Elucidation of the Reversible Self-Association Interface of a Diabody-Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling.

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

Foslevodopa/Foscarbidopa: A New Subcutaneous Treatment for Parkinson's Disease.

OBJECTIVE: The aim was to demonstrate that continuous s.c. infusion of a soluble levodopa (LD)/carbidopa (CD) phosphate prodrug combination effectively delivers stable LD exposure via a minimally invasive and convenient mode and has the potential to treat Parkinson's disease (PD) patients who are not well controlled on oral medication. METHODS: Foslevodopa and foscarbidopa were prepared and the equilibrium solubility and chemical stability examined in aqueous media with different values of pH. Solutions of foslevodopa/foscarbidopa (ratios ranging from 4:1 to 20:1) were prepared by dissolving pH-adjusted lyophilized materials in water and infused s.c. in healthy volunteers for ≤72 hours. Frequent blood samples were collected to measure LD and CD exposure, and safety was monitored throughout the study. RESULTS: Foslevodopa/foscarbidopa (ABBV-951) demonstrates high water solubility and excellent chemical stability near physiological pH, enabling continuous s.c. infusion therapy. After s.c. infusion, a stable LD pharmacokinetic (PK) profile was maintained for ≤72 hours, and the infusion was well tolerated. INTERPRETATION: Preparation of foslevodopa and foscarbidopa enables preclinical and clinical PK, safety, and tolerability studies in support of their advancement for the treatment of PD. In phase 1 clinical trials, foslevodopa/foscarbidopa demonstrates consistent and stable LD plasma exposure, supporting further studies of this treatment as a potentially transformational option for those suffering from PD. ANN NEUROL 2021;90:52-61.

14-3-3η Protein in serum and synovial fluid correlates with radiographic damage and progression in a longitudinal evaluation of patients with established rheumatoid arthritis.

Objectives: The aim of the study is to examine the association between 14-3-3η protein levels in both serum and synovial fluid (SF) with various parameters in a longitudinal cohort of patients with established rheumatoid arthritis (RA).Methods: Serum and SF samples were obtained from RA patients and 14-3-3η levels were measured. Radiological damage and progression were evaluated using Sharp/van der Heijde score (SHS) at study entry and at 2-years follow-up.Results: A total of 39 RA patients were included with a mean disease duration of 9.6 ± 8 years. Levels of 14-3-3η were two-fold higher in SF than in serum (mean of 3.7 versus 1.7 ng/mL, respectively). While no significant association was found between 14-3-3η levels with disease activity or other laboratory assessments, both serum and SF 14-3-3η levels positively correlated with radiographic damage at baseline (SHS; p < .001). SF, but not serum, 14-3-3η levels correlated with absolute progression (p < .03).Conclusion: 14-3-3η levels are significantly higher in RA SF than in serum in an established RA cohort. Serum and SF 14-3-3η levels correlate with radiographic damage at baseline and at 2-years follow-up. This study further substantiates the utility of 14-3-3η as a biomarker for mechanistic joint damage in established RA.

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen-Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.

Proteomic profiling of the plasma of Gambian children with cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection. A number of pathological findings have been correlated with pediatric CM including sequestration, platelet accumulation, petechial haemorrhage and retinopathy. However, the molecular mechanisms leading to death in CM are not yet fully understood. METHODS: A shotgun plasma proteomic study was conducted using samples form 52 Gambian children with CM admitted to hospital. Based on clinical outcome, children were assigned to two groups: reversible and fatal CM. Label-free liquid chromatography-tandem mass spectrometry was used to identify and compare plasma proteins that were differentially regulated in children who recovered from CM and those who died. Candidate biomarkers were validated using enzyme immunoassays. RESULTS: The plasma proteomic signature of children with CM identified 266 proteins differentially regulated in children with fatal CM. Proteins from the coagulation cascade were consistently decreased in fatal CM, whereas the plasma proteomic signature associated with fatal CM underscored the importance of endothelial activation, tissue damage, inflammation, haemolysis and glucose metabolism. The concentration of circulating proteasomes or PSMB9 in plasma was not significantly different in fatal CM when compared with survivors. Plasma PSMB9 concentration was higher in patients who presented with seizures and was significantly correlated with the number of seizures observed in patients with CM during admission. CONCLUSIONS: The results indicate that increased tissue damage and hypercoagulability may play an important role in fatal CM. The diagnostic value of this molecular signature to identify children at high risk of dying to optimize patient referral practices should be validated prospectively.

