Good manufacturing procedure production of [18 F]SynVesT-1, a radioligand for in vivo positron emission tomography imaging of synaptic vesicle glycoprotein 2A.

[18 F]SynVesT-1 (also known as [18 F]SDM-8 or [18 F]MNI-1126) is a potent and selective synaptic vesicle glycoprotein 2 (SV2A) positron emission tomography (PET) imaging agent. In order to fulfill the increasing clinical demand of an 18 F-labeled SV2A PET ligand, we have developed a fully automated procedure to provide a sterile and pyrogen-free good manufacturing procedure (GMP)-compliant product of [18 F]SynVesT-1 suitable for clinical studies in humans. [18 F]SynVesT-1 is synthesized via a rapid copper-mediated radiofluorination protocol. The procedure was developed and established on a commercially available module, TracerMaker (ScanSys Laboratorieteknik ApS, Copenhagen, Denmark), a synthesis platform originally developed to conduct carbon-11 radiochemistry. From ~130 GBq (end-of-bombardment), our newly developed procedure enabled us to prepare [18 F]SynVesT-1 in an isolated radioactivity yield of 14,220 ± 800 MBq (n = 3), which corresponds to a radiochemical yield (RCY) of 19.5 ± 0.5%. The radiochemical purity (RCP) and enantiomeric purity of each of the final formulated batches exceeded 98%. The overall synthesis time was 90 min and the molar activity was 330 ± 60 GBq/µmol (8.9 ± 1.6 Ci/µmol). The produced [18 F]SynVesT-1 was stable over 8 h at room temperature and is suitable for in vivo PET imaging studies in human subjects.

Inefficacy of a highly selective T-type calcium channel blocker in preventing atrial fibrillation related remodeling.

BACKGROUND: The T-type Ca(2+) channel (I(CaT)) blocker mibefradil prevents AF-promoting remodeling occurring with atrial tachycardia, an action that has been attributed to I(CaT) inhibition. However, mibefradil has other effects, including ability to inhibit L-type Ca(2+) channels, Na(+) channels and cytochromes. Thus, the relationship between I(CaT) inhibition and remodeling protection in AF is still unknown. OBJECTIVE: To assess the effects of a novel highly selective Cav3 (I(CaT)) blocker, AZ9112, on atrial remodeling induced by 1-week atrial tachypacing (AT-P) in dogs. METHODS: Mongrel dogs were subjected to AT-P at 400 bpm for 7 days, with atrioventricular-node ablation and right-ventricular demand pacing (80 bpm) to control ventricular rate. Four groups of dogs were studied in investigator-blinded fashion: (1) a sham group, instrumented but without tachypacing or drug therapy (n = 5); (2) a placebo group, tachypaced but receiving placebo (n = 6); (3) a positive control tachypacing group receiving mibefradil (n = 6); and (4) a test drug group, subjected to tachypacing during oral treatment with AZ9112 (n = 8). RESULTS: One-week AT-P decreased atrial effective refractory period (ERP) at 6 of 8 sites and diminished rate-dependent atrial ERP abbreviation. Mibefradil eliminated AT-P-induced ERP-abbreviation at 4 of these 6 sites, while AZ9112 failed to affect ERP at any. Neither drug significantly affected AF vulnerability or AF duration. CONCLUSIONS: I(CaT) blockade with the highly selective compound AZ9112 failed to prevent rate-related atrial remodeling. Thus, prevention of atrial electrophysiological remodeling by mibefradil cannot be attributed exclusively to I(CaT) blockade. These results indicate that I(CaT) inhibition is not likely to be a useful approach for AF therapy.

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP - up to 3% of TSP.

Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs.

An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein fivefold in the A. thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

The effect of exhalation flow on endogenous particle emission and phospholipid composition.

Exhaled particles constitute a micro-sample of respiratory tract lining fluid. Inhalations from low lung volumes generate particles in small airways by the airway re-opening mechanism. Forced exhalations are assumed to generate particles in central airways by mechanisms associated with high air velocities. To increase knowledge on how and where particles are formed, different breathing manoeuvres were compared in 11 healthy volunteers. Particles in the 0.41-4.55µm diameter range were characterised and sampled. The surfactant lipid dipalmitoylphosphatidylcholine (DPPC) was quantified by mass spectrometry. The mass of exhaled particles increased by 150% (95% CI 10-470) for the forced exhalation and by 470% (95% CI 150-1190) for the airway re-opening manoeuvre, compared to slow exhalations. DPPC weight percent concentration (wt%) in particles was 2.8wt% (95%CI 1.4-4.2) and 9.4wt% (95%CI 8.0-10.8) for the forced and the airway re-opening manoeuvres, respectively. In conclusion, forced exhalation and airway re-opening manoeuvres generate particles from different airway regions having different DPPC concentration.

