Strategies to Stabilize Dalbavancin in Aqueous Solutions; Section-1: the Effects of Metal Ions and Buffers.

PURPOSE: 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. METHOD: 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. RESULTS: 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. CONCLUSION: 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.

Strategies To Stabilize Dalbavancin in Aqueous Solutions; Section 3: The Effects of 2 Hydroxypropyl-ß-Cyclodextrin and Phosphate Buffer with and without Divalent Metal Ions.

PURPOSE: New formulations of the glycopeptide drug dalbavancin containing 2-hydroxpropyl-ß-cyclodextrin (2HPßCD) 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. METHOD: Recovery of dalbavancin from phosphate buffered solutions at pH 7.0 with different concentrations of 2HPßCD 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 2HPßCD and Mg2+ was carried out over six months at 5°C, 25°C, and 40°C using RP-HPLC. RESULTS: Dalbavancin solutions with either 5.5 mM or 55 mM 2HPßCD 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 2HPßCD and Mg2+ at 40°C for six months. CONCLUSION: The results suggest that 2HPßCD forms an inclusion complex with dalbavancin that slows the formation of the major degradant, mannosyl aglycone (MAG). The effect of 2HPßCD 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.

Effect of ingested lipids on drug dissolution and release with concurrent digestion: a modeling approach.

PURPOSE: To mechanistically study and model the effect of lipids, either from food or self-emulsifying drug delivery systems (SEDDS), on drug transport in the intestinal lumen. METHODS: Simultaneous lipid digestion, dissolution/release, and drug partitioning were experimentally studied and modeled for two dosing scenarios: solid drug with a food-associated lipid (soybean oil) and drug solubilized in amodel SEDDS (soybean oil and Tween 80 at 1:1 ratio). Rate constants for digestion, permeability of emulsion droplets, and partition coefficients in micellar and oil phases were measured, and used to numerically solve the developed model. RESULTS: Strong influence of lipid digestion on drug release from SEDDS and solid drug dissolution into food-associated lipid emulsion was observed and predicted by the developed model. Ninety minutes after introduction of SEDDS, there was 9% and 70% drug release in the absence and presence of digestion, respectively. However, overall drug dissolution in the presence of food-associated lipids occurred over a longer period than without digestion. CONCLUSION: A systems-based mechanistic model incorporating simultaneous dynamic processes occurring upon dosing of drug with lipids enabled prediction of aqueous drug concentration profile. This model, once incorporated with a pharmacokinetic model considering processes of drug absorption and drug lymphatic transport in the presence of lipids, could be highly useful for quantitative prediction of impact of lipids on bioavailability of drugs.

A Systematic Degradation Kinetics Study of Dalbavancin Hydrochloride Injection Solutions.

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.

Synthesis of a spin-labeled anti-estrogen as a dynamic motion probe for the estrogen receptor ligand binding domain.

The preparation and characterization of a novel nitroxide spin probe based on a steroidal anti-estrogen is described. The probe 5 demonstrated very high binding affinity for both the alpha and beta isoforms of the estrogen receptor-ligand binding domain. EPR spectrometric studies demonstrate conformational constraints for the ligand, consistent with the nitroxyl moiety occupying a position just beyond the receptor-solvent interface.

Glycopeptide antibiotic drug stability in aqueous solution.

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.

Significantly improved sensitivity of Q-band PELDOR/DEER experiments relative to X-band is observed in measuring the intercoil distance of a leucine zipper motif peptide (GCN4-LZ).

Pulsed electron double resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin-labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, we present for the first time a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and higher-quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.

Dynamic conformational responses of a human cannabinoid receptor-1 helix domain to its membrane environment.