In-Situ Molecular Vapor Composition Measurements During Lyophilization.

PURPOSE: Monitoring process conditions during lyophilization is essential to ensuring product quality for lyophilized pharmaceutical products. Residual gas analysis has been applied previously in lyophilization applications for leak detection, determination of endpoint in primary and secondary drying, monitoring sterilization processes, and measuring complex solvents. The purpose of this study is to investigate the temporal evolution of the process gas for various formulations during lyophilization to better understand the relative extraction rates of various molecular compounds over the course of primary drying. METHODS: In this study, residual gas analysis is used to monitor molecular composition of gases in the product chamber during lyophilization of aqueous formulations typical for pharmaceuticals. Residual gas analysis is also used in the determination of the primary drying endpoint and compared to the results obtained using the comparative pressure measurement technique. RESULTS: The dynamics of solvent vapors, those species dissolved therein, and the ballast gas (the gas supplied to maintain a set-point pressure in the product chamber) are observed throughout the course of lyophilization. In addition to water vapor and nitrogen, the two most abundant gases for all considered aqueous formulations are oxygen and carbon dioxide. In particular, it is observed that the relative concentrations of carbon dioxide and oxygen vary depending on the formulation, an observation which stems from the varying solubility of these species. This result has implications on product shelf life and stability during the lyophilization process. CONCLUSIONS: Chamber process gas composition during lyophilization is quantified for several representative formulations using residual gas analysis. The advantages of the technique lie in its ability to measure the relative concentration of various species during the lyophilization process. This feature gives residual gas analysis utility in a host of applications from endpoint determination to quality assurance. In contrast to other methods, residual gas analysis is able to determine oxygen and water vapor content in the process gas. These compounds have been shown to directly influence product shelf life. With these results, residual gas analysis technique presents a potential new method for real-time lyophilization process control and improved understanding of formulation and processing effects for lyophilized pharmaceutical products.

Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

PURPOSE: Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb). METHODS: Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies. RESULTS: mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried. CONCLUSIONS: The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS).

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the Fab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the CH2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.

Accelerated Storage for Shelf-Life Prediction of Lyophiles: Temperature Dependence of Degradation of Amorphous Small Molecular Weight Drugs and Proteins.

Prediction of lyophilized product shelf-life using accelerated stability data requires understanding the temperature dependence of the degradation rate. Despite the abundance of published studies on stability of freeze-dried formulations and other amorphous materials, there are no definitive conclusions on the type of pattern one can expect for the temperature dependence of degradation. This lack of consensus represents a significant gap which may impact development and regulatory acceptance of freeze-dried pharmaceuticals and biopharmaceuticals. Review of the literature demonstrates that the temperature dependence of degradation rate constants in lyophiles can be represented by the Arrhenius equation in most cases. In some instances there is a break in the Arrhenius plot around the glass transition temperature or a related characteristic temperature. The majority of the activation energies (Ea), which are reported for various degradation pathways in lyophiles, falls in the range of 8 to 25 kcal/mol. The degradation Ea values for lyophiles are compared with the Ea for relaxation processes and diffusion in glasses, as wells as solution chemical reactions. Collectively, analysis of the literature demonstrates that the Arrhenius equation represents a reasonable empirical tool for analysis, presentation, and extrapolation of stability data for lyophiles, provided that specific conditions are met.

Value of tumor necrosis factor-alpha and high-sensitive cardiac troponin-I as early predictors of subclinical atherosclerosis and their relation to disease activity in patients with rheumatoid arthritis: A case-control study.

