Minimization of artifact protein aggregation using tetradecyl sulfate and hexadecyl sulfate in capillary gel electrophoresis under reducing conditions.

In the biopharmaceutical industry, CE-SDS assesses the purity, heterogeneity, and stability of therapeutic proteins. However, for mAb-1 and mAb-2, typical CE-SDS under reducing conditions produced atypical protein peak profiles, which led to biased purity results, thus were not acceptable for biologics manufacturing. This bias was caused by the formation of method-induced higher molecular weight artifacts, the levels of which correlated with protein concentration. Here we show that adding sodium tetradecyl and hexadecyl sulfates to the sample and the sieving gel buffer solutions was required to prevent formation of aggregate artifacts and to maintain detergent:protein uniformity, suggesting their importance during the sample preparation steps of heat denaturation and subsequent cooling as well as during capillary migration. For these proteins, we show that this uniformity was likely due to the ability of these detergents to bind proteins with markedly higher affinities compared to SDS. "CE-SCX S" methods (where CE-SCX S is CGE using detergent composed of a sodium sulfate head group and a hydrocarbon tail, with "CX " representing various tail lengths), were developed with a sodium tetradecyl sulfate sample buffer and a sodium hexadecyl sulfate containing sieving gel buffer that minimized artifacts and provided robust characterization and release results for mAb-1 and mAb-2.

Synthesis and application of ß-carbolines as novel multi-functional anti-Alzheimer's disease agents.

The design, synthesis and assessment of ß-carboline core-based compounds as potential multifunctional agents against several processes that are believed to play a significant role in Alzheimer's disease (AD) pathology, are described. The activity of the compounds was determined in Aß self-assembly (fibril and oligomer formation) and cholinesterase (AChE, BuChE) activity inhibition, and their antioxidant properties were also assessed. To obtain insight into the mode of action of the compounds, HR-MS studies were carried out on the inhibitor-Aß complex formation and molecular docking was performed on inhibitor-BuChE interactions. While several compounds exhibited strong activities in individual assays, compound 14 emerged as a promising multi-target lead for the further structure-activity relationship studies.

Sulfonamides as multifunctional agents for Alzheimer's disease.

Sulfonamide linker-based inhibitors with extended linear structure were designed and synthesized with the aim of producing multifunctional agents against several processes involved in the pathology of Alzheimer's disease (AD). The potency of the compounds were assessed in the inhibition of Aß self-assembly (fibril and oligomer formation), in modulating cholinesterase (AChE, BuChE) activity, and scavenging free radicals. Several compounds exhibited promising Aß self-assembly and cholinesterase inhibition and in parallel, showed good free radical scavenging properties. The investigation of the scaffold described in this study resulted in the identification of three compounds (14, 19 and 26) as promising leads for the further design of multifunctional drug candidates for AD.

Diaryl hydrazones as multifunctional inhibitors of amyloid self-assembly.

The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of amyloid ß (Aß) fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Because the structure of the hydrazone-based inhibitors mimics the redox features of the antioxidant resveratrol, the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had a considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aß species, as well. Atomic force microscopy was also applied to monitor the morphology of Aß deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed a radical scavenging effect equal to or better than that of resveratrol or ascorbic acid.

Design, synthesis and biological activity of multifunctional α,ß-unsaturated carbonyl scaffolds for Alzheimer's disease.

A series of compounds containing an α,ß-unsaturated carbonyl moiety, such as chalcones and coumarins were designed, synthesized and tested in a variety of assays to assess their potential as anti-Alzheimer's disease (AD) agents. The investigations included the inhibition of cholinesterases (AChE, BuChE), the inhibition of amyloid beta (Aß) self-assembly and the disassembly of preformed Aß oligomers. Several compounds showed excellent potential as multifunctional compounds for AD. Docking studies for 16 that performed well in all the assays gave a clear interpretation of various interactions in the gorge of AChE. Based on the results, the long-chain coumarin scaffold appears to be a promising structural template for further AD drug development.

Enhancement of covalent aggregate quantification of protein therapeutics by non-reducing capillary gel electrophoresis using sodium hexadecyl sulfate (CE-SHS).

