Recombinant Insulin Research Articles

Nanomedicine Innovations for Diabetes Management: Revolutionizing Diagnosis, Treatment, and Monitoring

Diabetes mellitus, a widespread metabolic disease characterized by high blood sugar levels, affects many people globally. The limitations of conventional diagnostic and therapeutic approaches necessitate exploring innovative strategies. Nanotechnology shows remarkable potential for revolutionizing the field of diabetes theranostics (combined diagnosis and treatment) by enabling accurate diagnosis and precise treatment delivery. This article provides a comprehensive review of the latest advancements in nanomaterials for diagnosing and treating diabetes. It explores the applications of various nanomaterials, including inorganic and organic nanoparticles, nanocomposites, and nanostructured biosensors, in biomarker detection, glucose monitoring, insulin delivery, and addressing diabetes-related complications. The study focuses on the synthesis and functionalization of nanomaterials for diabetes, covering both traditional and environmentally friendly synthesis methods. This study looks into how nanomaterials can be used to carry natural antidiabetic extracts, recombinant insulin, and other antidiabetic drugs, to make them more bioavailable, targetable, and effective. However, the review also talks about the problems that come with using nanosensors to diagnose diabetes. It also looks at the newest developments in nanosensors for biomarker detection, implantable devices, and continuous glucose monitoring. Additionally, the review examines the potential of nanomaterials in the management of diabetic sequelae, including diabetic nephropathy, cardiovascular disorders, retinopathy, and wound healing. We underscore the significance of nanomaterials in islet transplantation, as they provide immunological protection and enhance the viability and efficacy of islets. This study provides useful insights into the prospects and challenges connected with the rapidly growing field of nanotechnology in diabetic theranostics through a comprehensive examination of the current landscape. Academics, clinicians, and stakeholders engaged in developing innovative nanomaterial-based approaches to accurately diagnose and effectively manage diabetes will find this resource highly helpful.

Fluorescence-Based Study of Oligonucleotide Interactions With Recombinant Proteins: Insulin, Interferon α2-β, Somatotropin, and Their Receptors

Background. Oligonucleotides (OLNs) can participate in a wide range of protein-ligand interactions and perform numerous cellular functions by forming structures that enable specific interactions with DNA, RNA, and proteins, what is crucial for many biological processes. Advances in understanding these interactions could lead to the development of new technologies for treating various diseases. However, the mechanism of interaction between proteins and OLNs is complex and still requires detailed study. More research is needed to fully elucidate this process and enhance our understanding of these biomolecular interactions. Objective. The aim of this study was to synthesize, purify, and investigate the interaction of OLNs with recombinant signaling proteins interferon α2-β and insulin with their receptors and somatropin by assessing binding strength using fluorescence spectroscopy. Methods. The interactions were analyzed using the Stern–Volmer equation in both general and modified forms, as well as the Hill equation. OLNs were synthesized via the solid-phase phosphoramidite method, purified through solid-phase extraction, and subsequently verified with a spectrophotometer. Results. Fluorometric titration revealed that OLNs bind to proteins within the medium affinity range, forming non-fluorescent complexes, with the most active interactions observed with shorter OLN. Positive cooperative binding of interferon to G20 and T20, and negative cooperative binding of insulin to C20 and A20, were identified. Additionally, negative cooperative binding of somatropin to C20 was observed. Conclusions. The study demonstrated the interaction between OLNs and recombinant signaling proteins and receptors through various binding mechanisms, which could potentially affect their conformation and biolo­gical activity. These findings have implications for the therapeutic use of OLNs in the context of signaling proteins and receptors.

Efficacy, safety, and immunogenicity of recombinant insulin aspart (BioGenomics Limited) and NovoRapid® (Novo Nordisk) in adults with type 2 diabetes mellitus: a randomized, open-label, multicenter, phase-3 study

Background and purpose: To compare the efficacy, safety, and immunogenicity of recombinant insulin aspart 100 U/mL manufactured by BioGenomics Limited (BGL-ASP) with innovator NovoRapid® in type 2 diabetes mellitus patients (T2 DM). Experimental approach: This was a multicenter, open-label, randomized, parallel-group study in T2 DM patients, on premix human insulin therapy ± oral anti-diabetics. Besides self-monitored plasma glucose, fasting and post-prandial plasma glucose (FPG and PPG) were tested at baseline, week 12, and week 24. Anti-insulin aspart antibodies measured immunogenicity at 12 and 24 weeks. Findings/Results: 160 patients out of 320 patients randomly received BGL-ASP and the remaining patients received NovoRapid®. The changes in glycated hemoglobin (HbA1c) from baseline to weeks 12 and 24 for the BGL-ASP group were -0.8 ± 0.83 and -0.8 ± 0.81, respectively, while for the NovoRapid®group was -0.8 ± 1.01 and -0.9 ± 0.89, respectively. Changes in FPG and PPG were comparable between the treatment groups after 12 weeks and 24 weeks. The incidence of detectable antibodies at baseline, weeks 12, and 24 were comparable between treatment groups. Eighteen (11.3%) patients in the BGL-ASP group and 23 (14.4%) in the NovoRapid®group reported adverse events. Conclusion and implications: BGL-ASP and NovoRapid®were comparable and equally effective in lowering HbA1c, FPG, and PPG levels, with similar immunogenicity and safety profiles.

