Dispersity determination of poly(ethylene glycol)s using supercritical fluid chromatography-mass spectrometry and different mass analysers.

RATIONALE: Dispersity values are considered critical quality attributes for the quality control of poly(ethylene glycol) formulations due to the direct impact on drug performance. However, when these polymers are analysed using mass spectrometry, the design of the mass analyser can impact the oligomer response and affect the obtained dispersity values, so further understanding is needed. METHODS: The deconvoluted electrospray ionisation mass spectra of poly(ethylene glycol)s obtained using supercritical fluid chromatography (SFC) hyphenated to different mass analysers were compared, and visualisation diagrams were used to understand the differences in the dispersity value calculations. Five calibration approaches based on a surrogate single oligomer that represents the whole distribution, or the whole distribution itself, for response selection, were used to evaluate ionisation efficiency prior to quantitation. The impact of using an internal standard (ISTD) on the expanded uncertainty was also assessed. RESULTS: Although there were challenges related to the resolution of multiply charged species when low-resolution instruments were used, similar quantitation capabilities were obtained to those when high-resolution mass analysers were used. Evaluation of approaches using a surrogate oligomer or the whole distribution suggested the independence of both approaches and a constant ionisation efficiency across the oligomer chain length. The higher degree of chromatographic resolution of SFC allowed incorporating a monodispersed ISTD to improve the accuracy and precision of the method. CONCLUSIONS: The use of low resolution mass analysers was sufficient to provide accurate and precise dispersity values; however, higher resolution instruments were recommended for characterisation due to the improved mass resolution of ions. The introduction of a monodispersed ISTD improved precision without compromising the calculated dispersity value due to the lack of analyte suppression.

Palinopsia in the Setting of Normal Pressure Hydrocephalus.

Normal pressure hydrocephalus (NPH) is characterized by pathologic ventriculomegaly with normal opening pressures on lumbar puncture. It commonly presents with a triad of gait disturbance, cognitive impairment, and urinary bladder detrusor dysfunction. Its pathogenesis is complex but is thought to arise in the setting of imbalanced cerebrospinal fluid (CSF) secretion and absorption. Given that intracranial pressure often remains normal in the setting of NPH, visual symptoms are quite uncommon. Here we present a case of a 70-year-old female with a subacute history of visual aberration described as a seconds-long persistent recurrence of visual images after the stimulus was removed from the visual field in the setting of slowed and unstable gait, urinary urgency, and cognitive impairment. This patient was evaluated and ultimately diagnosed with NPH before undergoing definitive treatment with ventriculoperitoneal shunt implantation. She has shown persistent responsiveness to shunting of the CSF as manifested by sustained improvement in gait speed and stability, urinary bladder urgency, and palinopsia resolution at the six-month follow-up assessment.

Protocol for a mixed-methods study to develop and feasibility test a digital system for the capture of patient-reported outcomes (PROs) in patients receiving chimeric antigen receptor T-cell (CAR-T) therapies (the PRO-CAR-T study).

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapies are novel, potentially curative therapies for haematological malignancies. CAR T-cell therapies are associated with severe toxicities, meaning patients require monitoring during acute and postacute treatment phases. Electronic patient-reported outcomes (ePROs), self-reports of health status provided via online questionnaires, can complement clinician observation with potential to improve patient outcomes. This study will develop and evaluate feasibility of a new ePRO system for CAR-T patients in routine care. METHODS AND ANALYSIS: Multiphase, mixed-methods study involving multiple stakeholder groups (patients, family members, carers, clinicians, academics/researchers and policy-makers). The intervention development phase comprises a Delphi study to select PRO measures for the digital system, a codesign workshop and consensus meetings to establish thresholds for notifications to the clinical team if a patient reports severe symptoms or side effects. Usability testing will evaluate how users interact with the digital system and, lastly, we will evaluate ePRO system feasibility with 30 CAR-T patients (adults aged 18+ years) when used in addition to usual care. Feasibility study participants will use the ePRO system to submit self-reports of symptoms, treatment tolerability and quality of life at specific time points. The CAR-T clinical team will respond to system notifications triggered by patients' submitted responses with actions in line with standard clinical practice. Feasibility measures will be collected at prespecified time points following CAR T-cell infusion. A qualitative substudy involving patients and clinical team members will explore acceptability of the ePRO system. ETHICS AND DISSEMINATION: Favourable ethical opinion was granted by the Health and Social Care Research Ethics Committee B(HSC REC B) (ref: 23/NI/0104) on 28 September 2023. Findings will be submitted for publication in high-quality, peer-reviewed journals. Summaries of results, codeveloped with the Blood and Transplant Research Unit Patient and Public Involvement and Engagement group, will be disseminated to all interested groups. TRIAL REGISTRATION NUMBER: ISCTRN11232653.

