Pharmaceutical Research

Besheer, Ahmed; Mahler, Hanns-Christian

doi: 10.1007/s11095-020-02956-zpmid: 33078256

Bor Tekdemir, Zeynep; Seckin, Ali Ihsan; Kacar, Turgay; Yilmaz, Emine; Bekiroglu, Somer

doi: 10.1007/s11095-020-02932-7pmid: 33026512

PurposeGranulocyte colony stimulating factor (GCSF; also known as filgrastim) is a growth factor used to induce production of granulocytes. As the first locally developed and approved biosimilar medicine of Turkey, Fraven® being a biosimilar of filgrastim has been ab initio manufactured from cell to finished product at two different production facilities. Comprehensive structural, biological and functional characterization studies were performed to compare Fraven® from two different production sites and its reference product Neupogen® sourced from Turkey.MethodsPrimary and higher-order protein structures were analyzed by high performance liquid chromatography electrospray ionization-time of flight mass spectrometry, circular dichroism, and two-dimensional nuclear magnetic resonance spectroscopy. Isoelectric focusing, SDS-Page, size exclusion chromatography, and related proteins analyses were used to compare impurities. In order to assess functional similarity, surface plasmon resonance (SPR) was used. In vitro cell proliferation assay was also performed to show dose related drug response in NFS-60 cell line.ResultsPrimary, secondary and tertiary structures of biosimilar Fraven® manufactured at both sites were found to be highly similar to the reference Neupogen®. Product related substances and impurities were also highly similar to the reference. Comparability of GCSF receptor binding affinities of each product was shown using the KD values of SPR analysis. In vitro cell proliferation similarity was also evaluated and proven by PLA.ConclusionBased on the similarity assessment, despite being manufactured at two different production sites, biosimilar Fraven® is highly similar to the reference product Turkey originated Neupogen®.

Alshehri, Sameer; Fan, Wei; Zhang, Wenting; Garrison, Jered C.

doi: 10.1007/s11095-020-02952-3pmid: 33098043

PurposeThe development of diagnostic and therapeutic agents utilizing small peptides (e.g., bombesin (BBN)) to target the overexpression of the gastrin-releasing peptide receptor (GRPR) in cancers has been widely investigated. Herein, we examine the capabilities of BBN-modified HPMA copolymers to target the GRPR.MethodsFour positive, four negative, and two zwitterionic BBN HPMA copolymer conjugates of varying peptide content and charge were synthesized. In vitro and in vivo studies were conducted in a GRPR-overexpressing prostate cancer cell line (PC-3) and a normal CF-1 mouse model, respectively.ResultsCellular uptake of the conjugates were found to be charge and BBN density dependent. The positively-charged conjugates illustrated a direct relationship between the extent of cellular internalization, ranging from 0.7 to 20%, and BBN-incorporation density. The negative and zwitterionic conjugates showed low PC-3 uptake values. Blocking studies confirmed the GRPR-targeting effect of the positively-charged constructs. In vivo studies of the positively-charged copolymers resulted in rapid blood clearance by the mononuclear phagocyte system (MPS)-associated tissues (e.g., liver and spleen).ConclusionPositively-charged BBN-HPMA copolymer conjugates demonstrated good GRPR-targeting and internalization in vitro. However, the impact of peptide density and charge on in vivo MPS recognition are parameters that must be optimized in future agent development.

Shen, Bin-Bin; Zhang, Zhongwei; Yuan, Jun-Jie; Zheng, Aiping; Zeng, Su; Gao, Jian-Qing; Bao, Wenhan; Barnard, James; Wang, Haibin; Fang, Wei-Jie

doi: 10.1007/s11095-020-02947-0pmid: 33098017

PurposesThe main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon.MethodsA series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques.ResultsThe formation of the impurity as observed in CE-SDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LC-MS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS.ConclusionsOur study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.

Mohylyuk, Valentyn; Goldoozian, Seyedreza; Andrews, Gavin P.; Dashevskiy, Andriy

doi: 10.1007/s11095-020-02940-7pmid: 33094368

PurposeWhen establishing IVIVC, a special problem arises by interpretation of averaged in vivo profiles insight of considerable individual variations in term of time and number of mechanical stress events in GI-tract. The objective of the study was to investigate and forecast the effect of mechanical stress on in vivo behavior in human of hydrophilic matrix tablets.MethodsDissolution profiles for the marketed products were obtained at different conditions (stirring speed, single- or repeatable mechanical stress applied) and convoluted into C-t profiles. Vice versa, published in vivo C-t profiles of the products were deconvoluted into absorption profiles and compared with dissolution profiles by similarity factor.ResultsInvestigated hydrophilic matrix tablets varied in term of their resistance against hydrodynamic stress or single stress during the dissolution. Different scenarios, including repeatable mechanical stress, were investigated on mostly prone Seroquel® XR 50 mg. None of the particular scenarios fits to the published in vivo C-t profile of Seroquel® XR 50 mg representing, however, the average of individual profiles related to scenarios differing by number, frequency and time of contraction stress. When different scenarios were combined in different proportions, the profiles became closer to the original in vivo profile including a burst between 4 and 5 h, probably, due to stress-events in GI-tract.ConclusionFor establishing IVIVC of oral dosage forms susceptible mechanical stress, a comparison of the deconvoluted individual in vivo profiles with in vitro profiles of different dissolution scenarios can be recommended.

