Drug-plasticizer interactions causing solid state transitions of rifaximin.

Solid phase interactions are often the reason for incompatibilities in solid dosage forms. A special situation occurs, if the incompatible compounds are able to migrate within the solid matrix. This study describes for the first time the migration of a plasticizer from the coating into the core and its interaction with the active ingredient located there. This behavior was observed in rifaximin gastro-resistant granules and resulted in the formation of solvates with altered dissolution behavior. For a detailed study, rifaximin was incubated with five plasticizers of different solubility and miscibility as well as different molecular geometry (linear vs branched), (dibutyl sebacate, tributyl citrate, triacetin, polyethylene glycol 400, and propylene glycol). The resulting solid states were analyzed by means of PXRD, supported by thermogravimetric analysis, infrared spectroscopy, and quantitative H NMR. No direct correlation could be demonstrated between the resulting type of solvate/hydrate and the affinity of rifaximin with the respective plasticizers. Interestingly all plasticizers that are able to form type I solvates/hydrates have linear structures. This common feature, which distinguishes them from the more bulky TAC and TBC, seems to be a key characteristic. Rifaximin-PG-solvate formation was not only detected after direct incubation trials, but also observed in enteric coated granules.

Needle clogging of protein solutions in prefilled syringes: A two-stage process with various determinants.

Clogging of staked-in-needle prefilled syringes (PFS) is a sporadic and scarcely predictable event, which occurs particularly in highly concentrated protein solutions and can result in the injection of incomplete doses, especially if autoinjector devices are used for administration. A systematic screening of possible causes and triggers was performed in order to find the crucial factors of influence, the underlying mechanisms and possible measures for prevention. An essential prerequisite for the formation of a solidified clog in the needle is the ingress of liquid from the barrel, which was investigated and quantified by means of neutron imaging after storage of prefilled syringes under various conditions. The needle filling ratio increases with both the storage temperature and the storage period, as a result of atmospheric gas diffusion through the needle shield. While the air pocket in the needle is reduced by this process, diffusion of water vapor does not affect the filling ratio but instead increases the protein concentration in the needle lumen, leading to an exponential rise of the viscosity and finally to the solidification of the protein solution. Maintaining the air pocket in the needle by avoiding any diffusion promoting pressure gradient is therefore the most effective protection from clogging.

Characterization of the aggregation propensity of charge variants of recombinant human growth hormone.

Biopharmaceutical products are subject to in depth analysis to ensure and improve their safety and efficacy. As part of this effort the stability and aggregation mechanisms of the therapeutic protein is characterized over the whole life cycle. The stability and aggregation behavior of single charge variants present in biopharmaceuticals were hardly investigated. In this study we applied a previously established methodology to assess the charge variants of the drug substance (DS) of human growth hormone (hGH). We assessed the stability and aggregation propensity of an acidic variant which forms in DS at a larger extent during short time storage at elevated temperatures. We developed a semi-preparative method to separate and analyze the charge species. Thermal and colloidal stability of this variant was analyzed by light scattering methods and a stability testing in different buffer formulations. The acidic variant showed slightly attractive self-interaction at lower pH. Thermal stress did not result in increased aggregation propensity or decreased stability compared to the DS. Thus, the methodology enabled to assess the risk of a single protein variant within the DS of hGH. The approach can also be utilized for other protein drugs as previously shown for a monoclonal antibody.

Identification of monoclonal antibody variants involved in aggregate formation - Part 2: Hydrophobicity variants.

Monoclonal antibodies (mAbs) are valuable tools both in therapy and in diagnostic. Their tendency to aggregate is a serious concern. Since a mAb drug substance (DS) is composed of different variants, it is important for manufacturers to know the behavior and stability not only of the mAb as a whole, but also of the variants contained in the product. We present a method to separate hydrophobicity variants of a mAb and subsequently analyzed these variants for stability and aggregation propensity. We identified a potentially aggregation prone hydrophilic variant which is interrelated with another previously identified aggregation prone acidic charge variant. Additionally, we assessed the risk posed by the aggregation prone variant to the DS by spiking hydrophobicity variants into DS and did not observe an enhanced aggregation propensity. Thus we present an approach to separate, characterize and analyze the criticality of aggregation prone variants in protein DS which is a step forward to further assure drug safety.

