Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Cost Effectiveness of First-Line Oral Therapies for Pulmonary Arterial Hypertension: A Modelling Study.

BACKGROUND: In recent years, a significant number of costly oral therapies have become available for the treatment of pulmonary arterial hypertension (PAH). Funding decisions for these therapies requires weighing up their effectiveness and costs. OBJECTIVE: The aim of this study was to assess the cost effectiveness of monotherapy with oral PAH-specific therapies versus supportive care as initial therapy for patients with functional class (FC) II and III PAH in Canada. METHODS: A cost-utility analysis, from the perspective of a healthcare system and based on a Markov model, was designed to estimate the costs and quality-adjusted life-years (QALYs) associated with bosentan, ambrisentan, riociguat, tadalafil, sildenafil and supportive care for PAH in treatment-naïve patients. Separate analyses were conducted for cohorts of patients commencing therapy at FC II and III PAH. Transition probabilities, based on the relative risk of improving and worsening in FC with treatment versus placebo, were derived from a recent network meta-analysis. Utility values and costs were obtained from published data and clinical expert opinion. Extensive sensitivity analyses were conducted. RESULTS: Analysis suggests that sildenafil is the most cost-effective therapy for PAH in patients with FC II or III. Sildenafil was both the least costly and most effective therapy, thereby dominating all other treatments. Tadalafil was also less costly and more effective than supportive care in FC II and III; however, sildenafil was dominant over tadalafil. Even given the uncertainty within the clinical inputs, the probabilistic sensitivity analysis showed that apart from sildenafil and tadalafil, the other PAH therapies had negligible probability of being the most cost effective. CONCLUSION: The results show that initiation of therapy with sildenafil is likely the most cost-effective strategy in PAH patients with either FC II or III disease.

A United Kingdom-based economic evaluation of ranibizumab for patients with retinal vein occlusion (RVO).

OBJECTIVE: This study compares the cost-effectiveness of intravitreal ranibizumab vs observation and/or laser photocoagulation for treatment of macular edema secondary to retinal vein occlusion in a UK-based model. METHODS: A Markov model was constructed using transition probabilities and frequency of adverse events derived using data from the BRAVO, CRUISE, and HORIZON trials. Outcomes associated with treatments and health states were combined to predict overall health costs and outcomes for cohorts treated with each option. RESULTS: In branch retinal vein occlusion, ranibizumab produced a gain of 0.518 quality-adjusted life years at an incremental cost of £8141, compared with laser photocoagulation. The incremental cost-effectiveness ratio was £15,710 per quality-adjusted life year, and the incremental cost per month free from blindness was £658. In central retinal vein occlusion, ranibizumab produced a gain of 0.539 quality-adjusted life years at an incremental cost of £9216, compared with observation only. The incremental cost-effectiveness ratio was £17,103, and the incremental cost per month free from blindness was £423. CONCLUSIONS: These incremental cost-effectiveness ratios are below the £20,000-30,000 range typically accepted as a threshold for cost-effectiveness. This suggests that ranibizumab may be regarded as a cost-effective therapy for patients with macular edema secondary to retinal vein occlusion, relative to grid laser photocoagulation (for BRVO) and observation (for CRVO). Limitations include sparse data for utilities associated with the severity of visual impairment in the WSE in patients with RVO. A lack of direct comparative evidence between ranibizumab and the dexamethasone intravitreal implant for the treatment of BRVO and CRVO and the infeasibility of an indirect comparison due to significant heterogeneity in trial designs prevented the inclusion of this treatment as a comparator in the Markov model.

Incidence and Characteristics of Patients with Visual Impairment due to Macular Edema Secondary to Retinal Vein Occlusion in a Representative Canadian Cohort.

Retinal vein occlusion (RVO) is an obstruction of the retinal venous system, and macular edema (ME) is a complication of RVO that can lead to blindness. The Canadian incidence of visual impairment (VI) due to ME secondary to RVO is unknown. This observational, retrospective study used records from the Southwestern Ontario database to observe the annual incidence, demographics, and comorbidity characteristics of patients with VI due to ME secondary to RVO. From 47,166 patients, 73 with RVO (>40 years old) were identified: 53 with branch retinal vein occlusion (BRVO), 20 with central retinal vein occlusion (CRVO). The annual incidence of VI (visual acuity <20/40 in Snellen equivalent) due to ME secondary to BRVO was (mean (95%CI)) 0.056% (0.011-0.072), and to CRVO was 0.021% (0.008-0.081). Furthermore, a greater proportion of RVO patients had hypertension (68% versus 14%) or dyslipidemia (16% versus 10%), when compared to a healthy control cohort of 76,077 subjects (P < 0.05). This study presents a description of the characteristics of patients with VI due to ME secondary to RVO in a real-world Canadian setting. The results demonstrate that BRVO was more frequent than CRVO, and that RVO in this patient population was associated with several vascular comorbidities.

Development of homogeneous nonradioactive methyltransferase and demethylase assays targeting histone H3 lysine 4.

