Gene expression patterns in peripheral blood correlate with the extent of coronary artery disease.

Systemic and local inflammation plays a prominent role in the pathogenesis of atherosclerotic coronary artery disease, but the relationship of whole blood gene expression changes with coronary disease remains unclear. We have investigated whether gene expression patterns in peripheral blood correlate with the severity of coronary disease and whether these patterns correlate with the extent of atherosclerosis in the vascular wall. Patients were selected according to their coronary artery disease index (CADi), a validated angiographical measure of the extent of coronary atherosclerosis that correlates with outcome. RNA was extracted from blood of 120 patients with at least a stenosis greater than 50% (CADi > or = 23) and from 121 controls without evidence of coronary stenosis (CADi = 0). 160 individual genes were found to correlate with CADi (rho > 0.2, P<0.003). Prominent differential expression was observed especially in genes involved in cell growth, apoptosis and inflammation. Using these 160 genes, a partial least squares multivariate regression model resulted in a highly predictive model (r(2) = 0.776, P<0.0001). The expression pattern of these 160 genes in aortic tissue also predicted the severity of atherosclerosis in human aortas, showing that peripheral blood gene expression associated with coronary atherosclerosis mirrors gene expression changes in atherosclerotic arteries. In conclusion, the simultaneous expression pattern of 160 genes in whole blood correlates with the severity of coronary artery disease and mirrors expression changes in the atherosclerotic vascular wall.

Discovery of proteins related to coronary artery disease using industrial-scale proteomics analysis of pooled plasma.

BACKGROUND: Relating a disease state to an entire population of proteins provides an opportunity to gain new insights into a disease. METHODS: Male populations of 53 patients with angiographic coronary artery disease and 53 control subjects without coronary disease from the Duke Databank for Cardiovascular Disease were established and matched for age and race as well as extremes of risk factors. Major plasma protein abnormalities were excluded. Plasma samples of each group were pooled to make large volumes (6 L each) to identify low-abundance proteins. After removal of albumin as well as immunoglobulins and enrichment of smaller proteins (<20-40 kDa), samples were separated into 12,960 fractions by cation exchange and 2 reversed-phase chromatography steps. Proteins were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. RESULTS: There were 731 plasma proteins or fragments identified. Of these proteins, 95 were differentially displayed in the case versus control populations. These represent broad categories of proteins involved with natural defenses, inflammation, growth, and coagulation. CONCLUSION: We identified a large number of proteins that differ in abundance in populations with and those without angiographic coronary disease. These proteins now comprise candidates for validation studies in individual patients and in larger clinical data sets to better define disease pathways and establish novel markers for disease.

High-performance liquid chromatography of lactose with evaporative light scattering detection, applied to determine fine particle dose of carrier in dry powder inhalation products.

A method for quantification of the fine particle dose of lactose is described, using a hydrophilic interaction chromatography (HILIC) method and evaporative light scattering detection. The HILIC method used an aminopropyl column and a mobile phase consisting of acetonitril/water (80/20, v/v) for isocratic elution. Sensitive chromatography was obtained using a low concentration of water in the extraction solvent. The detection limit (RSD<10%) at an injection volume of 10 microL is 10 microg/mL. Linearity was obtained in the range of 10-80 microg/mL (R(2)>0.99). A relative standard deviation (RSD) of 0.5% (N=6) demonstrated good precision of the optimized method.

Conditioning following powder micronization: influence on particle growth of salbutamol sulfate.

Micronization is a high-energy process that induces changes in the crystallinity of materials. As a result, the crystalline structures on the particles' surface are being destroyed and amorphous areas are formed. After micronization of salbutamol sulfate to be used in dry powder inhalers, only small amounts of amorphous material are produced. Nevertheless, even these small amounts can have important effects on the physical stability of the powder. The amorphous state is thermodynamically unstable and tends to convert to the stable, crystalline state. The recrystallization process of disordered regions on the particles' surface leads to particle growth of milled particles. In this case, bridges of solid material are being formed between the individual particles, which leads to particle growth. This is an undesirable process, because particles for pulmonary administration are designed to range between 1 and 10 microm in diameter to exert respirative effect. In the present investigation, salbutamol sulfate is micronized by an air jet mill, and the generated products are exposed to different conditions. Thereafter, the best possible conditioning parameters and storage conditions for the micronized salbutamol sulfate are worked out and rated. The aim of this treatise is to demonstrate the importance of conditioning following micronization.

Influence of mechanical activation on the physical stability of salbutamol sulphate.

In order to obtain the optimal particle size distribution for pharmaceutical powders in dry powder inhalers the particles have to be micronised. In most cases the process of micronisation is connected with a high input of energy which induces disorder and defects on the surface of the drug particles and as a result changes in the crystallinity. Consequently, changes in the physical stability of the powders may occur. To investigate changes on the physical stability of the powder, different analytical methods are used in the present investigation: laser diffraction, Differential Scanning Calorimetry (DSC), isothermal microcalorimetry and DVS-method.Air-jet-milling is one of the most frequently used techniques in the pharmaceutical industry, in order to obtain particles of respirable size. In the treatise described here the influence of the critical parameters of the process, i.e. feed pressure, grind pressure and feed rate is assessed for salbutamol sulphate. The grind pressure is of utmost importance with respect to particle size distribution and the physical powder stability. For salbutamol sulphate, ground with a MC Jetmill 50, a grind pressure of 6 bar has been found optimal. Pressures below 6 bar are not sufficient to produce the required reduction in particle size. The feed pressure and rate have negligible influence on the powder quality. Furthermore, the micronisation process is optimised to achieve respirable particles while minimising the amorphous content. A correlation between mechanical activation and the amount of the amorphous regions is showed clearly.Air-jet-milling has been compared to ball milling in this investigation. In pilot tests ball milling was not suitable to achieve the needed particle size distribution, however, it generates a specific quantity of amorphous material. With the help of specific amorphous regions in the powder, the sensitivity of the used methods for salbutamol sulphate can be examined.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Population pharmacodynamic analysis of octreotide in acromegalic patients.

INTRODUCTION: Octreotide is an octapeptide analog of somatostatin used to normalize growth hormone levels in acromegaly. This article presents a population analysis of the relationship between octreotide and growth hormone concentrations in 94 patients with acromegaly, including 10 patients responding incompletely to subcutaneous treatment (poor responders). METHODS: Growth hormone and octreotide concentrations were recorded hourly over 12-hour time periods during long-term subcutaneous treatment. Twelve-hour profiles were also collected on different days up to 2 months after intramuscular injection of the long-acting formulation. We modeled the inhibition of growth hormone secretion by octreotide with a direct maximum inhibition model. A joint analysis of both formulations was performed with NONMEM (GloboMax, LLC, Hanover, Md). During model building, we examined the relationships between parameters and demographic covariates or formulations with the use of likelihood ratio tests. RESULTS: The baseline growth hormone level was higher in poor responders and was best described by a bimodal distribution. The maximum inhibition was common to both formulations and had a mean of 90%, with low interindividual variability. Sensitivity to octreotide (50% inhibitory concentration) was found to be slightly lower on average with intramuscular administration than with subcutaneous administration. CONCLUSION: Given adequate doses of octreotide, in 72% of 94 patients, growth hormone would decrease to levels below 2.5 ng. mL(-1), considered to be a desirable target concentration in acromegaly. This study provides a way to identify poor responders during subcutaneous treatment, allowing an early clinical decision to be made to switch nonresponders to alternative therapies.