Oxidation-Induced Destabilization of Model Antibody-Drug Conjugates

Oxidation of biopharmaceutics represents a major degradation pathway, which may impact bioactivity, serum half-life, and colloidal stability. This study focused on the quantification of oxidation and its effects on structure and colloidal stability for a model antibody and its lysine (ADC-L) and cysteine (ADC-C) conjugates. The effects of oxidation were evaluated by a forced degradation study using H₂O₂ and a shelf-life simulation, which used degrading polysorbate 80 as source for reactive oxygen species. Differential scanning fluorimetry revealed decreasing transition temperatures of the CH₂ domain with rising oxidation, resulting in a loss of colloidal stability as assessed by size-exclusion high pressure liquid chromatography. The conjugation technique influences structural changes of the monoclonal antibody (mAb) and subsequently alters the impact of oxidation. ADC-C was most effected by oxidation as the CH₂ domain showed the biggest destabilization on conjugation compared to the mAb and ADC-L. Quantification of Fc methionine oxidation by analytical protein A chromatography revealed 4-fold higher oxidation after 8 weeks for the ADC-C compared to the mAb. Payload degradation was observed independently of the conjugation technique used or if free in solution by ultraviolet-visible. In addition, adding antioxidants can be a suitable approach to prevent oxidation and achieve baseline stabilization of the proteins.     

Quantitative Prediction of Oral Bioavailability of a Lipophilic Antineoplastic Drug Bexarotene Administered in Lipidic Formulation Using a Combined In Vitro Lipolysis/Microsomal Metabolism Approach

For performance assessment of the lipid-based drug delivery systems (LBDDSs), in vitro lipolysis is commonly applied because traditional dissolution tests do not reflect the complicated in vivo micellar formation and solubilization processes. Much of previous research on in vitro lipolysis has mostly focused on rank-ordering formulations for their predicted performances. In this study, we have incorporated in vitro lipolysis with microsomal stability to quantitatively predict the oral bioavailability of a lipophilic antineoplastic drug bexarotene (BEX) administered in LBDDS. Two types of LBDDS were applied: lipid solution and lipid suspension. The predicted oral bioavailability values of BEX from linking in vitro lipolysis with microsomal stability for lipid solution and lipid suspension were 34.2 ± 1.6% and 36.2 ± 2.6%, respectively, whereas the in vivo oral bioavailability of BEX was tested as 31.5 ± 13.4% and 31.4 ± 5.2%, respectively. The predicted oral bioavailability corresponded well with the oral bioavailability for both formulations, demonstrating that the combination of in vitro lipolysis and microsomal stability can quantitatively predict oral bioavailability of BEX. In vivo intestinal lymphatic uptake was also assessed for the formulations and resulted in <1% of the dose, which confirmed that liver microsomal stability was necessary for correct prediction of the bioavailability.     

Large effect of membrane tension on the fluid–solid phase transitions of two-component phosphatidylcholine vesicles