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

Prevalence, Demographics, and Treatment Characteristics of Visual Impairment due to Diabetic Macular Edema in a Representative Canadian Cohort.

Diabetic macular edema (DME) is the leading cause of blindness in the diabetic population. However, there is limited understanding of the epidemiology of DME with visual impairment (VI) and treatment in patients with diabetes in Canada. This observational, retrospective study used records from the Southwestern Ontario database to observe the demographics, prevalence, and treatment characteristics of VI due to DME compared to a healthy population in a real-world Canadian setting. Data was compared between a cohort of 8,368 diabetic (type 1 or 2) patients, who were ≥18 years old and had a diagnosis of DME with VI (visual acuity <20/40 in Snellen equivalent), and 76,077 age- and gender-matched subjects representing a healthy population. Among diabetic patients, prevalence of DME was 15.7%, and prevalence of VI due to DME was 2.56%. Laser monotherapy was the most frequently used treatment. Public funding covered costs for 85% of persons with DME while 18% were paid for with private funds. This study provides insight into the demographics, prevalence, and treatment of VI due to DME in a representative Canadian cohort. This data can help to inform evaluation of current DME treatment patterns and of proposed new treatment on drug plan budgets in Canada.

Catalytic specificity of human protein tyrosine kinases revealed by Peptide substrate profiling.

Out of the 90 human protein tyrosine kinases, 81 were assayed with short peptides derived from well-characterized [CDK1(Tyr15), IRS1(Tyr983), and JAK1(Tyr1023)] or generic [polyGlu:Tyr(4:1) and poly-Glu:Ala:Tyr(1:1:1)] substrates. As expected, the CDK1 peptide is a substrate for all Src family kinases. On the other hand, some of the activities are novel and lead to a better understanding of the function of certain kinases. Specifically, the CDK1 peptide is a substrate for many of the Eph family members. Interestingly, profiling of nearly all the human protein tyrosine kinases revealed a distinct pattern of selectivity towards the CDK1 and IRS1 peptides.

Impact of noncompliance with alendronate and risedronate on the incidence of nonvertebral osteoporotic fractures in elderly women.

AIMS: To evaluate the association between noncompliance with alendronate and risedronate and the risk of nonvertebral osteoporotic fracture in community-dwelling elderly women. METHODS: A nested case-control study was conducted using the Quebec administrative health databases. To be included in the cohort, women needed to be aged > or = 68 years and to have initiated treatment with alendronate or risedronate between 1 January 2002 and 31 March 2005. Cases consisted of all women with an incident nonvertebral osteoporotic fracture occurring > or = 1 year after initiation of therapy. Each case was matched with up to 20 controls using incidence density sampling, according to age (+/- 1 year) and follow-up duration. A woman was noncompliant if she had a medication possession ratio (MPR) <80% for total follow-up duration. Rate ratios (RR) for fracture were estimated through conditional logistic regression analysis, adjusting for potential confounders. RESULTS: Among the 30 259 women included in the cohort, 1036 nonvertebral fracture cases were identified and were matched to 20 069 controls. Compared with women with a MPR > or = 80%, those with a MPR < 80% had a greater risk of nonvertebral fracture [adjusted RR 1.27, 95% confidence interval (CI) 1.12, 1.44]. Considering hip fracture only, the multivariate model yielded similar results, (adjusted RR 1.28, 95% CI 1.02, 1.61). CONCLUSIONS: Among community-dwelling elderly women, noncompliance with alendronate or risedronate is associated with an increased risk of nonvertebral fracture.

Intraoperative floppy-iris syndrome associated with alpha1-adrenoreceptors: comparison of tamsulosin and alfuzosin.

PURPOSE: To compare the incidence of intraoperative floppy-iris syndrome (IFIS) in men exposed to tamsulosin and men exposed to alfuzosin and evaluate the effect of IFIS on the complication rate of cataract surgery. SETTING: Tertiary care hospital, Chicoutimi, Quebec, Canada. METHODS: The medical charts of 64 men (92 eyes) who had phacoemulsification cataract surgery between June 2005 and July 2006 and reported having used tamsulosin or alfuzosin at their initial visit for cataract evaluation were reviewed. The presence or absence of IFIS, potential confounding clinical covariates, duration of surgery, and complications were noted. The history of taking an alpha1-antagonist was verified. To address the main objective of the study, only patients who had exclusively used tamsulosin or alfuzosin were included. For the secondary objective, all eligible patients were included even if they had received more than one alpha1-antagonist in the past. RESULTS: Of men exclusively exposed to tamsulosin (22) or alfuzosin (13), 86.4% and 15.4%, respectively, developed IFIS (P<.001). The adjusted odds ratio of IFIS in patients exposed to tamsulosin compared to those exposed to alfuzosin was 32.15 (95% confidence interval, 2.74-377.11). Eyes with IFIS had a higher risk for complications (focal iris stromal atrophy, transient postoperative hypertension, major iris trauma, posterior capsule break with vitreous loss, zonular dehiscence, postoperative cystoid macular edema) than eyes without IFIS (P<.001). CONCLUSIONS: Men exposed to tamsulosin had a significantly higher risk for developing IFIS than men exposed to alfuzosin. Intraoperative floppy-iris syndrome significantly increased the complication rate of cataract surgery.