Identification of monoclonal antibody variants involved in aggregate formation - Part 1: Charge variants.

Biopharmaceutical products contain conformational and chemical variants, that are typically well characterized regarding identity and activity. However, little is known about their self-interaction propensity and tendency to unfold, which are critical characteristics for drug stability and safety. This study aimed to separate and compare charge variants of a monoclonal antibody (mAb) and to identify aggregation prone species. We show a semi-preparative cation exchange method, that we developed to separate the individual acidic and basic variants from the naïve mAb. Additionally, we demonstrate, that the yield and purity of the fractionated charge species, extracted by that method, were sufficient for subsequent analysis of aggregate content, conformation stability and self-interaction. Our analysis revealed a differently behaving acidic variant and confirmed its increased aggregation propensity by molecular modeling. During a stability study, the potentially aggregation prone charge variant posed a limited risk to the drug substance (DS). We are the first to look at the stability of single charge variants of biopharmaceuticals, and thus present manufacturers and regulatory authorities with a method to enhance drug safety.

Permeability changes in response to NONOate and NONOate prodrug derived nitric oxide in a blood-brain barrier model formed by primary porcine endothelial cells.

The influence of nitric oxide (NO) and NO-donors on the permeability of the blood-brain barrier (BBB) is still not well understood and the literature about this is quite controversial. Some studies suggest increasing, others decreasing or even no effects of NO-donors on the BBB permeability. In this work we report about the influence of three diazeniumdiolates, which release NO spontaneously and three different diazeniumdiolate prodrugs, which have to be cleaved chemically or enzymatically before releasing NO, on the permeability of an in vitro BBB-model formed by primary porcine endothelial cells. By measuring the flux of a small polar molecule (carboxyfluorescein: CF) we could show, that the NO-releasers PHEPIPERAZI/NO (sodium 1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-diolate), DBA/NO (sodium 1-(N,N-dibutylamino)diazen-1-ium-1,2-diolate) and DETA/NO (1-N,N-di-(2-aminoethyl)amino)diazen-1-ium-1,2-diolate) reduced the BBB-model permeability. In contrast, the NO-prodrugs Et-PHEPIPERAZI/NO (O(2)-Ethyl-1-(phenylpiperazin-1-yl)diazen-1-ium-1,2-diolate) and TOSYL-PYRRO/NO (O(2)-(p-Methylbenzen-sulfonyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate) increased the permeability in all investigated concentrations, whereas the prodrug Et-BUPIPERAZI/NO (O(2)-Ethyl-1-(butylpiperazin-1-yl)diazen-1-ium-1,2-diolate) reduced it at the lowest investigated concentration of 100 microM, at the higher concentrations it increased the permeability. Blocking the effect of the BBB-model permeability reducing compounds could be done by methylene blue, whereas permeability increasing effects could not be blocked.

Using a modified shepards method for optimization of a nanoparticulate cyclosporine a formulation prepared by a static mixer technique.

An innovative methodology has been used for the formulation development of Cyclosporine A (CyA) nanoparticles. In the present study the static mixer technique, which is a novel method for producing nanoparticles, was employed. The formulation optimum was calculated by the modified Shepard's method (MSM), an advanced data analysis technique not adopted so far in pharmaceutical applications. Controlled precipitation was achieved injecting the organic CyA solution rapidly into an aqueous protective solution by means of a static mixer. Furthermore the computer based MSM was implemented for data analysis, visualization, and application development. For the optimization studies, the gelatin/lipoid S75 amounts and the organic/aqueous phase were selected as independent variables while the obtained particle size as a dependent variable. The optimum predicted formulation was characterized by cryo-TEM microscopy, particle size measurements, stability, and in vitro release. The produced nanoparticles contain drug in amorphous state and decreased amounts of stabilizing agents. The dissolution rate of the lyophilized powder was significantly enhanced in the first 2 h. MSM was proved capable to interpret in detail and to predict with high accuracy the optimum formulation. The mixer technique was proved capable to develop CyA nanoparticulate formulations.

A systematic evaluation of mechanisms, material effects, and protein-dependent differences on friction-related protein particle formation in formulation and filling steps.