Clonidine reduces diarrhea and sodium loss in patients with proximal jejunostomy: a controlled study.

BACKGROUND: Patients with short bowel syndrome have significant fluid losses. This represents a significant management problem, especially in patients with minimal residual intestine. We determined whether clonidine, an alpha2-adrenergic agonist, is effective in decreasing fecal water and sodium (Na) losses in patients with proximal jejunostomy. Eight parenteral nutrition (PN)-dependent subjects (3 men, 5 women), aged 49.9+/-10.2 years, with a residual small bowel length of 71.8+/-152.0 cm that ended in a jejunostomy, were studied. METHODS: Subjects were admitted to the North-western General Clinical Research Center (GCRC) for a 2-day equilibrium period while receiving a self-selected 100 g fat diet with protein 1.5 g/kg/d and 30 kcal/kg/d and 1 L/d of oral rehydration solution. A D-xylose test was performed after an overnight fast. On days 3-5, all stool and urine were collected for volume, weight, fat, nitrogen, energy, sodium, magnesium, potassium, and calcium. Meals were provided in duplicate and the equivalent portions consumed by each patient were analyzed for fluid volume, fat, nitrogen, energy, sodium, magnesium, calcium, and potassium in order to calculate nutrient balances. At the conclusion of the stool and urine collections (day 6), a clonidine (0.3 mg) patch was applied to the shoulder. Subjects were restudied after 1 week. RESULTS: Daily fecal volume and weight were 4.514+/-1.769 L/d and 4394+/-1727 g/d, respectively, at baseline. Five subjects were net "secretors" in that excreted fecal volume exceeded oral intake. Fecal volume decreased by 427+/-562 mL/d (8.9%, p=.07). Fecal weight decreased by 438+/-527 g/d (9.4%, p=.05). Urine volume correspondingly increased by 747+/-1934 mL (18.9%, p=not significant [NS]). The increase in urine output was weakly and negatively correlated with the decrease in fecal volume and weight (r=-0.37 and -0.41, respectively, p=NS). Oral fluid intake decreased slightly from 3.328+/-1.246 L/d baseline to 3.203+/-1.119 L/d with clonidine therapy (-3.8%, p=NS). Fecal Na loss was significantly decreased from baseline (887+/-996 mg/d, 11.2+/-12.3%; p=.036). This was not related to decreased oral Na intake, which actually increased from baseline (3.799+/-2.271 g/d) to 3.933+/-1.314 g/d after clonidine therapy (p=NS). No patient developed hypotension. CONCLUSIONS: Our results show the transdermal administration of clonidine is associated with a modest but clinically significant decrease in fecal output in patients with short bowel syndrome and high-output proximal jejunostomy that require chronic parenteral fluid infusion. This is accompanied by decreased fecal Na loss.

A higher dose requirement of tacrolimus in active Crohn's disease may be related to a high intestinal P-glycoprotein content.

Tacrolimus, a relatively new therapeutic option for patients with corticosteroid-refractory Crohn's disease or ulcerative colitis, is a substrate for the apically directed efflux transporter P-glycoprotein (P-gp). Duodenal biopsy specimens obtained from a patient with corticosteroid-refractory Crohn's disease and with significantly higher-than-average tacrolimus dose requirements were analyzed for P-gp by Western blot. The P-gp content in this patient was more than double that in specimens obtained from 9 of 10 healthy subjects. Elevated intestinal P-gp could have resulted in decreased tacrolimus absorption, thereby leading to decreased blood concentration and decreased efficacy in this patient. The cause and prevalence of this phenomenon are unknown.

Videocapsule endoscopy renders obscure gastrointestinal bleeding no longer obscure.

