Optimization of the experimental parameters of fluticasone propionate microparticles for pulmonary delivery using a box behenken design.

The objective of this study was to examine extensively the influences of formulation and process variables on the microparticles. The microparticles were generated by the spray-drying technique using polymer chitosan, mannitol along with L-leucine. The effects of various experimental parameters such as polymer concentration, inlet temperature, and feed flow rate on particle size and production yields were evaluated by means of experimental box-behnken design. Multiple regression analysis was carried out and response surfaces were obtained. Optimized formulation and check points batches were selected by feasibility and grid search. Experimental design it was evaluated that inlet temperature and polymer concentration influence on the production yield. Feed flow rate impact on particle size. Results showed that spray drying technique yield 985 to 4060 nm indicate micro size range and production yield was found in between 27.01-52.96%. The selection of appropriate parameters yielded spray-dried microparticles characterized by narrow dimensional distribution. In our present work, prepared microparticles using the spray-drying technique and systematically estimated their feasibility for the pulmonary delivery of microparticles by careful investigations of their characteristics and aerosolization properties. Spray drying technique yield optimum size for deposition beyond the narrow airway into the alveoli and suitable for respiratory deposition.

Effects of spray drying conditions on the physicochemical properties of the Tramadol-Hcl microparticles containing Eudragit(®) RS and RL.

The preparation of Tramadol-HCL spray-dried microspheres can be affected by the long drug recrystallization time. Polymer type and drug-polymer ratio as well as manufacturing parameters affect the preparation. The purpose of this work was to evaluate the possibility to obtain tramadol spray-dried microspheres using the Eudragit(®) RS and RL; the influence of the spray-drying parameters on morphology, dimension, and physical stability of microspheres was studied. The effects of matrix composition on microparticle properties were characterized by Laser Light scattering, differential scanning calorimetry (DSC), X-ray diffraction study, FT-infrared and UV-visible spectroscopy. The spray-dried microparticles were evaluated in terms of shape (SEM), size distribution (Laser light scattering method), production yield, drug content, initial drug loding and encapsulation efficiency. The results of X-ray diffraction and thermal analysis reveals the conversion of crystalline drug to amorphous. FTIR analysis confirmed the absence of any drug polymer interaction. The results indicated that the entrapment efficiency (EE), and product yield were depended on polymeric composition and polymeric ratios of the microspheres prepared. Tramadol microspheres based on Eudragit(®) blend can be prepared by spray-drying and the nebulization parameters do not influence significantly on particle properties.

Assessment of dissolution profile of marketed aceclofenac formulations.

Statistical comparison of dissolution profiles under a variety of conditions relating to formulation characteristics, lot-to-lot, and brand-to-brand variation attracts interest of pharmaceutical scientist. The objective of this work is to apply several profile comparison approaches to the dissolution data of five-marketed aceclofenac tablet formulations. Model-independent approaches including ANOVA-based procedures, ratio test procedure, and pair wise procedure. The ratio test includes percentage, area under the curve, mean dissolution time, while the pair wise procedure includes difference factor (f(1)), similarity factor (f(2)), and Rescigno index. In the model-dependent approach, zero order, first order, Hixson-Crowell, Higuchi, and Weibull models were applied to the utilization of fit factors. All the approaches were applicable and useful. ANOVA with multiple comparison tests was found to be sensitive and discriminating for comparing the profiles. Weibull parameters were more sensitive to the difference between two release kinetic data in terms of curve shape and level.

Mathematical evaluation of similarity factor using various weighing approaches on aceclofenac marketed formulations by model-independent method.

The US Food and Drug Administration's (FDA's) guidance for industry on dissolution testing of immediate-release solid oral dosage forms describes that drug dissolution may be the rate limiting step for drug absorption in the case of low solubility/high permeability drugs (BCS class II drugs). US FDA Guidance describes the model-independent mathematical approach proposed by Moore and Flanner for calculating a similarity factor (f2) of dissolution across a suitable time interval. In the present study, the similarity factor was calculated on dissolution data of two marketed aceclofenac tablets (a BCS class II drug) using various weighing approaches proposed by Gohel et al. The proposed approaches were compared with a conventional approach (W = 1). On the basis of consideration of variability, preference is given in the order of approach 3 > approach 2 > approach 1 as approach 3 considers batch-to-batch as well as within-samples variability and shows best similarity profile. Approach 2 considers batch-to batch variability with higher specificity than approach 1.