Patients with rheumatoid arthritis (RA) have a higher risk of cardiovascular disease (CVD) compared to the general population, which leads to increased morbidity and mortality. Inflammation is the key in RA and CVD. Our study aimed to refine cardiovascular (CV) risk assessment in RA patients by using carotid intima-media thickness (cIMT) as a marker of subclinical atherosclerosis. We also explored whether proinflammatory cytokines represented by tumor necrosis factor-alpha (TNF-α) and high-sensitivity cardiac troponin I (hs-cTnI), a biomarker of myocardial injury, could be correlated in RA patients. The study included 80 RA patients and 80 control subjects. TNF-α and hs-cTnI levels were measured. Subclinical atherosclerosis was evaluated by cIMT by means of carotid ultrasound. Disease activity score 28 (DAS28) was used to evaluate disease activity. hs-cTnI and TNF-α serum levels were higher in RA patients compared to controls (p=0.001). There was a significant difference in the median of cIMT between cases and controls (median (IQR) 0.9 (0.2) for cases, 0.7 (0.1) for controls, (p=0.001). A significant correlation was found between the level of TNF-α and hs-cTnI (p=0.002). Also, there was a significant correlation between the cIMT level and TNF-α and hs-cTnI (p=0.003 and p=0.002, respectively). Significant correlation was found between cIMT, TNF-α, and hs-cTnI in relation to the DAS28 score (p < 0.001, p < 0.001, and p=0.001, respectively). In conclusion, patients with RA are more likely to develop subclinical atherosclerosis, as reflected in increased cIMT. Higher levels of hs-cTnI in RA patients may correlate with the presence of occult cardiovascular disease. TNF-α and hs-cTnI correlations can reveal the interplay between disease activity and CVD. Thus, inflammation must be the primary target of various therapeutic approaches.

End-to-End Approach to Surfactant Selection, Risk Mitigation, and Control Strategies for Protein-Based Therapeutics.

A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions.

Association of hand grip strength with disease activity, disability and quality of life in children and adolescents with Juvenile Idiopathic Arthritis.

BACKGROUND: Juvenile idiopathic arthritis (JIA) affects wrist and hand joints leading to decrease hand function and patients' daily living activities. The assessment of hand grip strength (HGS) in children and adolescents with JIA is of major importance, and the association of HGS with JIA disease activity, disability and quality of life has not been explored. The primary objective of this study was to evaluate hand grip strength (HGS) in children and adolescents with Juvenile Idiopathic Arthritis (JIA) compared to matched healthy peers. The secondary objective was to explore the relationship between HGS and JIA disease activity, disability, and quality of life. METHODS: This study involved 23 patients with JIA and 46 age and sex matched healthy controls. Hand held dynamometer was used to evaluate HGS for all study participants. Anthropometric parameters for all study participants were measured. Disease activity, physical function, and quality of life were assessed for the JIA group using juvenile arthritis disease activity score (JADAS-27), juvenile arthritis functionality scale (JAFS), and pediatric quality of life inventory (PedsQL) respectively. Laboratory marker of inflammation, erythrocyte sedimentation rate (ESR), and plain radiography of hands were performed for all patients. RESULTS: Hand grip strength of children and adolescents with JIA was significantly weaker compared to matched controls (p < 0.001). Hand grip strength had a significant inverse correlation with JADAS-27 (r = - 0.467, p = 0.025), JAFS (r = - 0.650, p = 0.001) and a significant direct correlation with PedsQL (r = 0.438, p = 0.036). In addition, HGS was negatively correlated with ESR and duration of morning stiffness (r = - 0.489, p = 0.018 and r = - 0.201, p = 0.359, respectively). HGS was detected as an independent predictor of disease activity, disability, and quality of life in JIA patients in multivariate linear regression. CONCLUSIONS: Assessment of HGS could be a simple non-invasive tool for assessing disease activity, disability and quality of life in JIA patients in clinical practice.

Predictive models of lyophilization process for development, scale-up/tech transfer and manufacturing.