Impact of Probiotics on the Glycemic Control of Pediatric and Adolescent Individuals with Type 1 Diabetes: A Systematic Review and Meta-Analysis.

Human recombinant insulin is currently the only therapy for children and adolescents with type 1 diabetes (T1D), although not always efficient for the glycemic control of these individuals. The interrelation between the gut microbiome and the glycemic control of apparently healthy populations, as well as various populations with diabetes, is undeniable. Probiotics are biotherapeutics that deliver active components to various targets, primarily the gastrointestinal tract. This systematic review and meta-analysis examined the effect of the administration of probiotics on the glycemic control of pediatric and adolescent individuals with T1D. Randomized controlled trials employing the administration of probiotics in children and adolescents with T1D (with ≥10 individuals per treatment arm), written in English, providing parameters of glycemic control, such as mean glucose concentrations and glycosylated hemoglobin (HbA1c), were deemed eligible. The search strategy resulted in six papers with contradictory findings. Ultimately, five studies of acceptable quality, comprising 388 children and adolescents with T1D, were included in the meta-analysis. Employing a random and fixed effects model revealed statistically significant negative effect sizes of probiotics on the glycemic control of those individuals, i.e., higher concentrations of glucose and HbA1c than controls. Children and adolescents with T1D who received probiotics demonstrated worse glycemic control than controls after the intervention. Adequately powered studies, with extended follow-up periods, along with monitoring of compliance and employing the proper strains, are required to unravel the mechanisms of action and the relative effects of probiotics, particularly concerning diabetes-related complications and metabolic outcomes.

Insulin restores retinal ganglion cell functional connectivity and promotes visual recovery in glaucoma.

Dendrite pathology and synaptic loss result in neural circuit dysfunction, a common feature of neurodegenerative diseases. There is a lack of strategies that target dendritic and synaptic regeneration to promote neurorecovery. We show that daily human recombinant insulin eye drops stimulate retinal ganglion cell (RGC) dendrite and synapse regeneration during ocular hypertension, a risk factor to develop glaucoma. We demonstrate that the ribosomal protein p70S6 kinase (S6K) is essential for insulin-dependent dendritic regrowth. Furthermore, S6K phosphorylation of the stress-activated protein kinase-interacting protein 1 (SIN1), a link between the mammalian target of rapamycin complexes 1 and 2 (mTORC1/2), is required for insulin-induced dendritic regeneration. Using two-photon microscopy live retinal imaging, we show that insulin rescues single-RGC light-evoked calcium (Ca2+) dynamics. We further demonstrate that insulin enhances neuronal survival and retina-brain connectivity leading to improved optomotor reflex-elicited behaviors. Our data support that insulin is a compelling pro-regenerative strategy with potential clinical implications for the treatment and management of glaucoma.

4S-fluorination of ProB29 in insulin lispro slows fibril formation

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by circular dichroism, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo, and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

High-activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane.

Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6±20mgL-1 with a specific activity of 96±0.32 Umg-1, resulting in a total enzyme activity of 19137±1131UL-1, demonstrating significant potential for industrial applications.

Pharmacological and Benefit-Risk Profile of Once-Weekly Basal Insulin Administration (Icodec): Addressing Patients' Unmet Needs and Exploring Future Applications.

Diabetes mellitus (DM) is a chronic metabolic disease affecting over 500 million people worldwide, which leads to severe complications and to millions of deaths yearly. When therapeutic goals are not reached with diet, physical activity, or non-insulin drugs, starting/adding insulin treatment is recommended by international guidelines. A novel recombinant insulin is icodec, a once-weekly insulin that successfully completed phase III trials and that has recently obtained the marketing authorization approval from the European Medicines Agency. This narrative review aims to assess icodec pharmacological and clinical features concerning evidence on benefit-risk profile, as compared to other basal insulins, addressing the potential impact on patients' unmet needs. Icodec is a full agonist, recombinant human insulin analogue characterized by an ultra-long half-life (196 h), enabling its use in once-weekly administration. Phase III randomized clinical trials involving more than 4000 diabetic patients, mostly type 2 DM, documented non-inferiority of icodec, as compared to currently available basal insulins, in terms of estimated mean reduction of glycated hemoglobin levels; a superiority of icodec, compared to control, was confirmed in insulin-naïve patients (ONWARDS 1, 3, and 5), and in patients previously treated with basal insulin (ONWARDS 2). Icodec safety profile was comparable to the currently available basal insulins. Once-weekly icodec has the potential to improve patients' adherence, thus positively influencing patients' treatment satisfaction as well as quality of life, especially in type 2 DM insulin-naïve patients. An improved adherence might positively influence glycemic target achievement, reduce overall healthcare costs and overcome some of the unmet patients' needs. Icodec has the potential to emerge as a landmark achievement in the evolution of insulin therapy, with a positive impact also for the National Health Services and the whole society.

Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats.

