Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin® Biosimilar Candidate and Its Innovator: A Comparative Study.

Avastin® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants' in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact.

Combination of Paclitaxel and R-flurbiprofen loaded PLGA nanoparticles suppresses glioblastoma growth on systemic administration.

Malignant gliomas are highly lethal. Delivering chemotherapeutic drugs to the brain in sufficient concentration is the major limitation in their treatment due to the blood-brain barrier (BBB). Drug delivery systems may overcome this limitation and can improve the transportation through the BBB. Paclitaxel is an antimicrotubule agent with effective anticancer activity but limited BBB permeability. R-Flurbiprofen is a nonsteroidal antienflammatory drug and has potential anticancer activity. Accordingly, we designed an approach combining R-flurbiprofen and paclitaxel and positively-charged chitosan-modified poly-lactide-co-glycolic acid (PLGA) nanoparticles (NPs) and to transport them to glioma tissue. NPs were characterized and, cytotoxicity and cellular uptake studies were carried out in vitro. The in vivo efficacy of the combination and formulations were evaluated using a rat RG2 glioma tumor model. Polyethylene glycol (PEG) modified and chitosan-coated PLGA NPs demonstrated efficient cytotoxic activity and were internalized by the tumor cells in RG2 cell culture. In vivo studies showed that the chitosan-coated and PEGylated NPs loaded with paclitaxel and R-flurbiprofen exhibited significantly higher therapeutic activity against glioma. In conclusion, PLGA NPs can efficiently carry their payloads to glioma tissue and the combined use of anticancer and anti-inflammatory drugs may exert additional anti-tumor activity.

Preclinical evaluation of a biobetter candidate: Pharmacokinetics and pharmacodynamics of GX-G3 in healthy and neutropenia-induced rats.

Neutropenia is a condition of an abnormally low number of neutrophils which render patients more susceptible to infections, especially to bacterial infections, as the condition may become life threatening and deadly without prompt medical attention. Various factors such as, anticancer drugs, radiotherapy, infectious diseases, congenital defects, or vitamin B12/B9 deficiency can trigger neutropenia. GX-G3, a human hybrid (hy) Fc-fused granulocyte colony stimulating factor (G-CSF), was developed as next-generation G-CSF for the treatment of cancer therapy-induced neutropenia. In this study, with the aim of investigating this promising potential next-generation G-CSF, comparative pharmacokinetic and pharmacodynamic studies were conducted in healthy and neutropenia-induced rats. It was found that t1/2 of GX-G3 is longer than same mass injection of filgrastim and pegfilgrastim and AUEClast (area under theeffect-time curve from time zero to the last measurable ANC level) of absolute neutrophil count showed a significant increase after GX-G3 injection compared with filgrastim and pegfilgrastim in healthy rats. Besides, in duration of neutropenia after the same mass injection GX-G3 showed about 3.3 days of reduction effect compared with that of filgrastim, and 1.3 days of reduction effect compared with that of pegfilgrastim in neutropenia-induced rats. These results demonstrate that the half-life of GX-G3 is longer than pegfilgrastim and GX-G3 is more effective than filgrastim and pegfilgrastim in neutropenia-induced rats.

Preparation and Characterization of Biocompatible Chitosan Nanoparticles for Targeted Brain Delivery of Peptides.

Here, we describe a nanocarrier system that can transfer chitosan nanoparticles loaded with either small peptides such as the caspase inhibitor Z-DEVD-FMK or a large peptide like basic fibroblast growth factor across the blood-brain barrier. The nanoparticles are selectively directed to the brain and are not measurably taken up by the liver and spleen. Intravital fluorescent microscopy provides an opportunity to study the penetration kinetics of nanoparticles loaded with fluorescent agents such as Nile red. Nanoparticles functionalized with anti-transferrin antibody and loaded with peptides efficiently provided neuroprotection when systemically administered either as a formulation bearing a single peptide or a mixture of them. Failure of brain permeation of the nanoparticles after inhibition of vesicular transcytosis by imatinib as well as when nanoparticles were not functionalized with anti-transferrin antibody indicates that this nanomedicine formulation is rapidly transported across the blood-brain barrier by receptor-mediated transcytosis.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

Development and evaluation of paclitaxel nanoparticles using a quality-by-design approach.

