Immunoglobulin G particles manufacturing by spray drying process for pressurised metered dose inhaler formulations.

OBJECTIVE: The objective of this work is to show the feasibility of manufacturing from a spray drying process particles containing immunoglobulin G capable of being administered by inhalation via a pressurized metered dose inhaler. METHODS: Spray drying were made from aqueous solutions containing IgG and two types of excipients, mannitol and trehalose, with two ratios: 25% w/w and 75%w/w. The physicochemical and aerodynamic properties of the powders obtained were characterized just after manufacturing and after 1 month of storage at 40°C/75% RH according to criteria defined as needed to satisfy an inhaled formulation with a pressurized metered dose inhaler. Maintain of the biological activity and the structure of IgG after atomization was also tested by slot blot and circular dichroism. RESULTS: All spray-dried powders presented a median diameter lower than 5µm. The powders atomized with trehalose showed a solid state more stable than those atomized with mannitol. All atomized powders were in the form of wrinkled particles regardless the nature and the ratios of excipients. The results showed that the aerosolisation properties were compliant with the target, independently of the excipient used at a ratio of 25% w/w IgG-excipient. Moreover, the addition of excipient during the atomization process the denaturation of IgG was limited. CONCLUSION: This study showed that the use of trehalose as excipient could satisfy the requirements of an inhaled formulation with a pressurized metered dose inhaler.

On the dimorphism and the pressure-temperature state diagram of racemic m-nisoldipine, a dihydropyridine calcium ion antagonist.

The pressure-temperature phase diagram of the dimorphism of racemic m-nisoldipine is constructed using temperatures and enthalpies of fusion of forms A and B. At ordinary pressure, the transition from form B to form A is found to occur around 192K, which indicates that these polymorphs are enantiotropically related and that form A is stable at room temperature. Nevertheless, the phase relationship turns to be monotropic when pressures become greater than about 100MPa, which indicates that form B becomes the sole stable phase.

The topological pressure-temperature phase diagram and crystal structures of the dimorphic system spiperone.

The topological pressure-temperature phase diagram for the dimorphism of spiperone, a potent neuroleptic drug, has been constructed using literature data and improved crystal structures obtained with new crystallographic data from single-crystal X-ray diffraction at various temperatures. It is inferred that form II, which is the more dense form and exhibits the lower melting temperature, becomes the more stable phase under pressure. Under ambient conditions, form I is more stable.

[Crystallization and structural investigation of supramolecular compounds with modified cyclodextrins. Application to chiral discrimination]. / Cristallisation et analyse structurale de composés supramoléculaires formés avec des cyclodextrines modifiées. Application à la discrimination chirale.

Among the numerous applications of cyclodextrins in separative sciences, organic synthesis, pharmacy and biotechnology, supramolecular host-guest complexation plays a major role due to the macrocyclic geometry of the host and the hydrophobic properties of the inner cavity. A less developed application of native or chemically modified cyclodextrins is their use for the preparative enantioseparation using co-crystallization methods. The intrinsic chirality of cyclodextrins enables the crystallization of diastereomeric compounds when the macrocyclic host interacts with a racemic mixture of the guest molecule. The preferential nucleation of one of these compounds, the amplification of the enantiomeric enrichment during the crystal growth and successive recrystallizations may lead to complete chiral separations. This alternative method of chiral discrimination is illustrated here with permethylated beta-cyclodextrin and (+/-)-1-(4-bromophenyl)ethanol. The results could be partially rationalized thanks to crystal growth studies and through the systematic determination of crystal structures by X-ray diffraction on single crystals. This study has shown that both inclusion geometries and crystal packing features determine the capability of modified cyclodextrins for chiral discrimination by crystallization.

[Resolution of chiral molecules of pharmaceutical interest by means of preferential crystallization]. / Dédoublement de molécules pharmaceutiques chirales par cristallisation préférentielle.

SUMMARY: Various aspects of the chiral discrimination in the solid state are examined. The interests of the conglomerate are illustrated by two applications: the preparative enantiomeric purification and the preferential crystallization. The latter process is described by a careful examination of the heterogeneous equilibria that govern the crystallization and its selectivity. Two variants of the preferential crystallization are detailed. A "good" example illustrates the productivity at the laboratory scale. The ratio between homochiral interaction energies and heterochiral interaction energies at different (hkl) interfaces are involved in the "difficult" cases where the entrainment effect is limited.

Influence of ageing, grinding and preheating on the thermal behaviour of alpha-lactose monohydrate.