Cerebellar Stroke in a Young Adult Following an Amusement Park Ride.

The incidence of stroke in young individuals has been showing an increasing trend. In such cases, atypical mechanisms of stroke should be considered. Here, we report a case of a 37-year-old healthy female who presented with complaints of right-sided ataxia and clumsiness that started after an amusement park ride where she was swung rapidly. Imaging revealed an infarct in the right cerebellar hemisphere. This case report highlights certain mechanisms that can cause a stroke in this situation, with special emphasis on recognition, timely medical management, public awareness of such risk factors, and the prevention of complications.

Controlling the positive ion electrospray ionization of poly(ethylene glycols) when using ultra-high-performance supercritical fluid chromatography-mass spectrometry.

Poly(ethylene glycols) are complex polymers often added to pharmaceutical formulations to improve drug solubility and delivery. One of the main challenges when using chromatographic techniques coupled to mass spectrometry is the unselective ionization of poly(ethylene glycols) oligomers. Additionally, when the chain length is large enough, multiple charged species are formed, further complicating the mass spectra and processing. This study uses the advanced oligomer separation provided by supercritical fluid chromatography with a mass spectrometry approach that selectively ionizes poly(ethylene glycols) as ammoniated molecules to simplify data analysis and facilitate batch-to-batch comparisons. Several visual representations of the response of the ionization events based on the polymer molecular weight and the repeating unit were used to elucidate trends in ionization. Evaluation of the influence of the oligomer length and end-group on the electrospray ionization of the polymer allowed the development of a process to enable selective ionization for these complex polymers.

Venetoclax ramp-up strategies for chronic lymphocytic leukaemia in the United Kingdom: a real world multicentre retrospective study.

This retrospective, observational study evaluated patterns of inpatient versus outpatient tumour lysis syndrome (TLS) monitoring during venetoclax ramp-up in 170 patients with chronic lymphocytic leukaemia. The primary outcome was clinical/biochemical TLS. Two clinical and four biochemical TLS occurred (4.1%). Five of the six events occurred in high-risk patients, four occurred at 20 mg dose and three at the 6-h time-point. Inpatient versus outpatient TLS rates within the high-risk subgroup were 15% and 8%. Risk category was the only predictor of TLS events in multivariate analysis. Outpatient escalation did not associate with clinically meaningful TLS events, suggesting outpatient escalation has manageable associated TLS risks, including in high-risk cohorts. These observations require confirmation in larger studies.

Improving retention-time prediction in supercritical-fluid chromatography by multivariate modelling.

The prediction of chromatographic retention under supercritical-fluid chromatography (SFC) conditions was studied, using established and novel theoretical models over ranges of modifier content, pressure and temperature. Whereas retention models used for liquid chromatography often only consider the modifier fraction, retention in SFC depends much more strongly on pressure and temperature. The viability of combining several retention models into surfaces that describe the effects of both modifier fraction and pressure was investigated. The ability of commonly used retention models to describe retention as a function of modifier fraction, expressed either as mass or volume fraction, pressure and density was assessed. Using the multivariate surfaces, retention-time prediction for isocratic separations at constant temperature improved significantly compared to univariate modelling when both pressure and modifier fractions were changed. The "mixed-mode" model with an additional exponential pressure or density parameter was able to predict retention times within 5%, with the majority of the predictions within 2%. The use of mass fraction and density further improves retention modelling compared to volume fraction and pressure. These variables however, do require extra computations.

DNA and modified vaccinia Ankara prime-boost vaccination generates strong CD8+ T cell responses against minor histocompatibility antigen HA-1.

Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.

Towards improved characterisation of complex polyethylene glycol excipients using supercritical fluid chromatography-evaporative light scattering detection-mass spectrometry and comparison with size exclusion chromatography-triple detection array.