Particle formation by physical degradation during the compounding step of biopharmaceuticals is a common concern and found in vessels with bottom mounted stirrers. It was potentially linked to sliding bearings, however, the exact mechanism was still unclear. In this study, custom designed small scale bearings in combination with an IgG1 antibody as model protein were used for investigations of the degradation mechanism inside a bearing. Thereby, abrasion of adsorbed proteins by contact sliding was identified as prevailing protein degradation mechanism and was quantified by an increase in turbidity and by monomer loss. As the protein degradation was highly dependent on combinations of the material of the bearing and the buffer solution, a test system was introduced which allowed to study these effects. Results from the test system using IgG1 and recombinant human growth hormone confirmed a protective effect of Polysorbate 80 by a reduction of protein adsorption, which was strongest in combination with a highly hydrophobic sliding material (PTFE). Finally, a comparison of degradation products from various stresses by ATR-FTIR revealed a high similarity between friction-related degradation products. Therefore, abrasion of adsorbed proteins is very likely the prevailing physical degradation mechanism in processing steps where contact sliding occurs.

The Effect of Shear on the Structural Conformation of rhGH and IgG1 in Free Solution.

The effect of hydrodynamic forces on proteins in free solution, also referred to as shear stress in multiple drug substance and drug product processing steps, was investigated by means of in situ and inline biophysical measurements. The use of a quartz Couette cell in combination with a circular dichroism spectrometer allowed simultaneously the creation of simple shear flow and direct measurements of the proteins' secondary and tertiary structure. Recombinant human growth hormone and an IgG1 mAb were chosen as model proteins. Under the exclusion of interfacial effects by the addition of a surfactant, no unfolding was observed due to shearing for 30 min up to the highest possible shear rate under laminar flow (3840 s(-1)). In another experiment, guanidine hydrochloride was added to a surfactant-protected and sheared sample to lower the thermodynamic and mechanical stability of the proteins. However, even under these destabilizing conditions, the proteins showed no change in their secondary and tertiary structure. We conclude that shear stress in terms of velocity gradients is unlikely to unfold the investigated proteins in free solution up to shear rates of at least 10(4) s(-1).

A detailed view of microparticle formation by in-process monitoring of the glass transition temperature.

Biodegradable poly(D,L-lactide-co-glycolide) microspheres were prepared by a well-controlled emulsion solvent extraction/evaporation process. The objective of this study was to investigate how drug release can be modified by changing the morphology of the polymer matrix. The matrix structure was controlled by the preparation temperature which was varied between 10 and 35 °C, thus changing the 4 weeks release pattern from almost linear kinetics to a sigmoidal profile with a distinct lag phase and furthermore decreasing the encapsulation efficiency. By monitoring the glass transition temperature during the extraction process, it was shown that the preparation temperature determines the particle morphology by influencing the time span in which the polymer chains were mobile and flexible during the extraction process. Further factors determining drug release were found to be the molecular weight of the polymer and the rate of solvent removal. The latter, however, has also influence on the encapsulation efficiency with slow removal causing a higher drug loss. A secondary modification of the outer particle structure could be achieved by ethanolic post-treatment of the particles, which caused an extension of the lag phase and subsequently an accelerated drug release.

Understanding reflection behavior as a key for interpreting complex signals in FBRM monitoring of microparticle preparation processes.

The application of focused beam reflectance measurement (FBRM) was studied in a larger scale PLGA microparticle preparation process for monitoring changes of the particle size and the particles' surface properties. Further understanding how these parameters determine the chord length distribution (CLD) was gained by means of single object measurements and data of monodisperse microparticles. It was evaluated how the FBRM signal is influenced by the surface characteristics of the tested materials and the measuring conditions. Particles with good scattering properties provided comparable values for the CLD and the particle size distribution. Translucent particles caused an overestimation of the particle size by FBRM, whereas the values for transparent emulsion droplets were too low. Despite a strong dependence of FBRM results on the optical properties of the samples, it is a beneficial technique for online monitoring of microparticle preparation processes. The study demonstrated how changing reflection properties can be used to monitor structural changes during the solidification of emulsion droplets and to detect process instabilities by FBRM.

Cellular uptake and degradation behaviour of biodegradable poly(ethylene glycol-graft-methyl methacrylate) nanoparticles crosslinked with dimethacryloyl hydroxylamine.