It is shown that the onset temperature and the magnitude of thermal events observed during DSC analyses of alpha-lactose monohydrate can be strongly affected by various treatments such as ageing, manual grinding and preheating (cycle of preliminary dehydration and rehydration). In the case of grinding and preheating, the change of dehydration pathways was further investigated by using a suitable combination of characterization techniques, including X-ray powder diffraction (XRPD) performed with a synchrotron source (allowing an accurate Rietveld analysis), scanning electron microscopy (SEM), laser particle size measurements, FTIR spectroscopy and (1)H NMR for the determination of beta-lactose contents in samples. It appeared that the dehydration mechanism is affected not only by a smaller particle size distribution, but also by residual anisotropic lattice distortions and by the formation of surface defects or high energy surfaces. The fusion-recrystallization process occurring between anhydrous forms of alpha-lactose at ca. 170 degrees C is not significantly affected by grinding, whereas a preheating treatment induces an unexpected large increase of the enthalpy associated with this transition. Our observations and interpretations confirm the important role of water molecules in the crystal cohesion of the title compound and illustrate the necessity to consider the history of each sample for a satisfactory understanding of the physical properties and the behaviour of this important pharmaceutical excipient.

Inhibition of the Vapor-Mediated Phase Transition of the High Temperature Form of Pyrazinamide.

Tailor-made additives can prove an effective method to prolong the lifetime of metastable forms of pharmaceutical compounds by surface stabilization. Pyrazinamide (PZA) is a pharmaceutical compound with four polymorphic forms. The high temperature γ form, which can be produced by spray drying or sublimation growth, is metastable at room temperature and transforms within days when produced by spray drying, and within several months up to years for single crystals produced by sublimation. However, when PZA is cospray dried with 1,3-dimethylurea (DMU), it has been reported to remain in its γ form for several years. Scanning electron microscopy (SEM) images reveal that the phase transition from γ-PZA to the low temperature forms involves a vapor-mediated recrystallization, while the reverse phase transition upon heating is a nucleation-and-growth solid-solid phase transition. The lifetime-extending effect of DMU on spray-dried PZA has been investigated in more detail and compared with high-energy ball milling of sublimation-grown γ-PZA crystals. Co-ball milling of PZA and DMU is found to extend the lifetime of the high temperature form of PZA to a few months, while separate ball milling leads to an extension of merely a few weeks. DMU acts as an additive that most likely stabilizes the surface of γ-PZA, which would reduce the vapor pressure of PZA, thereby reducing the transition rate. Alternatively, DMU could prevent nucleation of low temperature forms of PZA.

Modeling of anti-nucleosome immunoglobulin Fv domains: analysis of electrostatic interactions.

Three-dimensional structural models of six murine anti-(H2A-H2B) monoclonal autoantibody variable fragments were built by comparative molecular modeling using the COMPOSER software. Analysis of the antibody combining sites is based on the hypothesis that ionic and/or electrostatic interactions predominate in antigen antibody binding, as suggested by the cationic nature of histones and the amino acid sequences of the antibody hypervariable regions. The study of the electrostatic potentials of their combining site surfaces, computed with the MOLCAD software, and the comparison with the electrostatic potentials of 13 selected control mAbs show the lack of a unique electrostatic pattern. One group of three mAbs expresses a strong and large electronegative area, supporting the hypothesis that ionic interactions predominate in antigen recognition. The second group, containing the other three mAbs, exhibits an alternation of electropositive and electronegative areas. All, however, present a localized electronegative area in the vicinity of H-CDR1 and H-CDR2 loops that is generated by the presence of at least one acidic residue. The model suggesting that the binding activity may depend on charged residues at the same site is reminiscent of what was previously reported in anti-DNA mAbs. In addition, the alternation of electropositive areas and electronegative areas in second group mAbs is also frequently observed in certain anti-DNA mAbs. These data argue for the existence of relationships between these two autoantibody populations and suggest that they share a common immunogenic particle formed by anionic and cationic components, such as a nucleosome.

Inhibition of the spontaneous polymorphic transition of pyrazinamide γ form at room temperature by co-spray drying with 1,3-dimethylurea.

