The development of stable biopharmaceutical formulations, such as monoclonal antibodies, poses a great challenge in the pharmaceutical industry. This study investigated the stabilizing effect of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in liquid and solid formulations of infliximab during processing and storage. The solid formulation was produced by a scaled-up high-speed electrospinning method, resulting in a product suitable for reconstitution with excellent dissolution properties. Liquid formulations contained exclusively HP-β-CD as a stabilizing excipient at different concentrations. Both types of formulations were stored under various conditions for up to 6 months, and infliximab stability was compared based on monomer recovery and the analysis of fragments and aggregates. The solid formulation stored at low (-18 °C, 4 °C) temperatures and lower humidity conditions show the most promising results. Moreover, the monomer loss was less than 10 % even at room temperature storage after 2 months, indicating a potentially good temperature tolerance. Increasing HP-β-CD content in liquid formulations significantly improved the long-term stability of infliximab while enhancing protection against mechanical stress. Furthermore, to better understand the stabilizing mechanism of HP-β-CD on infliximab, the molecular interactions were investigated by bio-layer interferometry and isothermal titration calorimetry methods. The experiments proved that the presence of HP-β-CD decreased infliximab self-interaction, correlating with the aggregation-suppressing effect of HP-β-CD observed during the stress- and long-term stability studies. The results demonstrate the potential of HP-β-CD as a stabilizing excipient in liquid and solid formulations of infliximab.

Psoriasis, a chronic inflammatory skin disease, poses a significant burden on patients' quality of life and healthcare systems. While mild-to-moderate cases are treated topically, usually combined with phototherapy, severe cases require systemic treatment with immunosuppressants, retinoids or biologics. However, all available treatments have drawbacks in terms of efficiency and side effects. Drawing from studies linking depression treatment to psoriasis improvement, we investigated whether topical formulations of selective serotonin reuptake inhibitors (SSRIs) could offer a viable therapy for psoriasis. Five SSRIs (sertraline, fluoxetine, paroxetine, escitalopram, fluvoxamine) were evaluated for their in vitro cytotoxicity, in human keratinocytes and THP-1 monocytes. Their anti-inflammatory action was tested using cell differentiation assays and immunoassays of pro-inflammatory cytokines in THP-1 monocytes. The results obtained with sertraline, escitalopram, and fluvoxamine suggested further evaluation in vivo. Anti-inflammatory effects were evaluated by skin parameter monitoring and histopathology, in an imiquimod-induced psoriasis-like inflammation mice model, and the best results were obtained for fluvoxamine. These findings were further supported by in silico molecular docking studies of the structural interaction between the serotonergic receptors and the drugs. Future research will focus on developing and characterizing of topical fluvoxamine formulations, like emulsions and penetration-enhancer vesicles, which offer advantages over the gels used herein.

Access of drugs to the central nervous system is limited by the blood-brain barrier, and this in turn affects drug efficacy/toxicity. To date, most drug discovery optimization paradigms have relied heavily on in vitro transporter assays and preclinical species pharmacokinetic evaluation to provide a qualitative assessment of human brain penetration. Because of the lack of human brain pharmacokinetic data, mechanistic models for preclinical species, combined with in vitro and in silico data, are useful for translation to human. These models require transporter expression data to be measured in both in vitro and in vivo systems. The purpose of this work was to quantify transporter expression and generate scaling factors (SFs) to enable in vitro in vivo extrapolation (IVIVE) of transporter-mediated processes and to support the development of a PBPK model of the brain in rats. SF represents the ratio of abundance of the relevant transporters in the tissue relative to transporter expressing cells. Using quantitative proteomics with QconCAT technology, the expression of human and rat P-gp (ABCB1/Abcb1) and BCRP/Bcrp (ABCG2/Abcg2) was measured in rat brain microvessels, mock and transfected cell lines including, (Madin-Darby canine kidney I (MDCK I), Madin-Darby canine kidney II (MDCK II) and pig kidney epithelial cells (LLC-PK1). P-gp expression ranged from 32 to 71 pmol/mg in rat brain microvessels, exceeding literature values of 14.1-25.2 pmol/mg microvessels proteins. Conversely, Bcrp expression ranged between 0.02-0.27 pmol/mg,proteinslower than the literature range (2-6.2 pmol/mg of proteins). P-gp expression in MDCK I and LLC-PK1 cells transfected with rat Mdr1a was similar (within 1.5-fold) as was human P-gp expression in MDR1 transfected LLC-PK1 and MDCK II cells. The generated SFs were 34.4 and 50.4 for brain P-gp (depending on the cell line used) and 0.53 for brain Bcrp. Endogenous P-gp transporter was detected in MDCK II cell lines when protein expression was measured using a surrogate peptide that was shared across species. The current work provides a framework for proteomics-informed translation of in vitro P-gp and BCRP-related kinetics of drugs and supports the development of PBPK models to predict drug disposition in the brain.