The application of supercritical fluid chromatography (SFC) coupled to an evaporative light scattering detector (ELSD) and mass spectrometer (MS) was evaluated for the characterisation of three analogues of functionalised polyethylene glycol (PEG) 2000 (m-PEG-OH, m-PEG-cm and cm-PEG-cm (where m = OCH3 and cm = OCH2COOH)). These polymers are common excipients in drug product formulations for pharmaceuticals as they help provide the desired pharmacokinetic profile for successful drug delivery. A SFC-ELSD-MS method was developed which was selective to all three polymers, and allowed visualisation of these low UV chromophore materials. The method provided baseline resolution of the individual oligomers which allowed facile calculation of the polymer dispersity. A number of molecular weight characteristics were calculated, which showed the SFC-ELSD-MS methodology to be comparable with the current standard of analysis using size exclusion chromatography (SEC) with a triple detector array (TDA). The increased resolving power of SFC compared to SEC revealed a bimodal distribution of oligomers in the cm-PEG-cm 2000 polymer, which was not observed using SEC-TDA and exemplified SFC-ELSD as an orthogonal approach for polymer characterisation with the potential for much simpler, reduced sample and instrument preparation, calibration-less dispersity determination. When combined with SEC-TDA data, this combination allows a more complete characterisation of complex formulations excipients.

Cervical Spine Ischemic Stroke Complicated by Spastic Quadriparesis and Ogilvie Syndrome: A Case Report and Literature Review.

Infarction or ischemia of the spinal cord is a rare entity and is often misdiagnosed as inflammatory myelopathy in acute settings. Atherosclerotic disease can affect spinal arteries, leading to cord ischemia with clinical presentation mixed with myelopathy. We present a case of a 66-year-old male who came to the hospital with unsteady gait and numbness of all extremities without associated pain for the past 48 hours. The neurological examination on admission directed the diagnosis towards myelopathy of the cervical spine. However, the initial magnetic resonance imaging (MRI) of the cervical spine demonstrated gliosis and restricted diffusion of the cord with multilevel neuroforaminal stenosis but without central canal stenosis or cord compression. The MRI brain, cerebrospinal fluid analysis, and rheumatologic evaluation were unremarkable. Four days into the clinical course, the patient developed weakness and spasticity of all extremities prompting further evaluation. Computed tomography angiography (CTA) scan of the head and neck revealed right vertebral artery occlusion and intracranial atherosclerotic disease. He was started on aspirin and clopidogrel for secondary risk reduction. The hospital course was further complicated by Ogilvie syndrome (OS), and the patient underwent uncomplicated cecostomy.

Psychosocial and Physiologic Characteristics of Patients with Non-epileptic Events: A Retrospective Study.

Background The main focus of this study is to aid early identification of psychogenic non-epileptic seizure (PNES) patients by identifying physical and psychosocial characteristics to reduce the health care burden, to reduce the unnecessary use of anti-epileptic medications and their side effects, and maximizing cost-effective use of video electroencephalography (VEEG). Methods We analyzed PNES subject data from VEEG monitoring performed at the Epilepsy Center at the Marshall University School of Medicine. We reviewed more than 360 episodes in 54 subjects older than 18 years (mean age ± standard deviation (SD): 48 ± 12.97 years; 83% female). Results We found that most of our PNES patients were older than 45 years of age (66.7%), females (83.3%); obese (66.6%) or overweight (18.5%); either single (33.3%), separated (7.4%), divorced (22.2%), or widowed (14.8%); of low education, unemployed (either received government assistance (83.3%) or disability benefits (57.4%)) with associated physical illness (85.2%) and psychiatric illness (96.3%).  Conclusion Our study adds to the current knowledge of the sociodemographic and sociocultural variability of PNES. It might enable early diagnosis and management of patients with PNES.

Developmental vascular anomaly associated hemi facial spasms and botox injections therapy.

Hemifacial spasm (HFS) is characterized by involuntary synchronous contractions or spasms of one side of the face, usually beginning around the eye. They are typically brief, irregular clonic movements but are occasionally tonic. We present a case of a 41-year-old female who presented to the neurology clinic with complaints of recurrent right facial spasms. These involuntary spontaneous movements had affected her quality of life. The neuroimaging revealed the vascular malformation right cranial nerves (CN) VII/VIII complex. It was considered to be responsible for the patient's HFSs. The patient responded well symptomatically to the botox injections without any neurovascular decompression.

Combination Lenalidomide and Azacitidine: A Novel Salvage Therapy in Patients Who Relapse After Allogeneic Stem-Cell Transplantation for Acute Myeloid Leukemia.