The present study focuses on the ability of excipients to induce the crystallization of a specific polymorphic form of pyrazinamide (PZA) and more interestingly, to block the irreversible solid-solid transition of the metastable forms of the PZA to the stable form at room temperature. We outline an experimental protocol for the production of a structurally pure γ form of PZA by means of spray drying. Without any particular treatment, phase transition to δ form was detected after 14 days of storage under ambient conditions. In order to prevent this irreversible phase transition, different excipients were co-spray dried with PZA. By co-spray drying 5% in mass of 1,3-dimethylurea (DMU) with PZA, we noticed its ability in preventing phase transitions and thus to maintain PZA under its γ form up to 12 months of storage at room temperature. Raman spectroscopy evidenced how DMU crystals surround particles of γ PZA which suggest that DMU might interact with the surface of PZA particles, thus blocking the phase transition. On the other hand, the co-spray drying of PZA with the polymerpolyvinylpyrrolidone (PVP) resulted in the crystallization of δ form of PZA. The physical mixture was intact over 12 months of storage at room temperature.

[Recurrent familial neuropathy due to compression (author's transl)]. / Neuropathie récurrente par compression. Etude d'une famille de 13 membres.

The authors describe a family, of which 13 members had peripheral neuropathy manifesting as episodes of paralysies usually provoked by minor compression of nerve trunks. Nerve and muscle biopsies in two patients, demonstrated many thickenings of the myelin sheath ("tomacula"). Electron microscopy investigations are used as a basis for discussing the origin and place of this affection among other hereditary neuropathies.

Biomimetic layer-by-layer templates for calcium phosphate biomineralization.

Carboxylated, sulfated and/or phosphorylated surfaces are admitted as potential optimal templates for biomimetic deposition of calcium phosphate (CaP) coatings in view of improving implants' osseointegration. Layer-by-layer films were built up consisting of anionic chondroitin sulfate (ChS), a biological carboxylated and sulfated polysaccharide and cationic poly(l-lysine) (PLL). The films were used as soft matrices to immobilize a model phosphoprotein, phosvitin (PhV). The respective roles of ChS, PLL and PhV terminal layers on the heterogeneous nucleation kinetics and the structure of CaP deposits obtained from supersaturated solutions were inspected. Critical supersaturation ratios and induction times preceding heterogeneous nucleation were precisely determined and interpreted within the framework of classical nucleation theory in order to derive the effective interfacial energies of CaP crystals. It was found that the potency of terminal layers toward CaP nucleation increased in the order: PLL

Review on the heterogeneous equilibria between condensed phases in binary systems of enantiomers.

In contrast to what is often expressed in the literature, reversible and irreversible solid(s)-solid(s) transitions in binary system of non-racemizable enantiomers are not always referred to as polymorphism. Order-disorder phenomena, miscibility gaps with critical temperatures and transitions in which three-phases or even four-phases are involved can also occur. In the case of reversibility, three-phase invariants such as eutectoid or peritectoid corresponding to the reversible formation or decomposition of racemic compounds are described. The heterogeneous equilibria are reviewed with or without miscibility in the solid state. Several four-phase invariants and order-disorder transitions are predicted.

Structural models of antibody variable fragments: a method for investigating binding mechanisms.

The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that antinucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nucleosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

Chiral recognition of binaphthyl derivatives: a chiral recognition model on the basis of chromatography, spectroscopy, and molecular mechanistic calculations for the enantioseparation of 1,1'-binaphthyl derivatives on cholic acid-bonded stationary phases.

In an effort to elucidate the mechanism of chiral discrimination of cholic acid-based stationary phases, the enantiomeric recognition ability of six chiral stationary phases (CSPs), prepared from differently substituted cholic acid derivatives, was evaluated in normal phase high-performance liquid chromatography (HPLC) with a series of 1,1'-binaphthyl compounds. The influence of structural variations of analytes on retention and enantioselectivity was investigated. Particularly high values of enantioselectivity were observed for the binaphthol enantiomers on a CSP prepared from the allyl 7 alpha,12 alpha-dihydroxy-3 alpha-phenylcarbamoyloxy-5 beta-cholan-24-oate. The complexes of this chiral selector with both enantiomers of binaphthol were studied as models for the interactions responsible for the enantioseparation with the cholic acid-based stationary phases. The 1:1 stoichiometry of the complex in solution was determined by UV titration. The chiral selector dissolved in chloroform exhibited a chiral discrimination for the binaphthol in (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopies. Some aromatic proton and carbon resonances of binaphthol were clearly separated into a pair of peaks due to enantiomers in the presence of the chiral selector. Moreover, on the basis of molecular mechanics calculation, a chiral discrimination model was proposed which nicely explains the relevant chromatographic behavior of the 1,1'-binaphthyl derivatives.