DUF3328 is a protein family widely found in fungal natural product biosynthesis pathways. Although DUF3328 proteins have long been implicated in diverse modifications of inert C(sp3)─H bonds, including halogenation, hydroxylation, and macrocyclization, the biochemical properties and catalytic mechanisms of DUF3328 proteins remain elusive. Here, we report the characterization of the DUF3328 protein CctR, which catalyzes C(sp3)─H hydroxylation of fungal cyclic peptide cyclochlorotine. Through AlphaFold modeling, in vitro biochemical characterization, and spectroscopic analysis, we demonstrate that CctR is a membrane-associated copper-dependent enzyme that functions as a homodimer. The dimerization of CctR is mediated by its transmembrane helix, a four-helix coiled coil, and C-terminal disulfide bonds. The conserved HxxHC(x)nHxxHC motif, characteristic of the DUF3328 superfamily, is anchored on the dimerization interface and forms a binuclear copper coordination center. Moreover, we show that CctR is dioxygen-dependent and requires electron input for the hydroxylation reaction. Together, these findings define DUF3328 as a previously unrecognized family of binuclear copper-dependent metalloenzymes, capable of catalyzing diverse chemical transformations, and lay the groundwork for future discovery of novel biocatalysts within this widespread enzyme class.

Objectives: The effect of 2-hydroxpropyl-β-cyclodextrin (2HPβCD) with or without divalent metal ions (Ca2+, Mg2+, and Zn2+) on the stability of dalbavancin in acetate buffer was investigated. Methods: Dalbavancin recovery from formulations with 2HPβCD and divalent metal ions after four weeks of storage at 5 °C and 55 °C was measured by RP-HPLC and HP-SEC; a longer-term study was carried out over six months at 5 °C, 25 °C, and 40 °C. Binding of 2HPβCD was characterized by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). Results: The stability of the dalbavancin formulations after 4 weeks at 55 °C in 10 mM acetate buffer was significantly improved with 0.6 mM, 5.5 mM, and 55 mM 2HPβCD relative to without 2HPβCD. No further improvement was observed with the addition of any of the divalent cations. Dalbavancin in a 1:10 molar ratio with 2HPβCD was more stable at a concentration of 1 mg/mL than at 20 mg/mL under accelerated conditions at 40 °C for six months. ITC revealed two 2HPβCD binding sites to dalbavancin in 10 mM acetate: one with a 1:1 stoichiometry and thermodynamics consistent with known cyclodextrin–drug interactions, and a second with 0.1:1 stoichiometry, a positive binding enthalpy, and an unusually large entropy of binding. NMR spectroscopy indicates that dalbavancin exhibits aggregation in acetate buffer that is disrupted by 2HPβCD binding. Conclusions: 2HPβCD significantly improves the short- and long-term heat stability of dalbavancin in pH 4.5 acetate buffer at and above molar ratios of 1:1. The strong 1:1 binding of 2HPβCD to dalbavancin demonstrated by ITC confirms that this stability is conferred by the formation of a stable complex. This observation, combined with the NMR results, points to the aliphatic hydrocarbon chain of the glycone moiety as the most likely site of 2HPβCD–dalbavancin interaction.

The effect of monovalent (Na+ and K+) and divalent (Ca2+, Mg2+, and Zn2+) metal ions combined with citrate or acetate buffers (pH 4.5) on the stability of dalbavancin in aqueous solutions was investigated. RP-HPLC and HP-SEC were used to evaluate the stability of aqueous solutions of dalbavancin in different combinations of buffers and metal ions after four weeks of storage at 5°C and 55°C. A long-term study of formulations with divalent metal ions was conducted over six months at 5°C., 25°C and 40°C using RP-HPLC. All formulations in citrate buffered solutions precipitated. Dalbavancin solutions in 10 mM acetate buffer at 55°C were more stable in 10 mM CaCl2, 5 mM ZnCl2 and 10 mM MgCl2 than those containing 2 mM NaCl or 5 mM KCl, although the MgCl2 formulations precipitated slightly. No significant effect was observed for any of the divalent metal ions at 40°C for six months. Dalbavancin’s stability in solution was improved by a combination of acetate and divalent metal ions at 55°C for four weeks. No effect was observed with acetate or metal ions alone, and no effect was observed after six months at 40°C suggesting that acetate and divalent metal ions together interact with dalbavancin via a thermally activated step to inhibit hydrolysis of the drug.