Dry eye disease is a multifactorial condition characterized by a loss of homeostasis of the tear film. Among the various treatment approaches, the application of ophthalmic oil-in-water nanoemulsions with incorporated anti-inflammatory drugs represents one of the most advanced approaches. However, the liquid nature of nanoemulsions limits their retention time at the ocular surface. Transforming the nanoemulsions into a dry form that would disperse rapidly in the tear fluid would improve the retention of the drug at the ocular surface. The aim of this study was to investigate electrospinning as a method for the preparation of a solid eye preparation based on nanoemulsion loaded with the anti-inflammatory drug loteprednol etabonate. Four nanoemulsions differing in oil-to-surfactant ratios were incorporated in hydrophilic nanofibers based on polyethylene oxide, poloxamer 188, and Soluplus®. The dried nanoemulsions in the form of nanofibers dispersed readily on contact with aqueous medium, resulting in a dispersion of nanometre-sized droplets with average size comparable to the average droplet size of the initial nanoemulsions. A rheological study revealed the predominant elastic behavior of the dispersed nanofibers, which indicates the formation of a weak gel after the dispersion of the dried nanoemulsion in tear fluid at the ocular surface. The biocompatibility of the dried nanoemulsions in the form of nanofibers after a single and multiple-dose application was confirmed using the 3D HCE-T model of the stratified epithelium of the human cornea, suggesting that this innovative solid eye preparation could represent a new approach to the treatment of dry eye disease.

Triol-type ginsenoside Re (GS-Re) exhibits potent anti-myocardial ischemia-reperfusion effects, but its clinical use is hindered by poor bioavailability. This study evaluates the impact of β-cyclodextrin (β-CD) inclusion on GS-Re bioavailability and tissue dynamics in rat models. The GS-Re-β-CD complex was prepared using aqueous stirring and characterized. Male Wistar rats (200 ± 20 g) were administered GS-Re at a dose of 500 mg/kg. Plasma concentrations were quantified using UHPLC-MS/MS to evaluate pharmacokinetics and analyze metabolites in tissues and feces. Compared to the group receiving GS-Re alone, the GS-Re-β-CD inclusion complex exhibited significantly improved pharmacokinetic characteristics in rats: Maximum concentration (Cmax) increased by 1.86-fold. Area under the curve (AUC) increased by 2.09-fold. Time to reach peak concentration (T) was reduced, while the half-life (t) was extended, suggesting a faster and prolonged absorption of GS-Re. Metabolite analysis showed higher concentrations of Rg1, Rg2, Rh1, F1, PPT, and Re in tissues with GS-Re-β-CD, while metabolite types remained unchanged. The inclusion of β-CD significantly enhanced the bioavailability and tissue concentration of GS-Re, as demonstrated by increased C and AUC, along with a shorter T and longer t. These findings suggest that β-CD inclusion could be an effective strategy to improve the clinical applicability of GS-Re, providing valuable pharmacokinetic and tissue concentration insights for further development.

We evaluated algorithms designed to extrapolate extractables data for predicting process equipment-related leachables (PERLs) and assessing PERL exposure in single-use systems (SUSs) and their assemblies. The robustness and sensitivity of these algorithms were tested against variations in input data, including extrapolation algorithms for both short and long contact time extractables data obtained from the standardized extractables protocol provided in USP 〈665〉. Our findings demonstrate that extrapolated data for SUS and assemblies are suitable for safety assessments. Extrapolated and aggregated data do not systematically underestimate potential PERL exposure values, provided that the extractables data originate from experiments with a higher surface area to contact liquid volume ratio and/or a low liquid to material volume ratio compared to the use scenario. The algorithms are non-sensitive to deviations in input data, as these deviations are propagated decreasingly into extrapolated data and parameters. The quality and significance of PERL exposure calculations can be enhanced by incorporating extractables study data from experiments using a semipolar organic solution, such as ethanol.

