Polymorphic properties of micronized carbamazepine produced by RESS.

Carbamazepine microparticles were produced by the rapid expansion of supercritical carbon dioxide solutions (RESS) method. The characteristics of the resulting particles were studied by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and image analysis. X-ray diffractograms and SEM photomicrographs revealed that the crystalline nature of the produced carbamazepine microparticles depended on operating pressure and temperature conditions. Different polymorphs were obtained under various operating conditions. Under certain temperature (below 40 degrees C) and pressure (below 240 bar) conditions, it was possible to form primarily the carbamazepine polymorph stipulated by US Pharmacopeia. A significant reduction was observed in the particle size and size distribution range of carbamazepine produced by RESS. The processed particles had a mean diameter smaller than 3 microm and a size distribution range between 0.5 and 2.5 microm compared to unprocessed starting material with a mean diameter of approximately 85 microm and a size distribution range between 15 and 336 microm. Thus, this study demonstrates that the polymorphic characteristics of carbamazepine microparticles produced by the RESS method can be controlled by varying operating pressure and temperature parameters.

Translocational status of ApoB in the presence of an inhibitor of microsomal triglyceride transfer protein.

Despite numerous studies demonstrating that microsomal triglyceride transfer protein (MTP) activity is critical to apoB secretion, there is still controversy as to whether MTP directly facilitates the translocation of apoB across the membrane of the endoplasmic reticulum (ER) through either the recruitment of lipids and/or chaperone activity. In the present study, a specific inhibitor of MTP (BMS 197636) was utilized in HepG2 cells to investigate whether a direct relationship exists between the translocation of apoB across the ER membrane and the lipid-transferring activity of MTP. Inhibition of MTP (with 10 and 50 nmol/L of the inhibitor) did not significantly affect the translocation of newly synthesized apoB (P = 0.77) or the translocational efficiency of the steady-state apoB mass (P = 0.45), despite a 49% decrease in apoB secretion and increased proteosomal degradation. These results compared well with subcellular fractionation experiments which showed no significant change in the fraction of apoB accumulated in the lumen of isolated microsomes in MTP-treated cells (P = 0.35). In summary, MTP lipid transfer activity does not appear to influence translocational status of apoB, but its inhibition is associated with an increased susceptibility to proteasome-mediated degradation and reduced assembly and secretion of apoB lipoprotein particles.

Determination of the viscosity of an amorphous drug using thermomechanical analysis (TMA).

PURPOSE: To evaluate thermomechanical analysis (TMA) as a technique for determining the viscosity of amorphous pharmaceutical materials. This property of amorphous drugs and excipients is related to their average rate of molecular mobility and thus to their physical and chemical stability. METHODS: Indomethacin was selected as a model amorphous drug whose viscosity has previously been reported in the literature. A Seiko TMA 120C thermomechanical analyzer was utilized in isothermal penetration mode to determine the viscosity of the amorphous drug over the maximum possible range of temperatures. RESULTS: Using a cylindrical penetration geometry it was possible to accurately determine the viscosity of amorphous indomethacin samples by TMA over the temperature range from 35 to 75 degrees C. The results were consistent with those reported in the literature using a controlled strain rheometer over the range 44-75 degrees C. The limiting lower experimental temperature for the TMA technique was extended to significantly below the calorimetric glass transition temperature (Tg approximately 42 degrees C), thus allowing a direct experimental determination of the viscosity at Tg to be made. CONCLUSIONS: Thermomechanical analysis can be used to accurately determine the viscosity of amorphous pharmaceutical materials at temperatures near and above their calorimetric glass transition temperatures.

Influence of the Fractal Character of Model Substances on their Reactivity at Solid-Liquid Interfaces.

