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Cartilage tissue has a relatively low regenerative capacity which leads to a need for disease‐modifying active pharmaceutical ingredients (APIs) which can stimulate its regeneration and more urgently, effective ways of introducing APIs into the cartilage defects. Herein, an integrated system is proposed in which a hyaluronic acid (HA)‐based injectable hydrogel scaffold is embedded with API‐loaded poly(d,l‐lactide‐co‐glycolide) microspheres (PLGA MPs). This new injectable drug delivery system can successfully deliver APIs locally, increase drug retention time, and promote endogenous cartilage regeneration. In this study, resveratrol (RSV), a small hydrophobic compound is introduced as the selected API. PLGAs with differing copolymer ratios and endgroups are evaluated to study their effects on particle formation, RSV encapsulation, and drug release kinetics. This system is optimized as a 1 month sustained release formulation. In addition, the long‐acting chondrogenic induction of the RSV‐loaded system is evaluated by coculturing with cartilage‐derived progenitor cells in vitro. PLGA–RSV MPs stimulated the expression of cartilage‐related genes and the production of the cartilage‐specific extracellular matrix. This integrated system shows great potential as an injectable formulation for long‐acting chondrogenesis and endogenous cartilage tissue regeneration. An integrated system which consists of a hyaluronic acid (HA)‐based injectable hydrogel scaffold embedded with resveratrol (RSV)‐loaded poly(d,l‐lactide‐co‐glycolide) microspheres (PLGA MPs) is developed. This novel injectable system can provide controlled drug release and stimulating chondrogenesis in vitro for 4 weeks, exhibiting great potential as a long‐acting formulation for fast filling of cartilage defects and facilitating hyaline cartilage generation.

The aim of this study was to develop a hydrophilic oral controlled release system (CRS) using the amorphous form of gliclazide, a BCS class II compound, listed on the WHO list of essential medicines. For this purpose, spray-dried dispersions (SDDs) of gliclazide were produced using various grades of hydroxypropyl methylcellulose acetate succinate (HPMCAS) or copovidone as carrier under fully automated conditions. The solid-state properties of prepared SDDs were characterized using X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), modulated differential scanning calorimetry (MDSC), and Fourier transform infrared spectroscopy (FTIR). Supersaturated micro-dissolution testing of SDDs in fasted state-simulated intestinal fluid showed prolonged supersaturation state, with solubility increases of 1.5- to 4.0-fold. Solubility and stability characteristics of the most desirable SDDs in terms of relative dissolution area under the curves (AUCs) (AUC(SDD)/AUC(crystalline)) and stable supersaturated state concentration ratio up to 180 min (C180/Cmax) were determined. The optimized gliclazide-SDD amorphous forms were included into matrix tablets with HPMC blends using compaction simulator. Developed matrix systems were subjected to standard USP dissolution testing. Dissolution profiles obtained were linear with different slopes indicating varying rates of dissolution. Six-month storage stability testing was performed, and dissolution profiles remained stable with “similarity factor” (f2 = 85). Results show that the use of various HPMCAS as a drug carrier in the spray-drying process produces homogeneous single-phase SDDs which are stable and promising for inclusion into HPMC-based hydrophilic matrix systems.

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is associated with secondary hyperparathyroidism (HPT) and serum elevations in the phosphaturic hormone FGF23, which may be maladaptive and lead to increased morbidity and mortality. To determine the role of FGF23 in the pathogenesis of CKD-MBD and development of secondary HPT, we developed a monoclonal FGF23 antibody to evaluate the impact of chronic FGF23 neutralization on CKD-MBD, secondary HPT, and associated comorbidities in a rat model of CKD-MBD. CKD-MBD rats fed a high-phosphate diet were treated with low or high doses of FGF23-Ab or an isotype control antibody. Neutralization of FGF23 led to sustained reductions in secondary HPT, including decreased parathyroid hormone, increased vitamin D, increased serum calcium, and normalization of bone markers such as cancellous bone volume, trabecular number, osteoblast surface, osteoid surface, and bone-formation rate. In addition, we observed dose-dependent increases in serum phosphate and aortic calcification associated with increased risk of mortality in CKD-MBD rats treated with FGF23-Ab. Thus, mineral disturbances caused by neutralization of FGF23 limited the efficacy of FGF23-Ab and likely contributed to the increased mortality observed in this CKD-MBD rat model.

Background and PurposeThere is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses.Methods and ResultsHighly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials.ConclusionThe ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

In a formulation, traces of peroxides in copovidone can impact the stability of drug substances that are prone to oxidation. The present study aimed to investigate the impact of peroxides in novel Plasdone™ S630 Ultra and compare it with regular Plasdone™ S630 on the oxidative degradation of quetiapine fumarate amorphous solid dispersions prepared via hot-melt extrusion technique. The miscibility of copovidones with drug was determined using the Hansen solubility parameter, and the results indicated a miscible drug–polymer system. Melt viscosity as a function of temperature was determined for the drug–polymer physical mixture to identify the suitable hot-melt extrusion processing temperature. The binary drug and polymer (30:70 weight ratio) amorphous solid dispersions were prepared at a processing temperature of 160°C. Differential scanning calorimetry and Fourier transform infrared spectroscopy studies of amorphous solid dispersions revealed the formation of a single-phase amorphous system with intermolecular hydrogen bonding between the drug and polymer. The milled extrudates were compressed into tablets by using extragranular components and evaluated for tabletability. Stability studies of the milled extrudates and tablet formulations were performed to monitor the oxidative degradation impurity (N-oxide). The N-oxide impurity levels in the quetiapine fumarate - Plasdone™ S630 Ultra milled extrudates and tablet formulations were reduced by 2- and 3-folds, respectively, compared to those in quetiapine fumarate - Plasdone™ S630. The reduced oxidative degradation and improved hot-melt extrusion processability of Plasdone™ S630 Ultra make it a better choice for oxidation-labile drugs over Plasdone™ S630 copovidone.

This study investigated the effect of binder level on the physicochemical changes and tabletability of acetaminophen (APAP)-hydroxypropyl cellulose (HPC) granulated using twin-screw melt granulation. Even at 5% HPC level, the tablet tensile strength achieved up to 3.5 MPa. A minimum of 10% HPC was required for the process robustness. However, 20% HPC led to tabletability loss, attributable to the high mechanical strength of APAP granules. The over-granulated APAP granules had thick connected HPC scaffold and low porosity. Consequently, these granules were so strong that they underwent a lower degree of fracture under compression and higher elastic recovery during decompression. HPC was enriched on the surface of APAP extrudates at all HPC levels. Amorphous APAP was also observed on the extrudate surface at 20% HPC level, and it recrystallized within 24 h storage. To achieve a robust process and optimal improvement in APAP tabletability, the preferred HPC level was 10 to 15%.

Marshall Crew, PhD, President & CEO, Agere Pharmaceuticals, Inc., continues his multiple-part series discussing today's most challenging issues in solubility.

Marshall Crew, PhD, President & CEO, Agere Pharmaceuticals, Inc., continues his multiple-part series discussing today's most challenging issues in solubility.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling.

COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID 50 of SARS-CoV-2, respectively. Mice infected with 10 5 TCID 50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19.