One of the main concerns with formulations containing amorphous solid dispersions (ASDs) is their physical stability. Stability can be compromised if a formulation contains any residual crystallinity of an active pharmaceutical ingredient (API) that could act as seeds for further crystallisation. This study presents four methods for crystalline amlodipine maleate quantification in ASD, which were developed using one Raman and three NIR process analysers. A preliminary analysis revealed distinct differences between amorphous and non-amorphous forms of the API, both in the API alone and in the formulation. These differences laid the foundation for model development in subsequent steps. The development of four partial least squares (PLS) models proceeded through two stages, initially using a single granulation batch dataset for training, and then expanding to include three batches. Their predictability was evaluated on an additional batch dataset. Models were evaluated primarily using root mean square error of prediction (RMSEP), residual prediction deviation (RPD), and limit of detection along with other metrics. To the best of authors' knowledge, this is the first study that focuses on process monitoring of fluidized bed granulation used in preparation of ASD. The results of this study and their interpretations present novel aspects of Raman and NIR process analyser applications in combination with PLS.

In the endocrine system, anaplastic thyroid cancer (ATC) is extremely aggressive since it inhibits the majority of medications and treatments. Therefore, there is an immediate demand to identify new treatment approaches or drugs to deal with ATC. Recently, amino acid Schiff base copper complexes have received great attention due to their excellent anti-tumor activity. In this research, three copper(II) complexes, [Cu(o-van-D-Trp)(phen)](1), [Cu(o-van-D-Trp)(bipy)](2), [Cu(naph-D-Trp)(bipy)](3), [D-Trp = D-tryptophan; o-van = o-vanillin; naph = 2‑hydroxy-1- naphthaldehyde; phen = 1,10-phenanthroline; bipy = 2,2-biprydine], have been synthesized and investigated as potential anticancer agents. The crystal structure data of the complexes demonstrate that the central copper (II) atom forms a twisted polyhedral environment with nitrogen and oxygen atoms. The MTT results demonstrated that three complexes exhibited superior cytotoxicity against five cell lines of thyroid cancer (Cal-62 cells, ARO cells, KHM-5 m cells, BHP10-3 cells and K1 cells), especially complex 1 with the IC values of 0.59±0.05 μM, 2.36±0.47 μM, 1.10±0.87 μM, 0.75±0.09 μM, 1.72±0.06 μM, when cisplatin was used as a control. Research on antitumor mechanisms has demonstrated that complex 1 can significantly reduce the mitochondrial membrane potential, raise autophagy, and produce reactive oxygen species (ROS) in ARO cells in a dose-dependent manner. RNA sequencing study reveals that complex 1 may cause apoptosis in ARO cells and exhibit anticancer efficacy in vitro through ROS-mediated downregulation of Akt and p38 MAPK activation.

The main purpose of this study was to optimize a cyclodextrin-based nanogel of flurbiprofen (FP) for prolonged dermal administration and evaluate its stability, in vitro release, ex vivo skin permeation, and in vivo pharmacokinetic profile.

Elevated intraocular pressure (IOP) is implicated in the structural and functional damage to the retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). Topical IOP lowering agents provide short-term relief, necessitating frequent dosing. Moreover, non-adherence to frequent eyedrops administration contributes significantly to visual field loss and worsens the disease outcome. We optimized the poly (lactic-co-glycolic acid) (PLGA) nanoparticles encapsulation of hybrid antioxidant-nitric oxide donor SA-2 (SA-2NP), investigated its bioavailability, duration of IOP lowering efficacy, and effects on retinal function in the microbead model of ocular hypertension (OHT). SA-2 was bioavailable in the anterior and posterior segments after 1, 8, and 24 h post-single topical eyedrop administration. SA-2NP significantly lowered IOP (∼25-34%) and preserved the RGC function after weekly eyedrop administration for 3 weeks in C57BL/6J mice. In conclusion, the optimized SA-2NP formulation demonstrated optimal bioavailability, ocular safety, and prolonged IOP-lowering efficacy in the mouse microbead occlusion model of OHT.