Heat stress studies have been conducted in support of developing a heat-stable liquid solution of dalbavancin. The degradation products that form in heat-stressed buffered dalbavancin solutions have been identified, including the known major degradation product, mannosyl aglycone (MAG), and four previously uncharacterized compounds. Liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS) was used to identify the degradation products of dalbavancin in acetate- and phosphate-buffered solutions under thermal stress at 70 °C and the changes in the degradation pattern in the presence of 2HPβCD and divalent metal ions. Although Ca 2+ , Mg 2+ , and Zn 2+ did not reduce dalbavancin degradation under thermal stress in acetate buffer, 2HPβCD significantly reduced its overall degradation, in particular, the formation of MAG. This protective effect was enhanced by the addition of Ca 2+ to the formulation. In phosphate buffer, MAG formation was also reduced by the addition of 2HPβCD, although significant increases in other degradation products were observed in this case. The addition of Mg 2+ to 2HPβCD significantly reduced the overall degradation while increasing MAG formation somewhat. The results strongly suggest that 2HPβCD forms a complex with the hydrophobic glycone tail of dalbavancin, suppressing hydrolysis of the glycosidic bond.

Heisenberg spin exchange between nitroxide (Tempone) spin probes has been measured as a function of concentration in the aqueous phase of the hydrated ion exchange membrane Nafion 117. The observed fast-motional electron paramagnetic resonance spectra were analyzed in terms of the stochastic Liouville equation lineshape calculation of Freed and coworkers and the “new paradigm” for interpreting spin exchange effects proposed by Salikhov. Differences between the effective spin exchange measured from the spectrum by these methods are presented and compared, and indicate that dipolar interactions make a significant contribution to spin exchange in this system. In acidic Nafion membranes, the spin probes are deactivated over time, allowing simultaneous measurement of the decay kinetics and spin exchange as a function of paramagnetic probe concentration. Both these processes deviate from the behavior that would be expected from classical chemical kinetics in isotropic media. The results are discussed in terms of currently available models for diffusion and reaction in a percolation network.

New formulations of the glycopeptide drug dalbavancin containing 2-hydroxpropyl-β-cyclodextrin (2HPβCD) with or without divalent metal ions in phosphate buffer (pH 7.0) were tested to evaluate whether these excipients influence the aqueous solution stability of dalbavancin. Recovery of dalbavancin from phosphate buffered solutions at pH 7.0 with different concentrations of 2HPβCD and a divalent metal ion (Ca2+, Mg2+, or Zn2+) was evaluated by RP-HPLC and HP-SEC after four weeks of storage at 5°C and 55°C. A long-term study of formulations with 2HPβCD and Mg2+ was carried out over six months at 5°C, 25°C, and 40°C using RP-HPLC. Dalbavancin solutions with either 5.5 mM or 55 mM 2HPβCD were significantly more stable with Mg2+ than with the other divalent metal ions, both at 55°C for four weeks and at 40°C for six months. Dalbavancin was found to be more stable in aqueous solutions at a concentration of 1 mg/mL than at 20 mg/mL with 2HPβCD and Mg2+ at 40°C for six months. The results suggest that 2HPβCD forms an inclusion complex with dalbavancin that slows the formation of the major degradant, mannosyl aglycone (MAG). The effect of 2HPβCD is increased in the presence of Mg2+ and phosphate at pH 7.0, and the complex is more stable at a dalbavancin concentration of 1 mg/mL than at 20 mg/mL. These observations point towards the possibility of formulating a dalbavancin injection solution with a long shelf life at room temperature and physiological pH.

