The use of 3-dimensional imaging of Holstein cows to estimate body weight and monitor the composition of body weight change throughout lactation.

Three-dimensional (3D) imaging offers new possibilities in animal phenotyping. Here, we investigated how this technology can be used to study the morphological changes that occur in dairy cows over the course of a single lactation. First, we estimated the individual body weight (BW) of dairy cows using traits measured with 3D images. To improve the quality of prediction, we monitored body growth (via 3D imaging), gut fill (via individual dry matter intake), and body reserves (via body condition score) throughout lactation. A group of 16 Holstein cows-8 in their first lactation, 4 in their second lactation, and 4 in their third or higher lactation-was scanned in 3D once a month for an entire lactation. Values of morphological traits (e.g., chest depth or hip width) increased continuously with parity, but cows in their first lactation experienced the largest increase during the monitoring period. Values of partial volume, estimated from point of shoulder to pin bone, predicted BW with an error of 25.4 kg (R2 = 0.92), which was reduced to 14.3 kg when the individual effect of cows was added to the estimation model. The model was further improved by the addition of partial surface area (from point of shoulder to pin bone), hip width, chest depth, diagonal length, and heart girth, which increased the R2 of BW prediction to 0.94 and decreased root mean square error to 22.1 kg. The different slopes for individual cows were partly explained by body condition score and morphological traits, indicating that they may have reflected differences in body density among animals. Changes in BW over the course of lactation were mostly due to changes in growth, which accounted for around two-thirds of BW gain regardless of parity. Body reserves and gut fill had smaller but still notable effects on body composition, with a higher gain in body reserves and gut fill for cows in their first lactation compared with multiparous cows. This work demonstrated the potential for rapid and low-cost 3D imaging to facilitate the monitoring of several traits of high interest in dairy livestock farming.

Rear shape in 3 dimensions summarized by principal component analysis is a good predictor of body condition score in Holstein dairy cows.

Body condition is an indirect estimation of the level of body reserves, and its variation reflects cumulative variation in energy balance. It interacts with reproductive and health performance, which are important to consider in dairy production but not easy to monitor. The commonly used body condition score (BCS) is time consuming, subjective, and not very sensitive. The aim was therefore to develop and validate a method assessing BCS with 3-dimensional (3D) surfaces of the cow's rear. A camera captured 3D shapes 2 m from the floor in a weigh station at the milking parlor exit. The BCS was scored by 3 experts on the same day as 3D imaging. Four anatomical landmarks had to be identified manually on each 3D surface to define a space centered on the cow's rear. A set of 57 3D surfaces from 56 Holstein dairy cows was selected to cover a large BCS range (from 0.5 to 4.75 on a 0 to 5 scale) to calibrate 3D surfaces on BCS. After performing a principal component analysis on this data set, multiple linear regression was fitted on the coordinates of these surfaces in the principal components' space to assess BCS. The validation was performed on 2 external data sets: one with cows used for calibration, but at a different lactation stage, and one with cows not used for calibration. Additionally, 6 cows were scanned once and their surfaces processed 8 times each for repeatability and then these cows were scanned 8 times each the same day for reproducibility. The selected model showed perfect calibration and a good but weaker validation (root mean square error=0.31 for the data set with cows used for calibration; 0.32 for the data set with cows not used for calibration). Assessing BCS with 3D surfaces was 3 times more repeatable (standard error=0.075 versus 0.210 for BCS) and 2.8 times more reproducible than manually scored BCS (standard error=0.103 versus 0.280 for BCS). The prediction error was similar for both validation data sets, indicating that the method is not less efficient for cows not used for calibration. The major part of reproducibility error incorporates repeatability error. An automation of the anatomical landmarks identification is required, first to allow broadband measures of body condition and second to improve repeatability and consequently reproducibility. Assessing BCS using 3D imaging coupled with principal component analysis appears to be a very promising means of improving precision and feasibility of this trait measurement.