This study investigates the effects of disintegrants sodium starch glycolate (SSG) and crospovidone (CP) on the printability, rheological properties, and disintegration time of agar and hydroxypropyl methylcellulose (HPMC)-based formulations designed for semi-solid extrusion. Printability was assessed by measuring the dimensional accuracy of manually extruded filaments. Rheological analysis was performed using oscillatory measurements. Principal component analysis (PCA) and Spearman correlation analysis identified three key components (phase angle, critical strain, and elastic modulus) that explained the total variance in the rheological dataset. A 2 × 3 factorial design was employed to evaluate the impact of CP, SSG, and HPMC on these rheological parameters, as well as on printability and disintegration time. Results indicated that formulations containing HPMC and SSG generally exhibited better printability. Formulations containing CP achieved satisfactory printability only when SSG or HPMC was included. The optimal printability and rheological properties were achieved with formulations containing 5 % CP and 10 % SSG. Linear regression models correlated geometric volumes of the model and pycnometric volumes of printed objects, with validation showing that predicted masses were within a 95 % confidence interval of measured values for various shapes. All formulations demonstrated immediate-release properties, confirming the successful fabrication of personalised immediate-release dosage forms using semi-solid extrusion technology.

Zonisamide exhibits significant pharmacokinetic variability, demanding for the development of population pharmacokinetic (PopPK) models to identify key factors influencing drug disposition. This study aimed to develop and validate a PopPK model to optimize zonisamide posology in patients with refractory epilepsy. A total of 114 plasma concentrations of zonisamide, obtained from 64 patients, were used for PopPK model development, employing the nonlinear mixed-effects modelling approach. The final model was evaluated by visually inspecting the goodness-of-fit plots and the visual predictive check plot and by the bootstrap resampling method. A one-compartment model with first-order elimination was the one that best described the pharmacokinetic profile of zonisamide. Between-patient variability (BPV) was included on clearance (CL/F), volume of distribution (Vd/F) and absorption rate constant (ka). The residual error (RE) was modeled as proportional. The final model estimates for CL/F, Vd/F and ka were 0.761 L/h, 48.10 L and 0.671 h⁻¹, respectively. The BPV associated with CL/F, Vd/F, and ka was 43.93%, 52.06%, and 91.27%, respectively, while the proportional RE was 7.18%. The concomitant administration of enzyme-inducing antiseizure drugs (EIASDs), included in the model as inducer drug load (INDDL), significantly accounted for BPV associated with CL/F and led to increased CL/F in patients receiving EIASDs compared to the others. Consequently, patients receiving EIASDs require higher daily doses of zonisamide to achieve therapeutic plasma concentrations compared to those not treated with EIASDs. Model validation, using bootstrap and visual predictive checks, confirmed its stability and robustness, making it a valuable tool for individualized zonisamide dosing in adults with refractory epilepsy.

Premature drug release is the primary hindrance to the effective function of the lyso-thermosensitive liposomes (LTSLs) of doxorubicin (Dox), known as ThermoDox® for the treatment of cancer. Herein, we have optimized LTSLs by using a combination of phospholipids (PLs) with high transition temperatures (Tm) to improve the therapeutic outcome in an assisted ultrasound approach. For this, several Dox LTSLs were prepared using the remote loading method at varying molar ratios (0 to 90 %) of DPPC (Tm 41 °C) and HSPC (Tm 54.5 °C), as well as a constant molar ratio of MSPC (10 %), DSPE-mPEG (4 %). The treatment efficacy was explored by using ultrasound as external hyperthermia (HT) (40-42℃) in mice bearing C26 murine colon carcinoma. All the formulations had an average diameter of around 110 nm, PDI ≤ 0.15, zeta potential of around -12 mV, and Dox encapsulation of >90 %. The cytotoxicity results indicated a higher IC value of Dox-LTSLs compared to the ThermoDox® (F0: DPPC:MSPC:DSPE-mPEG, 90:10:4), attributed to the faster Dox release in F0 formulation devoid of HSPC. Among various formulations, F25 (DPPC: MSPC: DSPE-mPEG: HSPC, 65:10:4:25) showed the highest cellular uptake at 42℃ and significantly improved the antitumor and survival efficacy in mice bearing C26 colon carcinoma in combination with ultrasonic HT compared to F0. Collectively, results demonstrated that optimizing the rigidity of the liposomal bilayers through the combinatorial selection of PLs of different transition temperatures could improve the plasma stability of the liposome, and hence ameliorate the outcome of therapy in assistance with an effective HT approach.