Model phospholipid membranes and vesicles have long provided insight into the nature of confined materials and membranes while also providing a platform for drug delivery. The rich thermodynamic behavior and interesting domain shapes in these membranes have previously been mapped in extensive studies that vary temperature and composition; however, the thermodynamic impact of tension on bilayers has been restricted to recent reports of subtly reduced fluid–fluid transition temperatures. In two-component phosphatidylcholine unilamellar vesicles [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)], we report a dramatic influence of tension on the fluid–solid transition and resulting phases: At fixed composition, systematic variations in tension produce differently shaped solid domains (striped or irregular hexagons), shift fluid–solid transition temperatures, and produce a triple-point–like intersection of coexistence curves at elevated tensions, about 3 mN/m for 30% DOPC/70% DPPC. Tension therefore represents a potential switch of microstructure in responsive engineered materials; it is an important morphology-determining variable in confined systems, and, in biological membranes, it may provide a means to regulate dynamic structure.Phospholipid vesicles, capsular lamellar assemblies of phospholipid amphiphiles, are model systems that have advanced our perceptions of material surfaces and thin films, facilitated drug delivery technologies, and anchored the understanding of biological membranes to fundamental physics. Extensive studies of phase transitions in phospholipid bilayers and vesicles have focused almost exclusively on temperature and composition, revealing complex phase behavior (1–6) and beautiful patterns in the domain shapes within vesicle membranes (4, 6, 7). Tension has been mostly neglected as a thermodynamic variable and is unspecified in phase diagrams of vesicle membranes, although in analogous studies of phospholipid monolayers, surface pressure is known to drive transitions between gas-like layers, liquid fluids, and ordered crystals, which are sometimes polymorphic (8–10). Besides its fundamental thermodynamic importance, membrane tension may be biologically important, because stresses on cells can dominate their interactions (11–14) and fates (15). Indeed, tension has been proposed to regulate the dynamic structure of the cellular surface, for instance through coupling with curvature (16) or by clustering proteins in “rafts” (17, 18).Used as a mechanical variable, tension can stretch or bend uniform bilayers (19, 20). In multicomponent vesicles containing coexisting fluid domains, coupling of line tension with membrane bending determines vesicle shapes and drives budding transitions (21–24). Tension has also been hypothesized, but not confirmed, to influence the shapes of solid domains (25). Relevant to the current focus on the thermodynamic role of tension, tension was shown reduce the liquid–liquid coexistence temperature only slightly (a fraction of a Celsius degree for each 0.1 mN/m in tension) in cholesterol-containing phospholipid vesicles (26, 27).In the current work, we systematically examine the broader thermodynamic impact of membrane tension on the phase behavior of two-component phosphatidylcholine (PC) lipid vesicles: PC amphiphiles are an important class of molecules because of their prominence in cell membranes. Important to note, their relatively large hydrated head groups enable PC lipids, in single-component membranes, to order into solid bilayers of differing molecular areas [by about 10% (19)], such as ripple (P_β′) and tilt (“gel” or L_β′) phases (19, 28–31).To quantitatively probe the impact of membrane tension on the fluid–solid transition(s) of two-component PC bilayers, giant unilamellar vesicles containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were chosen as model membranes. This work focuses on the impact of tension and temperature at the single-membrane composition of 30 mol% DOPC/70 mol% DPPC, taking a constant-composition slice through thermodynamic space, which qualitatively reflects behaviors at other compositions. Well-hydrated DOPC lamellae melt at Tm = −17 ± 1 °C (32), whereas hydrated DPPC bilayers have a higher main transition Tm = 41.5 ± 0.5 °C and a so-called “pretransition” at Tp = 35.5 ± 0.5 °C (28, 33). The ripple solid is found below the main transition temperature, whereas a tilt gel phase is observed below the pretransition temperature; however, many studies of DPPC-containing mixtures cannot distinguish these two solids (6, 34). Cooling mixed vesicles from the miscible fluid regime at elevated temperatures produces solid-like membrane domains comprised predominantly of DPPC (3, 34, 35). Indeed, cooling from the one phase region is the typical protocol for reliably creating solid domains and measuring transition temperatures.We report here that in two-component phosphatidylcholine membranes, even though the main fluid–solid transition temperature is only mildly reduced with increased tension, consistent with first principles, tension alters the equilibrium and the nature of the solid domains within the fluid membrane. The latter include the molecular ordering and shapes of the domains. Additionally, by shifting a second coexistence line in the opposite direction from the main transition curve, tension produces a triple-point–like feature at their intersection. Tension can thus be manipulated to select which solid phase forms on cooling, in turn directing domain morphology.     