The influence of membrane environment on human cannabinoid 1 (hCB(1)) receptor transmembrane helix (TMH) conformational dynamics was investigated by solid-state NMR and site-directed spin labeling/EPR with a synthetic peptide, hCB(1)(T377-E416), corresponding to the receptor's C-terminal component, i.e., TMH7 and its intracellular alpha-helical extension (H8) (TMH7/H8). Solid-state NMR experiments with mechanically aligned hCB(1)(T377-E416) specifically (2)H- or (15)N-labeled at Ala380 and reconstituted in membrane-mimetic dimyristoylphosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) bilayers demonstrate that the conformation of the TMH7/H8 peptide is more heterogeneous in the thinner DMPC bilayer than in the thicker POPC bilayer. As revealed by EPR studies on hCB(1)(T377-E416) spin-labeled at Cys382 and reconstituted into the phospholipid bilayers, the spin label partitions actively between hydrophobic and hydrophilic environments. In the DMPC bilayer, the hydrophobic component dominates, regardless of temperature. Mobility parameters (DeltaH(0)(-1)) are 0.3 and 0.73 G for the peptide in the DMPC or POPC bilayer environment, respectively. Interspin distances of doubly labeled hCB(1)(T377-E416) peptide reconstituted into a TFE/H(2)O mixture or a POPC or DMPC bilayer were estimated to be 10.6 +/- 0.5, 16.8 +/- 1, and 11.6 +/- 0.8 A, respectively. The extent of coupling (>or=50%) between spin labels located at i and i + 4 in a TFE/H(2)O mixture or a POPC bilayer is indicative of an alpha-helical TMH conformation, whereas the much lower coupling (14%) when the peptide is in a DMPC bilayer suggests a high degree of peptide conformational heterogeneity. These data demonstrate that hCB(1)(T377-E416) backbone dynamics as well as spin-label rotameric freedom are sensitive to and altered by the peptide's phospholipid bilayer environment, which exerts a dynamic influence on the conformation of a TMH critical to signal transmission by the hCB(1) receptor.

Molecular-scale force measurement in a coiled-coil peptide dimer by electron spin resonance.

A new method for measuring forces between small protein domains based on double electron-electron resonance (DEER) spectroscopy is demonstrated using a model peptide derived from the alpha-helical coiled-coil leucine zipper of yeast transcriptional activator GCN4. The equilibrium distribution of distances between two nitroxide spin labels rigidly attached to the helices of the dimer was determined by DEER and yielded a closing force of 100 +/- 10 pN between monomers, in excellent agreement with theoretical predictions.

Investigation of water and methanol sorption in monovalent- and multivalent-ion-exchanged nafion membranes using electron spin resonance.

Electron spin resonance (ESR) spectroscopy was used to monitor the local environment of 2,2,6,6-tetramethyl-4-piperidone N-oxide (TEMPONE) spin probe in Li(+), Ca(2+), and Al(3+) ion-exchanged Nafion 117 membranes swollen with mixed methanol/water solvent at varying compositions. The (14)N hyperfine splitting, a(N), which reflects the local polarity of the nitroxide probe, remains nearly steady at higher solvent contents but increases substantially at lower solvent contents, reflecting close contact with the ions. The rotational rate (R) of the probe increased with solvent content, depending strongly on the amount of solvent at low contents but increasing more gradually at higher solvent contents, similar to the behavior of previously measured solvent translation diffusion coefficients. The rotational rate data from water-containing membranes were fitted using the Fujita free-volume diffusion model, which indicated that multivalent ions tend to increase the free volume fraction of the polymer while decreasing that of the solvent phase. Methanol-containing membranes exhibited greater variation with different exchange ions, but the data could not be fit using the free-volume model, suggesting that the assumption of two phases underlying the free-volume model might not apply to this case. The difference in the trends of swelling between water and methanol is consistent with previous results that have indicated different patterns of penetration for the two solvents. The results are interpreted in terms of changes in membrane morphology with higher-valence ions.

Electron spin resonance investigation of microscopic viscosity, ordering, and polarity in Nafion membranes containing methanol-water mixtures.