Historically, the primary result of an EPR experiment is the CW EPR spectrum, typically displayed as the first derivative of the absorption spectrum as a function of the magnetic field. Beyond very qualitative assessments, the detailed analysis of an experimental EPR spectrum is a difficult inverse problem. Given a set of parameters and a model, it is easy to calculate a spectrum, but given an EPR spectrum, it is a challenge to decide on the correct model and find all defining parameters of interest. Programs to simulate and fit CW EPR spectra have been around for a long time. Except for a very well-defined model system, an experimental spectrum of a spin labeled protein is typically a mix of multiple states. This article focuses on the analysis of the CW spectrum in several stages of detail, from qualitative to detailed. The use of the EPR lineshape fitting program MultiComponent developed in the Hubbell lab is used to illustrate common approaches to extract information relevant to protein structure, function, dynamics, and thermodynamics.

The degradation kinetics of the glycopeptide antibiotic dalbavancin in solution are systematically evaluated over the pH range 1–12 at 70°C. The decomposition rate of dalbavancin was measured as a function of pH, buffer composition, temperature, ionic strength, and drug concentration. A pH-rate profile was constructed using pseudo first-order kinetics at 70°C after correcting for buffer effects; the observed pH-rate profile could be fitted with standard pseudo first order rate laws. The degradation reactions of dalbavancin were found to be strongly dependent on pH and were catalyzed by protons or hydroxyl groups at extreme pH values. Dalbavancin shows maximum stability in the pH region 4–5. Based on the Arrhenius equation, dalbavancin solution at pH 4.5 is predicted to have a maximum stability of thirteen years under refrigerated conditions, eight months at room temperature and one month at 40°C. Mannosyl Aglycone (MAG), the major thermal and acid degradation product, and DB-R6, an additional acid degradation product, were formed in dalbavancin solutions at 70°C due to hydrolytic cleavage at the anomeric carbons of the sugars. Through deamination and hydrolytic cleavage of dalbavancin, a small amount of DB-Iso-DP2 (RRT-1.22) degradation product was also formed under thermal stress at 70°C. A greater amount of the base degradation product DB-R2 forms under basic conditions at 70°C due to epimerization of the alpha carbon of phenylglycine residue 3.

Glycopeptide antimicrobials are a class of naturally occurring or semi-synthetic glycosylated products that have shown antibacterial activity against gram-positive organisms by inhibiting cell-wall synthesis. In most cases, these drugs are prepared in dry powder (lyophilized) form due to chemical and physical instability in aqueous solution; however, from an economic and practical point of view, liquid formulations are preferred. Researchers have recently found ways to formulate some glycopeptide antibiotic therapeutic drugs in aqueous solution at refrigerated or room temperature. Chemical degradation can be significantly slowed by formulating them at a defined pH with specific buffers, avoiding oxygen reactive species, and minimizing solvent exposure. Sugars, amino acids, polyols, and surfactants can reduce physical degradation by restricting glycopeptide mobility and reducing solvent interaction. This review focuses on recent studies on glycopeptide antibiotic drug stability in aqueous solution. It is organized into three sections: (i) glycopeptide antibiotic instability due to chemical and physical degradation, (ii) strategies to improve glycopeptide antibiotic stability in aqueous solution, and (iii) a survey of glycopeptide antibiotic drugs currently available in the market and their stability based on published literature and patents. Antimicrobial resistance deaths are expected to increase by 2050, making heat-stable glycopeptides in aqueous solution an important treatment option for multidrug-resistant and extensively drug-resistant pathogens. In conclusion, it should be possible to formulate heat stable glycopeptide drugs in aqueous solution by understanding the degradation mechanisms of this class of therapeutic drugs in greater detail, making them easily accessible to developing countries with a lack of cold chains.