Multiple patterns of polymer gels in microspheres due to the interplay among phase separation,wetting, and gelation

We report the spontaneous patterning of polymer microgels by confining a polymer blend within microspheres. A poly(ethylene glycol) (PEG) and gelatin solution was confined inside water-in-oil (W/O) microdroplets coated with a layer of zwitterionic lipids: dioleoylphosphatidylethanolamine (PE) and dioleoylphosphatidylcholine (PC). The droplet confinement affected the kinetics of the phase separation, wetting, and gelation after a temperature quench, which determined the final microgel pattern. The gelatin-rich phase completely wetted to the PE membrane and formed a hollow microcapsule as a stable state in the PE droplets. Gelation during phase separation varied the relation between the droplet size and thickness of the capsule wall. In the case of the PC droplets, phase separation was completed only for the smaller droplets, wherein the microgel partially wetted the PC membrane and had a hemisphere shape. In addition, the temperature decrease below the gelation point increased the interfacial tension between the PEG/gelatin phases and triggered a dewetting transition. Interestingly, the accompanying shape deformation to minimize the interfacial area was only observed for the smaller PC droplets. The critical size decreased as the gelatin concentration increased, indicating the role of the gel elasticity as an inhibitor of the deformation. Furthermore, variously patterned microgels with spherically asymmetric shapes, such as discs and stars, were produced as kinetically trapped states by regulating the incubation time, polymer composition, and droplet size. These findings demonstrate a way to regulate the complex shapes of microgels using the interplay among phase separation, wetting, and gelation of confined polymer blends in microdroplets.The regulation of the 3D shapes of biopolymer gels at the mesoscale has numerous applications in the biomedical, cosmetic, and food materials industries, among others (1). Recently, top-down and bottom-up approaches have been reported to control the mesoscopic patterns of polymer gels. For example, photolithography and two-photon polymerization allow the regulation of gel patterns at the mesoscale (2–4). The advanced design of the molecules enables polymerization with a self-assembly and produces nonspherical microgels: spherical particles with a cavity, capsules, and cubic particles (5–7). However, these methods require highly specialized equipment and are generally difficult to adapt for biopolymer gels.Dynamical coupling between phase separation and sol–gel transition in polymer blends has also been investigated for the spontaneous formation of spherical microgels and a porous gel (8, 9). Ma et al. (10) and Choi et al. (11) confined aqueous two-phase systems (ATPSs) in microdroplets and fabricated microgels by selective polymerization. In such a confined space, phase separation accompanies wetting of a polymer to the substrate (12–15). Although the selective polymerization of phase-separated polymers in microdroplets has a great potential to produce variously shaped microgels, the dynamical coupling among the phase separation, wetting, and gelation of polymers in a confined space remains unclear (16). If it was better understood, the shapes of polymer microgels could be regulated in a self-organized manner.In the present work, we used gelatin, one of the most popular biopolymer gels, and poly(ethylene glycol) (PEG) as the desolvating agent because PEG leads to phase separation for various biopolymers, such as proteins and DNA (17). The gelatin/PEG solution was confined in water-in-oil (W/O) microdroplets coated by a lipid layer, wherein the phase separation and sol–gel transition of the gelatin occur with a decrease in the temperature (18–20). This process led to gelation after and during the phase separation in the presence of the interactions between the polymers and lipid membranes. We analyzed the pattern formation of the gelatin microgel as a function of the temperature history, droplet size, and polymer composition. We found that variously shaped microgels appeared as stable states and kinetically trapped states. These findings yield a method to regulate the shapes of polymer microgels using the interplay among the interfacial tensions, elastic properties of the gels, and interactions between the polymers and the surfaces of the droplets.     

中枢神经系统感染患儿脑脊液中利奈唑胺浓度测定方法的初步研究

目的：建立测定利奈唑胺在中枢神经系统感染患儿脑脊液中浓度的高效液相色谱法（HPLC）。方法：采用Syncronis C18柱（5 μm,4.6 mm×250 mm）;流动相为乙腈:水（含0.2%磷酸）=27∶73;流速1.0 mL/min;检测波长254 nm;柱温30 ℃。结果：利奈唑胺在脑脊液浓度为0.2~40 μg/mL范围内浓度和峰面积线性关系良好,标准曲线为As=0.553 9C-0.019 3（r=0.999 9,n=5）。最低定量限、低浓度、中浓度、高浓度的日内和日间变异RSD均<9%,准确度范围为99%~112%,提取回收率在85%~107%。稳定性考察RSD<8%。结论：该方法准确性高、灵敏度好且操作简便,适用于监测利奈唑胺在中枢神经系统感染患儿脑脊液中的浓度,进而为利奈唑胺的个体化用药提供依据。     