The fractal theory has been applied to study surface complexity where surface aspects influence physical properties and play a role in controlling heterogeneous reactions at interfaces. In this work, the influence of the fractal character of some selected antacids was investigated in regard to their neutralizing activity. The materials used were magnesium trisilicate, magnesium hydroxide, and heavy and light magnesium oxide, each from three different manufacturers. Surface area, total pore volume, and particle size were measured. Fractal dimension was determined from gas adsorption data according to pore size distribution, the Frenkel-Halsey-Hill, and thermodynamic methods. The results obtained show a correlation between neutralization activity and fractal character rather than total surface area or particle size. Also, the effect of porosity in terms of total pore volume was modified by the structure of the porous network. The complexity of the pore network played a major role in controlling reactivity. However, the effect of surface roughness was only demonstrated when adsorption on the surface was a rate-limiting step in the reaction. Copyright 1999 Academic Press.

Carbamylated hemoglobin and carbamylated plasma protein in hemodialyzed patients.

The carbamylation reaction in vivo involves the nonenzymatic, covalent attachment of isocyanic acid, the spontaneous dissociation product of urea, to proteins. Carbamylated proteins have been proposed as markers of uremia and indicators of uremic control. However, the utility of measuring carbamylated proteins has not been investigated adequately. Therefore, this study was done to determine the relationship between the carbamylation of long-lived protein (hemoglobin) with that of short-lived proteins (plasma proteins) in hemodialyzed patients. Significantly higher carbamylated hemoglobin (CHb; 157 +/- 40 microg valine hydantoin/g Hb) and carbamylated protein (CTP; 0.117 +/- 0.011 absorbance/mg protein) concentrations were found in hemodialyzed patients (N = 13) as compared to normal individuals (N = 9, 53 +/- 20 microg valine hydantoin/g Hb and 0.08 +/- 0.01 absorbance/mg protein, respectively). A high correlation was found between CHb and CTP concentrations (r = 0.87, P < 0.0001), demonstrating a strong relationship between these two different half-lived proteins. A six-month longitudinal study of seven hemodialyzed patients showed that the between subject correlations were significant for CHb versus CTP as well as CHb versus pre-dialysis urea. Correlations were not significant for CTP versus pre-dialysis urea or Kt/V, nor CHb versus Kt/V. Carbamylated hemoglobin fluctuated the most over this time period (30.1% +/- 20.2%), pre-dialysis urea and CTP varied less (18.3% +/- 13.4% and 14.9% +/- 7.5%, respectively), and Kt/V varied the least (6.3% +/- 3.3%). Within subject correlations were not significant between any two tests. It is unclear whether the lack of correlations found is real or a function of the small sample size. However, these data do show that CHb and CTP are positively associated and reflect the degree of urea exposure in the blood, but their usefulness for patients on maintenance hemodialysis is not clear.

The choice of jet nebulizer, nebulizing flow, and addition of albuterol affects the output of tobramycin aerosols.

The use of inhaled antibiotics in the treatment of cystic fibrosis has become widespread despite controversy in the literature as to the appropriate dosing regimen and its effectiveness. This study compared two tobramycin (T) preparations (one with and one without the addition of albuterol) using two different jet nebulizers in order to determine if drug output would be affected. Using calibrated flows from a dry compressed gas source of 6 and 8 L/min as well as a specific compressor (Pulmo-Aide), the Hudson 1720 nebulizer was compared with the newer disposable Hudson 1730. The albuterol preparation used in this study was the Ventolin (albuterol) Respirator Solution (VRS). The nebulizers were charged with (1) 2 mL T (80 mg/2 mL) with 0.5 mL VRS (5 mg/mL) and normal saline solution to make the total nebulizer charge of 3 or 4 mL, or (2) 2 mL T and either 1 or 2 mL normal saline solution. A laser diffraction analyzer (Malvern 2600) was used to determine the aerosol particle size distribution. From the distribution, the respirable fraction, which is the fraction of aerosol that could enter and remain in the lungs, was calculated. For all solutions and each particular flow, the Hudson 1730 had a larger respirable fraction of T. The addition of VRS lowered the surface tension of the solution in the nebulizer and resulted in a greater output of T. This effect was most apparent for the 3-mL volume fills of the Hudson 1720. The greatest differences were between the 3-mL nebulizer charges of T using the Hudson 1720 driven by a flow of 6 L/min, which produced 8 mg of T in the respirable fraction, compared with 35 mg produced by the Hudson 1730 driven by a flow of 8 L/min. These results suggest that different nebulizers, different nebulizer solutions, and different techniques of nebulization may result in very different amounts of T aerosol output in the respirable fraction.