Discontinuation of antiresorptive therapies: a comparison between 1998-2001 and 2002-2004 among osteoporotic women.

CONTEXT: Studies having reported high rates of discontinuation of antiresorptive therapies (ART) may not reflect their actual use. OBJECTIVES: We compared probability of discontinuation among women aged 70 yr or older with a diagnosis of osteoporosis or recent osteoporotic fracture having started ART (alendronate, risedronate, cyclical etidronate, raloxifene, nasal calcitonin) between 1998-2001 or 2002-2004. PATIENTS AND METHODS: We constructed two cohorts of women using Régie de l'Assurance Maladie du Québec databases. Discontinuation was defined as a lapse of 30 d or longer after completion of a refill. Switching from one ART to another was allowed. Probability of discontinuation was estimated using Kaplan-Meier analysis. Multivariate Cox models were used to identify potential determinants of ART discontinuation over 1 yr. RESULTS: After 1 yr, probability of discontinuation was slightly lower in the 2002-2004 cohort than the 1998-2001 cohort (52.2 vs. 57.5%; P < 0.001). This difference remained significant after adjusting for determinants [adjusted rate ratio (RR) 0.92, 95% confidence interval (CI) 0.87-0.98]. Significant determinants of ART discontinuation within 1 yr included bone mineral density testing (RR 0.77; CI 0.73-0.82) performed within 2 yr prior to initiation of therapy and having consulted more than two pharmacies (RR 1.15; CI 1.06-1.25) in the year before starting therapy. In the 2002-2004 cohort, when switching was allowed, women initiating a once-weekly regimen of alendronate or risedronate did not show a 1-yr risk of discontinuation different from women initiating daily regimens of the same drugs (RR 0.90; CI 0.82-1.00). CONCLUSIONS: Even if new dosing regimens were introduced, discontinuation of ART among osteoporotic women remains high.

Trends and determinants of antiresorptive drug use for osteoporosis among elderly women.

AIM: It has been established that women who have had a first osteoporotic fracture are at a significantly greater risk of future fractures. Effective antiresorptive treatments (ART) are available to reduce this risk, yet little information is available on trends in ART drug use among the elderly. The objective is to estimate the rate ratio (RR) of having an ART prescription filled among elderly women and its relation to selected determinants from 1995 through 2001. METHOD: A cohort design was used. Through random sampling, we selected 40% of the women aged 70 years and older listed in the Régie de l'assurance maladie du Québec (RAMQ) health database. The women were grouped into four cohorts (for 1995, 1996, 1998 and 2000). January 1 was established as the index date within each cohort (1995, 1996, 1998 and 2000). The dependent variable was the RR of having at least one prescription of ART drugs filled during the year following the index date among women with and without prior use. ART users were divided in two groups: bone-specific drugs (bisphosphonates, calcitonin, raloxifen) and HRT (hormone replacement therapy). The independent variable was whether or not (control) there had been an osteoporotic-related fracture. The RR was determined for having at least one prescription of bone-specific drugs or of HRT filled during the year following the index date using a Cox regression adjusted for age, chronic disease score (CDS) and prior bone mineral density (BMD) test. RESULTS: Crude rates of BMD testing (per 500 person-years) ranged from 20.4 (1995) to 41.1 (2000) in women who had had an osteoporotic-related fracture, and from 4.4 to 15.3 in controls. The crude rate of women (per 100 person-years) who had had an osteoporotic-related fracture and who took at least one bone-specific drug during follow-up ranged from 1.9 in 1995 to 31 in 2000 among those with prior osteoporotic-related fracture, and from 0.5 in 1995 to 11 in 2000 for controls; the corresponding figures for HRT ranged 6.7 in 1995 to 13 in 2000, and from 8.4 in 1995 to 11 in 2000 respectively. BMD test is the only major factor affecting the adjusted RR of having a prescription filled for bone-specific drugs (RR of 10.44; 6.91-15.79 in 1995 and RR of 3.68; 3.30-4.10 in 2000) or HRT (RR of 2.08; 1.64-2.64 in 1995 and RR of 1.44; 1.17-1.77 in 2000), particularly among women who had not had prior use. The fact of having a fracture status does significantly affect the RR of having at least one bone-specific drug prescription filled only among women without prior use (RR of 1.71; 1.26-2.33 in 1996 and RR of 1.77; 1.44-2.19 in 2000). The fact of being younger did not affect the RR of having at least one prescription of bone-specific drugs filled, but being younger increased the RR of filling a prescription of HRT. CONCLUSIONS: Significant change was seen over time in the number of BMD tests ordered and ART use. Effective osteoporosis interventions are not optimal in the treatment of elderly women in a Canadian health-care system who have had an osteoporotic fracture, given that approximately 25% of women who had had an osteoporotic-related fracture were users of ART.