Molecular Dynamics Modeling Based Investigation of the Effect of Freezing Rate on Lysozyme Stability.

PURPOSE: The stability of protein drug products frozen during fill finish operations is greatly affected by the freezing rate applied. Non-optimal freezing rates may lead to the denaturation of protein's complex macromolecular conformation. However, limited work has been done to address the effect of different freezing rates on protein stability at nano-scale level. METHODS: The stability of a model protein, lysozyme, was investigated at atomic and molecular scale under varying freezing rates and moving ice-water interface. Ice seeding approach was adopted to initiate ice formation in this present simulation. RESULTS: The faster freezing rate (11-12 K/490 ns) applied resulted in overall smaller ice fraction within the simulation box with a larger freeze-concentrated liquid (FCL) region. Consequently, the faster freezing rate better maintained protein stability with less secondary structure deviations, higher hydration level and structural compactness, and less fluctuations at individual residues than observed following slow (5-6 K/490 ns) and medium (7-8 K/490 ns) freezing rates. The present study also identified the residues near and within helices 3, 6, 7, and 8 dominate the structural instability of the lysozyme at 247 K freezing temperature. CONCLUSIONS: For the first time, ice formation in therapeutic protein solution was studied "non-isothermally" at different freezing rates using molecular dynamics simulations. Thus, a good understanding of freezing rates on protein instability was revealed by applying the developed computational model.

Use of a Design of Experiments (DoE) Approach to Optimize Large-Scale Freeze-Thaw Process of Biologics.

Large volumes of protein solutions are commonly stored in a frozen state before further drug product fill and finish. This study aimed to establish a design space to perform large-scale freeze-thaw (F/T) processes of biotherapeutics without inducing protein destabilization. A response surface model was designed to evaluate the following main factors and interactions: fill volume of the protein solution in 1-L containers, distance among nine containers during both F/T, freezer set temperature, and a novel forced air flow methodology during thawing. The analysis from 46 experimental runs indicated over 4-fold increase in the freezing rate by lowering the freezing temperature from -20 to -80°C, and the forced air flow at 98 fpm doubled the thawing rate. Furthermore, multivariate linear regression modeling revealed the significant impact of all main factors investigated on lactate dehydrogenase (LDH) quality attributes. The factor that most strongly affected the retention of LDH activity was the loading distance: ≥ 5 cm among containers positively affected the LDH activity response in 50.6%. The factor that most strongly retained the LDH tetramers was the set freezer temperature towards the lower range of -80°C (2.2% higher tetramer retention compared to -20°C freezing, due to faster freezing rate). In summary, this DoE-based systematic analysis increased F/T process understanding at large scale, identified critical F/T process parameters, and confirmed the feasibility of applying faster freezing and forced air thawing procedures to maintain the stability of LDH solutions subject to large-scale F/T.

Impact of Porous Excipients on the Manufacturability and Product Performance of Solid Self-Emulsifying Drug Delivery Systems.

FDA-approved self-emulsifying medicines rely on liquid-based formulations, which can exhibit limited stability and short shelf-lives. Solid self-emulsifying drug delivery systems (SEDDS) can improve such issues, but there is still a great need for identifying suitable porous carriers to convert liquid SEDDS into solids without impairing their mechanical properties, functionality, and industrial feasibility. The impact of SEDDS adsorption on tableting is also poorly understood. Therefore, solid SEDDS were prepared by adsorbing liquid SEDDS onto ten commercially available porous excipients. Products were assessed with respect to mechanical behavior, tabletability, and product performance. Adsorbing SEDDS onto porous excipients led to satisfactory stability, with the exception of Zeopharm® 600 due to its high alkalinity, and Neusilin® US2/UFL2, which caused quercetin to crystallize out of the liquid concentrate. SEDDS adsorption reduced the elastic recovery of most excipients, making tableting achievable using Aeroperl® 300 and Aerosil® 200/300. The impact of SEDDS on elastic recovery provides additional understanding on solid SEDDS manufacture process. Acceptable tablets were made via direct compression but with slow disintegration. Addition of a superdisintegrant (crospovidone 5% w/w) ensured tablet manufacturing without impairment of product performance. Solid SEDDS displayed several technical advantages over their liquid counterparts, but attention must be given to the properties of the porous excipient chosen. Drug-excipient interactions play a significant role in drug degradation and crystallization in solid SEDDS. Improved mechanical behavior upon adsorption led to well-composed tablets that performed satisfactorily in vitro upon addition of a superdisintegrant. This study provides an insight on excipient-oriented rational development of solid SEDDS.

Mechanistic investigation of mixing and segregation of ordered mixtures: experiments and numerical simulations.