INTRODUCTION: Hemorrhage arising from inaccessible areas of the gastrointestinal tract has long been an enigma in gastroenterology. The advent of the Given M2A videocapsule endoscope now permits direct visualization of small bowel mucosa. The purpose of this study is to compare the diagnostic yield of the Given M2A videocapsule endoscope to conventional push enteroscopy. METHODS: Twenty consecutively referred patients (9 men aged 54.8 +/- 21.7 years, 11 women aged 65.6 +/- 16.6 years) who had previously had 1.6 +/- 0.8 EGDs, 1.6 +/- 0.8 colonoscopies, at least 1 normal small bowel radiographic study, and who had received 6.2 +/- 3.9 units of blood were studied. Patients underwent videocapsule endoscopy and subsequently push enteroscopy within 1 week. The endoscopist was blinded to the results of the videocapsule study. RESULTS: Videocapsule endoscopy determined the source of bleeding in 12/20 (60%) of patients versus 15% for push enteroscopy (McNemara chi2, P = 0.02). Videocapsule endoscopy found a source of bleeding in 9/13 patients in whom enteroscopy was negative. Three patients had surgical resections for vascular ectasias (2) and a hamartoma (1) based on the videocapsule endoscopy results. CONCLUSION: The Given M2A videocapsule endoscope has superior diagnostic utility for the evaluation of gastrointestinal bleeding when compared with standard push enteroscopy. The Given M2A videocapsule endoscope can be used to direct appropriate therapy in addition to avoiding the use of unnecessary conventional endoscopic and radiologic procedures.

Videocapsule endoscopy versus barium contrast studies for the diagnosis of Crohn's disease recurrence involving the small intestine.

OBJECTIVES: Historically, suspected Crohn's disease (CD) has been evaluated with small bowel follow-through (SBFT) or enteroclysis (equally accurate). This study was undertaken to determine the accuracy of videocapsule endoscopy (VCE) in the diagnosis of CD relative to SBFT and clinical/laboratory indices of CD activity. Previous investigations have used VCE for the diagnosis of suspected CD in patients presenting with a variety of gastrointestinal symptoms. This is the first study to evaluate the occurrence of active disease in patients with known CD. METHODS: Thirty subjects (22 female, 8 male, aged 36.9 +/- 14.2 yr); all with prior CD diagnosis made on the basis of standard criteria (5.5 +/- 6.5 yr prior to study), in whom recurrent CD was suspected based on abdominal pain, diarrhea, anemia, and/or arthralgias. Subjects were studied in a prospective, blinded evaluation of VCE versus SBFT. SBFT was performed first; those with stricture and proximal bowel dilation were excluded from further study. For SBFT, studies were graded as grade 0 (normal), grade 1 (minimal nodularity, ulcerations, normal luminal diameter, < 5 cm involved), grade 2 (more extensive ulcers, minimal luminal narrowing, 5-10 cm involved), or grade 3 (fistula, skip areas, extensive ulceration, >10 cm involved). VCE was performed within 1 wk of SBFT. Serum was obtained for ESR (erythrocyte sedimentation rate) and CRP (C-reactive protein), stool was obtained for alpha-1 antitrypsin, and the Harvey Bradshaw index of CD severity was calculated. VCE (digitalized video) was graded as grade 0 (normal), grade 1 (erythema, isolated villi loss), grade 2 (erosion, no ulcer), or grade 3 (ulcers, spontaneous bleeding, and/or stricture). RESULTS: Twelve patients were excluded for small bowel obstruction. VCE and SBFT scores highly correlated (r = 0.65; p= 0.001). Active CD was visualized in 21 of 30 patients with videocapsule endoscopy and in 20 of 30 patients with SBFT. Complete agreement occurred in 13 of 30 studies; 13 of 17 studies differed by one grade. SBFT found mucosal disease in 20 of 30 patients and VCE found mucosal disease in 21 of 30 patients. VCE found mucosal disease in 6 patients (5 in grade 1, 1 in grade 3) with normal SBFT. SBFT showed CD in 5 patients (all grade 1) with normal VCE. Neither VCE nor SBFT scores correlated with biological or clinical indices. Patient satisfaction was superior for VCE. CONCLUSIONS: VCE and SBFT are complementary for the diagnosis of CD. SBFT may be required to detect strictures as the videocapsule may not pass. However, some strictures may also be missed with SBFT. VCE is less invasive, less time-consuming for the patient than SBFT, and avoids radiation exposure, although reading time is greater for the gastroenterologist than the radiologist. Given that patients with clinically suspected CD recurrence may not have active disease, unnecessary and potentially harmful empiric therapy is not warranted without imaging.

Strictures from Crohn's disease diagnosed by video capsule endoscopy.

Video capsule endoscopy is a new diagnostic modality that allows imaging of the entire small intestine. We report on a patient with a presumed but undocumented case of Crohn's disease who was found to have 9 ileal strictures by video capsule endoscopy. These strictures were undetected by small intestinal contrast studies and required surgical intervention. This case report suggests that small bowel radiographic studies may not be as sensitive for the detection of clinically significant luminal lesions as once thought.