Electron spin resonance (ESR) was used to monitor the local environment of 2,2,6,6-tetramethyl-4-piperidone N-oxide (Tempone) spin probe in water and methanol mixtures in solution and in Li(+) ion exchanged Nafion 117 membranes. Solution spectra were analyzed using the standard fast-motion line width parameters, while membrane spectra were fitted using the microscopic order macroscopic disorder (MOMD) slow-motional line shape program of Freed and co-workers. The (14)N hyperfine splitting, aN, which reflects the local polarity of the nitroxide probe, decreases with increasing methanol concentration, consistent with the decrease in solvent polarity. The polarity depended only weakly on composition in the Nafion membrane, but was noticeably more temperature-dependent. The microviscosity of the membrane aqueous phase as reflected by the rotational correlation time (tauc) of the probe, was nearly 2 orders of magnitude longer in the membrane than in solution and varied by an order of magnitude over the composition range studied. The probe exhibits significant local ordering in the aqueous phase of Nafion membranes that is diminished with increasing methanol concentration.

Effect of sorbed methanol, current, and temperature on multicomponent transport in nafion-based direct methanol fuel cells.

The CO2 in the cathode exhaust of a liquid feed direct methanol fuel cell (DMFC) has two sources: methanol diffuses through the membrane electrode assembly (MEA) to the cathode where it is catalytically oxidized to CO2; additionally, a portion of the CO2 produced at the anode diffuses through the MEA to the cathode. The potential-dependent CO2 exhaust from the cathode was monitored by online electrochemical mass spectrometry (ECMS) with air and with H2 at the cathode. The precise determination of the crossover rates of methanol and CO2, enabled by the subtractive normalization of the methanol/air to the methanol/H2 ECMS data, shows that methanol decreases the membrane viscosity and thus increases the diffusion coefficients of sorbed membrane components. The crossover of CO2 initially increases linearly with the Faradaic oxidation of methanol, reaches a temperature-dependent maximum, and then decreases. The membrane viscosity progressively increases as methanol is electrochemically depleted from the anode/electrolyte interface. The crossover maximum occurs when the current dependence of the diffusion coefficients and membrane CO2 solubility dominate over the Faradaic production of CO2. The plasticizing effect of methanol is corroborated by measurements of the rotational diffusion of TEMPONE (2,2,6,6-tetramethyl-4-piperidone N-oxide) spin probe by electron spin resonance spectroscopy. A linear inverse relationship between the methanol crossover rate and current density confirms the absence of methanol electro-osmotic drag at concentrations relevant to operating DMFCs. The purely diffusive transport of methanol is explained in terms of current proton solvation and methanol-water incomplete mixing theories.

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

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.

Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides.

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.

Statistical criteria for the identification of protein active sites using Theoretical Microscopic Titration Curves.

Theoretical Microscopic Titration Curves (THEMATICS) may be used to identify chemically important residues in active sites of enzymes by characteristic deviations from the normal, sigmoidal Henderson-Hasselbalch titration behavior. Clusters of such deviant residues in physical proximity constitute reliable predictors of the location of the active site. Originally the residues with deviant predicted behavior were identified by human observation of the computed titration curves. However, it is preferable to select the unusual residues by mathematically well-defined criteria, in order to reduce the chance of error, eliminate any possible biases, and substantially speed up the selection process. Here we present some simple statistical tests that constitute such selection criteria. The first derivatives of the predicted titration curves resemble distribution functions and are normalized. The moments of these first derivative functions are computed. It is shown that the third and fourth moments, measures of asymmetry and kurtosis, respectively, are good measures of the deviations from normal behavior. Results are presented for 44 different enzymes. Detailed results are given for 4 enzymes with 4 different types of chemistry: arginine kinase from Limulus polyphemus (horseshoe crab); beta-lactamase from Escherichia coli; glutamate racemase from Aquifex pyrophilus; and 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans. The relationship between the statistical measures of nonsigmoidal behavior in the predicted titration curves and the catalytic activity of the residue is discussed.