PURPOSE: Salvage options for patients who relapse after allogeneic stem-cell transplantation (allo-SCT) for acute myeloid leukemia (AML) and myelodysplasia (MDS) remain limited, and novel treatment strategies are required. Both lenalidomide (LEN) and azacitidine (AZA) possess significant antitumor activity effect in AML. Administration of LEN post-transplantation is associated with excessive rates of graft-versus-host disease (GVHD), but AZA has been shown to ameliorate GVHD in murine transplantation models. We therefore examined the tolerability and activity of combined LEN/AZA administration in post-transplantation relapse. PATIENTS AND METHODS: Twenty-nine patients who had relapsed after allo-SCT for AML (n = 24) or MDS (n = 5) were treated with sequential AZA (75 mg/m2 for 7 days) followed by escalating doses of LEN on days 10 to 30. Dose allocation and maximum tolerated dose (MTD) estimation were guided by a modified Bayesian continuous reassessment method (CRM). RESULTS: Sequential AZA and LEN therapy was well tolerated. The MTD of post-transplantation LEN, in combination with AZA, was determined as 25 mg daily. Three patients developed grade 2 to 4 GVHD. There was no GVHD-related mortality. Seven of 15 (47%) patients achieved a major clinical response after LEN/AZA therapy. CD8+ T cells demonstrated impaired interferon-γ/tumor necrosis factor-α production at relapse, which was not reversed during LEN/AZA administration. CONCLUSION: We conclude LEN can be administered safely post-allograft in conjunction with AZA, and this combination demonstrates clinical activity in relapsed AML/MDS without reversing biologic features of T-cell exhaustion. The use of a CRM model delivered improved efficiency in MTD assessment and provided additional flexibility. Combined LEN/AZA therapy represents a novel and active salvage therapy in patients who had relapsed post-allograft.

Disseminated histoplasmosis in pediatric kidney transplant recipients-A report of six cases and review of the literature.

BACKGROUND: We report a case series of histoplasmosis in KTx patients in a children's hospital in an endemic area. METHODS: All KTx cases from January 1, 2002, to August 31, 2016, were reviewed to identify those with disseminated histoplasmosis. RESULTS: The attack rate of histoplasmosis among our KTx patients was 6.9 per 100 cases. The median age at the time of diagnosis was 16 years (11-18). Comorbidities included glomerulosclerosis (3), medullary cystic disease (1), and obstructive uropathy (2) and HIV (1). There were 5 deceased and 1 living-related donor transplants, and no patient had a history of rejection prior to histoplasmosis. Median time from transplant to histoplasmosis was 14.8 months (IQR 2.2-38.3) and 33% occurred in the first year after transplant. Urine and/or serum antigens were positive in all patients. They were either treated with amphotericin B and transitioned to an azole or received azole monotherapy. Most (83%) received chronic suppression with itraconazole. No patients died and relapse occurred in 1 patient after repeat transplant. CONCLUSIONS: KTx patients in endemic areas are at risk for disseminated histoplasmosis. Further study is needed to determine which factors portend the need for fungal prophylaxis in this subset of patients.

Case Report: Glatiramer Acetate-Induced Serum Sickness.

Multiple sclerosis (MS) is a central nervous system demyelinating disease with a prevalence of approximately 400,000 individuals in the United States. Glatiramer acetate is a frequently prescribed disease-modifying therapy used for the management of relapsing forms of the disease. A 40-year-old woman with relapsing-remitting MS presented with symptomatic concerns of vomiting, fever, diffuse rash, joint and low back pain, and distal lower-limb paresthesia and was subsequently admitted to the hospital for investigation and treatment. She was discharged initially after conservative management with intravenous methylprednisolone and diphenhydramine. She was restarted on glatiramer acetate 3 weeks later and required rehospitalization for similar symptoms 3 days after resumption of the disease-modifying therapy and was diagnosed as having serum sickness.

Outcome of Azacitidine Therapy in Acute Myeloid Leukemia Is not Improved by Concurrent Vorinostat Therapy but Is Predicted by a Diagnostic Molecular Signature.