Congenital diaphragmatic hernia.

Over the last two decades there has been a constant improvement in the understanding of the pathophysiology of Congenital Diaphragmatic Hernia (CDH) and its management. However, the ideal treatment remains elusive. The earlier management strategy of immediate surgery is replaced by the principle of physiological stabilisation and delayed surgery. Conventional mechanical ventilatory techniques, with high pressures and hyperventilation to reverse ductal shunting and cause alkalinization, are being questioned because of the risks of barotrauma and consequent broncho-pulmonary dysplasia. It has also been shown that paralysis with pancuronium bromide for patients on conventional mechanical ventilation results in increased incidence of sensorineural hearing loss in childhood survivors of CDH. With the introduction of the concept of permissive hypercapnia and high frequency oscillation ventilation, the complications of pulmonary barotrauma are circumvented. Although ECMO therapy is invasive, yet has improved survival by about 15% independently, especially in critically ill infants who have the predictive mortality rate of more than 80%. Further insights into the pathophysiology of CDH and the introduction of less invasive therapeutic techniques in the form of high frequency oscillation ventilation, inhalation nitric oxide, surfactant, and perfluorocarbon liquid ventilation may even make the need for ECMO redundant.

[Inherited factor VII deficiency and pregnancy. Apropos of 1 case]. / Déficit constitutionnel en facteur VII et grossesse. A propos d'un cas.

Hereditary factor VII deficiency is a rare autosomal recessive condition. Factor VII's level elevates during pregnancy in normal patients, as well in deficient individuals for some authors. Various treatments (fresh frozen plasma, prothrombin complex or factor VII concentrate) have been used to lessen the peri-partum hemorrhage in those factor VII-deficient pregnant women. We report the case of a pregnant woman presenting a factor VII deficiency (level 4%), without variation of level during her pregnancy. The single infusion of factor VII concentrate, prior to delivery, has elevated factor VII's level at 17% and has likely permitted minimal post-partum bleeding. The peripartum management of factor VII deficiency is discussed.

HDL phospholipid content and composition as a major factor determining cholesterol efflux capacity from Fu5AH cells to human serum.

The relationships of cell cholesterol efflux to HDL phospholipid (PL) content and composition in human serum were analyzed in two groups of subjects selected on the basis of their HDL cholesterol (HDL-C) levels: a norm-HDL group (1.10 mmol/L < HDL-C < 1.50 mmol/L) and a high-HDL group (HDL-C > 1.75 mmol/L). In the high-HDL group, the relative fractional efflux was significantly higher than in the norm-HDL group, and in both groups, fractional efflux was correlated with a number of lipoprotein parameters, the best correlation and the only one that remained significant after multivariate analysis being with HDL phospholipid (HDL-PL). Analysis of the HDL-PL subclasses revealed that HDL in the high-HDL sera was enriched with phosphatidylethanolamine (HDL-PE) and relatively deficient in sphingomyelin (HDL-SM) compared with norm-HDL sera. Moreover, the fractional efflux values in the high-HDL group were negatively correlated with the proportion of HDL-PE (r = -.64, P < .0001) and positively correlated with the proportion of HDL-SM (r = .43, P < .01). Thus, this study provides evidence that HDL-PL concentration can be used to predict the capacity of serum to accept cellular cholesterol. Among the differences described between norm-HDL and high-HDL sera, the variability in PE to SM ratio might reflect changes in serum cholesterol acceptors that modulate the first step of reverse cholesterol transport.

Plasma and erythrocyte vitamin E content in asymptomatic hypercholesterolemic subjects.

