Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Antibodies are molecules that exhibit diverse conformational changes on different timescales, and there is ongoing interest to better understand the relationship between antibody conformational dynamics and storage stability. Physical stability data for an IgG4 monoclonal antibody (mAb-D) were gathered through traditional forced degradation (temperature and stirring stresses) and accelerated stability studies, in the presence of different additives and solution conditions, as measured by differential scanning calorimetry, size exclusion chromatography, and microflow imaging. The results were correlated with hydrogen exchange mass spectrometry (HX-MS) data gathered for mAb-D in the same formulations. Certain parameters of the HX-MS data, including hydrogen exchange in specific peptide segments in the C_H2 domain, were found to correlate with stabilization and destabilization of additives on mAb-D during thermal stress. No such correlations between mAb physical stability and HX-MS readouts were observed under agitation stress. These results demonstrate that HX-MS can be set up as a streamlined methodology (using minimal material and focusing on key peptide segments at key time points) to screen excipients for their ability to physically stabilize mAbs. However, useful correlations between HX-MS and either accelerated or real-time stability studies will be dependent on a particular mAb's degradation pathway(s) and the type of stresses used.     

Prediction of Human Brain Penetration of P-glycoprotein and Breast Cancer Resistance Protein Substrates Using In Vitro Transporter Studies and Animal Models

Four P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates with human cerebrospinal fluid (CSF) concentrations and preclinical neuropharmacokinetics were used to assess in vitro–in vivo extrapolation of brain penetration in preclinical species and the ability to predict human brain penetration. Unbound brain (C_b,u), unbound plasma (C_p,u), and CSF compound concentrations (C_CSF) were measured in rats and nonhuman primates (NHPs), and the unbound partition coefficients (C_b,u/C_p,u and C_CSF/C_p,u) were used to assess brain penetration. The results indicated that for P-gp and BCRP dual substrates, brain penetration was severally impaired in all species. In comparison, for P-gp substrates that are weak or non-BCRP substrates, improved brain penetration was observed in NHPs and humans than in rats. Overall, NHP appears to be more predictive of human brain penetration for P-gp substrates with weak or no interaction with BCRP than rat. Although C_CSF does not quantitatively correspond to C_b,u for efflux transporter substrates, it is mostly within 3-fold higher of C_b,u in rat and NHP, suggesting that C_CSF can be used as a surrogate for C_b,u. Taken together, a holistic approach including both in vitro transporter and in vivo neuropharmacokinetics data enables a better estimation of human brain penetration of P-gp/BCRP substrates.     

Long-Acting Profile of 4 Drugs in 1 Anti-HIV Nanosuspension in Nonhuman Primates for 5 Weeks After a Single Subcutaneous Injection

Daily oral antiretroviral therapy regimens produce limited drug exposure in tissues where residual HIV persists and suffer from poor patient adherence and disparate drug kinetics, which all negatively impact outcomes. To address this, we developed a tissue- and cell-targeted long-acting 4-in-1 nanosuspension composed of lopinavir (LPV), ritonavir, tenofovir (TFV), and lamivudine (3TC). In 4 macaques dosed subcutaneously, drug levels over 5 weeks in plasma, lymph node mononuclear cells (LNMCs), and peripheral blood mononuclear cells (PBMCs) were analyzed by liquid chromatography–tandem mass spectrometry. Plasma and PBMC levels of the active drugs (LPV, TFV, and 3TC) were sustained for 5 weeks; PBMC exposures to LPV, ritonavir, and 3TC were 12-, 16-, 42-fold higher than those in plasma. Apparent T_1/2z of LPV, TFV, and 3TC were 219.1, 63.1, and 136.3 h in plasma; 1045.7, 105.9, and 127.7 h in PBMCs. At day 8, LPV, TFV, and 3TC levels in LNMCs were 4.1-, 5.0-, and 1.9-fold higher than in those in PBMCs and much higher than in plasma. Therefore, 1 dose of a 4-drug nanosuspension exhibited persistent drug levels in LNMCs, PBMCs, and plasma for 5 weeks. With interspecies scaling and dose adjustment, this 4-in-1 HIV drug-combination could be a long-acting treatment with the potential to target residual virus in tissues and improve patient adherence.     

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.     

Exploring motivations behind pollution-mask use in a sample of young adults in urban China

Background

Wearing a pollution mask is an effective, practical, and economic way to prevent the inhalation of dangerous particulate matter (PM). However, it is not uncommon to observe negligence in adopting such behaviour, and this especially among young segments of the population. Using the Theory of Planned Behaviour (TPB) as conceptual framework, this study explores the role of socio-cognitive factors that affect the decision of wearing a pollution mask in the context of young educated people. This is done by selecting a sample of college students in urban China, a country that has seen air quality as one of the major challenges in the last decades. While young urban college students might be expected to be receptive to standard attempts to be influenced through reason-based cognitive stimuli, it is often found that this is not the case. The empirical analysis was articulated it in two steps. Structural Equation Modelling (SEM) was first used to examine the relationships among the conceptual constructs derived from the TPB conceptual model, and second Step-Wise Ordinary Least Squares Regressions (SWOLS) were employed to observe the partial effect played by each item on the decision to wear a mask.

Results

Results show that, while reason-based stimuli play a role, attitude, social norm, and self-efficacy were the most important predictors of the behavioural intention (p?<?0.01). The role of past behaviour was also acknowledged as strongly associated with the dependent variable (p?<?0.01). Overall, the likelihood of wearing a pollution mask increases with the importance of others socio-cognitive and psychological factors, which could help understand behavioural biases, and explain the relative role of several mechanisms behind the decision to wear a mask.

Conclusions

While tackling pollution requires multiple and synergic approaches, encouraging self-prevention using pollution mask is a simple and effective action, implementable at negligible costs. Resistance among younger, well-educated cohorts to wear masks can be overcome by stressing the social desirability of action and the sense of empowerment derived from its usage. This study has the potential to inform policies aimed at changing suboptimal behavioural attitudes by identifying triggers for change, and it could serve in improving the tailoring of health promotion messages aimed at nudging healthy behaviour.

     

Delayed Urinary Symptoms Induced by Ketamine

One of the side-effects of ketamine abuse is genito-urinary damage. This report describes a case of a former ketamine user who presented with urinary symptoms associated with ketamine years after stopping consumption. This was a 26-year-old male with a history of ketamine abuse. He started treatment for alcohol dependence at age 19. He smoked marijuana daily and denied any other drug use. During the follow-up, urinary symptoms were evidenced (dysuria, frequency, urgency, incontinence, nocturia, hematuria, and suprapubic pain). Urinary symptoms started two years ago and worsened over time. The patient was referred to a urologist. A cystoscopy revealed lesions compatible with interstitial cystitis like the ones that appear in some ketamine abusers. Given the medical history, the urologist asked him about ketamine consumption and the patient declared a daily use of 50 milligrams intranasally from age 15 to age 17. Given these findings, not reported previously in the medical literature, future research should follow up patients who at some point in their life made an abusive consumption of ketamine in order to understand the pathogenesis and to be able to intervene before clinical disease manifests itself.