A comparison of background membrane imaging versus flow technologies for subvisible particle analysis of biologics.

A recently developed high-throughput background membrane imaging (BMI) technique, the HORIZON, was assessed for its ability to quantify subvisible particulate (SVP) generated during protein therapeutic development. The HORIZON platform method was optimized and compared to three well-characterized SVP counting techniques: light obscuration, micro-flow imaging (MFI), and FlowCam®. A head-to-head comparison was performed for precision, linearity, SVP concentration, and morphological output of BMI compared to the other three techniques using two unique enzymes under investigation. We found that dilution requirements for BMI are protein-specific, and membrane coverage is the critical instrument parameter to monitor for dilution suitability. The precision of BMI ranked similarly to all other techniques. Analysis of the same sample dilution, run in triplicate, across all four techniques indicated the BMI technique provides SVP concentrations that are comparable with the flow imaging techniques. Morphological information from BMI was generally less practical when compared with flow microscopy. The major drawback of BMI was that the current software indiscriminately clips large particles, potentially resulting in a misrepresentation of SVP size distribution. Despite this phenomenon, the concentration and size data generated corresponds well with current flow imaging techniques while decreasing time, cost, and sample requirements for SVP quantification.

In vivo and in vitro sustained release of ranibizumab from a nanoporous thin-film device.

Current administration of ranibizumab and other therapeutic macromolecules to the vitreous and retina carries ocular risks, a high patient treatment burden, and compliance barriers that can lead to suboptimal treatment. Here we introduce a device that produces sustained release of ranibizumab in the vitreous cavity over the course of several months. Composed of twin nanoporous polymer thin films surrounding a ranibizumab reservoir, these devices provide release of ranibizumab over 16 weeks in vitro and 12 weeks in vivo, without exhausting the initial drug payload. Following implantation in vivo, devices were well-tolerated and showed no sign of immune response. This platform presents a potential solution to the challenge of delivering protein therapeutics to the vitreous and retina for sustained periods of time.

Polycaprolactone thin-film drug delivery systems: Empirical and predictive models for device design.

PURPOSE: To define empirical models and parameters based on theoretical equations to describe drug release profiles from two polycaprolactone thin-film drug delivery systems. Additionally, to develop a predictive model for empirical parameters based on drugs' physicochemical properties. METHODS: Release profiles from a selection of drugs representing the standard pharmaceutical space in both polycaprolactone matrix and reservoir systems were determined experimentally. The proposed models were used to calculate empirical parameters describing drug diffusion and release. Observed correlations between empirical parameters and drug properties were used to develop equations to predict parameters based on drug properties. Predictive and empirical models were evaluated in the design of three prototype devices: a levonorgestrel matrix system for on-demand locally administered contraception, a timolol-maleate reservoir system for glaucoma treatment, and a primaquine-bisphosphate reservoir system for malaria prophylaxis. RESULTS: Proposed empirical equations accurately fit experimental data. Experimentally derived empirical parameters show significant correlations with LogP, molecular weight, and solubility. Empirical models based on predicted parameters accurately predict experimental release data for three prototype systems, demonstrating the accuracy and utility of these models. CONCLUSION: The proposed empirical models can be used to design polycaprolactone thin-film devices for target geometries and release rates. Empirical parameters can be predicted based on drug properties. Together, these models provide tools for preliminary evaluation and design of controlled-release delivery systems.

Planar microdevices for enhanced in vivo retention and oral bioavailability of poorly permeable drugs.

The development of novel oral drug delivery platforms for administering therapeutics in a safe and effective manner through the harsh gastrointestinal environment is of great importance. Here, the use of engineered thin planar poly(methyl methacrylate) (PMMA) microdevices is tested to enhance oral bioavailability of acyclovir, a poorly permeable drug. Acyclovir is loaded into the unidirectional drug releasing microdevice reservoirs using a drug entrapping photocross-linkable hydrogel matrix. An increase in acyclovir permeation across in vitro caco-2 monolayer is seen in the presence of microdevices as compared with acyclovir-entrapped hydrogels or free acyclovir solution. Cell proliferation studies show that microdevices are relatively nontoxic in nature for use in in vivo studies. Enhanced in vivo retention of microdevices is observed as their thin side walls experience minimal peristaltic shear stress as compared with spherical microparticles. Unidirectional acyclovir release and enhanced retention of microdevices achieve a 4.5-fold increase in bioavailability in vivo as compared with an oral gavage of acyclovir solution with the same drug mass. The enhanced oral bioavailability results suggest that thin, planar, bioadhesive, and unidirectional drug releasing microdevices will significantly improve the systemic and localized delivery of a broad range of oral therapeutics in the near future.

Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.

Nanostructured thin film polymer devices for constant-rate protein delivery.

Herein long-term delivery of proteins from biodegradable thin film devices is demonstrated, where a nanostructured polymer membrane controls release. Protein was sealed between two poly(caprolactone) films, which generated the thin film devices. Protein release for 210 days was shown in vitro, and stable activity was established through 70 days with a model protein. These thin film devices present a promising delivery platform for biologic therapeutics, particularly for application in constrained spaces.

Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter.

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.

Effects of disulfide bond formation and protein helicity on the aggregation of activating transcription factor 5.

Amorphous aggregation is a major problem for protein biopharmaceuticals, and aggregate formation in a drug formulation can have serious health implications for the patient. In many cases, an immunogenic response is generated from the administration of a drug product containing aggregated protein. This becomes especially significant when the patient requires long-term or repeated administration of the drug, because the likelihood of a severe immune response increases. While the prevention of protein aggregation is critically important for the future of protein pharmaceuticals, the mechanism of amorphous aggregation is still poorly understood. The lack of understanding regarding nonfibrillar aggregation is largely due to the fact that assembly is difficult to study. In particular the role that various structural features (i.e., alpha-helix, beta-structure, disulfide bonds) play in the aggregation process varies with the amino acid sequence and is dependent upon tertiary structure and solution conditions. Well-structured proteins do not readily aggregate in solution, whereas partially unfolded proteins tend to aggregate rapidly and often become insoluble. Here, we present a unique and simple system for studying amorphous protein aggregation. We have previously reported the isolation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5), a protein notable for its potential as a pharmaceutical target for treatment of glioblastoma multiforme. This domain consists of a single alpha-helix and possesses a single cysteine residue. It is only partially structured and displays marginal stability in solution under physiological conditions. We have modulated solution conditions that affect backbone solubility and the oxidation state of the thiol to successfully investigate the role that alpha-helical structure and disulfide bond formation play in protein stability. Our data indicate that covalent cross-linking helps to retain ATF5's helicity, which inhibits the formation of large aggregates. These studies have led to the identification of stabilizing conditions for ATF5, which will enable further study of the protein as a pharmaceutical target. Moreover, this work has general implications for analyzing stability of helical proteins in vitro as well as the specific atomic-level interactions in ATF5 that contribute to instability and self-association.

High-yield expression in E. coli and refolding of the bZIP domain of activating transcription factor 5.

Activating transcription factor 5 (ATF5) recently has been demonstrated to play a critical role in promoting the survival of human glioblastoma cells. Interference with the function of ATF5 in an in vivo rat model caused glioma cell death in primary tumors but did not affect the status of normal cells surrounding the tumor, suggesting ATF5 may prove an ideal target for anti-cancer therapy. In order to examine ATF5 as a pharmaceutical target, the protein must be produced and purified to sufficient quantity to begin analyses. Here, a procedure for expressing and refolding the bZIP domain of ATF5 in sufficient yield and final concentration to permit assay development and structural characterization of this target using solution NMR is reported. Two-dimensional NMR and circular dichroism analyses indicate the protein exists in the partially alpha-helical, monomeric x-form conformation with only a small fraction of ATF5 participating in formation of higher-order structure, presumably coiled-coil homodimerization. Despite the persistence of monomers in solution even at high concentration, an electrophoretic mobility shift assay showed that ATF5 is able to bind to the cAMP response element (CRE) DNA motif. Polyacrylamide gel electrophoresis and mass spectrometry were used to confirm that ATF5 can participate in homodimer formation and that this dimerization is mediated by disulfide bond formation.

Involvement of endothelin in morphine tolerance in neuroblastoma (SH-SY5Y) cells.

Long-term use of morphine in pain management leads to adverse effects, such as development of antinociceptive tolerance. We have previously shown the involvement of central endothelin (ET) mechanisms in morphine analgesia and development of tolerance in vivo. The present study was conducted to investigate the in vitro mechanism of interaction of the ET(A) receptor antagonist, BMS182874, and morphine during acute and chronic morphine tolerance in SH-SY5Y cells. SH-SY5Y cells were exposed to acute and chronic treatment with vehicle, morphine, ET-1, BMS182874, or morphine plus BMS182874. Activation of G-protein-coupled receptors in SH-SY5Y cells was determined using [35S]GTPgammaS binding assays. Acute morphine treatment produced a concentration-dependent increase in GTP binding. Median effective concentration (EC50) values were significantly decreased after acute morphine treament, suggesting sensitization of opioid receptors. Chronic morphine treatment produced a lower maximal response of GTP binding compared with both control (vehicle treated) and acute morphine treatment, indicating uncoupling of G-proteins. Acute and chronic exposure of cells to ET-1 did not affect changes in ET-1-induced GTP binding. BMS182874 treatment alone (acute or chronic) did not produce G-protein activation. However, in cells chronically cotreated with 10 microM morphine and 1 microM BMS182874, morphine-induced GTP stimulation was significantly higher than control (vehicle treated). The EC50 value after control treatment was 414 nM, and was significantly increased in chronically morphine-treated cells (>1000 nM ). However, the EC50 value in cells receiving a chronic treatment of BMS182874 and 63 nM morphine was significantly reduced compared with control (vehicle treated) and chronic morphine treatment. ET(A) antagonists significantly enhance the coupling of G-protein to opioid receptors. Therefore, we propose that restoration of morphine antinociception by ET(A) antagonists in morphine-tolerant animals is likely via a G-protein mediated mechanism.