The present study was designed to assess plasma and erythrocyte vitamin E concentrations in 57 asymptomatic hypercholesterolemic (HC) men compared with 56 normocholesterolemic (NC) men. Vitamin E concentrations were determined by using a reversed-phase HPLC method. Compared with NC subjects, HC men had a significantly lower red blood cell (RBC) vitamin E content in spite of their normal plasma vitamin E concentration. This study demonstrates that total plasma vitamin E concentration is not a suitable predictor of cell vitamin E status and suggests an abnormal transfer of tocopherol between plasma and RBCs in HC men. Moreover, the RBCs of HC men were more susceptible to a peroxidative stress. The strong correlation between RBC susceptibility to oxidation and RBC vitamin E content suggests that the low RBC vitamin E content found in HC men has physiological consequences on the RBC oxidation.

Post-prandial metabolism of triglyceride-rich lipoproteins in non-insulin-dependent diabetic patients before and after bezafibrate treatment.

Retarded post-prandial (pp) lipid clearance is potentially a major component of the increased cardiovascular risk incurred by hypertriglyceridaemic non-insulin-dependent diabetic mellitus (NIDDM) patients. The effect of bezafibrate (Bz, 400 mg per day for 5 weeks on chylomicron (CM) and remnant clearance after loads of 100 g of fat and vitamin A was therefore explored in 10 male patients (glycaemia 11.9 +/- 3.3 TG 4.5 +/- 2.4 mmol L(-1)). In all subjects CM-TG and retinyl palmitate (RP) were reduced by 50%, but 8-h non-CM (remnant) RP decreased only in initially mildly hypertriglyceridaemic subjects (-35%, P < 0.05), while in three patients with very elevated initial TG (epsilon3/3, epsilon3/2 and epsilon2/2 genotypes) 8-h remnant RP increased by 100%. The decrease in pp CM-TG correlated with that of fasting Sf 20-400 (r = 0.686, P = 0.026), suggesting that improved lipolysis was a major determinant of hypolipidaemic effect. Apo CIII synthesis is known to be depressed by Bz: concentrations were lower under Bz (P < 0.05). A positive correlation (r = 0.880, P < 0.001) with fasting TG before treatment and its disappearance after treatment suggested an involvement of high concentrations with hypertriglyceridaemia. Post-prandial non-esterified fatty acids were decreased by 35 in correlation with a significant (-19%, P < 0.05) improvement in fasting glycaemia (r = 0.801, P < 0.005). These results suggest that Bz acts both on lipolysis and on removal of CM remnants, but that removal can become saturated when lipolysis is massively improved.

Fatty acid composition of an oral load affects chylomicron size in human subjects.

HDL-phospholipids are determinants in reverse cholesterol transport. They are mostly derived from triacylglycerol (TG)-rich lipoproteins. Chylomicron size is important, therefore, because it is related to the ratio surface phospholipids: core TG and, thus, determines the availability of postprandial phospholipids for transfer to HDL. Eleven healthy young women each ingested four different fat loads supplemented with retinyl palmitate and containing 60 g sunflower oil (SO), oleic-sunflower oil (OSO), mixed oil (MO; (g/kg) linoleic acid 480, oleic acid 380, linolenic acid 13) or beef tallow (BT). At the peak of TG absorption for all loads (4 h) chylomicron diameters, determined by agarose-gel filtration, were larger after SO compared with OSO (P < 0.05) and BT (P = 0.06) and after MO compared with BT (P < 0.05). At 6 h chylomicron size was larger after the vegetable oils compared with BT (P < 0.05 in each case). After each fat load chylomicron size decreased at 6 and 8 h compared with that at 4 h (P < 0.05) except for OSO. Retinyl ester and TG concentrations were lower in chylomicrons after BT than after the other fats but not in the chylomicron-free serum (containing chylomicron remnants), suggesting absorption in the form of very small particles. Compared with the fasting value, the concentration of the Svedberg unit of flotation 20-400 fraction, which contains VLDL and chylomicron remnants, was lower 8 h after MO, the only fat to contain significant amounts of linolenic acid. We conclude that chylomicron size is dependent on the fatty acid composition of ingested fats and the time-course of digestion, being larger for polyunsaturated fatty acid-rich fats and in the early phase of digestion. On the basis of retinyl ester concentration there were no differences between fats in chylomicron-remnant clearance.