Controlling magnetization dynamics is imperative for developing ultrafast spintronics and tunable microwave devices. However, the previous research has demonstrated limited electric-field modulation of the effective magnetic damping, a parameter that governs the magnetization dynamics. Here, we propose an approach to manipulate the damping by using the large damping enhancement induced by the two-magnon scattering and a nonlocal spin relaxation process in which spin currents are resonantly transported from antiferromagnetic domains to ferromagnetic matrix in a mixed-phased metallic alloy FeRh. This damping enhancement in FeRh is sensitive to its fraction of antiferromagnetic and ferromagnetic phases, which can be dynamically tuned by electric fields through a strain-mediated magnetoelectric coupling. In a heterostructure of FeRh and piezoelectric PMN-PT, we demonstrated a more than 120% modulation of the effective damping by electric fields during the antiferromagnetic-to-ferromagnetic phase transition. Our results demonstrate an efficient approach to controlling the magnetization dynamics, thus enabling low-power tunable electronics.

Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD' dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuD', but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

Magnetoelectric effect, arising from the interfacial coupling between magnetic and electrical order parameters, has recently emerged as a robust means to electrically manipulate the magnetic properties in multiferroic heterostructures. Challenge remains as finding an energy efficient way to modify the distinct magnetic states in a reliable, reversible, and non-volatile manner. Here we report ferroelectric switching of ferromagnetic resonance in multiferroic bilayers consisting of ultrathin ferromagnetic NiFe and ferroelectric Pb0.92La0.08Zr0.52Ti0.48O3 (PLZT) films, where the magnetic anisotropy of NiFe can be electrically modified by low voltages. Ferromagnetic resonance measurements confirm that the interfacial charge-mediated magnetoelectric effect is dominant in NiFe/PLZT heterostructures. Nonvolatile modification of ferromagnetic resonance field is demonstrated by applying voltage pulses. The ferroelectric switching of magnetic anisotropy exhibits extensive applications in energy-efficient electronic devices such as magnetoelectric random access memories, magnetic field sensors, and tunable radio frequency (RF)/microwave devices.

Electric-field modulation of magnetism in strain-mediated multiferroic heterostructures is considered a promising scheme for enabling memory and magnetic microwave devices with ultralow power consumption. However, it is not well understood how electric-field-induced strain influences magnetic relaxation, an important physical process for device applications. Here, we investigate resonant magnetization dynamics in ferromagnet/ferroelectric multiferroic heterostructures, FeGaB/PMN-PT and NiFe/PMN-PT, in two distinct strain states provided by electric-field-induced ferroelectric phase transition. The strain not only modifies magnetic anisotropy but also magnetic relaxation. In FeGaB/PMN-PT, we observe a nearly two-fold change in intrinsic Gilbert damping by electric field, which is attributed to strain-induced tuning of spin-orbit coupling. By contrast, a small but measurable change in extrinsic linewidth broadening is attributed to inhomogeneous ferroelastic domain switching during the phase transition of the PMN-PT substrate.

We present a simple technique using a cavity-based resonance spectrometer to quantify the anti-damping torque due to the spin Hall effect. Modification of ferromagnetic resonance is observed as a function of small DC current in sub-mm-wide strips of bilayers, consisting of magnetically soft FeGaB and strong spin-Hall metal Ta. From the detected current-induced linewidth change, we obtain an effective spin Hall angle of 0.08–0.09 independent of the magnetic layer thickness. Our results demonstrate that a sensitive resonance spectrometer can be a general tool to investigate spin Hall effects in various material systems, even those with vanishingly low conductivity and magnetoresistance.

