlation (IVIVC). The relative bioavailability for the linear, extended release profiles was about 85% which is similar to other extended release administrations of metoprolol. There was an excellent agreement between the predetermined release profiles and the in vivo release for these two administrations. For IntelliCap® CR capsules programmed to deliver 2 distinct and equal drug pulses, the first pulse was delivered as expected whereas only about half of the second dose was released. Thus, it is concluded that the IntelliCap® system is well suited for the fast and reliable generation of in vivo pharmacokinetic data for extended release drug profiles, e.g. in context of regional drug absorption investigations. For immediate release pulses delivered in the distal GI tract this version of the device appears however less suitable.

Gastrointestinal pH and Transit Time Profiling in Healthy Volunteers Using the IntelliCap System Confirms Ileo-Colonic Release of ColoPulse Tablets.

INTRODUCTION: ColoPulse tablets are an innovative development in the field of oral dosage forms characterized by a distal ileum and colon-specific release. Previous studies in humans showed release in the ileo-colonic region, but the relationship between gastrointestinal pH and release was not experimentally proven in vivo. This information will complete the in vivo release-profile of ColoPulse tablets. MATERIALS AND METHODS: Release from ColoPulse tablets was studied in 16 healthy volunteers using the dual label isotope strategy. To determine gastrointestinal pH profiles and transit times the IntelliCap system was used. A ColoPulse tablet containing 13C-urea and an uncoated, immediate release tablet containing 15N2-urea were taken simultaneously followed by a standardized breakfast after three hours. Five minutes after intake of the tablets the IntelliCap capsule was swallowed and pH was measured until excretion in the feces. Breath and urine samples were collected for isotope analysis. RESULTS: Full analysis could be performed in 12 subjects. Median bioavailability of 13C -urea was 82% (95% CI 74-94%, range 61-114%). The median lag time (5% release of 13C) was 5:42 h (95% CI 5:18-6:18 h, range 2:36-6:36 h,) There was no statistically significant difference between lag time based on isotope signal and colon arrival time (CAT) based on pH (median 5:42 vs 5:31 h p = 0.903). In all subjects an intestinal pH value of 7.0 was reached before release of 13C from the ColoPulse tablet occurred. DISCUSSION AND CONCLUSIONS: From the combined data from the IntelliCap system and the 13C -isotope signal it can be concluded that release from a ColoPulse tablet in vivo is not related to transit times but occurs in the ileo-colonic region after pH 7.0 is reached. This supports our earlier findings and confirms that the ColoPulse system is a promising delivery system for targeting the distal ileum and colon. TRIAL REGISTRATION: ISRCTN Registry 18301880.

Investigation of pH and Temperature Profiles in the GI Tract of Fasted Human Subjects Using the Intellicap(®) System.

Gastrointestinal (GI) pH and temperature profiles under fasted-state conditions were investigated in two studies with each 10 healthy human subjects using the IntelliCap(®) system. This telemetric drug delivery device enabled the determination of gastric emptying time, small bowel transit time, and colon arrival time by significant pH and temperature changes. The study results revealed high variability of GI pH and transit times. The gastric transit of IntelliCap(®) was characterized by high fluctuations of the pH with mean values ranging from pH 1.7 to pH 4.7. Gastric emptying was observed after 7-202 min (median: 30 min). During small bowel transit, which had a duration of 67-532 min (median: 247 min), pH values increased slightly from pH 5.9-6.3 in proximal parts to pH 7.4-7.8 in distal parts. Colonic pH conditions were characterized by values fluctuating mainly between pH 5 and pH 8. The pH profiles and transit times described in this work are highly relevant for the comprehension of drug delivery of solid oral dosage forms comprising ionizable drugs and excipients with pH-dependent solubility.

Novel orally swallowable IntelliCap(®) device to quantify regional drug absorption in human GI tract using diltiazem as model drug.