A comparison of pulmonary availability between Ventolin (albuterol) nebules and Ventolin (albuterol) Respirator Solution.

The two most common albuterol preparations used for nebulization are: (1) Ventolin (albuterol) respirator solution (Glaxo Canada Inc; Montreal, Canada) of which 2.5 mg (0.5 mL) is diluted with 2 mL of normal saline solution, and (2) the preservative-free, prediluted Ventolin (albuterol) Nebules PF (Glaxo) (2.5 mg/2.5 mL). The two preparations were compared using both a Hudson 1720 "T" up-draft Neb-U-Mist jet nebulizer and a Hudson 1730 "T" up-draft Neb-U-Mist II jet nebulizer (Hudson; Temecula, Calif), which were driven by a compressor (Pulmo-Aide; Devilbiss; Somerset, Pa) and by dry compressed air at 6 and 8 L/min. Particle size distribution was measured with a particle sizer (Malvern 2600; Malvern Instruments; Malvern, UK) and drug output for the nebulizer was calculated from the differences in predrug and postdrug volume and concentration. Drug availability was defined as the amount of drug carried in particles less than 5 microns in diameter. Drug availability was greater with the albuterol respiratory solution, due to the surface activity of the preservative benzalkonium chloride, for both nebulizers but particularly for the 1720. Differences in drug availability between nebulizers exceeded fourfold depending on the preparation, the nebulizer, and the nebulizing flow. These differences could not have been predicted from the manufacturer's specifications. The results suggest that prediction of drug availability must be based on measurements with the specific preparation and the specific nebulizer used.

Carbamylation of erythrocyte membrane aminophospholipids: an in vitro and in vivo study.

OBJECTIVES: To study the binding of cyanate to erythrocyte membrane aminophospholipids in vitro, and to investigate whether carbamylated aminophospholipids can be detected in the plasma membrane of native erythrocytes. DESIGN AND METHODS: For in vitro studies, the lipid components of 14C-carbamylated erythrocyte membranes were resolved by thin-layer chromatography (TLC). The covalent incorporation of cyanate was visualized by autoradiography and quantitated by phosphorus analysis. For the in vivo studies, phospholipid headgroups were enzymatically hydrolyzed by phospholipase D and subsequently reacted with diacetyl monoxime. RESULTS: Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) were covalently modified by [14C] cyanate; incorporating 15.76 +/- 0.09 and 13.34 +/- 0.81 mol%, respectively, following a 15-h incubation. Carbamylated PE (carb-PE) was resolved with PE by TLC in a solvent system consisting of chloroform/methanol/ammonia (65/35/5, v/v/v). Treatment of native erythrocyte membrane lipid micelles with phospholipase D, followed by reaction with diacetyl monoxime, suggests the presence of intrinsic carb-PE (2.85 +/- 0.65 percent of the total PE). CONCLUSIONS: Carbamylation of erythrocyte aminophospholipid may be involved in some of the hematological consequences of uremia on the erythrocyte.

Carbamylation of erythrocyte membrane proteins: an in vitro and in vivo study.

