Use of the channel fill level in defining a design space for twin screw wet granulation.

Twin screw wet granulation is a key process in the continuous manufacture of oral solid dosage forms. Previous research has qualitatively suggested that the channel fill level influences the granules produced. In this paper a quantitative measure of the total volumetric fraction of the conveying element channels of the screw filled with powder (φ) was used. Experimental results are shown which demonstrate that very similar particle size distributions can be obtained at the same φ with the same material and screw configuration but radically different solids feed rates and screw speeds. Morphology of the granules also correlates with φ. This is consistent with previous observations in the literature correlating granule attributes with powder feed rate and screw speed but also considers the two parameters in combination. A process design space approach based on φ is proposed. This can be determined empirically, and potentially has value in setting process control strategies, assuring process robustness and allowing process flexibility during the product lifecycle.

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin.

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.

Effect of the antibiotic azithromycin on thermotropic behavior of DOPC or DPPC bilayers.

Azithromycin is a macrolide antibiotic known to bind to lipids and to affect endocytosis probably by interacting with lipid membranes [Tyteca, D., Schanck, A., Dufrene, Y.F., Deleu, M., Courtoy, P.J., Tulkens, P.M., Mingeot-Leclercq, M.P., 2003. The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages. J. Membr. Biol. 192, 203-215]. In this work, we investigate the effect of azithromycin on lipid model membranes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Thermal transitions of both lipids in contact with azithromycin are studied by (31)P NMR and DSC on multilamellar vesicles. Concerning the DPPC, azithromycin induces a suppression of the pretransition whereas a phase separation between the DOPC and the antibiotic is observed. For both lipids, the enthalpy associated with the phase transition is strongly decreased with azithromycin. Such effects may be due to an increase of the available space between hydrophobic chains after insertion of azithromycin in lipids. The findings provide a molecular insight of the phase merging of DPPC gel in DOPC fluid matrix induced by azithromycin [Berquand, A., Mingeot-Leclercq, M.P., Dufrene, Y.F., 2004. Real-time imaging of drug-membrane interactions by atomic force microscopy. Biochim. Biophys. Acta 1664, 198-205] and could help to a better understanding of azithromycin-cell interaction.

Interaction of the macrolide antibiotic azithromycin with lipid bilayers: effect on membrane organization, fluidity, and permeability.

PURPOSE: To investigate the effect of a macrolide antibiotic, azithromycin, on the molecular organization of DPPC:DOPC, DPPE:DOPC, SM:DOPC, and SM:Chol:DOPC lipid vesicles as well as the effect of azithromycin on membrane fluidity and permeability. METHODS: The molecular organization of model membranes was characterized by atomic force microscopy (AFM), and the amount of azithromycin bound to lipid membranes was determined by equilibrium dialysis. The membrane fluidity and permeability were analyzed using fluorescence polarization studies and release of calcein-entrapped liposomes, respectively. RESULTS: In situ AFM images revealed that azithromycin leads to the erosion and disappearance of DPPC and DPPE gel domains, whereas no effect was noted on SM and SM:cholesterol domains. Although azithromycin did not alter the permeability of DPPC:DOPC, DPPE:DOPC, SM:DOPC, and SM:Chol:DOPC lipid vesicles, it increased the fluidity at the hydrophilic/hydrophobic interface in DPPC:DOPC and DPPE:DOPC models. This effect may be responsible for the ability of azithromycin to erode the DPPC and DPPE gel domains, as observed by AFM. CONCLUSIONS: This study shows the interest of both AFM and biophysical methods to characterize the drug-membrane interactions.

Rheology of giant vesicles: a micropipette study.

We develop a micropipette rheometer to study the effect of oscillatory shear flow on the spontaneous fluctuations of phospholipid bilayers. Our results on giant vesicles show that oscillatory shear flow leads to a suppression of membrane fluctuations. They also imply that the Helfrich equation is modified in the presence of the flow. This equation, a fundamental constitutive relation between the amount of area stored in the fluctuations and the membrane tension, must be supplemented under oscillatory shear by a flow excess function that we determine.

Spectrum of beta thalassemia mutations and their linkage to beta-globin gene haplotypes in the Indo-Mauritians.

The beta thalassemia alleles in 53 thalassemic Indo-Mauritian patients and their families consisting of 23 homozygous beta-thalassemia, 9 HbE/beta-thalassemia, 18 HbS/beta-thalassemia, 1 HbD/beta-thalassemia, 1 deltabeta/beta-thalassemia and 1 HbH/beta-thalassemia from the island of Mauritius were studied. Characterization by polymerase chain reaction-based reverse dot blot hybridization technique revealed that the IVS1-5 (G-->C) mutation accounted for 74% of the beta thalassemic alleles, while six other mutations occurred at much lower frequencies: HbE codon 26 (G-->A); 10.4%, codon 8/9 (+G); 3.5%, codon 30 (AGG-->ACG) also called IVSI (-1).G-->C; 3.5%, codon 15 (G-->A); 3.5%, codon 41/42 (-CTTT); 2.4% and -28 (A-->G); 2.4%. Association of these mutations to specific beta globin gene sequence framework and haplotype allowed to trace their ancestral link. These data are useful in future molecular screening of the population in view of implementing a thalassemia prevention and control program in Mauritius.

Three major G6PD-deficient polymorphic variants identified among the Mauritian population.

We report the results of the first epidemiological study investigating glucose 6-phosphate dehydrogenase (G6PD) deficiency among the heterogenous Mauritian population. Mauritius has a population of approximately 1 million, and of these 66.8% are Indo-Mauritian (of Indian origin), 27.9% are Creoles (of African ancestry) and 2.1% are Sino-Mauritian, predominantly of Chinese origin. Of the 1435 Mauritian males tested, 73 (5.1%) were G6PD deficient. However, the prevalence varied considerably between the two major ethnic groups: 35/1157 (3.0%) for Indo-Mauritians and 37/267 (13.9%) for Creoles. Molecular analysis revealed three major deficient polymorphic variants; G6PD Orissa, G6PD Mediterranean and G6PD A-. G6PD Orissa (nt 131 G-->C; residue 44 Ala-->Gly) was found to be the most common variant among Indo-Mauritians: this deficient variant was recently identified to be highly characteristic of the tribal groups in central India. In Creoles the most common deficient variant was G6PD A- (27/37). These data are consistent with the different ancestral contributions to the present gene pool of the Mauritian population. This study has provided further information as to the precise nature of G6PD deficiency at the molecular level among Indians, about whom previously there was scant information. The data presented suggest that G6PD Orissa is widespread in central and southern states of India. Additionally, the identification and frequency of G6PD-deficient alleles in Mauritius is of public-health importance.