Serum Levels of Alpha1-antitrypsin and Their Relationship With COPD in the General Spanish Population

BackgroundLow plasma level of alpha1-antitrypsin (AAT) is an established risk factor for early-onset chronic obstructive lung disease (COPD). However, less attention is given to the levels of AAT in the general population.MethodsThis is a part of a multicentre, population-based study conducted at 11 sites throughout Spain. Plasma levels of AAT were available for 837 persons with a mean (SD) age of 58.05 (11.3) years: 328-smokers, 272-ex-smokers and 237 non-smokers. Out of 837, 303 (36.2%) had a diagnosis of COPD, 222 (26.5%) had respiratory symptoms but no COPD, and 312 (37.3%) were healthy controls.ResultsIn the whole cohort, the mean level of plasma AAT was 1.51 (0.47) g/L. Levels were higher in COPD patients [1.55 (0.45) g/L] and individuals with respiratory symptoms [1.57 (0.47) g/L] than in controls [1.43 (0.47) g/L], p < 0.001, a finding which persisted after correction for age and CRP. Plasma AAT levels were negatively associated with FEV1/FVC ratio, after adjustment for age, sex, smoking status, CRP, TNFα, fibrinogen and albumin. The risk for COPD was significantly associated with higher AAT levels in univariate and multivariate models, with odds ratios of 1.8 and 1.5, respectively. In the univariate and multivariate models smoking status, gender, and CRP levels were also associated with COPD probability, demonstrating that they act independently.ConclusionIncreased circulating levels of AAT, similarly to CRP and other markers of systemic inflammation, is an important feature of COPD. Our results highlight a complex interrelationship between levels of AAT and health of respiratory system.     

Trying to fall asleep while catastrophising: what sleep-disordered adolescents think and feel

ObjectiveCatastrophising is a repetitive cognitive process related to sleep disturbance in adult insomnia patients. More recently catastrophising has been associated with increased sleep disturbances in community samples of children and adolescents, with this association mediated by anxiety and depression. However, there currently is no evidence of these processes outside of community samples; impeding our ability to draw clinical conclusions. Knowledge on such dysfunctional cognitive processes in adolescents experiencing sleep disturbance would be clinically beneficial in aetiology and intervention. Our research examined the link between catastrophising, anxiety, depression and sleep latency in a sample of sleep-disordered adolescents. We also explored specific catastrophising themes which may impact the sleep latency of these adolescents.MethodForty adolescents (age = 15.1 ± 1.5 years, 53% boys) diagnosed with delayed sleep phase disorder completed a 7-day sleep diary, along with measures of anxiety and depression and a catastrophising interview with a trained sleep therapist.ResultsSeveral catastrophisation themes were generated, the most common concerning interpersonal and performance aspects of school. Bootstrapping analyses showed depression did not mediate the relationship between catastrophising and sleep; however, an indirect relationship was found between catastrophising, anticipatory anxiety, and sleep latency.ConclusionThese findings have implications for the role of dysfunctional thinking in prolonging sleep onset for adolescents and providing a clinical framework for health professionals when assessing and treating adolescents with delayed sleep timing.     