This chapter reviews the range of methods currently available for calculating the electron paramagnetic resonance (EPR) spectrum of nitroxide spin-labeled biomolecules undergoing slow motion. The two major approaches include the stochastic Liouville equation (SLE) which represents the spin label motion using diffusion operators, and the dynamic trajectory (DT) approach, which enables the EPR spectrum to be calculated from molecular dynamics simulations. The basic model parameters needed for each approach are described, together with a broad outline of the theoretical approaches underlying the methods, sufficient to allow their comparison for different applications. Hybrid methods utilizing a combination of SLE and DT approaches are briefly discussed.

This chapter reviews the range of methods currently available for calculating the electron paramagnetic resonance (EPR) spectrum of nitroxide spin-labeled biomolecules undergoing slow motion. The two major approaches include the stochastic Liouville equation (SLE) which represents the spin label motion using diffusion operators, and the dynamic trajectory (DT) approach, which enables the EPR spectrum to be calculated from molecular dynamics simulations. The basic model parameters needed for each approach are described, together with a broad outline of the theoretical approaches underlying the methods, sufficient to allow their comparison for different applications. Hybrid methods utilizing a combination of SLE and DT approaches are briefly discussed.

SUMMARY Despite the significance of fat-rich food/drug interactions to oral bioavailability, there is an incomplete understanding and a lack of general in vitro and theoretical models able to predict a priori the in vivo performance of drug–lipid systems. The aim of this study is to quantitatively investigate and model the effects of ingested lipids on dissolution, partitioning and absorption of orally delivered drugs by means of updated in vitro models incorporating simulated intestinal fluids and the lipid digestion process. Kinetics of drug dissolution and partitioning between colloidal phases (oil, micellar, aqueous) have been studied by high performance liquid chromatography (HPLC) and electron paramagnetic resonance (EPR), respectively. A cell-based in vitro model reproducing the absorption properties of the intestinal wall has been employed to assess drug permeability in the presence of ingested lipids. INTRODUCTION Ingested lipids could offer valuable opportunities for enabling oral drug delivery, as approximately 40-70% of all new drug candidates have been estimated to have very poor water solubility and are expected to exhibit low bioavailability when orally dosed. For hydrophobic drug molecules, the dissolution process in water is likely the limiting step of overall oral absorption. Therefore, the influence of ingested lipids on oral absorption has been associated with complex and poorly characterized interactions between drugs and multiple colloidal structures, which are naturally present in the gastrointestinal fluids after food intake. In particular, emulsion droplets, micelles and vesicles influence dissolution kinetics, and they are able to maintain larger quantities of hydrophobic drugs in solution, increasing the solubilization power of the gastrointestinal (GI) tract contents. Furthermore, the distribution of drugs within oil, aqueous and micellar phases affects their absorption rate. However, despite the recognized capability of ingested lipids to impact several processes associated with the overall absorption of hydrophobic drugs, the fate of coadministered drugs remains unclear and unpredictable. An updated in vitro lipid digestion model incorporating simulated intestinal fluids was developed and utilized to quantitatively investigate and model the impacts of ingested lipids on the multiple simultaneous key processes involved in oral drug absorption namely, lipid digestion, drug dissolution, drug partitioning and drug permeability. Kinetics of drug dissolution and partitioning between colloidal phases (oil, micellar, aqueous) have been measured by high performance liquid chromatography (HPLC) and electron paramagnetic resonance (EPR), respectively. The latter is a noninvasive technique that has the capability of detecting and quantifying radicals acting as spin probes. Since EPR spectra are highly sensitive to changes in environment micropolarity and microviscosity, the relocation of a selected spin probe within different phases can be monitored in real-time and quantified. Drug permeability has been determined by means of a cell culture model based on Caco-2/HT29-MTX (intestinal epithelial and mucus-producing) co-cultures. MODEL DEVELOPMENT A systems-based mathematical model was developed based on kinetics of parallel processes occurring in the gastrointestinal tract upon co-dosing a drug with lipids (namely drug dissolution in presence of colloidal species, drug partitioning into colloidal species, lipid digestion, and absorption), and relationships between kinetics and chemical composition/structures of colloidal species within intestinal contents. A film-equilibrium drug dissolution model was utilized to predict drug dissolution kinetics in simulated intestinal fluids with and without lipids. In the model, the influence of colloids interacting