Sparing methylation of beta-cyclodextrin mitigates cytotoxicity and permeability induction in respiratory epithelial cell layers in vitro.

Cyclodextrins (CDs) are promising solubility enhancers for inhaled drug delivery. However, they have dose-dependent effects on the respiratory epithelium, which may have advantages for permeability enhancement but also gives rise to safety concerns. In this study, the methyl thiazol tetrazolium (MTT) assay was used to compare a new sparingly methylated beta-CD, Kleptose Crysmebeta (Crysmeb) with the more established CD derivatives hydroxypropyl-gamma-cyclodextrin (HPgammaCD), randomly methylated beta-cyclodextrin (Rameb) and hydroxypropyl-beta-cyclodextrin (HPbetaCD). The betaCD derivatives affected cell metabolism in A549 cells in a concentration dependent manner with LD(50) of 56, 31 and 11 mM obtained for HPbetaCD, Crysmeb and Rameb, respectively. Calu-3 cells were less susceptible to betaCD with an LD(50) of 25 mM being obtained for Rameb only. Permeability increases in Calu-3 cell layers were observed with betaCD derivatives and a concentration dependency shown. The mechanism of permeability enhancement and its reversibility was investigated. Rameb produced an irreversible loss of cell layer barrier function at > or = 25 mM, but perturbations of epithelial integrity were moderate and reversible in the case of HPbetaCD and Crysmeb (25-50 mM). Given its high solubilisation capacity, the low toxicity and transient absorption promoting properties, this study identifies Crysmeb as a promising adjuvant in formulations for inhalation.

Raman spectroscopy as a process analytical technology (PAT) tool for the in-line monitoring and understanding of a powder blending process.

The aim of this study is to propose a strategy to implement a PAT system in the blending step of pharmaceutical production processes. It was examined whether Raman spectroscopy can be used as PAT tool for the in-line and real-time endpoint monitoring and understanding of a powder blending process. A screening design was used to identify and understand the significant effects of two process variables (blending speed and loading of the blender) and of a formulation variable (concentration of active pharmaceutical ingredient (API): diltiazem hydrochloride) upon the required blending time (response variable). Interactions between the variables were investigated as well. A Soft Independent Modelling of Class Analogy (SIMCA) model was developed to determine the homogeneity of the blends in-line and real-time using Raman spectroscopy in combination with a fiber optical immersion probe. One blending experiment was monitored using Raman and NIR spectroscopy simultaneously. This was done to verify whether two independent monitoring tools can confirm each other's endpoint conclusions. The analysis of the experimental design results showed that the measured endpoints were excessively rounded due to the large measurement intervals relative to the first blending times. This resulted in effects and critical effects which cannot be interpreted properly. To be able to study the effects properly, the ratio between the blending times and the measurement intervals should be sufficiently high. In this study, it anyway was demonstrated that Raman spectroscopy is a suitable PAT tool for the endpoint control of a powder blending process. Raman spectroscopy not only allowed in-line and real-time monitoring of the blend homogeneity, but also helped to understand the process better in combination with experimental design. Furthermore, the correctness of the Raman endpoint conclusions was demonstrated for one process by using a second independent endpoint monitoring tool (NIR spectroscopy). Hence, the use of two independent techniques for the control of one response variable not only means a mutual confirmation of both methods, but also provides a higher certainty in the determined endpoint.

[The galenic pharmacy in the development of new drugs]. / La pharmacie galénique dans le développement de nouveaux médicaments.

After having regarded the drug dosage form as a simple presentation of the active substance, the pharmaceutical world is convinced that the drug substance activity is not only controlled by its molecular structure but also by the galenical formulation as well as the manufacturing methods of the drug dosage forms. The galenical research in universities and in pharmaceutical companies gave rise to a new generation of dosage forms called "drug delivery systems" which have often taken advantage of the potential of new administration routes and have definitely found an important place in both human and veterinary therapeutics.

Validation of manufacturing process of Diltiazem HCl tablets by NIR spectrophotometry (NIRS).