S-(−)equol producing status not associated with breast cancer risk among low isoflavone-consuming US postmenopausal women undergoing a physician-recommended breast biopsy

Soy foods are the richest sources of isoflavones, mainly daidzein and genistein. Soy isoflavones are structurally similar to the steroid hormone 17β-estradiol and may protect against breast cancer. S-(−)equol, a metabolite of the soy isoflavone daidzein, has a higher bioavailability and greater affinity for estrogen receptor β than daidzein. Approximately one-third of the Western population is able to produce S-(−)equol, and the ability is linked to certain gut microbes. We hypothesized that the prevalence of breast cancer, ductal hyperplasia, and overall breast pathology will be lower among S-(−)equol producing, as compared with nonproducing, postmenopausal women undergoing a breast biopsy. We tested our hypothesis using a cross-sectional study design. Usual diets of the participants were supplemented with 1 soy bar per day for 3 consecutive days. Liquid chromatography–multiple reaction ion monitoring mass spectrometry analysis of urine from 143 subjects revealed 25 (17.5%) as S-(−)equol producers. We found no statistically significant associations between S-(−)equol producing status and overall breast pathology (odds ratio [OR], 0.68; 95% confidence interval [CI], 0.23-1.89), ductal hyperplasia (OR, 0.84; 95% CI, 0.20-3.41), or breast cancer (OR, 0.56; 95% CI, 0.16-1.87). However, the mean dietary isoflavone intake was much lower (0.3 mg/d) than in previous reports. Given that the amount of S-(−)equol produced in the gut depends on the amount of daidzein exposure, the low soy intake coupled with lower prevalence of S-(−)equol producing status in the study population favors toward null associations. Findings from our study could be used for further investigations on S-(−)equol producing status and disease risk.     

Summary of knowledge gaps related to quality and efficacy of current influenza vaccines

Influenza viruses are a public health threat, as they are pathogenic, highly transmissible and prone to genetic changes. For decades vaccination strategies have been based on trivalent inactivated vaccines, which are regulated by specific guidelines. The progress in scientific knowledge and the lessons learned from the A(H1N1)2009 pandemic have highlighted further the need to improve current guidelines, including the immunogenicity criteria set by the CHMP in 1997, and to promote the discussion on the shortcomings encountered, e.g. the evaluation of vaccine efficacy in the paediatric and elderly populations, the measurement of the naivety of a population, the impact of prior immunity on subsequent vaccinations, and the technical issues with the serological assays for detection of immunity and immunogenicity.     

Significant disparity in base and sugar damage in DNA resulting from neutron and electron irradiation

In this study, a comparison of the effects of neutron and electron irradiation of aqueous DNA solutions was investigated to characterize potential neutron signatures in DNA damage induction. Ionizing radiation generates numerous lesions in DNA, including base and sugar lesions, lesions involving base–sugar combinations (e.g. 8,5′-cyclopurine-2′-deoxynucleosides) and DNA–protein cross-links, as well as single- and double-strand breaks and clustered damage. The characteristics of damage depend on the linear energy transfer (LET) of the incident radiation. Here we investigated DNA damage using aqueous DNA solutions in 10 mmol/l phosphate buffer from 0–80 Gy by low-LET electrons (10 Gy/min) and the specific high-LET (∼0.16 Gy/h) neutrons formed by spontaneous ²⁵²Cf decay fissions. 8-hydroxy-2′-deoxyguanosine (8-OH-dG), (5′R)-8,5′-cyclo-2′-deoxyadenosine (R-cdA) and (5′S)-8,5′-cyclo-2′-deoxyadenosine (S-cdA) were quantified using liquid chromatography–isotope-dilution tandem mass spectrometry to demonstrate a linear dose dependence for induction of 8-OH-dG by both types of radiation, although neutron irradiation was ∼50% less effective at a given dose compared with electron irradiation. Electron irradiation resulted in an exponential increase in S-cdA and R-cdA with dose, whereas neutron irradiation induced substantially less damage and the amount of damage increased only gradually with dose. Addition of 30 mmol/l 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), a free radical scavenger, to the DNA solution before irradiation reduced lesion induction to background levels for both types of radiation. These results provide insight into the mechanisms of DNA damage by high-LET ²⁵²Cf decay neutrons and low-LET electrons, leading to enhanced understanding of the potential biological effects of these types of irradiation.