Typically, colonic absorption of a drug is mandatory for a sustained release formulation to hold the drug's plasma level for more than 12 or 24 h above the minimum therapeutic plasma concentration (efficacy). According to Drugs@FDA, only 7.4% of the oral drugs are extended release forms probably showing colonic absorption. Therefore an early determination of a drug's colonic absorption using the IntelliCap® in animals or humans will provide the mandatory information to initiate or stop a SR form development. Diltiazem (60 mg) is used in the oral swallowable IntelliCap® and the marketed SR form from Mylan (coated beads). A human study with 14 healthy volunteers compared the Mylan formulation with the IntelliCap® device that releases the drug identical to the in-vitro dissolution of the Mylan product. The plasma profiles of IntelliCap® and Mylan formulation are highly similar. The mean AUC (bioequivalence fulfilled) and mean Cmax of IntelliCap® shows only a difference of +15% and -12%, respectively. But the PK profile of the Mylan formulation shows a broader peak around Cmax. About 81.8% diltiazem was absorbed in the colon (IntelliCap®) comparable to former publications. The Mylan is a SR diffusion coated beads form whereas the IntelliCap® is a monolithic capsule. The beads are transported in the gut and spread which results in a longer Tmax and a broader Cmax peak. The IntelliCap® device can quantitatively measure the colonic absorption of a drug in excellent accordance to a standard oral SR dosage form.

A novel ingestible electronic drug delivery and monitoring device.

BACKGROUND: We developed an ingestible electronic drug delivery and monitoring system. This system includes an electronic capsule comprising a drug reservoir, a pH and temperature sensor, a microprocessor and wireless transceiver, a stepper motor, and batteries. The location of the capsule in the gut derived from pH data can be monitored in real time. The stepper motor can be remotely actuated to expel the contents of the drug reservoir. OBJECTIVES: First human study. DESIGN: Two consecutive observational studies. SETTING: University medical center. SUBJECTS: Twenty healthy volunteers. INTERVENTIONS: Study I: Ingestion and passage of the capsule. Study II: Ingestion and passage of the capsule, loaded with (99m)technetium-pertechnetate ((99m)Tc); remotely actuated expulsion of (99m)Tc in the gut. MAIN OUTCOME MEASUREMENTS: Study I: Safety, tolerability, and functionality (wireless pH and temperature recording). Study II: Tracing of the capsule and expulsion and distribution of (99m)Tc from the drug reservoir by scintigraphy. Correlating location pH with scintigraphy. RESULTS: Study I: Ingestion and passage of the capsule was safe and well tolerated. Transmitted pH and temperature data were received by the recorder in 96.5% ± 3%. Study II: pH-determined passage of the esophagogastric, gastroduodenal, and ileocolonic junction correlated well with scintigraphy. Expulsion of (99m)Tc from the capsule was successful in 9 of 10 subjects. LIMITATIONS: Subjects with relatively low body mass index. CONCLUSIONS: This electronic drug delivery and monitoring system may be a promising tool for targeted delivery of substances to well-defined areas of the GI tract.

Gender differences in kidney function.

Sex hormones influence the development of female (F) and male (M) specific traits and primarily affect the structure and function of gender-specific organs. Recent studies also indicated their important roles in regulating structure and/or function of nearly every tissue and organ in the mammalian body, including the kidneys, causing gender differences in a variety of characteristics. Clinical observations in humans and studies in experimental animals in vivo and in models in vitro have shown that renal structure and functions under various physiological, pharmacological, and toxicological conditions are different in M and F, and that these differences may be related to the sex-hormone-regulated expression and action of transporters in the apical and basolateral membrane of nephron epithelial cells. In this review we have collected published data on gender differences in renal functions, transporters and other related parameters, and present our own microarray data on messenger RNA expression for various transporters in the kidney cortex of M and F rats. With these data we would like to emphasize the importance of sex hormones in regulation of a variety of renal transport functions and to initiate further studies of gender-related differences in kidney structure and functions, which would enable us to better understand occurrence and development of various renal diseases, pharmacotherapy, and drug-induced nephrotoxicity in humans and animals.

Generation of transducers for fluorescence-based microarrays with enhanced sensitivity and their application for gene expression profiling.