The goal of this study was to apply the Process Analytical Technology FDA's initiative in pharmaceutical tablets manufacturing. Near Infrared Spectrophotometry (NIRS) was used as a non-destructive, very fast technique requiring no sample preparation. Direct compression powder blends containing Diltiazem HCl as a model drug were pressed into tablets for the calibration and the validation steps. First, a partial least squares model was built to calibrate the NIR spectrometer. Then, this model was validated and compared with a validated UV spectrophotometry reference method. For this comparison, the Bland and Altman's statistical method was applied. The manufacturing process was validated by producing three batches at three different concentration levels. The NIR analysis of these batches was performed during 3 days. This study shows that NIRS can be used to validate the whole manufacturing process and not only as an analytical method for tablets assay. NIRS is an interesting tool to show possible variations during the manufacturing process which could lead the finished product to fall outside of specifications.

Study of the relationship between lipid binding properties of cyclodextrins and their effect on the integrity of liposomes.

It is well known that cyclodextrins are able to extract lipids constituting membranes, increasing their fluidity and permeability. This behaviour towards biological membranes is directly linked to the toxicological effects of methylated cyclodextrins. However, confusion is currently made in the literature between the different methylated cyclodextrin derivatives. Moreover, a new methylated cyclodextrin derivative recently occurred in the market, the Crysmeb. We wanted to compare and understand the effect of the most currently used cyclodextrins on a model membrane. We studied the influence of natural cyclodextrins (betaCD and gammaCD), methylated derivatives (2,6-dimethyl-betaCD (Dimeb), 2,3,6-trimethyl-betaCD (Trimeb) and randomly methylated-betaCD (Rameb), as well as the new derivative Crysmeb), hydroxypropylated derivatives (HPbetaCD of different substitution degrees and HPgammaCD) and the sulfobutylated derivative (SBEbetaCD) on the release of a fluorescent marker encapsulated in the inner cavity of liposomes. It was shown that the observed effect on calcein release can be directly related to the affinity of cyclodextrins for both lipid components of liposomes, cholesterol and phosphatidylcholine. From this relationship, we were able to determine, for each cyclodextrin, a theoretical concentration giving rise to 50% or 100% calcein release. This theoretical concentration was confirmed experimentally. We have also showed that cyclodextrins which provoke calcein release also induce large structure modifications of liposomes.

[Pharmaceutical applications of supercritical carbon dioxide]. / Applications pharmaceutiques du dioxyde de carbone supercritique.

The supercritical state of a fluid is intermediate between that of gases and liquids. Supercritical fluids exhibit some solvent power which is tunable in function of pressure and temperature. In the pharmaceutical field, supercritical carbon dioxide is by far the most commonly used fluid; of course, the first applications of supercritical fluids were the replacement of organic solvents in extraction processes; other applications appeared during the last twenty years: supercritical fluids are also used as eluents in chromatography, as solvents in organic synthesis or for the processing of solid dosage forms by drug micronization, by the production of nanospheres, of solid dispersions, of porous polymeric matrices containing different active substances. Supercritical carbon dioxide has been proposed for encapsulating both hydrophilic and hydrophobic drug substances into liposomes as well as for including different active substances into cyclodextrins. There are also future prospects for the use of pressurized carbon dioxide as a sterilizing agent.

Study of the physicochemical properties in aqueous medium and molecular modeling of tagitinin C/cyclodextrin complexes.