CW-EPR Spectral Simulations: Slow-Motion Regime.

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.

Toward Enediyne Mimics: Methanolysis of Azoesters and a Bisazoester.

Enediyne anticancer antibiotics have attracted tremendous interest in the past decade. The inherent difficulty in synthesizing these structurally complex natural products with the strained enediyne moiety has motivated a search for simpler molecules that mimic enediyne chemistry. The ultimate objective is to identify molecules that produce 1,4-benzenoid diradicals, which are known to induce DNA cleavage in the natural products. Toward this goal, several aromatic azoesters have been synthesized, and EPR reveals the presence of radical intermediates in their methanolysis. A 1,4-bisazoester has also been synthesized, and its methanolysis products have been studied by reversed-phase HPLC. The formation of 1,2-dicyanobenzene from the 1,4-bisazoester is consistent with the existence of a 1,4-diradical intermediate.

Multifrequency electron spin resonance detection of solid-state organic free radicals in HCN polymer and a Titan tholin.

Macromolecules derived from hydrogen cyanide (HCN) may be major components of the dark matter observed in bodies in the outer Solar System, which include comets and asteroids. HCN oligomers and polymers are readily formed at room temperature and react with water to produce polypeptides and alpha-amino acids or undergo pyrolysis to produce nitrogen heterocycles. Electron spin resonance (ESR) spectroscopy shows that HCN polymer mixtures contain a significant amount of long-lived organic free radicals that are primarily carbon-based. For comparison, we have also examined samples of tholins produced from experimental analogs of Titan aerosols, which has been shown by trace organic analysis to consist partly of HCN polymer. The "Titan tholin" exhibits at least two ESR signals that can be assigned to nitrogen- and carbon-centered radicals, although heating the sample eliminates the nitrogen centers and increases the signal from the carbon centers. This result suggests that the nitrogen-centered radicals may be thermodynamically less stable, but are kinetically trapped during the spark-discharge reactions that produce tholins from mixtures of gases such as methane and nitrogen. The results strongly support previous proposals of free radical mechanisms for HCN polymerization.

Electron spin labeling reveals the highly dynamic N-terminal arms of the SOS mutagenesis protein UmuD.

Electron paramagnetic resonance (EPR) spectroscopy was used to probe the conformational dynamics of the N-terminal arms of the umuD gene products. We determined that the arms of UmuD(2) display a large degree of motion, are largely unbound from the globular C-terminal domain, and that the free energy of dissociation is +2.1 kJ mol(-1).

Modeling the human intestinal mucin (MUC2) C-terminal cystine knot dimer.

Intestinal mucus, a viscous secretion that lines the mucosa, is believed to be a barrier to absorption of many therapeutic compounds and carriers, and is known to play an important physiological role in controlling pathogen invasion. Nevertheless, there is as yet no clear understanding of the barrier properties of mucus, such as the nature of the molecular interactions between drug molecules and mucus components as well as those that govern gel formation. Secretory mucins, large and complex glycoprotein molecules, are the principal determinants of the viscoelastic properties of intestinal mucus. Despite the important role that mucins play in controlling transport and in diseases such as cystic fibrosis, their structures remain poorly characterized. The major intestinal secretory mucin gene, MUC2, has been identified and fully sequenced. The present study was undertaken to determine a detailed structure of the cysteine-rich region within the C-terminal end of human intestinal mucin (MUC2) via homology modeling, and explore possible configurations of a dimer of this cysteine-rich region, which may play an important role in governing mucus gel formation. Based on sequence-structure alignments and three-dimensional modeling, a cystine knot tertiary structure homologous to that of human chorionic gonadotropin (HCG) is predicted at the C-terminus of MUC2. Dimers of this C-terminal cystine knot (CTCK) were modeled using sequence alignment based on HCG and TGF-beta, followed by molecular dynamics and simulated annealing. Results support the formation of a cystine knot dimer with a structure analogous to that of HCG.