Pulmonary delivery of cohesive and micronized drugs through dry powder inhalers (DPIs) is traditionally achieved through the formation of ordered mixtures. In order to improve the mechanistic understanding of formation of ordered mixtures, the system consisting of micronized lactose (AZFL, representative of an active pharmaceutical ingredient) and a coarse particle carrier (LH100) is investigated as a function of different process and material variables in a high shear mixer (HSM) and in a low shear double cone (DCN) blender, using both experimental and numerical methods. Process insight is developed using a Discrete Element Method (DEM) based numerical model which could predict the formation of ordered mixtures in the two blenders and was verified against experimental determinations. Spatial and temporal evolution of granular flow are visualized and quantified in silico to reveal distinguishing features of both blenders to aid in rational selection of blenders and process parameters.

Large-Scale Freeze-Thaw of Protein Solutions: Study of the Relative Contributions of Freeze-Concentration and Ice Surface Area on Stability of Lactate Dehydrogenase.

Although bulk biotherapeutics are often frozen during fill finish and shipping to improve their stability, they can undergo degradation leading to losses in biological activity during sub-optimal freeze-thaw (F/T) process. Except for a few small-scale studies, the relative contribution of various F/T stresses to the instability of proteins has not been addressed. Thus, the objective of this study was to determine the individual contributions of freeze-concentration, ice surface area, and processing time to protein destabilization at a practical manufacturing-scale. Lactate dehydrogenase (LDH) in histidine buffer solutions were frozen in 1L containers. The frozen solutions were sliced into representative samples and assessed for the ice specific surface area (SSA) and extent of solutes freeze-concentration. For the first time to our knowledge, ice SSA was measured in dried samples from large-volume protein solutions using volumetric nitrogen adsorption isotherms. SSA measurements of the freeze-dried cakes showed that the ice surface area increased with an increase in the freezing rate. The ice SSA was also impacted by the position of the sample within the container: samples closer to the active cooled surface of the container exhibited smaller ice surface area compared to ice-cored samples from the center of the bottle. The freeze-concentrate composition was determined by measuring LDH concentration in the ice-cored samples. The protein distributed more evenly throughout the frozen solution after fast freezing which also correlated with enhanced protein stability compared to slow freezing conditions. Overall, better protein stability parameters correlated with higher ice SSA and lower freeze-concentration extent which was achieved at a faster freezing rate. Thus, extended residence time of the protein at the freeze-concentrated microenvironment is the critical destabilizing factor during freezing of LDH in bulk histidine buffer system. This study expands the understanding of the relative contributions of freezing stresses which, coupled with the knowledge of cryoprotection mechanisms, is imperative to the development of optimized processes and formulations aiming stable frozen protein solutions.

Molecular Dynamics Simulation to Uncover the Mechanisms of Protein Instability During Freezing.

Freezing is a common process applied in the pharmaceutical industry to store and transport biotherapeutics. Herewith, multi-scale molecular dynamics simulations of Lactate dehydrogenase (LDH) protein in phosphate buffer with/without ice formation performed to uncover the still poorly understood mechanisms and molecular details of protein destabilization upon freezing. Both fast and slow ice growing conditions were simulated at 243 K from one or two-side of the simulation box, respectively. The rate of ice formation at all-atom simulations was crucial to LDH stability, as faster freezing rates resulted in enhanced structural stability maintained by a higher number of intramolecular hydrogen bonds, less flexible protein's residues, lower solvent accessibility and greater structural compactness. Further, protein aggregation investigated by coarse-grained simulations was verified to be initiated by extended protein structures and retained by electrostatic interactions of the salt bridges between charged residues and hydrogen bonds between polar residues of the protein. Lastly, the study of free energy of dissociation through steered molecular dynamics simulation revealed LDH was destabilized by the solvation of the hydrophobic core and the loss of hydrophobic interactions. For the first time, experimentally validated molecular simulations revealed the detailed mechanisms of LDH destabilization upon ice formation and cryoconcentration of solutes.

Phase-Field Modeling of Freeze Concentration of Protein Solutions.

Bulk solutions of therapeutic proteins are often frozen for long-term storage. During the freezing process, proteins in liquid solution redistribute and segregate in the interstitial space between ice crystals. This is due to solute exclusion from ice crystals, higher viscosity of the concentrated solution, and space confinement between crystals. Such segregation may have a negative impact on the native conformation of protein molecules. To better understand the mechanisms, we developed a phase-field model to describe the growth of ice crystals and the dynamics of freeze concentration at the mesoscale based on mean field approximation of solute concentration and the underlying heat, mass and momentum transport phenomena. The model focuses on evolution of the interfaces between liquid solution and ice crystals, and the degree of solute concentration due to partition, diffusive, and convective effects. The growth of crystals is driven by cooling of the bulk solution, but suppressed by a higher solute concentration due to increase of solution viscosity, decrease of freezing point, and the release of latent heat. The results demonstrate the interplay of solute exclusion, space confinement, heat transfer, coalescence of crystals, and the dynamic formation of narrow gaps between crystals and Plateau border areas along with correlations of thermophysical properties in the supercooled regime.