Scale-up and technology transfer of lyophilization processes remains a challenge that requires thorough characterization of the laboratory and larger scale lyophilizers. In this study, computational fluid dynamics (CFD) was employed to develop computer-based models of both laboratory and manufacturing scale lyophilizers in order to understand the differences in equipment performance arising from distinct designs. CFD coupled with steady state heat and mass transfer modeling of the vial were then utilized to study and predict independent variables such as shelf temperature and chamber pressure, and response variables such as product resistance, product temperature and primary drying time for a given formulation. The models were then verified experimentally for the different lyophilizers. Additionally, the models were applied to create and evaluate a design space for a lyophilized product in order to provide justification for the flexibility to operate within a certain range of process parameters without the need for validation.

Diffusion weighted magnetic resonance imaging in bladder cancer, is it time to replace biopsy?

INTRODUCTION: To assess if the apparent diffusion coefficient (ADC) value of magnetic resonance imaging (MRI) can discriminate between the cell type, histological grade and improve staging of urinary bladder cancer (BC). MATERIAL AND METHODS: 102 patients with urinary bladder masses underwent MRI using a 1.5 T machine. T2 weighted and diffusion weighted imaging (DWI) using b values of 0, 150, 500 and 1000 s/mm2 were done. The ADC values of bladder masses were measured. These values were correlated with the histopathologic results. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of T2WI, DWI and T2WI plus DWI for detecting bladder lesions were evaluated. RESULTS: The cut-off ADC value for diagnosing malignant bladder wall pathologies was ≤1 x 10-3 mm2/s with 94.5% sensitivity and 87.5% specificity. The mean ADC value of different malignant cell types was statistically insignificant. A significant difference in ADC values was found between G1 and G3 (P = 0.000), G2 and G3 (P = 0.045) but not between G1 and G2 (p = 0.066). Staging accuracy for differentiation between invasive and non-invasive lesions was nearly the same for all MRI data sets. For differentiation between organ confined (pT1-pT2) and non-organ confined lesions (pT3-pT4), staging accuracy was better in T2WI plus DWI (83%) as compared to DWI alone (77%) or T2WI alone (75%). CONCLUSIONS: Adding DWI and the ADC value to T2WI improve the accuracy of MRI in BC detection and staging. However, at this time point, MRI cannot replace transurethral resection (TUR) biopsy or distinguish sharply between all different histologic grades and cell types.

Immunogenicity of Therapeutic Protein Aggregates.

Therapeutic proteins have a propensity for aggregation during manufacturing, shipping, and storage. The presence of aggregates in protein drug products can induce adverse immune responses in patients that may affect safety and efficacy, and so it is of concern to both manufacturers and regulatory agencies. In this vein, there is a lack of understanding of the physicochemical determinants of immunological responses and a lack of standardized analytical methods to survey the molecular properties of aggregates associated with immune activation. In this review, we provide an overview of the basic immune mechanisms in the context of interactions with protein aggregates. We then critically examine the literature with emphasis on the underlying immune mechanisms as they relate to aggregate properties. Finally, we highlight the gaps in our current understanding of this issue and offer recommendations for future research.

Evaluation of penile vascular status in men with epilepsy with erectile dysfunction.

PURPOSE: Erectile dysfunction (ED) is common in males with epilepsy, likely of multifactorial etiology, including possible systemic vascular comorbidities and medication effects. Here we examined male patients for the possibility of a vasculogenic element of ED. METHODS: Research participants included 47 men with epilepsy (mean age=30.98 years; duration of illness=13.98 years) and 25 healthy matched men (mean age=30.36). Erectile function was assessed using the International Index of Erectile Function Questionnaire (IIEF-5). Penile blood flow was assessed using Duplex Ultrasonography (PDU) after intracavernous alprostadil injection. Penile peak systolic velocity (PSV), end-diastolic velocity (EDV) and resistance index (RI) were the functional parameters analyzed. Carotid artery intima media thickness (CA-IMT) was also measured. RESULTS: Thirteen of the 47 men with epilepsy (23.40% versus 0% for controls) reported ED, and of these patients, 11 (84.62%) had abnormal PDU [PSV=28.23 ± 6.1cm/s, P=0.0001; EDV=2.22 ± 5.71 cm/s, P=0.004; RI=0.89 ± 0.22, P=0.071] suggesting vasculogic ED. Penile arterial insufficiency was identified in 5 (45.45%), while 6 (54.54%) had mixed arterial insufficiency and venous leak. Compared to patients with high PSV, patients with low PSV had lower IIED-5 scores, higher EDV, lower RI, higher diastolic blood pressure and higher CA-IMT values. There were no differences in depression, anxiety or concentrations of sex hormones. Significant correlations were evident between PDU variables and duration of illness, depression and anxiety scores and CA-IMT values. In multivariate analysis, the association between PDU parameters and CA-IMT values remained significant even after adjustment for other confounding variables. CONCLUSIONS: Vasculogenic ED is frequent with epilepsy and its relationship to systemic atherosclerosis cannot be excluded.