with drug on drug transport rates was explicitly taken into account (Figure 1). An appropriate kinetic expression for the lipid digestion process was developed correlating the amount of FA produced to the lipid droplet surface area available to the lipase enzyme. Drug permeability was modeled based on the drug flux between donor and acceptor compartments in sink conditions. The mathematical expressions used in the model were incorporated into MATLAB® code, and a fixed-step Runge-Kutta method with one-second time steps was used to integrate the differential equation describing simultaneous lipid digestion, drug dissolution, drug partitioning, and drug permeability over time. EXPERIMENTAL METHODS The proposed simulated intestinal fluids consists of a bio-relevant medium, reflecting GI contents in the fed state, which was prepared in maleate buffer at pH 6.5 and included lecithin 4 mM and sodium taurodeoxycholate 12 mM as a model of human bile. Soybean oil (50 mM) and lipase extract were added to account for the lipid intake and to start the lipolysis process. Since only paramagnetic molecules are visible to EPR, the spin probe TEMPOL benzoate (TB) was selected as a model for poorly watersoluble moderately lipophilic drug. In dissolution experiments, samples were collected at specific time intervals, and analyzed for drug concentration by means of HPLC. In EPR measurements, TB was dissolved in the bio-relevant medium with and without lipids and tracked between phases in real time without any sample processing. EPR spectra of TB in separate environments (maleate buffer, bio-relevant medium, soybean oil) were recorded at 37°C and used to obtain simulation parameters for resolving EPR multi-component spectra of pre and post-lipolysis samples. In drug transport studies, Caco-2 and HT29-MTX cells were co-cultured on a 24 plate Transwell® permeable support (0.4 um pore size) for 27 days. Apical compartment solution was replaced with intestinal simulating fluids (with and without model drug TB) to expose the cell monolayers for three hours inside an incubator at 37°C. Samples were collected from the basal compartments over time and analyzed for drug content using a UV spectrophotomer. Experimental lipid digestion profiles, drug dissolution, partitioning, and permeability were then compared with theoretical predictions. RESULTS AND DISCUSSION The kinetics of drug dissolution in maleate buffer, and in the bio-relevant medium with and without addition of lipids, was investigated by HPLC. The dissolution profile of TB in the bio-relevant medium showed 8-fold higher drug solubility and a faster dissolution rate with respect to the dissolution tests performed in maleate buffer. The presence of mixed micelle-forming species, bile salts and lecithin, greatly increased the solubilization power of the bio-relevant medium, in agreement with the predictions of the film-equilibrium drug dissolution model. During the lipolysis process, the dissolution profile of TB indicated a slower dissolution rate in the first 30 minutes of testing relative to dissolution in biorelevant medium without lipids or lipolysis, but 10-fold higher solubility. This behavior observed during lipolysis was due to partitioning processes occurring between the drug and oil droplets/vesicles/micelles, as supported by further investigation via EPR. In order to determine the partitioning of the drug model TB between aqueous, micellar, and oil phases, real-time compound tracking measurements in simulated intestinal fluids at pH 6.5 and 37°C were conducted by means of EPR. The recorded EPR spectra were complex, representing up to three different components (aqueous, oil, micellar), which were resolved and quantified by spectral simulation. In the EPR spectrum of TB dissolved in the bio-relevant medium, 85% of the TB molecules were associated with mixed micelles. After the addition of soybean oil, EPR spectra indicated increasing partitioning of TB into the oil phase (up to 30%), while the amount of TB associated with mixed micelles decreased to 67% over a time scale similar to that of drug dissolution (3 hours). Therefore, EPR analysis of simultaneous drug partitioning processes indicate that kinetics of partitioning of drug into the oil phase should be taken into account to properly model the impact of lipids on drug dissolution. Drug transport studies performed by means of Caco-2/HT29-MTX cocultures showed that presence of lipids could affect the drug permeability. In addition, the co-cultures employed here represented a more physiological in vitro absorption model as it included the mucus-producing goblet cell subline HT29-MTX. CONCLUSION This study represented an important step in the development of a systems-based model incorporating all the key processes involved in oral drug absorption – namely drug dissolution, drug partitioning, drug permeability, and lipid digestion. Such systems-based models might enable prediction of the fate of orally administered drugs during the lipid digestion process and provide insight into their anticipated overall effect on oral absorption. The ability to predict the impact of lipids on oral drug delivery would shed considerable light on the “food effect” on oral drug absorption, a phenomenon of tremendous significance to the pharmaceutical industry and drug development.