OBJECTIVES: To establish the degree of erythrocyte membrane protein carbamylation in uremic and nonuremic patients, and to characterize the in vitro binding of cyanate to the individual proteins of the cytoskeletal matrix. DESIGN AND METHODS: For in vivo studies, erythrocyte ghosts were digested with proteinase K and the released peptides colorimetrically assayed for carbamylation, using the diacetyl monoxime reagent, and quantitated using homocitrulline. For in vitro studies, erythrocyte ghosts were incubated with [14C] cyanate, and the membrane proteins separated by SDS-PAGE. Cyanate incorporation was quantitated by liquid scintillation counting and imaging densitometry of the excised bands. RESULTS: Erythrocytes from uremic patients were found to have a greater level of carbamylation than those from nonuremic patients (47.09 +/- 7.80 and 25.89 +/- 6.92 nmol homocitrulline/mg proteolyzed protein released, respectively). In vitro incorporation of [14C] cyanate into membrane protein followed the sequence: spectrin > ankyrin > Band 4.1 > Band 3 > actin > Band 7. CONCLUSIONS: The increased level of erythrocyte membrane protein carbamylation in uremic compared to nonuremic patients may lead to membrane destabilization and contribute to the decreased erythrocyte survival time observed in uremia.

A competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100.

OBJECTIVES: We have developed a competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100 (apoB) and conducted a preliminary evaluation of the method. DESIGN AND METHODS: In the assay, apoB-rabbit immunoglobulin G (IgG) conjugate competes with apoB in the sample for binding to acridinium N-hydroxysuccinimide labelled anti-apoB antibody. Goat anti-rabbit-IgG immobilized to magnetic particles is used to separate the apoB-rabbit IgG conjugate bound to the labelled anti-apoB antibody. Chemiluminescence, measured using a Ciba Corning Magic-Lite chemiluminometer, is inversely proportional to the concentration of apoB in the sample. RESULTS: The assay is completed in 1-2 h. Within-run imprecision (n = 5) determined at 10, 100, and 200 micrograms/L of apoB was found to be 4.8%, 2.4%, and 3.5%, respectively. The between-run imprecision (n = 5) at the same concentrations of apoB was determined to be 14.2%, 9.5%, and 7.7%, respectively. The proposed assay showed reasonable correlation (r = 0.86) with a commercially available immunoturbidimetric method. The lower limit of detection was 2.2 micrograms/L (4.0 pmol/L). CONCLUSIONS: This assay may be useful in tissue culture studies where sensitive measurement of secreted and intracellular concentrations of apoB are required. Our preliminary evaluation of the assay appears to confirm is usefulness.

Low-density lipoprotein binding assay using the calf adrenocortical low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor was purified to a semipure solubilized form from calf adrenocortical tissue. This receptor was found to be a suitable substitute for the human LDL receptor for studying human LDL binding. The apparent dissociation constant of the receptor from calf adrenocortical cells, using human LDL as the ligand, was found to be 8.8 +/- 1.0 micrograms 125I-LDL/mL, similar to that reported for the human LDL receptor (4-10 micrograms LDL/mL). The calf adrenocortical LDL receptor demonstrated specificity toward human lipoprotein fractions that was identical with that of the human LDL receptor. A competitive binding assay was optimized using the semipurified solubilized calf adrenocortical receptor. This facilitated the study of nonenzymatically glycosylated human LDL by a binding assay that is much simpler and faster than previous studies, which used intact cultured cells. The present assay requires only a 1-h incubation of LDL with the receptor and a simple filtration procedure to remove unbound LDL. Using the present assay, it was shown that nonenzymatic glycosylation of LDL on the order of what is seen in diabetics, that is, modification of 2-5% of lysine residues, caused a decreased ability of the LDL to bind to the receptor.

Use of fractal dimension in the study of fractured surfaces of insulin zinc crystals.

The micromorphology of two types of insulin zinc crystals was characterized by computerized image analysis. It was demonstrated that one can identify the recombinant DNA crystals from those of animal origin in a blinded sample by perimeter fractal dimension analysis. Fractal dimension analysis was also applied in the study of irregularly fractured surfaces produced by thermal and mechanical stress. The results obtained suggest that the fractal dimension is related to the intensity of applied stress. The characterization of newly formed boundaries of insulin zinc crystals produced by mechanical or thermal stress could determine various surface-related kinetic processes.