High-density lipoprotein 3 physicochemical modifications induced by interaction with human polymorphonuclear leucocytes affect their ability to remove cholesterol from cells.

1. We have recently reported that a short incubation (60 min) in vitro of high-density lipoprotein (HDL) 3 with human polymorphonuclear leucocytes (PMNs) leads to a proteolytic cleavage of apolipoprotein (apo) AII and to a change in the distribution of apo AI isoforms [Cogny, Paul, Atger, Soni and Moatti (1994) Eur. J. Biochem. 222, 965-973]. Since PMNs have been observed to be present in the earliest atherosclerotic lesions for a number of days, we investigated the HDL3 physiochemical modifications induced by in vitro interaction for a long period of time (24 h) with PMNs and the consequences of the changes on the ability of HDL3 to remove cholesterol from cells. 2. The stimulated PMN modification of HDL3 over 24 h resulted in a partial loss of protein with no variation in lipid molar ratio and a loss of 50% of HDL alpha-tocopherol content. The decrease in total protein was due first to a complete degradation of apo AII, and secondly to a partial loss of apo AI. The apo AI remaining on the particles was in part hydrolysed and the apo AI-1 isoform was completely shifted to the apo AI-2 isoform. These apo changes were accompanied by a displacement of the native HDL3 apparent size toward predominantly larger particles. 3. The ability of PMN-modified HDL3 to remove 3H-labelled free cholesterol from cells was measured in two cell lines: Fu5AH rat hepatoma cells and J774 mouse macrophages. HDL3 which had only a limited contact with PMNs (60 min) showed only a small non-significant reduction in the efficiency of cholesterol efflux. On the other hand, compared with native HDL3, HDL3 modified by PMNs for 24 h had a markedly reduced ability to remove cholesterol from cells, regardless of the type of cell. 4. The results suggest that PMN-modified HDL3, if occurring in vivo, could contribute to acceleration of the atherogenic process by decreasing the cholesterol efflux from cells.

Modulation of low density lipoprotein subclasses by alimentary lipemia in control and normotriglyceridemic non-insulin-dependent diabetic subjects.

Conventional factors do not fully account for the increased cardiovascular risk in NIDDM but, because of the underlying disorders in lipid metabolism, the postprandial state can be expected to induce temporary changes of a potentially atherogenic nature. The response to a 1000-kcal meal (70% lipid; 100,000 IU vitamin A) over 8 h was compared in 10 normoponderal, normotriglyceridemic NIDDM male patients and 12 controls. In patients lipolysis was normal, but remnant clearance was delayed (P < 0.02) and apo E concentrations were lower. LDL-C decreased postprandially, more in patients (P < 0.05), while LDL-PL accumulated in controls but not in patients. As a result UC:PL decreased in controls (P < 0.05) not in patients. The distribution of LDL subclasses shifted towards large particles in controls (LDL-I, 42%; LDL-II, 50%; LDL-III, 7.6% at 6 h) and smaller ones in patients (LDL-I, 29%; LDL-II, 56%; LDL-III, 16% at 6 h). In controls only, the percentage of LDL-III correlated negatively with apo E (r = -0.97, P < 0.001) suggesting that apo E promotes removal of light particles before they reach LDL-III and may be a limiting factor in patients. We conclude that the postprandial state is potentially more atherogenic in normoponderal, normotriglyceridemic patients than in controls: remnant clearance is delayed, the UC:PL ratio of LDL fails to decrease postprandially as it does in controls, limiting the acceptor capacity of LDL for UC, and the distribution of LDL subclasses is shifted towards a more atherogenic profile.

Regulatory effects of galactose on galactose-1-phosphate uridyltransferase activity on human hepatoblastoma HepG2 cells.