The frozen storage of biopharmaceuticals brings new challenges to the primary packaging material. Due to an increasing demand and the downsides of standard type I glass vials, such as vial breakage, novel vial types for special applications of parenteral drug products have been introduced to the market in the past years. Mechanical stresses due to dimensional changes experienced during freezing and thawing could change the material properties, hence affecting the interaction with the drug product stored in the vial or functionality such as overall integrity. Therefore, we studied the suitability of different vial qualities related to the thermally induced mechanical stresses experienced during frozen drug product preparation and storage. First, the possible failure modes for each vial type were identified. The interaction between vial surface and drug product were investigated considering surface hydrophobicity, surface free energy and surface roughness as well as microscopically visible changes analyzed by confocal laser scanning microscopy. Differences in surface hydrophobicity, roughness and surface free energy between the vial types did not impact the performance upon freeze-thaw stress and did not change with the stress. Screening the vial content for particles originating from the container using light and electron scanning microscopy combined with energy-dispersive X-ray spectroscopy showed only rare cases of particles in coated glass vials. Under extreme stress conditions, including a drop-test in the frozen state, a low number of particles was also detected in coated polymer vials. No quality issues regarding the functionality were observed upon container closure integrity testing, while the oxygen permeability was slightly increased for uncoated and especially coated polymer vials. Overall, the results show that several vial types are appropriate for the frozen storage of drug products and selection should be based on the formulation and other product requirements.

Adeno-associated virus (AAV)-based vectors have emerged as an effective and widely used technology for somatic gene therapy approaches, including those targeting the retina. A major advantage of the AAV technology is the availability of a large number of serotypes that have either been isolated from nature or produced in the laboratory. These serotypes have different properties in terms of sensitivity to neutralizing antibodies, cellular transduction profile and efficiency. The infectivity of AAV vectors depends on the affinity to certain molecules on the cell surface, in particular to cellular glycosaminoglycans (GAGs) such as heparan sulfate proteoglycans (HSPGs). Here, we tested how altering HSPG affinity in AAV vectors affects cellular tropism and transduction efficiency. The previously developed AAV2.GL variant was used as a starting variant to alter or disrupt HSPG affinity. The HSPG-independent AAV9 serotype was used to introduce different HSPG-binding sites. As an indicator of HSPG affinity, we measured the binding strength of the vector variant on a heparin chromatography column. We show that modification of capsid-exposed residues has a strong impact on HSPG affinity, cellular tropism and transduction efficiency in HeLa cells and in vivo in mouse retina. Our study shows that key properties of AAV vectors can be tailored in different directions and used to improve tropism and efficiency.

Amid the COVID-19 pandemic, various public health measures were adopted to reduce the transmission risk, including the full reimbursement of SARS-CoV-2 professional rapid antigen detection tests (Ag-RDT) conducted in clinical pathology laboratories, community pharmacies (CPs), and other authorized entities. This study aimed to assess the impact of integrating CPs into the Portuguese National Health Service (NHS) testing strategy on the capacity of professional Ag-RDT delivery, and to compare the equity in testing access with and without CPs participation.

To evaluate the drug-drug interactions (DDI) of tunodafil (youkenafil), a novel phosphodiesterase type 5 inhibitor, its inhibitory effects on CYP450 enzymes in vitro and its clinical trials in combination with ritonavir or omeprazole were conducted.

Excipients are ingredients in pharmaceutical products other than the active ingredient, added to facilitate manufacturing, enhance stability or modulate release and bioavailability. Vitamins are diverse molecules essential for human nutrition that also can fulfil excipient functions. This review focuses on vitamins used as excipients and provides an overview of the functions of vitamins in various pharmaceutical formulations. A thorough search was conducted to understand the current use of vitamins in marketed drug products, concluding that many vitamins are already used as functional excipients. Vitamins are used widely in different dosage forms, including oral, parenteral, and topical formulations, and alongside a broad range of active pharmaceutical ingredients, biologics, and small molecules from different biopharmaceutical classification system classes. Many examples of the use of vitamins to improve the performance of the pharmaceutical formulation in which they are included are presented and the mode of action of vitamins as excipients in the product is reviewed. Furthermore, the potential for future uses of vitamins in pharmaceutical products is highlighted. Lastly, considerations for the use of vitamins as excipients in drug products as well as the regulatory framework are discussed.