Evaluation of the surface roughness of cystine stones using a visible laser diode scattering approach.

To understand the processes of fragmentation and the chemical reactivity of solids, proper characterization of surface topography is crucial. This paper describes a non-destructive technique of quantifying the surface roughness of cystine renal stones, using visible laser diode scattering and fractal geometry. Fragments of cystine stones were mounted on microscope slides and coated by a carbon-sputtering apparatus. The slides were placed under a dynamic active-vision system, using a visible laser diode to measure three-dimensional surface coordinates. The data obtained were analyzed by fractal geometry. Surface fractal dimensions were determined by the variation method. The results showed that the surface of a compact-size sample can be evaluated quantitatively. The technique is valuable for the accurate presentation of surfaces in three dimensions.

Glycated calmodulin from platelets as an index of glycemic control.

In an effort to test whether a significant fraction of calmodulin would become glycated within the life span of the platelet (10-14 days), we monitored the kinetics of calmodulin glycation in vitro. Under the conditions we used, the fraction of glycated calmodulin reached a maximum (approximately 21%) within 10 days. We then extended the studies to human subjects. The intraplatelet concentrations of calmodulin and glycated calmodulin from age-matched type I diabetic subjects were monitored by a combination of m-aminophenylboronate affinity chromatography and enzyme-linked immunosorbent assay. The results indicate that the concentrations of total intraplatelet calmodulin (nonglycated plus glycated) were not dependent on the glycemic state of the subjects. Data from control and diabetic subjects showed a poor correlation between the concentrations of glycohemoglobin and of glycated calmodulin. However, a better correlation was obtained when glycated calmodulin concentrations were compared with those of serum fructosamine. The fraction of glycated calmodulin in the control population (7.71% +/- 0.75%) was significantly (P < 0.05) different from that of the diabetic population (21.6% +/- 1.26%). Given that the clinical role of the fructosamine assay remains controversial, estimation of glycated calmodulin in platelets might be useful as a short time-window index of glycemic control.

Glutathione metabolic enzyme activities in diabetic platelets as a function of glycemic control.

Type 1 diabetic subjects categorized on the basis of the glycated haemoglobin content of their blood (low less than 7%; medium, greater than 7% and less than 11%; high, greater than 11%) were analyzed for total intraplatelet GSH as well as for the steady-state kinetic parameters (apparent KM and apparent Vmax) of some glutathione metabolic enzymes including glutathione reductase, glutathione peroxidase, gamma-glutamyltrans-peptidase and glutathione-S-transferase. This study indicates that intraplatelet GSH content of subjects with low glycated-haemoglobin is approximately 2-fold higher than those with medium glycated-haemoglobin. There was no further decrease in intraplatelet-GSH in subjects with high glycated-haemoglobin. The kinetic parameters of the platelet-enzymes studied (glutathione reductase, gamma-glutamyltranspeptidase and glutathione-S-transferase) were essentially independent of the glycation state of the subject. However, the apparent KM of glutathione peroxidase was approximately 4-fold higher in the subjects with high glycated-haemoglobin, in comparison to low subjects. This decrease in affinity could possibly result from the susceptibility of this enzyme to non-enzymatic glucosylation as purified samples of glutathione peroxidase incubated in vitro with glucose showed similar increases in apparent KM. These results are discussed in terms of the potential contribution of glutathione peroxidase impairment, to the hyperaggregability of the diabetic platelet.

Rapid screening method for polymorphism of group A apolipoproteins.