SUMMARY Predicting pharmacokinetic profiles of orally administered drugs requires detailed knowledge on the factors influencing their absorption in the intestine. In order to achieve this knowledge it is important to track the drug behavior starting from its first point of interaction with the gastrointestinal (GI) tract. The stomach is the first point of interest for orally delivered drugs to significantly interact with their environment. A thorough systematic characterization of the drug-digestive system interactions in the stomach post food ingestion has been performed in order to predict drug dissolution profiles in the presence of factors such as colloidal structural changes over time, drug partitioning into the different phases, and reaction rates of food components due to digestion. INTRODUCTION In addition to being the preferred method of drug administration, the oral route is also one of the most complicated paths from a pharmacokinetic point of view. Hence, understanding the phenomena that govern drug behavior in the GI tract is of the utmost importance. In particular, food is a crucial component that affects drug behavior significantly and ultimately bioavailability. However, its effect is not fully known to be able to quantitatively predict dosage needs. We have developed an in vitro dynamic system that mimics the digestion of food in the stomach. The system conditions have been carefully chosen based on available in.vivo literature data in humans. In an attempt to understand drug behavior in the stomach in the presence and absence of food we have chosen a poorly water-soluble model drug compound called TEMPO benzoate (logP = 2.5). Not only does this compound constitute an attractive model drug due to its hydrophobicity, it also contains an unpaired electron which allows the use of Electron Paramagnetic Resonance (EPR) for its detection. EPR is a non-invasive technique that provides crucial information on the molecule’s microenvironment, leading ultimately to information on the partitioning of the compound in real time in the colloidal structures formed and transformed as food undergoes digestion. Through the use of a reverse phase High Performance Liquid Chromatography (HPLC), we have measured the concentration of the drug compound dissolved over time with and without the concurrent digestion process. Given this information, a mathematical model is being developed that captures the phenomena occurring in the gastric phase following oral administration. EXPERIMENTAL METHODS To simulate gastric digestion in vitro, a thorough investigation of the gastric juice composition and of the concentration of the enzymes present in vivo was performed. The simulated gastric fluid was an aqueous solution consisting of salts at concentrations found in fasted state conditions in humans, specifically 67 mM NaCl, 13.9 mM KCl, and 0.6 mM CaCl2.[1] The enzymes used for digestion, namely pepsin and lipase, were from porcine gastric mucosa and from the Rhizopus Niveus fungus respectively as shown by Daikidou et al.[2] For the digestion experiments milk was chosen as a commonly used food model that is relatively simple to handle. To have a fully characterized system, we prepared a simplified simulated milk solution containing the three major ingredients in milk, lactose (53 g/L), casein (33 g/L), triolein (32 g/L) along with CaHPO4, a necessary compound that helps solubilize and stabilize the casein molecules into micellar structures as shown by De Kruif et al.[3] A 2 mg/mL concentration of TEMPO benzoate was added at the beginning of digestion in powder form after sieving at a 20 μm size cutoff. The samples taken at different time points were processed and then measured by HPLC to determine the drug’s concentration. The sizes of the structures over time were measured through Dynamic Light Scattering (DLS). The drug was introduced to the system simultaneously with the start of digestion. EPR was used to detect the percent of the drug that traveled to each phase over time. RESULTS AND DISCUSSION The dissolution results in our system in the presence of digestion are shown in Figure 1. Figure 1: Dissolution profile of TEMPO Benzoate (TB) in the presence of digestion vs. in a fasted state stomach The digestion process of milk causes some precipitation due to the isoelectric point of casein which is about 4. Hence, in the DLS results (Figure 2) the 7 min time point contains much larger particles. Figure 2: Mean diameter of casein micellar structures during digestion One limitation of the DLS is that it cannot detect particles larger than 1μm in diameter such as the oil droplets. However, they can be easily observed under a light microscope. Although the light microscope does not provide an accurate measurement for size determination, it is a good approximation. EPR results showed that 93% of the drug was found inside the oil droplets and 3% in the casein micelles during digestion. The values changed to 91% and 5% respectively towards the end of digestion. CONCLUSION In order to ultimately develop an absorption profile in the intestine a fully developed digestive system needs to be in place, starting from the stomach into the intestine. The digestion process in the stomach has been characterized in order to develop a systematic model that can predict drug behavior. This system will help obtain a representative starting material entering the intestine. A mathematical model is being developed that can predict the drug dissolution profile in the presence of milk digestion. REFERENCES 1. Di Maio, S. and R.L. Carrier, Gastrointestinal contents in fasted state and post-lipid ingestion: in vivo measurements and in vitro models for studying oral drug delivery. J Control Release, 2011. 151(2): p. 110-22. 2. Diakidou, A., et al., Simulation of gastric lipolysis and prediction of felodipine release from a matrix tablet in the fed stomach. Eur J Pharm Sci, 2009. 37(2): p. 133-40. 3. de Kruif, C.G., et al., Casein micelles and their internal structure. Advances in Colloid and Interface Science, 2012. 171–172(0): p. 36-52. ACKNOWLEDGMENTS This work was made possible by support from the National Institutes of Health. I would like to acknowledge Dr. Heather Clark for the use of DLS and Dr. Erin Cram for the use of the equipment to run PAGE gels for protein digestion quantification. 0.0 0.2 0.4 0.6 0.8 0 20 40 60 80 TB C on c ( m g/ m L) Time (min) During Digestion Fasted State 0 200 400 600 80