The aims of this study were to develop and characterize paclitaxel nanoparticles, to identify and control critical sources of variability in the process, and to understand the impact of formulation and process parameters on the critical quality attributes (CQAs) using a quality-by-design (QbD) approach. For this, a risk assessment study was performed with various formulation and process parameters to determine their impact on CQAs of nanoparticles, which were determined to be average particle size, zeta potential, and encapsulation efficiency. Potential risk factors were identified using an Ishikawa diagram and screened by Plackett-Burman design and finally nanoparticles were optimized using Box-Behnken design. The optimized formulation was further characterized by Fourier transform infrared spectroscopy, X-ray diffractometry, differential scanning calorimetry, scanning electron microscopy, atomic force microscopy, and gas chromatography. It was observed that paclitaxel transformed from crystalline state to amorphous state while totally encapsulating into the nanoparticles. The nanoparticles were spherical, smooth, and homogenous with no dichloromethane residue. In vitro cytotoxicity test showed that the developed nanoparticles are more efficient than free paclitaxel in terms of antitumor activity (more than 25%). In conclusion, this study demonstrated that understanding formulation and process parameters with the philosophy of QbD is useful for the optimization of complex drug delivery systems.

Transport of a caspase inhibitor across the blood-brain barrier by chitosan nanoparticles.

The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Several approaches taking advantage of molecular and physicochemical characteristics of the BBB have been developed recently to improve drug delivery to the brain. Here, we introduce a nanomedicine that can efficiently transport BBB-impermeable peptides to the brain. This nanomedicine is made of chitosan nanoparticles into which considerable amounts of a peptide can be incorporated. The nanoparticle surface is modified with polyethylene glycol to enhance the plasma residence time by preventing their capture by the reticuloendothelial system. Monoclonal antibodies against the transferrin receptor (TfR), which is highly expressed on the brain capillary endothelium, are conjugated to nanoparticles via biotin-streptavidin bonds. The activation of TfR by the nanoparticle-antibody complex induces transcytosis and thus delivers the loaded drug to the brain. Penetration of nanoparticles to the brain can be illustrated in vivo by intravital microscopy as well as ex vivo by fluorescence or electron microscopy. N-Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK)-loaded nanoparticles rapidly release their contents within brain parenchyma, inhibit ischemia-induced caspase-3 activity, and thereby provide neuroprotection.

Preparation and characterization of biocompatible chitosan nanoparticles for targeted brain delivery of peptides.

Here, we describe a nanocarrier system that can transfer chitosan nanoparticles loaded with either small peptides such as the caspase inhibitor Z-DEVD-FMK or a large peptide like basic fibroblast growth factor across the blood-brain barrier. The nanoparticles are selectively directed to the brain and are not measurably taken up by liver and spleen. Intravital fluorescent microscopy provides an opportunity to study the penetration kinetics of nanoparticles loaded with fluorescent agents such as Nile red, and has demonstrated that this nanomedicine formulation is rapidly transported across the blood-brain barrier.

Evaluation of drug-excipient interaction in the formulation of celecoxib tablets.

In the present study, the possible interactions between celecoxib and some excipients (colloidal silicon dioxide (Aerosil), microcrystalline cellulose (Avicel PH 102), lactose anhydrous, magnesium stearate, cross-povidone and talc) were evaluated by examining the pure drug or drug-excipient powder mixtures which were stored under different conditions (25 +/- 2 degrees C, 60% RH +/- 5% RH or 40 + 2 degrees C, 75% RH +/- 5% RH) and different period (30 or 60 days) using DSC, FT-IR and HPLC. In order to investigate the possibility of celecoxib-excipient interaction in aqueous medium, dispersions of the pure drug or drug in physical powder mixture (1:1 w/w) in water (1%, w/v) were also prepared and evaluated by FT-IR and HPLC at day 0 and day 7 (40 +/- 2 degrees C). The interaction between celecoxib and magnesium stearate or colloidal silicon dioxide were determined in the aqueous dispersions by FT-IR. Different tablet formulations with or without excipients tested were prepared, and assessed for drug dissolution and permeability.

A nanomedicine transports a peptide caspase-3 inhibitor across the blood-brain barrier and provides neuroprotection.

