Application of disaccharides alone and in combination,for the improvement of stability and particle properties of spray-freeze dried IgG

Purpose: Spray-freeze drying (SFD) is a recently applied method to develop pharmaceutical powders. This study aimed to analyze the competence of Trehalose, Mannitol, Lactose, and Sorbitol instability and aerosolization of Immunoglobulin G (IgG) via SFD.

Methods: Induced soluble aggregates were quantified at 0 and 3?months, and 45?°C using size-exclusion chromatography. Conformation and thermogravimetric assessments were done by Fourier transform infrared spectroscopy and differential scanning calorimetry. Laser light scattering was performed to determine the particle sizes. Aerodynamic features were characterized by twin stage impinger and scanning electron microscopy.

Results: Although sugars/polyols preferably stabilized IgG following the process, storage stabilization was achieved in Trehalose, Trehalose-Lactose, Lactose, and Trehalose-Mannitol-based powders with soluble aggregates <5%. The conformation of antibody was preserved with β sheet content from 66.28% to 76.37%. Particle sizes ranged from 5.23 to 8.12?µm. Mannitol exhibited the best aerodynamic behavior, fine particle fraction (FPF: 70%) but high degree of protein aggregation during storage.

Conclusions: SFD could favorably stabilize antibody using Trehalose and its combination with Lactose and Mannitol, and also, Lactose alone. Sorbitol disturbed IgG powder recovery. Incorporation of other types of excipient is required for efficient respiratory delivery of IgG molecules.     

Cangrelor dose titration using platelet function testing during cerebrovascular stent placement

BackgroundOptimal antiplatelet inhibition is vital during cerebrovascular stenting procedures, yet no standardized recommendation exists for antithrombotic therapy in these scenarios. Cangrelor is an intravenous P2Y12 inhibitor with a favorable pharmacokinetic profile for use during neuroendovascular stenting.MethodsA retrospective review of all neuroendovascular patients who underwent stenting between 1 January 2019 and 22 March 2020 and were treated with cangrelor was conducted. Thirty-seven patients met inclusion criteria.ResultsAll patients were administered a bolus of 5 mcg/kg of cangrelor followed by a maintenance infusion. Antiplatelet effects of cangrelor were monitored using platelet reactivity units (PRU). Based on the initial PRU, seven patients’ doses were adjusted with subsequent PRUs in or near the goal range of 50–150. One patient experienced an acute intraprocedural occlusion likely related to a subtherapeutic PRU which subsequently resolved with cangrelor dose adjustment and intra-arterial tirofiban administration, and one patient experienced a post-procedure stent occlusion which required a thrombectomy and intra-arterial tirofiban administration. No hemorrhagic complications occurred.DiscussionCangrelor utilization during neuroendovascular stenting with maintenance doses of <2 mcg/kg/min with dose adjustments based on platelet function testing has not been previously described. Cangrelor presents many advantages compared to standard therapy in patients undergoing stent placement related to its pharmacokinetic profile, rapid onset of action, ease of transition to oral P2Y₁₂ antiplatelet agents, and measurability.ConclusionCangrelor is a promising alternative to currently available therapies, especially in patients with a high hemorrhagic risk.     

The Avon Patellofemoral Joint Replacement: Mid-Term Prospective Results from an Independent Centre

Background

Approximately 10% of patients with osteoarthritis (OA) of the knee have unicompartmental OA confined to the patellofemoral joint (PFJ). The main surgical options are total knee replacement (TKR) and PFJ replacement (PFJR). PFJR has a number of advantages over TKR, including being less invasive, preserving the unaffected parts of the knee, allowing faster recovery and better range of motion and function. We report our prospective mid-term results of the Avon PFJR for established isolated PFJ arthritis in 61 consecutive procedures.

Methods

Sixty-one Avon PFJRs were performed in 57 patients. The outcome measures were the new Oxford knee score (OKS), Hungerford and Kenna score (HKS), and Crosby Insall knee scores. Only patients with severe isolated PFJ OA were included. The diagnosis was based on a combination of clinical, radiological and, where available, arthroscopic findings.

Results

Mean follow-up was 5.09 years (range, 12 to 124 years). There were 2 revisions in the first 5 years. The median HKS score was 80 (interquartile range, 70 to 95) and the mean OKS was 31.8 (± standard deviation, 8.7) at 5 years. These were significantly better (p < 0.001) than the preoperative scores.

Conclusions

The Avon prosthesis gives good functional outcomes in the medium term and survives well. Our data support other studies in the literature and is the largest independent prospective study to date.     

Mitochondrial function and energy metabolism in neuronal HT22 cells resistant to oxidative stress

Background and Purpose

The hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Extracellular glutamate depletes cellular glutathione by blocking the glutamate/cystine antiporter system xc−. Glutathione depletion induces a well-defined programme of cell death characterized by an increase in reactive oxygen species and mitochondrial dysfunction.

