A method to predict the equilibrium solubility of drugs in solid polymers near room temperature using thermal analysis.

A method is presented for determining the equilibrium solubility of a drug in a solid polymer at or near room temperature, which represents a typical storage temperature. The method is based on a thermodynamic model to calculate the Gibbs energy change ΔG(SS) associated with forming a binary drug-polymer solid solution from the unmixed polymer and solid drug. The model includes contributions from heat capacity differences between the solid solution and the corresponding unmixed components, breaking up of the solid drug structure, and drug-polymer mixing. Calculation of ΔG(SS) from thermal analysis data is demonstrated, and it is shown that minima of plots of ΔG(SS) versus the dissolved drug concentration represent the equilibrium drug solubility in the polymer. Solid solutions were produced for drug-polymer systems (griseofulvin, indomethacin, itraconazole; PVP K30, Eudragit L100, Eudragit E100) in drug weight fractions up to ∼25%. At 25°C, it was seen that heat capacity effects were important in determining the drug solubility. It was concluded that drug solubilities in solid polymers can be determined using thermal analysis, and must include heat capacity effects when evaluated near room temperature.

Phase I clinical study to select a novel oral formulation for ibandronate containing the excipient sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC).

The aim of this study was to select a novel oral formulation for ibandronate (IBN, CAS number: 13892619). In four cohorts of 28, 21, 19 and 29 healthy volunteers, the impact of the carrier molecule sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC, CAS number: 203787-91-1) on the bioavailability of IBN was investigated. Within each cohort different oral formulations with one dose of ibandronate (30 mg) and three different ratios of IBN:SNAC (1:5, 1:10 and 1:20) were compared to the approved oral IBN tablet formulations (150 and 50 mg IBN) in a 4-way cross-over design and a one week washout between the administrations. The highest mean IBN exposure was achieved with a capsule formulation containing drug-coated beadlets and an IBN:SNAC ratio of 1:5. AUC(last) and C(max) of IBN were approximately 1.3- and 2.2-fold higher compared to the reference treatment (150 mg IBN without SNAC). Increasing the post-dose fasting duration from 15 to 30 min resulted in a more than 2-fold increase in AUC(last), while superimposable IBN serum concentration-time profiles were achieved after a 30 and 60 min fast. The tolerability of the IBN/SNAC treatments in all cohorts was similar to that in the IBN reference groups and most adverse events (AEs) were of mild to moderate intensity.

Endoscopic hemostasis in acute diverticular bleeding.

BACKGROUND AND STUDY AIMS: Early reports of urgent colonoscopy in acute lower intestinal bleeding suggest a role for endoscopic therapy for bleeding colonic lesions, but scant data exist on bleeding diverticula. We report our experience with endoscopic hemostasis in acute diverticular bleeding. PATIENTS AND METHODS: Bleeding diverticula were identified on urgent diagnostic endoscopy in five patients with acute gastrointestinal bleeding, two in the duodenum, and three in the colon. All patients had co-morbid conditions preventing more conventional therapeutic approaches. The five cases are described, including the technique of endoscopic hemostasis and outcome. RESULTS: Endoscopic therapy using epinephrine injection, thermal cautery and/or laser therapy successfully induced hemostasis in all patients. One patient died of co-morbid illness during the hospital stay, while the remaining four patients had no recurrent bleeding over a mean follow-up period of 20.6 months. CONCLUSION: Endoscopic therapy of bleeding diverticula is technically possible when the culprit diverticulum can be identified. This therapeutic modality may have a place in debilitated patients in whom other more invasive procedures are contraindicated, but further experience is needed to establish its safety.

Enteroscopy-enteroclysis: experience with a combined endoscopic-radiographic technique.