Titania nanotubes have the potential to be employed in a wide range of energy-related applications such as solar energy-harvesting devices and hydrogen production. As the functionality of titania nanostructures is critically affected by their morphology and crystallinity, it is necessary to understand and control these factors in order to engineer useful materials for green applications. In this study, electrochemically-synthesized titania nanotube arrays were thermally processed in inert and reducing environments to isolate the role of post-synthesis processing conditions on the crystallization behavior, electronic structure and morphology development in titania nanotubes, correlated with the nanotube functionality. Structural and calorimetric studies revealed that as-synthesized amorphous nanotubes crystallize to form the anatase structure in a three-stage process that is facilitated by the creation of structural defects. It is concluded that processing in a reducing gas atmosphere versus in an inert environment provides a larger unit cell volume and a higher concentration of Ti3+ associated with oxygen vacancies, thereby reducing the activation energy of crystallization. Further, post-synthesis annealing in either reducing or inert atmospheres produces pronounced morphological changes, confirming that the nanotube arrays thermally transform into a porous morphology consisting of a fragmented tubular architecture surrounded by a network of connected nanoparticles. This study links explicit data concerning morphology, crystallization and defects, and shows that the annealing gas environment determines the details of the crystal structure, the electronic structure and the morphology of titania nanotubes. These factors, in turn, impact the charge transport and consequently the functionality of these nanotubes as photocatalysts.