The inclusion complexes of tagitinin C with beta-, 2,6-di-O-methyl-beta- and gamma-cyclodextrin (CyD) was investigated in aqueous medium. The stoichiometric ratios and stability constants (K(f)) which describe the extent of formation of the complexes have been determined by UV spectroscopy and direct current tast polarography (DC(tast)), respectively. For each complex, a 1:1 molar ratio was formed in solution and the trend of stability constants was K(f) (2,6-di-O-methyl-beta-CyD)>K(f) (gamma-CyD)>K(f) (beta-CyD). The effect of molecular encapsulation on the photochemical conversion of tagitinin C was evaluated. No significant protection efficacy was noticed with beta- and gamma-CyD for the complexed drug with the respect to the free one. On the other hand, the photochemical conversion rate was slowed in presence of 2,6-di-O-methyl-beta-CyD. Data from (1)H NMR and ROESY experiments provided a clear evidence of formation of inclusion complexes. The lactone, the ester and the unsaturated ketone parts of tagitinin C inserted into the wide rim of the CyDs torus. These experimental results were confirmed by the molecular modeling using semiempirical Austin Model 1 (AM1) method.

[Generic medications and prescription by non-proprietary name]. / Le point sur les médicaments génériques et sur la prescription sous dénomination commune.

Complementarily to a previous publication, this paper tries to answer a few commonly asked questions about--generic medications: pharmaceutical quality, bioequivalence and therapeutic equivalence, substitution, etc. A summary of recent regulations about reference price and prescription by non-proprietary name, aiming at the promotion of generic medication prescription is also presented.

Comparison of FT-NIR transmission and UV-vis spectrophotometry to follow the mixing kinetics and to assay low-dose tablets containing riboflavin.

For several years, near-infrared spectroscopy (NIRS) has become an analytical technique of great interest for the pharmaceutical industry, particularly for the non-destructive analysis of dosage forms. The goal of this study is to show the capacity of this new technique to assay the active ingredient in low-dosage tablets. NIR spectroscopy is a rapid, non-destructive technique and does not need any sample preparation. As an example, a binary mixture of microcrystalline cellulose and riboflavin was used to prepare tablets of different weights by direct compression. A prediction model was built by using a partial least square regression fit method. The NIR assay was performed by transmission. The results obtained by NIR spectroscopy were compared with a conventional UV-vis spectrophotometry method. The study showed that tablets can be individually analysed by NIR with high accuracy. It was shown that the variability of this new technique is less important than that of the conventional method which is the UV-vis spectrophotometry.

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography.

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred microl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively.

Cyclodextrins as a potential carrier in drug nebulization.

The inhalation route is widely studied for many drug applications focusing on either local or systemic distributions. One matter of concern is the solubilization of hydrophobic drugs. We have studied the feasibility of using different cyclodextrins (CDs) to elaborate pharmaceutical formulations for the inhalation route and tested the short-term toxicity of such formulations administered by inhalation to C57BL/6 mice. We have shown that HP-beta-CD, gamma-CD, as well as RAMEB aqueous solutions can undergo aerosolization and that the resulting droplet-size ranges are compatible with pulmonary deposition. In vivo, we have demonstrated that short-term exposure to inhaled HP-beta-CD, gamma-CD and RAMEB solutions are non-toxic after assessing bronchoalveolar lavage (BAL), lung and kidney histology, bronchial responsiveness to methacholine and blood urea. The only change noted is a slight increase in lymphocyte count in the BAL after HP-beta-CD and gamma-CD inhalation. We conclude that CDs are useful in significantly enhancing the solubility of apolar drugs with a view to inhalation therapy although an increase in lymphocyte counts in the BAL after CDs inhalations needs further investigations.

Skin compatibility of cyclodextrins and their derivatives: a comparative assessment using a corneoxenometry bioassay.

Few studies have been performed to assess the risk of skin damage by cyclodextrins (CD) and they have yielded contradictory results. The present study was conducted using the corneoxenometry bioassay on human stratum corneum to compare the skin compatibility of CD currently used in pharmaceutical preparations (betaCD, gammaCD, Rameb, Dimeb, Trimeb, HP-betaCD and HP-gammaCD) and that of new amphiphilic CD derivatives, namely, the phospholipidyl-CD (DMPE-Dimeb and DMPE-Trimeb). All the tested CD were well tolerated by the stratum corneum at a concentration of 5%. However, inter-individual reactivity was larger for DMPE-Dimeb, suggesting a more aggressive trend for this compound. Cutaneous Index of Mildness values obtained confirm that Dimeb is able to extract some skin components and shows that DMPE-Dimeb performs similarly.