Crosslinked polymers with hydrolytically cleavable linkages are highly interesting materials for the design of biodegradable drug carriers. The aim of this study was to investigate if nanoparticles made of such polymers have the potential to be used also for intracellular drug delivery. PEGylated nanoparticles were prepared by copolymerization of methacrylic acid esters and N,O-dimethacryloylhydroxylamine (DMHA). The particles were stable at pH 5.0. At pH 7.4 and 9.0 the degradation covered a time span of about 14 days, following first-order kinetics with higher crosslinked particles degrading slower. Cellular particle uptake and cytotoxicity were tested with L929 mouse fibroblasts. The particle uptake rate was found to correlate linearly with the surface charge and to increase as the zeta potential becomes less negative. Coating of the particle surface with polysorbate 80 drops the internalization rate close to zero and the charge dependence disappears. This indicates the existence of a second effect apart from surface charge. A similar pattern of correlation with zeta potential and coating was also found for the degree of membrane damage while there was no effect of polysorbate on the cell metabolism which increased as the negative charge decreased. It is discussed whether exocytotic processes may explain this behaviour.

Application of Hansen solubility parameters for understanding and prediction of drug distribution in microspheres.

In an emulsion solvent extraction/evaporation process for the preparation of microspheres the employed solvents have a tremendous influence on the characteristics of the resulting particles. Nevertheless the solvent selection is often based on empirical data rather than on calculated values. The purpose of this investigation was to use the concept of solubility parameters for interpretation and improved understanding of solvent effects in the process of microparticle preparation. Partial solubility parameters of 3-{2-[4-(6-Fluor-1,2-benzisoxazol-3-yl)piperidino]ethyl}-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-on, which was used as a model drug, were determined experimentally using an extended Hansen regression model. Poly(lactide-co-glycolide) microparticles were prepared with an emulsion solvent removal process employing methylene chloride and its mixtures with benzyl alcohol and n-butanol. It could be shown, that the encapsulation efficiency was influenced by the change of the solvent composition during the extraction process. Furthermore the solvent selection had an essential influence on the morphological state of the drug and it could be shown and explained, that by a decrease of the dissolving power a completely amorphous product was obtained.

New insights into the pore structure of poly(d,l-lactide-co-glycolide) microspheres.

The objective of this work was to develop a fast and significant method for the determination of the intraparticulate pore size distribution of microspheres. Poly(lactide-co-glycolide) (PLGA) microspheres prepared with a solvent extraction/evaporation process were studied. From the envelope and the skeletal volume of the microspheres the porosity was calculated. The skeletal volume was determined with nitrogen and helium pycnometry and mercury intrusion porosimetry. Based on single particle optical sensing (SPOS) a novel method was developed by which the envelope volume is calculated from the particle size distribution (PSD), provided that all particles have a spherical shape. The penetration capacity of the applied intrusion media is limited by their atomic or molecular diameter or by the surface tension and the pressure in case of mercury. A classification of the pore structure was obtained by comparing these different skeletal values with the values for the envelope volume. Two well separated pore fractions were found, a nanoporous fraction smaller than 0.36nm and a macroporous fraction larger than 3.9µm. The total porosity and the ratio between both fractions is controlled by the preparation process and was shown to depend on the solvent extraction temperature.

Preservation of liquid drug preparations for oral administration.

The aim of this study was to investigate functional interactions between sorbic acid, alcoholic cosolvents, and pH with respect to pharmacopeial requirements for the antimicrobial preservation of oral liquids. Twenty-seven test formulations of sugar-free syrups with varying amounts of glycerol (18-36%), propylene glycol (4-21%), and sorbic acid (0-0.15%) and with different pH values (5-8) were tested for antimicrobial preservation efficacy after inoculation with spores of Aspergillus niger. Multivariate data analysis revealed that at pH 5 the minimum concentration of sorbic acid necessary for a tenfold decrease of viable spores ranges between 0.08% and 0.10% exhibiting only minor dependence on the cosolvents concentration. Various interactions between sorbic acid and cosolvents could be observed and were discussed on basis of the degree of dissociation and distribution of sorbic acid. All tested preparations, even those free of sorbic acid, met the criteria for oral products with aqueous bases according to USP32-NF27 and JP XV which claim no increase from the initial spore count at 14 and 28 days. The EP requirement, not less than 1 log(10) reduction from the initial count at 14 days, was only met by preparations with pH 5 and not less than 0.15% sorbic acid.