Polymorphism of apolipoproteins AI and AII (apo AI and apo AII) can be easily investigated in plasma by a simple method involving a 30-min incubation of EDTA plasma in the presence of urea, dithiothreitol, and Nonidet P-40 followed by subsequent isoelectric focusing (IEF). The sample (2 microL) was applied to an ultrathin flat acrylamide gel of pH range 4-6, and focused using a Bio-Rad Mini IEF Cell for 1.5 h at a maximum of 500 V. Coomassie Blue R-250 was used to visualize the apolipoproteins. To verify the identity of the different apolipoproteins after IEF, the gel was immunofixed directly with anti-apo AI, or immunoblotted on polyvinylidene difluoride (PVDF) membrane using monospecific antibodies to apo AI and apo AII and an anti-immunoglobulin-alkaline phosphatase conjugate. High-density lipoprotein (HDL) was used as a standard for Apo AI variants. Employing these techniques, human plasma apo AI was resolved into one major band (apo AI0, pI 5.54), and four minor bands identified as apo AI+2 (pI 5.75), apo AI+1 (pI 5.66), apo AI-1 (pI 5.45), and apo AI-2 (pI 5.34). Apo AII was resolved into one major isoprotein designated as apo AII0 (pI 4.87), and two minor isoforms apo AII+1 and apo AII-1 which focused at pIs of 5.18 and 4.58, respectively. The results showed that these methods can be used to identify apo AI and AII isoforms without prior ultracentrifugation to isolate the HDL. The entire procedure, including IEF, fixation (chemical or immunofixation), and staining, can be accomplished in 5 h compared to 2 days using previously reported technique.(ABSTRACT TRUNCATED AT 250 WORDS)

The study of the surface geometry of renal stone fragments after shock wave and ultrasound disintegration.

Computerized image analysis was used for characterizing the irregular boundaries of calcium oxalate stone fragments resulting from shock wave and ultrasound disintegration. The complexity of the contour of the fragments was determined to evaluate the surface roughness of the rugged profile of the samples. Crack propagation on the crystal surface of the mineral phase was studied using fractal geometry. A significant difference was observed in the boundary variation of the calcium oxalate stone fragments treated by shock wave and ultrasound. Crack propagation in the mineral phase crystal was found to depend on the method of fragmentation used. There is also an experimental evidence that the surface topography of the stone fragments produced by shock wave depends on the microhardness of the stone material.

A simple micromethod for rapid assessment of the distribution of apolipoprotein C isoforms in very-low-density lipoprotein.

A simple and rapid isoelectric focusing method for quantifying Apo C isoforms of triglyceride-rich lipoprotein was developed. The very-low-density lipoprotein (VLDL) was isolated from 100 microL of EDTA plasma using a Beckman Airfuge ultracentrifuge. The delipidated VLDL was applied to an ultrathin flat acrylamide gel, and focused using a Bio-Rad Mini IEF Cell, for 1.5 h at a maximum of 500 V. Apo CII and Apo CIII in VLDL were resolved into four major bands, CIII0 (PI 4.91), CII (PI 4.78), CIII1 (PI 4.72), and CIII2 (PI 4.53). The method demonstrated within-run and between-run CVs of 2.7% to 11.9% and 4.4% to 12.2%, respectively. The relative percentage of C apoproteins and the ratio of CII to CIII found in VLDL from plasma of normal, chronic renal failure, and hyperlipidemic subjects agreed with previously published data.

Determination of non-enzymatically glycated apolipoprotein A1.

A procedure for the quantitation of non-enzymatically glycated apolipoprotein A1 (GApoA1) was developed and optimized. Glycated total protein was separated from plasma using m-aminophenyl-boronate affinity chromatography. Apolipoprotein A1 present in the glycated and non-glycated fractions of each sample was determined by rate nephelometry, and the percent glycated apo A1 calculated. The measuring range of the assay was 0.5-8.0% GApoA1. The within- and between-run CV's were less than 5.2 and 7.9%, respectively, and recoveries were greater than 92%. Free glucose did not affect the results. In a group of female non-insulin diabetic subjects the mean GApoA1 was 3.8 +/- 1.6% (mean +/- SD). In non-diabetic subjects the mean level of GApoA1 was 2.1 +/- 0.8% (mean +/- SD).