Development of a Rational Design Space for Optimizing Mixing Conditions for Formation of Adhesive Mixtures for Dry-Powder Inhaler Formulations.

The purpose of the present study was to develop guidance toward rational choice of blenders and processing conditions to make robust and high performing adhesive mixtures for dry-powder inhalers and to develop quantitative experimental approaches for optimizing the process. Mixing behavior of carrier (LH100) and AstraZeneca fine lactose in high-shear and low-shear double cone blenders was systematically investigated. Process variables impacting the mixing performance were evaluated for both blenders. The performance of the blenders with respect to the mixing time, press-on forces, static charging, and abrasion of carrier fines was monitored, and for some of the parameters, distinct differences could be detected. A comparison table is presented, which can be used as a guidance to enable rational choice of blender and process parameters based on the user requirements. Segregation of adhesive mixtures during hopper discharge was also investigated.

Study on the developmental toxicity of combined artesunate and mefloquine antimalarial drugs on rats.

Antimalarial drug combinations containing artemisinins (ACTs) have become first choice therapies for Plasmodium falciparum malaria. Data on safety of ACTs in pregnancy are limited and no previous study has been conducted on the developmental toxicity of artesunate-mefloquine combinations on the first trimester of gestation. To evaluate the developmental toxicity of an artesunate/mefloquine combination, pregnant rats were treated orally with artesunate (15 and 40 mg/kg bwt/day), mefloquine (30 and 80 mg/kg bwt/day) and artesunate/mefloquine (15/30 and 40/80 mg/kg bwt/day) on gestation days 9-11. Dams were C-sectioned on day 20, and their uteri and fetuses removed and examined for soft tissue and skeleton abnormalities. Artesunate increased embryolethality and the incidence of limb long bone malformations on the absence of overt maternal toxicity. Mefloquine (80 mg/kg bwt/day) was maternally toxic and enhanced fetal variations. Combination of artesunate and mefloquine did not enhance their toxicity compared to the toxicity observed after its separate administration. Embryotoxicity of artesunate was apparently attenuated when it is co-administered with mefloquine.

Screening for in vivo (anti)estrogenic and (anti)androgenic activities of Tropaeolum majus L. and its effect on uterine contractility.

ETHNOPHARMACOLOGICAL RELEVANCE: Tropaeolum majus L. (Tropaeolaceae) is a medicinal herb popularly used in Brazil for treatment of inflammatory and cardiovascular diseases. Despite some published data on its efficacy, there are still few toxicological data describing the safety of this plant. The aim of this study was to evaluate the (anti)estrogenic and (anti)androgenic activity of the hydroethanolic extract obtained from Tropaeolum majus L. (HETM), as well as its possible effects on uterine contractility. MATERIALS AND METHODS: Three experimental protocols were performed, (a) uterotrophic assay, (b) Hershberger assay and (c) an ex vivo test to investigate the effects of maternal administration of HETM on uterine contractility at the end of pregnancy. In all protocols three doses of the HETM were administered to Wistar rats: 3, 30 and 300mg/kg. RESULTS: In vivo tests for detection of (anti)androgenic and (anti)estrogenic activities did not show any significant alterations. Similarly, no alterations were observed on uterine contractility induced by oxytocin and arachidonic acid. CONCLUSIONS: HETM was unable to produce (anti)estrogenic or (anti)androgenic activities in the short-term in vivo screening assays performed. In addition, there was no evidence that HETM can affect uterine contractility following gestational exposure of rats.

In vivo and in vitro estrogenic activity of the antidepressant fluoxetine.

Recent years have seen an increase in the use of antidepressant drugs, especially fluoxetine (FLX), in sensitive populations, such as pregnant and lactating women. Although some evidence suggests a possible endocrine action of FLX, no specific studies have been performed to investigate this hypothesis. In the present study, we investigated the possible (anti)androgenic and (anti)estrogenic actions of FLX using Hershberger, uterotrophic (0.4, 1.7, and 17mg/kg), and reporter gene (7.6-129µM) assays. In the Hershberger assay, no differences were observed in androgen-dependent organ weights. However, the uterotrophic and gene reporter assays indicated a possible estrogenic action of FLX. Uterine weight increased in the 1.7 and 17mg/kg/day groups in the 3-day uterotrophic assay in immature rats. Additionally, noncytotoxic concentrations of FLX induced estrogenic responses and increased the estrogenic response of estradiol in MCF-7 breast cancer cells transfected with luciferase.