Kuilamu, Esther; Subasic, Christopher; Cowin, Gary J; Simpson, Fiona; Minchin, Rodney F; Kaminskas, Lisa M

doi: 10.1007/s11095-020-02945-2pmid: 33078255

PurposeThe aim of this work was to identify whether biochemical and physiological sources of mAb pharmacokinetic sex-effects could be identified in the rat model where target-mediated disposition is avoided.MethodsPlasma and lymphatic pharmacokinetics of the humanised anti-EGFR antibody cetuximab, along with potential physiological and biochemical drivers of pharmacokinetic sex differences, were examined in male and female rats. Cetuximab was used as a model mAb since plasma clearance is slower in female patients.ResultsWhen plasma concentrations were normalised to dose, female rats displayed slower plasma clearance than males, but no significant differences were observed in liver and spleen biodistribution. Sex differences in apparent plasma clearance, however, were abolished after normalisation to body weight, surface area or fat-free mass. Significant sex differences were observed in plasma testosterone, endogenous IgG and fat free mass, but did not correlate with apparent clearance. Females did, however, show two-fold higher lymphatic exposure compared to males.ConclusionsThese data suggested that mAbs more efficiently access lymph in females, but this does not affect plasma pharmacokinetics or biodistribution. Further, the data suggest that sex differences observed in humans could be a function of antigen density.

Miranda, Margarida; Cova, Tânia; Augusto, Cátia; Pais, Alberto A. C. C.; Cardoso, Catarina; Vitorino, Carla

doi: 10.1007/s11095-020-02911-ypmid: 33037479

PurposeFollowing the recent European Medicine Agency (EMA) draft guideline on quality and equivalence of topical products, a modular framework for bioequivalence assessment is proposed, wherein the qualitative, quantitative, microstructure and product performance sameness is demanded to support generic applications. Strict regulatory limits are now imposed, but, the suitability of these limits has been subject of intense debate. In this context, this paper aims to address these issues by characterizing a panel of 8 reference blockbuster semisolid topical products.MethodsFor each product, three batches were selected and, whenever possible, batches retrieved from different manufacturing sites were considered. Product microstructure was evaluated in terms of globule size, pH, rheological attributes and, if required, the thermal behaviour was also assessed. Performance was evaluated through in vitro release testing (IVRT). Finally, an integrated multivariate analysis was performed to highlight the features that most contribute for product variability.ResultsMarked differences were registered within reference products. Statistical analysis demonstrated that if EMA criteria are applied, none of the same product batches can be considered as equivalent. Rheological parameters as well as IVRT indicators account for the majority of batch-to-batch differences.ConclusionsSemisolid dosage forms exhibit intrinsic variability. This calls for the attention to the need of establishing reasonable equivalence criteria applied to generic drug products.Graphical abstract[graphic not available: see fulltext]

Agbana, Preye; Lee, Min Jae; Rychahou, Piotr; Kim, Kyung-Bo; Bae, Younsoo

doi: 10.1007/s11095-020-02922-9pmid: 33025286

PurposeTo develop a new nanoparticle formulation for a proteasome inhibitor Carfilzomib (CFZ) to improve its stability and efficacy for future in vivo applications.MethodsCFZ-loaded ternary polypeptide nanoparticles (CFZ/tPNPs) were prepared by using heptakis(6-amino-6-deoxy)-β-cyclodextrin(hepta-hydrochloride) (HaβCD) and azido-poly(ethylene glycol)-block-poly(L-glutamic acid sodium salt) (N3-PEG-PLE). The process involved ternary (hydrophobic/ionic/supramolecular) interactions in three steps: 1) CFZ was entrapped in the cavity of HaβCD by hydrophobic interaction, 2) the drug-cyclodextrin inclusion complexes were mixed with N3-PEG-PLE to form polyion complex nanoparticles, and 3) the nanoparticles were modified with fluorescent dyes (AFDye 647) for imaging and/or epithelial cell adhesion molecule (EpCAM) antibodies for cancer cell targeting. CFZ/tPNPs were characterized for particle size, surface charge, drug release, stability, intracellular uptake, proteasome inhibition, and in vitro cytotoxicity.ResultstPNPs maintained an average particle size of 50 nm after CFZ entrapment, EpCAM conjugation, and freeze drying. tPNPs achieved high aqueous solubility of CFZ (>1 mg/mL), sustained drug release (t1/2 = 6.46 h), and EpCAM-mediated cell targeting, which resulted in increased intracellular drug accumulation, prolonged proteasome inhibition, and enhanced cytotoxicity of CFZ in drug-resistant DLD-1 colorectal cancer cells.ConclusionstPNPs improved stability and efficacy of CFZ in vitro, and these results potentiate effective cancer treatment using CFZ/tPNPs in future vivo studies.