Experimental Approach

We compared the mitochondrial shape, the abundance of mitochondrial complexes and the mitochondrial respiration of HT22 cells, selected based on their resistance to glutamate, with those of the glutamate-sensitive parental cell line.

Key Results

Glutamate-resistant mitochondria were less fragmented and displayed seemingly contradictory features: mitochondrial calcium and superoxide were increased while high-resolution respirometry suggested a reduction in mitochondrial respiration. This was interpreted as a reverse activity of the ATP synthase under oxidative stress, leading to hydrolysis of ATP to maintain or even elevate the mitochondrial membrane potential, suggesting these cells endure ineffective energy metabolism to protect their membrane potential. Glutamate-resistant cells were also resistant to oligomycin, an inhibitor of the ATP synthase, but sensitive to deoxyglucose, an inhibitor of hexokinases. Exchanging glucose with galactose rendered resistant cells 1000-fold more sensitive to oligomycin. These results, together with a strong increase in cytosolic hexokinase 1 and 2, a reduced lactate production and an increased activity of glucose-6-phosphate dehydrogenase, suggest that glutamate-resistant HT22 cells shuttle most available glucose towards the hexose monophosphate shunt to increase glutathione recovery.

Conclusions and Implications

These results indicate that mitochondrial and metabolic adaptations play an important role in the resistance of cells to oxidative stress.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Characterization of six novel mutations in CYBA: the gene causing autosomal recessive chronic granulomatous disease

One of the rarest forms of chronic granulomatous disease (CGD) is caused by mutations in CYBA, which encodes the p22-phox subunit of the phagocyte NADPH oxidase, leading to defective intracellular killing. This study investigated eight patients (six males and two females) from seven consanguineous, unrelated families with clinical CGD, positive family history and p22-phox deficiency. Mutation analysis of CYBA showed six different novel mutations: deletion of exons 3, 4 and 5; a missense mutation in exon 6 (c.373G>A); a splice site mutation in intron 5 (c.369+1G>A); a frameshift in exon 6 (c.385delGAGC); a frameshift in exon 3 (c.174delG); and a frameshift in exon 4 (c.223delC).     

Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphoblastic leukemia and is correlated with p53 gene mutations

Although dihydrofolate reductase (DHFR) gene amplification is a common mechanism of resistance to methotrexate (MTX) in tumor cell lines, with the exception of a few case reports, the incidence of this phenomenon as a mechanism of MTX resistance in the clinic has not been reported. We studied 38 untreated patients and 29 patients in relapse with acute lymphoblastic leukemia (ALL) for gene amplification and p53 gene mutations. Three patients were studied both at diagnosis and at each of two relapses after treatment with MTX. Nine of 29 relapsed patients (31%) had low-level DHFR gene amplification (two to four gene copies) associated with increased levels of DHFR mRNA and enzyme activity. Of significance was a correlation of gene amplification with p53 mutations in seven of nine relapsed patients (P < .001). Low-level DHFR gene amplification may be an important cause of MTX resistance in ALL and strengthens the concept that mutations in the p53 gene may lead to gene amplification as a consequence of defective cell cycle control.     

Open reduction internal fixation of lateral humeral condyle fractures in children. A series of 105 fractures from a single institution

Lateral humeral condyle fractures account for 17 % of the distal humeral condyle fractures. Displaced and/or rotated fractures require appropriate reduction and stabilisation. There are, however, a number of controversies in the surgical management of these patients. The aim of the present study was to review the results of patients with a displaced lateral humeral condyle fracture treated with open reduction and internal fixation (ORIF). We retrospectively reviewed children treated with ORIF of lateral humeral condyle fractures at a single institution over a period of 13 years. All cases were identified through the trauma register. Case notes and radiographs were retrieved. Fracture classification, mode of fixation, time to union, and final outcomes at the latest follow-up were reviewed. One hundred and five lateral condyle fractures were identified in 76 male and 29 female patients. Average age was 6.2 years. Ninety-two were Milch type II and 13 Milch type I. According to the Jacob’s classification, 38 were type II and 67 type III. All fractures were treated with open reduction and fixation with K-wires. Average time to radiological union was 33 days. Follow-up ranged between 2 and 8 years (average 3.2 years). Radiological hypertrophy of the lateral condyle was present in 45 cases (42 %). Three patients developed a pseudo-cubitus varus deformity. Further four patients developed a true cubitus varus. There was one case of superficial infection of the K-wires and one case of delayed union. At the latest follow-up, 96 % of the patients achieved an excellent final result and 4 % a good final result. Our results demonstrate that fracture union and excellent final outcomes can be expected in all patients using our protocol, whereby all patients with a displaced fracture are managed by ORIF with K-wire fixation, with the wires only being removed after there is evidence of radiological union. Compared to recent reports of closed reduction internal fixation, this series demonstrates good results with no complications directly relating to the open reduction technique. Level of evidence Case series, Level IV.