Horizon Therapeutics (United Kingdom)

Hydroxyapatite (HA) is a mixed-mode media that has been used extensively for the purification of proteins and DNA since the 1950s. Hydroxyapatite possesses a distinctive selectivity that may be applied in the purification of a wide range of biomolecules: immunoglobulins, alkaline proteins, acidic proteins, and DNA. The functional groups of HA can both attract and repel the carboxyl and amino groups on target molecules. This unique selectivity is due to the modalities that can be employed, which are not possible with traditional anion-exchange and cation-exchange chromatography. HA is a powerful chromatography step for reducing host cell-derived impurities and aggregated product, where a 2-4 log reduction in host cell proteins, aggregates, endotoxin, and viruses are routinely achieved. This chapter describes the procedures for: efficiently packing and evaluating a HA column, purifying IgG and acidic proteins respectively using HA chromatography.

To assess the impact of long-term inebilizumab, an anti-CD19, B-cell depleting antibody, on pain and quality of life (QoL) in patients with neuromyelitis optica spectrum disorder (NMOSD).

AIM: Neuromyelitis optica spectrum disorders (NMOSD) is an autoantibody-mediated, B cell-driven disease. Inebilizumab is a humanized, affinity-optimized, afucosylated IgG1 kappa monoclonal antibody that binds to the B cell specific surface antigen CD19, resulting in rapid, profound, and sustained depletion of circulating peripheral B cells in NMOSD subjects (pivotal study). The objective of this study was to conduct population modeling of B cell response following inebilizumab treatment in adult subjects with NMOSD, and to assess the impact of drug exposure to outcome. METHODS: A hematopoietic transit model was developed to describe the joint effects of reducing influx from pro-B cells and accelerating CD20+ B cell depletion in the blood by inebilizumab. Furthermore, the relationships between inebilizumab pharmacokinetic (PK) exposure and the primary efficacy endpoint and key secondary efficacy endpoints were evaluated. KEY RESULTS: At the 300 mg dose, there was no apparent relationship between efficacy (reduction in disease attack risk, risk of worsening from baseline in Expanded Disability Status Scale, cumulative total active MRI lesions, and the number of NMOSD-related in-patient hospitalizations) and PK exposure. Subjects with low, medium, and high PK exposure had a similar hazard ratio of NMOSD attack vs placebo group. CONCLUSIONS: The pharmacodynamic modeling confirmed effective depletion of B cells is achieved with a 300 mg intravenous dose of inebilizumab administered on Day 1 and Day 15 and every 6 months thereafter. The PK variability between patients had no apparent effect on clinical efficacy.

Abstract Background: Esophageal cancer is the 6th leading cause of cancer mortality worldwide with poor prognosis and 5-year survival rate as low as 18%. It is an unmet need to develop more effective chemotherapeutic drugs to improve the treatment outcome. We previously reported that the expression of Orai1, a calcium channel, was elevated in esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer, and the high expression is associated with poor survival rate in patients. Orai1-mediated store-operated calcium entry (SOCE) has been demonstrated to contribute to cancer cell proliferation, migration, and invasion in ESCC and other cancers. RP4010 is a potent inhibitor for Orai1 channel (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. Here we evaluated the anticancer effect of RP4010 in cultured ESCC cells and xenograft animals. Materials and Method: Orai channel inhibitory function was analyzed in ESCC cells loaded with fluorescent calcium indicator Fura-2. The antiproliferative effect was evaluated by MTT assay in various human ESCC cell lines, e.g., KYSE-30, KYSE-150, KYSE-790, and KYSE-70. Quantitative real-time RT-PCR, Western blot, and flow cytometry were used to analyze the changes in cell cycle, changes in mRNAs and proteins of some key molecules in calcium signaling pathway. The antitumor effects of RP4010 were monitored in ESCC xenografted nude mice. Result: Compared to commonly used Orai1 channel blockers, such as BTP-2, RP4010 is more potent in blocking Orai1-mediated SOCE and cell proliferation in ESCC cells. The IC50 in cell viability analysis for RP4010 were calculated as 1.2, 1.4, 1.4, and 5.5 μM for KYSE-30, KYSE-70, KYSE-150, and KYSE-790, respectively, while IC50 for BTP-2 was 17.7-17.8 μM for these cells. Treatment of RP4010 (10 μM) halted intracellular calcium oscillations, inhibited calcium-dependent activation of Akt/NF-κB and ERK1/2 pathways, and resulted in cell cycles arrested at G0/G1 phase. Interestingly, RP4010 did not induce significant cell death even at rather high concentrations. In vivo study revealed that the mice tolerated daily I.P. injection of 20 mg/kg RP4010 without body weight loss or noticeable adverse effects. At this dose, RP4010 significantly reduced tumor growth compared to vehicle treated control group. Conclusion: R4010 is a novel potent inhibitor for Orai1-mediated SOCE with demonstrated potential in animal models of esophageal cancer. Data indicate a therapeutic potential of the molecule in ESCC. Citation Format: Chaochu Cui, Yan Chang, Xiaoli Zhang, Kumar V. Penmetsa, Srikant Viswanadha, Liwu Fu, Zui Pan. Targeting Orai1-mediated store-operated Ca2+ entry by a novel compound RP4010 for antiproliferative activity against esophagus squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B062.

The objective of this study was to develop European League Against Rheumatism/American College of Rheumatology classification criteria for polymyalgia rheumatica (PMR). Candidate criteria were evaluated in a 6-month prospective cohort study of 125 patients with new-onset PMR and 169 non-PMR comparison subjects with conditions mimicking PMR. A scoring algorithm was developed based on morning stiffness >45 minutes (2 points), hip pain/limited range of motion (1 point), absence of rheumatoid factor and/or anti-citrullinated protein antibody (2 points), and absence of peripheral joint pain (1 point). A score ≥4 had 68% sensitivity and 78% specificity for discriminating all comparison subjects from PMR. The specificity was higher (88%) for discriminating shoulder conditions from PMR and lower (65%) for discriminating RA from PMR. Adding ultrasound, a score ≥5 had increased sensitivity to 66% and specificity to 81%. According to these provisional classification criteria, patients ≥50 years old presenting with bilateral shoulder pain, not better explained by an alternative pathology, can be classified as having PMR in the presence of morning stiffness >45 minutes, elevated C-reactive protein and/or erythrocyte sedimentation rate, and new hip pain. These criteria are not meant for diagnostic purposes.