Tacrolimus is extensively used for the prevention of graft rejection following solid organ transplantation in pregnant women. However, knowledge gaps in the dosage of tacrolimus for pregnant patients with different CYP3A5 genotypes and infection conditions have been identified. This study aimed to develop a pregnant physiologically based pharmacokinetic (PBPK) model to characterize the maternal and fetal pharmacokinetics of tacrolimus during pregnancy and explore and provide dosage adjustments. We developed PBPK models for nonpregnant patients and validated them via data from previous clinical studies using PK-Sim and Mobi software. To extrapolate to pregnancy, we considered anatomical, physiological, and metabolic alterations and simulated tacrolimus by adding six groups of IL-6 concentrations (0, 5, 25, 50, 500, and 5000 pg/mL). Models were verified by assessing goodness-of-fit plots and ratios of predicted-to-observed pharmacokinetic parameters. The developed PBPK models adequately describe the available clinical data; the fold errors of the predicted and observed values of the area under the curve and peak plasma concentration were between 0.59 and 1.64, and the average folding error and the absolute average folding error values for all concentration-time data points were 1.15 and 1.36, respectively. The simulation results indicated that the area under the steady-state concentration‒time curve and trough concentrations decreased from the first to the third trimester of pregnancy. The trough concentrations were not within the therapeutic range (4-11 ng/mL) in pregnant patients with the CYP3A5 genotype for most of the infection conditions and exceeded its effective concentration in all the CYP3A5 nonexpressers. Based on the model-derived dosing regimen, the tacrolimus trough concentration in pregnant patients with different CYP3A5 genotypes could fall into the therapeutic window, which provided a clinical practice reference for dosage adjustments during pregnancy.

The purpose of this study was to evaluate EpiColon, a novel human organotypic 3D colon microtissue prototype, developed to assess colonic drug disposition, with a particular focus on permeability ranking, and compare its performance to Caco-2 monolayers. EpiColon was characterized for barrier function using transepithelial electrical resistance (TEER), morphology via histology and immunohistochemistry, and functionality through drug transport studies measuring apparent permeability (P). Cutoff thresholds for the permeability of FITC-dextran 4 kDa (FD4), FITC-dextran 10 kDa (FD10S), and [C]mannitol were established to monitor microtissue integrity. Permeability of EpiColon for 20 benchmark drugs was compared with Caco-2 data, and the activity of pivotal efflux transporters, including multidrug resistance protein 1/P-glycoprotein (MDR1/P-gp), along with multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP), was evaluated using selective substrates. EpiColon exhibited a physiological barrier function (272.0 ± 53.05 Ω x cm) and effectively discriminated between high (e.g., budesonide and [H]metoprolol) and low permeable compounds (e.g., [H]atenolol and [C]mannitol). The model demonstrated functional activity for key efflux transporters, with efflux ratios of 2.32 for [H]digoxin (MDR1/P-gp) and 3.34 for sulfasalazine (MRP2 and BCRP). Notably, EpiColon showed an enhanced dynamic range in the low permeability range, differentiating P between FD4 and FD10S, in contrast to Caco-2 monolayers. Significant positive correlations were observed between human fraction absorbed (f) and logarithmically transformed P [AP-BL] values for both EpiColon (r = 0.68) and Caco-2 (r = 0.68). Furthermore, EpiColon recapitulates some essential phenotypic and cellular features of the human colon, including the expression of critical marker genes (Pan-Cytokeratin: epithelial/colonocytes, Vimentin: mesenchymal/fibroblast, and Alcian Blue: goblet cell/mucus). In conclusion, EpiColon is a promising platform that offers a valuable complement to conventional Caco-2 monolayers for studying colonic drug disposition. However, the presence of flat and some cuboidal cells, along with low throughput, must be addressed to improve its applicability in both academic research and pharmaceutical industry.