Purpose: Azacitidine (AZA) is a novel therapeutic option in older patients with acute myeloid leukemia (AML), but its rational utilization is compromised by the fact that neither the determinants of clinical response nor its mechanism of action are defined. Co-administration of histone deacetylase inhibitors, such as vorinostat (VOR), is reported to improve the clinical activity of AZA, but this has not been prospectively studied in patients with AML.Experimental Design: We compared outcomes in 259 adults with AML (n = 217) and MDS (n = 42) randomized to receive either AZA monotherapy (75 mg/m2 × 7 days every 28 days) or AZA combined with VOR 300 mg twice a day on days 3 to 9 orally. Next-generation sequencing was performed in 250 patients on 41 genes commonly mutated in AML. Serial immunophenotyping of progenitor cells was performed in 47 patients.Results: Co-administration of VOR did not increase the overall response rate (P = 0.84) or overall survival (OS; P = 0.32). Specifically, no benefit was identified in either de novo or relapsed AML. Mutations in the genes CDKN2A (P = 0.0001), IDH1 (P = 0.004), and TP53 (P = 0.003) were associated with reduced OS. Lymphoid multipotential progenitor populations were greatly expanded at diagnosis and although reduced in size in responding patients remained detectable throughout treatment.Conclusions: This study demonstrates no benefit of concurrent administration of VOR with AZA but identifies a mutational signature predictive of outcome after AZA-based therapy. The correlation between heterozygous loss of function CDKN2A mutations and decreased OS implicates induction of cell-cycle arrest as a mechanism by which AZA exerts its clinical activity. Clin Cancer Res; 23(21); 6430-40. ©2017 AACR.

An operational definition of primary refractory acute myeloid leukemia allowing early identification of patients who may benefit from allogeneic stem cell transplantation.

Up to 30% of adults with acute myeloid leukemia fail to achieve a complete remission after induction chemotherapy - termed primary refractory acute myeloid leukemia. There is no universally agreed definition of primary refractory disease, nor have the optimal treatment modalities been defined. We studied 8907 patients with newly diagnosed acute myeloid leukemia, and examined outcomes in patients with refractory disease defined using differing criteria which have previously been proposed. These included failure to achieve complete remission after one cycle of induction chemotherapy (RES), less than a 50% reduction in blast numbers with >15% residual blasts after one cycle of induction chemotherapy (REF1) and failure to achieve complete remission after two courses of induction chemotherapy (REF2). 5-year overall survival was decreased in patients fulfilling any criteria for refractory disease, compared with patients achieving a complete remission after one cycle of induction chemotherapy: 9% and 8% in patients with REF1 and REF2 versus 40% (P<0.0001). Allogeneic stem cell transplantation improved survival in the REF1 (HR 0.58 (0.46-0.74), P=0.00001) and REF2 (HR 0.55 (0.41-0.74), P=0.0001) cohorts. The utilization of REF1 criteria permits the early identification of patients whose outcome after one course of induction chemotherapy is very poor, and informs a novel definition of primary refractory acute myeloid leukemia. Furthermore, these data demonstrate that allogeneic stem cell transplantation represents an effective therapeutic modality in selected patients with primary refractory acute myeloid leukemia.

Small Molecule Docking Supports Broad and Narrow Spectrum Potential for the Inhibition of the Novel Antibiotic Target Bacterial Pth1.

Peptidyl-tRNA hydrolases (Pths) play ancillary yet essential roles in protein biosynthesis by recycling peptidyl-tRNA. In E. coli, inhibition of bacterial Pth1 leads to accumulation of peptidyl-tRNA, depletion of aminoacyl-tRNA, and cell death. Eukaryotes have multiple Pths and Pth1 knock out was shown to have no effect on viability in yeast. Thereby, bacterial Pth1 is a promising target for novel antibiotic development. With the abundance of Pth1 structural data, molecular docking was used for virtual screening of existing, commercially available antibiotics to map potential interactions with Pth enzymes. Overall, 83 compounds were docked to eight different bacterial Pth1 and three different Pth2 structures. A variety of compounds demonstrated favorable docking with Pths. Whereas, some compounds interacted favorably with all Pths (potential broad spectrum inhibition), more selective interactions were observed for Pth1 or Pth2 and even specificity for individual Pth1s. While the correlation between computational docking and experimentation still remains unknown, these findings support broad spectrum inhibition, but also point to the possibility of narrow spectrum Pth1 inhibition. Also suggested is that Pth1 can be distinguished from Pth2 by small molecule inhibitors. The findings support continued development of Pth1 as an antibiotic target.