Galactose-1-phosphate uridyltransferase (GALT) deficiency results in galactosemia in man. We have studied the regulation of the GALT gene expression on the HepG2 cell line by growing the cells in glucose or galactose medium. No difference of Km values was observed in glucose or galactose media but the Vmax value with galactose was 50% higher than that with glucose. Also in galactose medium, an increased GALT specific activity was detected suggesting the production of more enzyme proteins. Yet, slot dot quantification of GALT mRNA revealed a decreased amount of these transcripts in cells cultured with galactose or inosine while Northern blot analysis revealed the normal 1.4 kb transcript in all culture media used. Finally, IEF gel analysis displayed different isozymic patterns for the GALT enzyme in cells grown in glucose, galactose or inosine media. With glucose-free media, the major band of GALT corresponds to that found in human liver. Altogether, these results suggest that the control of GALT gene expression in HepG2 cells is located at the post-transcriptional level and correlated to the growth rate of the cell.

Metabolic effects of galactose on human HepG2 hepatoblastoma cells.

HepG2 cells were used as a model system to study the effects of galactose overload on the liver, a target organ of galactose toxicity in patients suffering from transferase-deficient galactosemia. In the presence of galactose, HepG2 cell growth was slow and the pattern of gene expression remained characteristic of liver cells (secretion of alpha-fetoprotein [AFP] albumin, and transferrin). Galactose-1-phosphate (Gal-1-P) accumulated, as it does in galactosemic cells, but did not affect the energetic status of the cells (no adenosine triphosphate [ATP] depletion). However, the substitution of galactose for glucose as the sole hexose in the medium affected the specific activities of the galactose-metabolizing enzymes. Galactokinase (GALK) activity was decreased, and those of galactose-1-phosphate uridyltransferase (GALT), phosphoglucomutase, and glucose-6-phosphate dehydrogenase (G6PDH) were increased. The conversion of radiolabeled galactose to glucose (CO2 production and glycogen level) was greater in galactose medium than in glucose medium after a 7-day culture. Therefore, the culture of HepG2 cells in galactose medium indicates that the enhanced utilization of this hexose is due to the increased enzyme activities regulating its own metabolism. Hence, HepG2 cells constitute a good model for the study of modulation of galactose-metabolizing enzymes by galactose.

Structural changes of high-density-lipoprotein apolipoproteins following incubation with human polymorphonuclear cells.

Based on the analogy in mechanisms and events between the pathogenesis of atherosclerosis and the inflammatory reaction, we investigated the impact of human polymorphonuclear leukocyte (PMN) degranulation and oxidative process on high-density-lipoprotein (HDL) structure. HDL were incubated (37 degrees C) with PMN at a physiological ratio (370 nmol cholesterol-HDL/ml with 2 x 10(6) PMN/ml) for 15, 30 and 60 min with or without stimulating agent. PMN activation was assessed by measurement of superoxide anion generation and elastase production, which both reached peak concentration at 15 min. HDL apolipoproteins (apo) analysed by immunoblotting after SDS/PAGE and electrofocusing evidenced the following modifications: (a) a slow hydrolysis of apo AII and apo Cs; (b) a rapid hydrolysis of apo E; (c) a change in apo AI isoform distribution with an increase in the most acidic isoform (AI-2) at the expense of a less acidic form (AI-1); (d) a shift of the major apo AII isoform into two more basic forms. In contrast, no quantifiable lipid modification nor lipid oxidation, assessed by thiobarbituric-acid-reactive substances (TBARS) were noted. Despite a lack of variation of TBARS, a decrease in HDL vitamin E content by 80% was observed. Since this decrease was prevented by addition of superoxide dismutase in the medium, we concluded the occurrence of an oxidative process affecting HDL. Experiments with proteolytic inhibitors showed that elastase caused the proteolytic cleavage of apolipoprotein E, AII and Cs. In contrast, apo AI modification might involve both oxidative and proteolytic processes.

[Vitamin E: metabolism and role in atherosclerosis]. / Vitamine E: métabolisme et rôle dans l'athérosclérose.