Seminal fluid analysis and testicular volume in adults with epilepsy receiving valproate.

Epilepsy and its medications adversely affect reproductive and sexual functions and fertility. This study aimed to assess sperm parameters and testicular volume in men with epilepsy on valproate (VPA). Included were 55 patients with idiopathic epilepsy with a mean age of 31.86 ± standard deviation (SD) 6.55 years, mean illness duration of 12.50 ± SD 5.10 years, and a mean treatment time of 9.55 ± SD 0.85 years. Sex hormone profile, semen analysis, testicular volume and total seminal plasma carnitine were determined. Compared to controls, patients had lower levels of free testosterone (p<0.01), sperm concentration (p<0.0001) and count (p<0.0001), carnitine (p<0.01), and testicular volume (p<0.01), and higher rates of immotile sperm (p<0.001) and abnormal forms (p<0.0001). Significant correlations were identified between sperm count, motility, immotile sperm, abnormal forms, testicular volume, carnitine levels and duration of illness, duration of treatment with VPA and VPA dose. Multivariable analysis demonstrated that duration of treatment with VPA, sperm count, motility and abnormal forms were significantly associated with seminal plasma carnitine. Long-term VPA treatment is adversely associated with reduced sperm count and motility, increased abnormal sperm count and reduced testicular volume. This may contribute to reduced fertility.

Bone status in patients with epilepsy: relationship to markers of bone remodeling.

Patients with epilepsy and treated with antiepileptic drugs (AEDs) may develop metabolic bone disease; however, the exact pathogenesis of bone loss with AEDs is still unclear. Included were 75 adults with epilepsy (mean age: 31.90 ± 5.62 years; duration of treatment with AEDs: 10.57 ± 3.55 years) and 40 matched healthy controls. Bone mineral content (BMC) and bone mineral densities (BMD) of the femoral neck and lumbar spine were measured using dual-energy X-ray absorptiometry (DEXA). Blood samples were analyzed for calcium, magnesium, phosphate, alkaline phosphatase (ALP), 25-hydroxy vitamin D (25OHD), soluble receptor activator of nuclear factor-kappa B ligand (sRANKL), osteoprotegerin (OPG), and OPG/RANKL ratio (markers of bone remodeling). Compared to controls, patients had lower BMD, BMC, Z-score, and T-score at the femoral neck and lumbar spine (all p < 0.001). Seventy-two percent and 29.33% of patients had osteoporosis of the lumbar spine and femoral neck. Patients had significantly lower serum calcium, 25(OH)D, and OPG and higher ALP, sRANKL levels, and sRANKL/OPG (all p < 0.001). Fifty-two percent of patients had hypocalcemia, 93% had hypovitaminosis D, 31% had high levels of sRANKL, and 49% had low levels of OPG. No differences were identified between DEXA and laboratory results in relation to the type, dose, or serum levels of AEDs. BMD at the femoral neck and lumbar spine were found to be correlated with the duration of illness (p = 0.043; p = 0.010), duration of treatment with AEDs (p < 0.001; p = 0.012), and serum levels of 25(OH)D (p = 0.042; p = 0.010), sRANKLs (p = 0.005; p = 0.01), and OPG (p = 0.006; p = 0.01). In linear regression analysis and after adjusting for gender, age, weight, duration, and number of AEDs, we observed an association between BMD, 25(OH)D (p = 0.04) and sRANKL (p = 0.03) concentrations. We conclude that AEDs may compromise bone health through disturbance of mineral metabolism and acceleration of bone turnover mechanisms.