Systematic characterization of oil-in-water emulsions for formulation design.

Oil-in-water emulsions varying in surfactant concentration and manufacturing process were prepared. About 10 experiments were performed to characterize them. The goal of this research was to find out which tests should systematically be carried out to assess efficiently the stability and the properties of an emulsified preparation. Thus, formulation design requires at least the measurement of the droplet size, the determination of the zeta potential, a TurbiScan analysis, the investigation of the stability under centrifugation and freeze/thaw cycles. If the emulsion contains an active substance, stability under storage at 4 degrees C and microscopic analysis are relevant. Quality control should be improved by measurements of viscosity and pH.

Oral bioavailability in sheep of albendazole from a suspension and from a solution containing hydroxypropyl-beta-cyclodextrin.

Albendazole (ABZ) is a benzimidazole derivative with a broad spectrum of activity against human and animal helminthe parasites. ABZ has a very poor aqueous solubility. This study shows that hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is able to form inclusion complexes with ABZ and that is able to increase its aqueous solubility. A synergistic effect exists between HP-beta-CD and citric acid. The combination of HP-beta-CD (200 mM) and citric acid (50 mM) allows dissolution of more than 1.5 mg of ABZ per ml. The aim of this study is the in vivo evaluation in sheep of a solution of the inclusion complex of ABZ with HP-beta-CD in comparison with a suspension of the same drug. A significant (P<0.05) increase in the relative bioavailability is obtained with the solution containing the ABZ-HP-beta-CD complex as measured by ABZSO plasma levels. The area under the curve (AUC(0--> proportional, variant )) of the solution is 37% higher than that obtained with the suspension. Likewise the peak plasma concentration (C(max)) is twice that of the solution while the time to reach C(max) (T(max)) is reduced.

Preparation of poly(D,L-lactide) nanoparticles assisted by amphiphilic poly(methyl methacrylate-co-methacrylic acid) copolymers.

When co-precipitated with amphiphilic copolymers from DMSO, poly(D,L-lactide) (PLA) can be readily converted into stable sub-200 nm nanoparticles by addition of an aqueous phase, free of any polymeric stabilizers such as poly(vinyl alcohol) or Poloxamer. In this work, the ability of random poly(methyl methacrylate-co-methacrylic acid) copolymers (PMMA-co-MA) to stabilize PLA nanoparticles was demonstrated, and the properties of PLA/PMMA-co-MA nanoparticles were investigated. When co-precipitated with PMMA-co-MA, PLA was totally converted into nanoparticles using a polymer concentration in DMSO (Cp) below 17.6 mg ml(-1), and a PMMA-co-MA proportion above a critical value depending on the content of MA repeating units (X). For instance, the lowest PMMA-co-MA proportion required was 0.9 mg mg(-1) PLA for X = 12%, and 0.5 mg mg(-1) PLA for X = 25% (for C(PLA) = 16 mg ml(-1) DMSO). The nanoparticle diameter was essentially independent of X, the proportion of PMMA-co-MA, and the PLA molecular weight, except for oligomers for which the nanoparticle diameter was smaller. It decreased when the organic phase was diluted (126 +/- 13 nm for Cp = 17.6 mg ml(-1), and 81 +/- 5 nm for C(P) = 5.6 mg ml(-1)). The time-dependence of the stability and the degradation of PLA/PMMA-co-MA nanoparticles was discussed. One of the main advantages of this technique is the ability to control surface properties and to bring functional groups to otherwise non-functionalized PLA nanoparticles. To illustrate this, a conjugate of PMMA-co-MA25 and biotin was synthesized, and used to prepare biotinylated nanoparticles that could be detected by fluorescence and transmission electron microscopy after infiltration into ligatured rat small intestine.