Vitamin E is the term used for eight naturally occurring fat-soluble nutrients. Alpha-tocopherol predominates in many species and has the highest biological activity. Vitamin E is absorbed via the lymphatic pathway and transported in association with CM. Vitamin E is carried in plasma by lipoproteins. It is secreted by the liver in nascent VLDL with a preferential incorporation of alpha-tocopherol. Most of the plasma vitamin E is in LDL and in HDL. Vitamin E is exchanged readily between lipoproteins: tocopherol in HDL readily transfers to apolipoprotein B-containing lipoproteins (VLDL, LDL), with little return of tocopherol from the apolipoprotein B-containing lipoproteins to HDL. The mechanisms of tissue uptake of vitamin E from the lipoproteins is poorly understood. This uptake may occur during catabolism of triacylglycerol-rich lipoproteins by the activity of lipoprotein lipase, via the LDL receptor or by nonreceptor-mediated uptake. Vitamin E may act to prevent the initiation/progression of spontaneous atherosclerosis. This concept is based on in-vitro data: vitamin E influences the responses of cells (vascular endothelial cells, leukocytes, vascular smooth muscle cells) and the modification of lipoproteins (especially LDL) which, at least in principle, could contribute to the initiation/progression of spontaneous atherosclerosis. In vivo studies are clearly required to establish the extent and mode of vitamin E's antiatherosclerotic impact and, hence, its therapeutic potential.

Lipid peroxidation abnormalities in hemodialyzed patients.

In order to test the existence of a possible oxidative damage during hemodialysis, plasma conjugated dienes (CD), plasma and red blood cell (RBC) thiobarbituric acid (TBA) reactants were investigated in 25 patients receiving regular dialysis treatment (RDT). The RBC TBA reactant concentration was significantly increased in RDT patients in comparison with healthy subjects. The extracellular antioxidant systems were evaluated by the assay of plasma antioxidant activity, plasma tocopherol, urate, transferrin, haptoglobin and ceruloplasmin levels. Except urate and transferrin, none of these parameters were different between the two groups. On the other hand, in RDT patients, RBC superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were significantly lower than in healthy subjects. There was an inverse correlation between decreased RBC GPX and RBC TBA reactant concentration. These results show in RDT patients the existence of an oxidizing stress, mainly intracellular, which could be due, in part, to a decrease in SOD and GPX activities.

Performance evaluation of 2-D adaptive prediction filters for detection of small objects in image data.

This work studies the performance of dimensional least mean square (TDLMS) adaptive filters as prewhitening filters for the detection of small objects in image data. The object of interest is assumed to have a very small spatial spread and is obscured by correlated clutter of much larger spatial extent. The correlated clutter is predicted and subtracted from the input signal, leaving components of the spatially small signal in the residual output. The receiver operating characteristics of a detection system augmented by a TDLMS prewhitening filter are plotted using Monte-Carlo techniques. It is shown that such a detector has better operating characteristics than a conventional matched filter in the presence of correlated clutter. For very low signal-to-background ratios, TDLMS-based detection systems show a considerable reduction in the number of false alarms. The output energy in both the residual and prediction channels of such filters is shown to be dependent on the correlation length of the various components in the input signal. False alarm reduction and detection gains obtained by using this detection scheme on thermal infrared sensor data with known object positions is presented.

Impaired hexose uptake by diploid skin fibroblasts from galactosaemic patients. Connection with cell growth and amino acid metabolism, and possible bearing on late-onset clinical symptoms.

In skin fibroblasts of patients presenting with galactosaemia, either from galactose 1-phosphate uridyltransferase or galactokinase deficiency, a deficit in extracellular glucose utilization was observed. This deficit was constant over 3 weeks of continuous cell growth in a medium containing 5.5 mmol/L glucose as the only hexose, and homologous serum. Levels of glucose utilization by deficient skin fibroblasts were stable at about 65-70% of the glucose utilization of control normal skin fibroblasts. Cell morphology was normal, and cell growth was subnormal during this period. However, the energy provision appeared sufficient for cellular needs since cell growth in this glucose medium was observed not to depend on the presence of extracellular glutamine. In contrast, glutamine was required for growth of galactosaemic fibroblasts cultured in medium containing 5.5 mmol/L galactose. If expressed in many cell types, this impaired glucose uptake would be expected seriously to damage highly glucose-dependent tissues such as the central nervous system. This might be of relevance to the persistent neurological damage observed in many galactosaemic patients in spite of their compliance with an early strict galactose-free diet.

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.