Caspases play an important role as mediators of cell death in acute and chronic neurological disorders. Although peptide inhibitors of caspases provide neuroprotection, they have to be administered intracerebroventricularly because they cannot cross the blood-brain barrier (BBB). Herein, we present a nanocarrier system that can transfer chitosan nanospheres loaded with N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK), a relatively specific caspase-3 inhibitor, across BBB. Caspase-3 was chosen as a pharmacological target because of its central role in cell death. Polyethylene glycol-coated nanospheres were conjugated to an anti-mouse transferrin receptor monoclonal antibody (TfRMAb) that selectively recognizes the TfR type 1 on the cerebral vasculature. We demonstrate with intravital microscopy that this nanomedicine is rapidly transported across the BBB without being measurably taken up by liver and spleen. Pre- or post-treatment (2 h) with intravenously injected Z-DEVD-FMK-loaded nanospheres dose dependently decreased the infarct volume, neurological deficit, and ischemia-induced caspase-3 activity in mice subjected to 2 h of MCA occlusion and 24 h of reperfusion, suggesting that they released an amount of peptide sufficient to inhibit caspase activity. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17 after closure of the BBB. Neither nanospheres functionalized with TfRMAb but not loaded with Z-DEVD-FMK nor nanospheres lacking TfRMAb but loaded with Z-DEVD-FMK had any effect on either paradigm, suggesting that inhibition of caspase activity and subsequent neuroprotection were due to efficient penetration of the peptide into brain. Thus, chitosan nanospheres open new and exciting opportunities for brain delivery of biologically active peptides that are useful for the treatment of CNS disorders.

Preparation and in vitro evaluation of bFGF-loaded chitosan nanoparticles.

The objective of our study was to prepare and characterize basic fibroblast growth factor (bFGF)-loaded nanoparticles. Protein-loaded chitosan nanoparticles were obtained by ionotropic gelation process based on the interaction between chitosan and tripolyphosphate (TPP). The protein-loading capacity and encapsulation efficiency were 0.021% and 27.388%, respectively. The bFGF-loaded nanoparticles have a mean diameter of 424 nm, a narrow size distribution, spherical shape and positive surface charges. In vitro release showed that the extent of release was 68% at 24 hr. The protein integrity was investigated by SDS-PAGE analysis that confirmed protein integrity was not affected by the encapsulation procedure and release conditions.

Treatment of malignant gliomas with mitoxantrone-loaded poly (lactide-co-glycolide) microspheres.

OBJECTIVE: Mitoxantrone (MTZ) has potent in vitro activity against malignant glioma cell lines, but it cannot be used effectively as a systemic agent for the treatment of brain tumors because of its poor central nervous system penetration. However, MTZ-loaded poly(lactide-co-glycolide) (PLGA) microspheres may be injected into the peritumoral area and into tumor tissue to provide effective and sustained local drug concentrations without causing systemic side effects. METHODS: Fisher rats were randomized into three groups. The first group (n = 9) was concomitantly implanted with rat glioma (RG2) cells and blank PLGA microspheres. The second group (n = 6) was implanted with RG2 cells and MTZ-loaded PLGA microspheres. The third group (n = 9) was implanted with RG2 cells, and MTZ-loaded PLGA microspheres were injected into the same area after 7 days. Animals were sacrificed on Day 15 or 35. Tumor volumes were measured after hematoxylin and eosin staining. Distribution kinetics of MTZ in the brain was determined by high-performance liquid chromatography in nine rats injected with MTZ-loaded microspheres. RESULTS: The tumor volumes were 76 +/- 11 and 107 +/- 11 mm (mean +/- standard error) on Days 15 (n = 6) and 35 (n = 3), respectively, in the control group. In rats treated with MTZ-loaded microspheres on Day 7, tumor volumes were significantly reduced to 17 +/- 4 and 23 +/- 2 mm on Days 15 (n = 6) and 35 (n = 3), respectively. No tumor formation was observed when glioma cells and MTZ-loaded PLGA microspheres were implanted concomitantly (n = 6). No systemic side effects or parenchymal inflammatory infiltration were observed in either group of rats. Brain MTZ concentration was highest at the injection site and declined with time and distance from the injection site and with time. CONCLUSION: These data demonstrate that MTZ-loaded PLGA microspheres can deliver therapeutic concentrations of drug to the tumor and prevent glioma growth without causing side effects. This treatment method may increase the efficiency of antineoplastic therapy and positively impact survival.