BACKGROUND: Video enteroscopy provides high-quality diagnostic and therapeutic capabilities in the proximal small bowel. Enteroclysis remains an essential diagnostic technique in the distal small bowel. We report our experience with the combination of these techniques. METHODS: Seventy-one patients with obscure gastrointestinal bleeding (group A, 54 patients) or abnormal radiologic studies (group B, 17 patients) were evaluated with enteroscopy. Enteroclysis via a tube inserted on withdrawal of the enteroscope was performed in all patients with nondiagnostic enteroscopy. RESULTS: Enteroscopy identified bleeding sites in 29 of 54 (54%) group A patients (12 angiodysplasia, 10 ulcers, 7 gastric erosions, 1 vessel, 1 aortoenteric fistula), and lesions in 11 of 17 (65%) group B patients (7 ulcers, 3 benign strictures, 2 radiation enteritis, 1 mass). In group A, 13 (24%) patients had findings detectable by standard esophagogastroduodenoscopy. Enteroclysis identified masses in 2 of 24 (8%) group A patients, and lesions in 5 of 10 (50%) group B patients (3 strictures, 1 mass, 1 large diverticulum). No complications occurred. CONCLUSIONS: The combination of enteroscopy and enteroclysis is safe and offers quality small bowel examinations in more comfortable and convenient single diagnostic sittings. This combination detected bleeding sources in 57% and lesions in 70% of patients. Though enteroclysis identified bleeding sources in only 8% of patients, this study excluded lesions other than angiodysplasia.

Endoscopic management of bile duct leaks after attempted laparoscopic cholecystectomy.

Fourteen patients with symptomatic bile duct leaks following laparoscopic cholecystectomy were treated using endotherapeutic techniques. Patients presented with abdominal pain, liver test abnormalities, jaundice, leukocytosis, and fever. Twelve leaks originated from cystic duct stumps and two from right posterior hepatic ducts. Distal biliary obstruction, which may have promoted leakage, was present in five patients. Treatment methods included stent insertion with endoscopic sphincterotomy (ES), stent insertion without ES, and nasobiliary tube (NBT) placement without ES. Eleven of 14 patients had prompt resolution of their bile leaks following initial endotherapy. Three patients with continued leakage underwent successful repeat endoscopic retrograde cholangiopancreatography 4-5 days after the initial examination. Cholangiographic evidence of leak closure was documented in all patients, and all remained asymptomatic during an average follow-up period of 18.5 months. Endoscopic therapy is safe and effective treatment for clinically significant bile leaks following laparoscopic cholecystectomy. In our small group of patients, NBT alone did not appear to be as effective as endoprostheses with or without ES. The ideal endoscopic treatment method has not yet been established but will likely vary depending on the site and specific nature of the injury and any concomitant biliary ductal pathology.

Fluorescence and chemiluminescence detection of oxazole-labelled amines and thiols.

Fluorescence and chemiluminescence analyses of amino acids and thiols derivatized with 2-fluoro-4,5-diphenyloxazole (DIFOX) and 2-chloro-4,5-bis(p-N,N-dimethylaminosulphonylphenyl)oxazole (SAOX-Cl) were investigated. Thirteen diphenyloxazole (DIOX)-derivatized amino acids were separated within 38 min by a linear gradient elution from 100% A [0.05 M phosphate (pH 7.0): CH3CN (75:25)] to 100% B [0.05 M phosphate (pH 7.0):CH3CN (1:1)] over 30 min and an isocratic elution of 100% B for 30 min. The detection limits (S/N = 2) with fluorescence detection were in the range of 19-64 fmol. Thiols derivatized with SAOX-Cl were separated by an isocratic elution using 0.1 M H3PO4:CH3CN (65:35) and detected fluorimetrically. The detection limits (S/N = 2) of reduced glutathione, N-acetylcysteine, 2-mercaptopropionylglycine, cysteine, homocysteine and captopril were 1.2, 1.5, 1.9, 5.7, 6.4 and 7.9 fmol, respectively. Peroxyoxalate chemiluminescence (CL) intensities of sulphonyl-5-N,N-dimethylaminonaphthalene (DNS), SAOX and DIOX derivatives were compared using three different oxalate esters (DFPO, TCPO and TDPO) by flow injection analysis. The relative chemiluminescence intensity (RCL) of SAOX-proline and DIOX-proline were 76-80% and 19-25% of DNS-proline (100%), respectively. Other SAOX and DIOX derivatives showed lower CL intensities (< 12%). Extremely low CL intensities were obtained for the fluorescent tagging reagents (< 0.11%) and their hydrolysis products (< 0.80%). Secondary amino acids and peptides, derivatized with DIFOX in aqueous media at room temperature for 1 h, were detected using DFPO/H2O2. TCPO/H2O2 and TDPO/H2O2 after separation by high performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF.

Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.

In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.

Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.

Glycosylation is not required for ligand or receptor binding by expressed rat intrinsic factor.

A cDNA clone encoding rat intrinsic factor (IF), pIFQ, has been inserted into the eukaryotic expression vector pSVL and used to transfect COS-1 cells. The IF produced by the transfected cells was secreted nearly exclusively into the medium at concentrations of 0.1-0.2 micrograms/ml. Tunicamycin treatment (1-10 micrograms/ml) completely blocked N-linked glycosylation but had no effect on IF secretion. The secreted glycosylated IF retained all the properties of native IF, i.e., high affinity for cobalamin (Cbl) and for the IF-Cbl receptor and relative resistance to low pH and to proteolysis. The nonglycosylated IF also retained these properties except that it was more protease sensitive. The protease degradation was prevented by the presence of the ligand Cbl. The presence of carbohydrate may play a role in protecting IF from digestion by pancreatic proteases in the intestinal lumen.

Oxalate/hydrogen peroxide chemiluminescence reaction. A 19F NMR probe of the reaction mechanism.

The mechanism of the oxalate/hydrogen peroxide chemiluminescence reaction has been examined by magnetic resonance techniques. Investigation of the reactive intermediates involved in chemiluminescence was carried out with bis(2,6-difluorophenyl)oxalate (DFPO) using 19F NMR to probe its reactions with aqueous hydrogen peroxide. Formation and reactions of the intermediate hydroperoxy oxalate ester B along with the formation of the half ester product C and difluorophenol D were monitored by 19F NMR. When the reaction of DFPO and aqueous hydrogen peroxide was carried out in the presence of dansylphenylalanine, a typical fluorescent analyte, the intensity of the resonance due to the intermediate B was diminished in direct proportion to the concentration of the analyte. Comparison of the time/intensity profile of the chemiluminescence emission with that of the 19F NMR transient suggests that the hydroperoxy oxalate ester B is the likely 'reactive' intermediate, capable of participating in a chemically initiated electron exchange luminescence mechanism.

Chemiluminescence: a sensitive and versatile method for analyte detection.

The generation of light from the oxidation of oxalic esters with hydrogen peroxide has been applied to the detection of luminescent materials. In order to improve the efficiency of this method, which is less than 0.1%, and to enhance the selectivity for target analytes, an in-depth investigation of the oxalate ester-hydrogen peroxide reaction has been conducted. A kinetic model has been developed based on the effects of catalysts, reagents and reaction conditions for maximum light production. Application of the model to liquid chromatography through the "time-dependent emission window" concept affords a predictable maximum sensitivity for selected analytes. Application to the detection and quantitation of met- and leu-enkephalins which have been labelled with naphthalene-2,3-dicarboxyaldehyde/cyanide provides support for this methodology. Other bioanalytical applications are presented.

In vitro proliferation and terminal differentiation of quail neural crest cells in a defined culture medium.

We report the formulation of a culture medium, medium MCDB202-21, that supports the in vitro proliferation of quail neural crest cells and their differentiation into melanocytes and adrenergic neuroblasts in the complete absence of serum and chick embryo extract. McKeehan & Ham's medium MCDB 202 was supplemented with hormones, stimulators of metabolism, vitamins, trophic factors, transport molecules, and small molecular nutrients.