The present paper describes a novel generation of microchips suitable for fluorescence-based assays, such as cDNA, oligonucleotide, or protein microarrays. The new transducers consist of a fully corrugated surface coated with a thin layer of Ta2O5 as a high refractive index material. Tuning of the incident excitation light beam to abnormal reflection geometry results in a confinement of the energy within the thin metal oxide layer. Consequently, strong evanescent fields are generated at the surface of these microchips and fluorophores located within the fields showed up to a 2 order of magnitude increase in fluorescence intensities relative to the epifluorescence signals. We have attributed this phenomenon as evanescent resonance (ER). Due to the surface architecture, propagation distances of the incident energy and fluorescence photons are in the micrometer range, thus preventing cross talk between adjacent regions. ER microchips offer a significant increase in fluorescence intensities in both "snapshot" fluorescence setups and commercial fluorescence scanners. The underlying principle of the novel chips is explained, and quantitative data on the fluorescence enhancement are provided. To demonstrate their potential, the novel chips are used to investigate the dependence of expression levels from metabolic genes in rat liver on drug treatment. In contrast to competitive hybridization, labeled samples were hybridized to individual ER microchips, and changes were observed by comparing with normalized data from different chips. Results obtained in gene expression profiling experiments with phenobarbital-treated rats are shown.

Evanescent resonator chips: a universal platform with superior sensitivity for fluorescence-based microarrays.

In the present paper, we introduce for the first time a novel generation of a universal fluorescence transducer, the so-called evanescent resonator (ER) platform. The device comprises a transparent substrate and a thin dielectric surface layer containing sub-micron corrugated structures. The ER chip exhibits an inherent physical signal amplification due to confinement of excitation energy in the thin surface layer. Energy confinement is based on interference effects created by the abnormal reflection geometry and leads to efficient excitation of surface-bound fluorophores in the evanescent field of the chip. The evanescent resonator platform has the potential to increase the fluorescence yield of labelled biomolecules to more than 100-fold when compared with conventional microarray chips. The new ER device has been developed for analysis of nucleic acids from different species. However, it can be used with all kinds of biomolecular affinity systems. The platform combines superior sensitivity with exceptional reproducibility and ease of use. The chips are compatible with commercially available laser scanners, confocal microscopes, and portable or miniaturised CCD read-out equipment.

Transcriptional program of mouse osteoclast differentiation governed by the macrophage colony-stimulating factor and the ligand for the receptor activator of NFkappa B.

Cytokines macrophage colony stimulating factor (M-CSF) and the receptor activator of NFkappaB ligand (RANKL) induce differentiation of bone marrow hematopoietic precursor cells into bone-resorbing osteoclasts without the requirement for stromal cells of mesenchymal origin. We used this recently described mouse cell system and oligonucleotide microarrays representing about 9,400 different genes to analyze gene expression in hematopoietic cells undergoing differentiation to osteoclasts. The ability of microarrays to detect the genes of interest was validated by showing expression and expected regulation of several osteoclast marker genes. In total 750 known transcripts were up-regulated by > or =2-fold, and 91% of them at an early time in culture, suggesting that almost the whole differentiation program is defined already in pre-osteoclasts. As expected, M-CSF alone induced the receptor for RANKL (RANK), but also, unexpectedly, other RANK/NFkappaB pathway components (TRAF2A, PI3-kinase, MEKK3, RIPK1), providing a molecular explanation for the synergy of M-CSF and RANKL. Furthermore, interleukins, interferons, and their receptors (IL-1alpha, IL-18, IFN-beta, IL-11Ralpha2, IL-6/11R gp130, IFNgammaR) were induced by M-CSF. Although interleukins are thought to regulate osteoclasts via modulation of M-CSF and RANKL expression in stromal cells, we showed that a mix of IL-1, IL-6, and IL-11 directly increased the activity of osteoclasts by 8.5-fold. RANKL induced about 70 novel target genes, including chemokines and growth factors (RANTES (regulated on activation, normal T cell expressed and secreted), PDGFalpha, IGF1), histamine, and alpha1A-adrenergic receptors, and three waves of distinct receptors, transcription factors, and signaling molecules. In conclusion, M-CSF induced genes necessary for a direct response to RANKL and interleukins, while RANKL directed a three-stage differentiation program and induced genes for interaction with osteoblasts and immune and nerve cells. Thus, global gene expression suggests a more dynamic role of osteoclasts in bone physiology than previously anticipated.