Capillary gel electrophoresis (CGE) using sodium dodecyl sulfate (CGE-SDS or CE-SDS) is commonly used in the biotechnology industry to assess the purity of a complex therapeutic during manufacturing process optimization and also for commercial release and stability testing. However, for therapeutic proteins mAb-1 and mAb-2, non-reducing (NR) CE-SDS yielded higher than expected % aggregate which considerably lowered its apparent purity relative to the purity reported by other complementary methods, such as Size Exclusion Chromatography (SEC). Furthermore, a strong protein load dependence on aggregate levels was observed which prevented any reasonable assessment of the true purity value. The solution was to supplement SDS with the relatively hydrophobic detergent sodium hexadecyl sulfate (SHS) in the sieving gel buffer matrix which virtually eliminated the protein load-dependence and reduced the % aggregate value to expected levels when compared to SEC. Analytical Ultracentrifugation (AUC) was used to help confirm the accuracy of the SEC results. This work underscored how using detergents other than SDS in CGE applications can be valuable in the commercial biologics space and provided an example of how SEC can be used to confirm the accuracy of CGE data.

Steroid-eluting contact lenses for corneal and intraocular inflammation.

Ocular inflammation is one of the leading causes of blindness worldwide, and steroids in topical ophthalmic solutions (e.g. dexamethasone eye drops) are the mainstay of therapy for ocular inflammation. For many non-infectious ocular inflammatory diseases, such as uveitis, eye drops are administered as often as once every hour. The high frequency of administration coupled with the side effects of eye drops leads to poor adherence for patients. Drug-eluting contact lenses have long been sought as a potentially superior alternative for sustained ocular drug delivery; but loading sufficient drug into contact lenses and control the release of the drug is still a challenge. A dexamethasone releasing contact lens (Dex-Lens) was previously developed by encapsulating a dexamethasone-polymer film within the periphery of a hydrogel-based contact lens. Here, we demonstrate safety and efficacy of the Dex-Lens in rabbit models in the treatment of anterior ocular inflammation. The Dex-Lens delivered drug for 7 days in vivo (rabbit model). In an ocular irritation study (Draize test) with Dex-Lens extracts, no adverse events were observed in normal rabbit eyes. Dex-Lenses effectively inhibited suture-induced corneal neovascularization and inflammation for 7 days and lipopolysaccharide-induced anterior uveitis for 5 days. The efficacy of Dex-Lenses was similar to that of hourly-administered dexamethasone eye drops. In the corneal neovascularization study, substantial corneal edema was observed in rabbit eyes that received no treatment and those that wore a vehicle lens as compared to rabbit eyes that wore the Dex-Lens. Throughout these studies, Dex-Lenses were well tolerated and did not exhibit signs of toxicity. Dexamethasone-eluting contact lenses may be an option for the treatment of ocular inflammation and a platform for ocular drug delivery. STATEMENT OF SIGNIFICANCE: Inflammation of the eye can happen either on the ocular surface (i.e. the cornea) or inside the eye, both of which can result in loss of vision or even blindness. Ocular inflammation is normally treated by steroid eye drops. Depending on the type and severity of inflammation, patients may have to take drops every hour for days at a time. Such severe dosing regimen can lead to patients missing doses. Also, more than 95% drug in an eye drop never goes inside the eye. Here we present a contact lens that release a steroid (dexamethasone) for seven days at a time. It is much more efficient than eye drops and a significant improvement since once worn, the patient will avoid missing doses.

Pharmacophore Modeling, virtual and in vitro screening for acetylcholinesterase inhibitors and their effects on amyloid-ß self- assembly.

One of the most promising methods of unveiling the pharmacology of marketed drugs is to screen them against new biological targets. In an attempt to find inhibitors for acetylcholinesterase (AChE), the Drug Bank Database and natural alkaloids with other known medicinal values were screened through a four-point pharmacophore built in this study. The development of the pharmacophore was based on a structurally diverse set of reported AChE inhibitors and was validated using a separate set of known inhibitors. The developed pharmacophore indicated that the presence of one H-acceptor motif, one H-donor motif, one positively charged group and one aromatic ring is needed for AChE inhibition. Selected hits were further investigated by molecular docking and in vitro testing. The assays revealed that the majority of these compounds showed reasonable inhibition, indicating that the developed pharmacophore can indeed reliably screen molecules for potential AChE inhibitors. It appears that several commercially available marketed drugs have further potential as AChE inhibitors. To extend our study the same compounds have been tested in the fibrillogenesis inhibition of amyloidß (Aß) peptide to explore the possibility of their dual-function therapeutic activity in Alzheimer's disease.