Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

Background: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio. Methodology/Principal Findings: A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes i

Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin

Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing

Relative receptor affinities, metabolic and mitogenic potencies, and IR off-rates [% of human insulin].

<p>All assays were performed in at least three independent experiments. Data are means ± SD and presented relative to human insulin. For human insulin, IR assay IC₅₀ values were in the picomolar affinity range, IGF-IR assay IC₅₀ values were in the nanomolar affinity range, rFFC assay EC₅₀ values were in the picomolar range, and mitogenic assay EC₅₀ values were in the nanomolar range (HMECs) and low nanomolar range (L6-hIR). The dissociation rate constant for human insulin was (3.7±0.3×10⁻² min⁻¹).</p

Representative dose-response profiles for mitogenic potency determination.

<p>Human insulin (•) or insulin analogue (B10D (♦), B10E (▪), or B10A (▾)) stimulated incorporation of [³H]-thymidine into DNA is shown in (<b>A</b>) L6-hIR cells and (<b>B</b>) HMECs. Data points are means ± SEM (n = 3).</p

Representative dissociation curves of [¹²⁵I]-labelled insulin or analogue from BHK-hIR cells.

<p>Dissociation was measured at different time points and the residual binding expressed as a percentage of initial binding. Dissociation of (<b>A</b>) B10D (♦) and B10E (▪); (<b>B</b>) B10W (▴), human insulin (•), and B10R (▾). Data points are means ± SEM (n = 3).</p

Relative receptor binding affinities.

<p>Receptor binding affinities for the solubilised IR-A (light teal), IR-B (dark teal) and IGF-IR (gray) relative to human insulin. The gray dotted line represents 100% binding affinity compared to that of human insulin. Data are means ± SD (n = 3).</p

An international observational study to assess the impact of the Omicron variant emergence on the clinical epidemiology of COVID-19 in hospitalised patients

Background: Whilst timely clinical characterisation of infections caused by novel SARS-CoV-2 variants is necessary for evidence-based policy response, individual-level data on infecting variants are typically only available for a minority of patients and settings. Methods: Here, we propose an innovative approach to study changes in COVID-19 hospital presentation and outcomes after the Omicron variant emergence using publicly available population-level data on variant relative frequency to infer SARS-CoV-2 variants likely responsible for clinical cases. We apply this method to data collected by a large international clinical consortium before and after the emergence of the Omicron variant in different countries. Results: Our analysis, that includes more than 100,000 patients from 28 countries, suggests that in many settings patients hospitalised with Omicron variant infection less often presented with commonly reported symptoms compared to patients infected with pre-Omicron variants. Patients with COVID-19 admitted to hospital after Omicron variant emergence had lower mortality compared to patients admitted during the period when Omicron variant was responsible for only a minority of infections (odds ratio in a mixed-effects logistic regression adjusted for likely confounders, 0.67 [95% confidence interval 0.61-0.75]). Qualitatively similar findings were observed in sensitivity analyses with different assumptions on population-level Omicron variant relative frequencies, and in analyses using available individual-level data on infecting variant for a subset of the study population. Conclusions: Although clinical studies with matching viral genomic information should remain a priority, our approach combining publicly available data on variant frequency and a multi-country clinical characterisation dataset with more than 100,000 records allowed analysis of data from a wide range of settings and novel insights on real-world heterogeneity of COVID-19 presentation and clinical outcome