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T0) at maturity. The T0 seeds were raised into the putative T1 plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T1 plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197µg/100mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.

Evolution of biosynthetic human insulin and its analogues for diabetes management.

Hormones play a crucial role in maintaining the normal human physiology. By acting as chemical messengers that facilitate the communication between different organs, tissues and cells of the body hormones assist in responding appropriately to external and internal stimuli that trigger growth, development and metabolic activities of the body. Any abnormalities in the hormonal composition and balance can lead to devastating health consequences. Hormones have been important therapeutic agents since the early 20th century, when it was realized that their exogenous supply could serve as a functional substitution for those hormones which are not produced enough or are completely lacking, endogenously. Insulin, the pivotal anabolic hormone in the body, was used for the treatment of diabetes mellitus, a metabolic disorder due to the absence or intolerance towards insulin, since 1921 and is the trailblazer in hormone therapeutics. At present the largest market share for therapeutic hormones is held by insulin. Many other hormones were introduced into clinical practice following the success with insulin. However, for the six decades following the introduction the first therapeutic hormone, there was no reliable method for producing human hormones. The most common source for hormones were animals, although semisynthetic and synthetic hormones were also developed. However, none of these were optimal because of their allergenicity, immunogenicity, lack of consistency in purity and most importantly, scalability. The advent of recombinant DNA technology was a game changer for hormone therapeutics. This revolutionary molecular biology tool made it possible to synthesize human hormones in microbial cell factories. The approach allowed for the synthesis of highly pure hormones which were structurally and biochemically identical to the human hormones. Further, the fermentation techniques utilized to produce recombinant hormones were highly scalable. Moreover, by employing tools such as site directed mutagenesis along with recombinant DNA technology, it became possible to amend the molecular structure of the hormones to achieve better efficacy and mimic the exact physiology of the endogenous hormone. The first recombinant hormone to be deployed in clinical practice was insulin. It was called biosynthetic human insulin to reflect the biological route of production. Subsequently, the biochemistry of recombinant insulin was modified using the possibilities of recombinant DNA technology and genetic engineering to produce analogues that better mimic physiological insulin. These analogues were tailored to exhibit pharmacokinetic and pharmacodynamic properties of the prandial and basal human insulins to achieve better glycemic control. The present chapter explores the principles of genetic engineering applied to therapeutic hormones by reviewing the evolution of therapeutic insulin and its analogues. It also focuses on how recombinant analogues account for the better management of diabetes mellitus.

Comparative assessment of immunogenicity of recombinant insulin Aspart from BioGenomics and its originator NovoRapid® in adult patients with type 2 diabetes mellitus.

To assess and compare the immunogenicity of recombinant Insulin Aspart [manufactured by BioGenomics Limited (BGL-ASP)] with its originator NovoRapid® (manufactured by Novo Nordisk) in adult patients with type 2 diabetes mellitus. BGL-IA-CTP301 study was a randomized, open label, parallel group, multicenter phase-III clinical study to compare the efficacy and safety of recombinant Insulin Aspart 100U/mL [manufactured by BioGenomics Limited (BGL-ASP)] with its reference medicinal product (RMP); NovoRapid® [manufactured by Novo Nordisk], in adult patients with Type 2 diabetes mellitus(T2DM). The primary objective of the study was to compare the immunogenicity of BGL-ASP and RMP; NovoRapid® in patient serum samples collected from phase-III clinical study. Immunogenicity was studied as the incidence of patients positive for anti-insulin Aspart (AIA) antibodies, developed against BGL-ASP/RMP at baseline, end of 12week and end of 24week of the treatment period. The changes in incidence of patients positive for AIA antibodies post-baseline were also studied to assess and compare the treatment-emergent antibody response (TEAR) between the treatment groups (BGL-ASP and RMP). Statistical evaluation was done by Fisher's exact test to compare the overall incidence of patients positive for AIA antibodies and the TEAR positives observed post-baseline in both the treated groups. An in-vitro neutralizing antibody assay (Nab assay) was also performed to study the effect of AIA antibodies in neutralizing the biological activity/metabolic function of the insulin. The neutralizing potential of AIA was studied by its effect on %glucose uptake. We also evaluated the association between AIA antibody levels and its impact on biological activity by studying the correlation between them. Analysis of immunogenicity data suggested that the percentage of patients positive for AIA antibodies until week 24 was similar and comparable in both the treatment groups, BGL-ASP and RMP; NovoRapid®. The changes in incidence of patients positive for AIA post-baseline in terms of TEAR positives were also similar and comparable between the treatment groups. The results of the Nab assay with confirmed positive AIA samples from BGL-ASP- and RMP-treated groups did not have any negative impact on %glucose uptake by the cells in Nab assay, confirming the absence of neutralizing antibodies in both the treatment groups. The correlation studies also showed absence of association between AIA antibody levels and percentage glucose uptake in both BGL-ASP and RMP-NovoRapid® treatment groups. CONCLUSIONS: The immunogenicity assessment based on the overall incidence of patients positive for AIA, changes in incidence of patients positive for AIA post-baseline, TEAR rates and absence of neutralizing antibodies, were found to be apparently similar and comparable in both the treatment groups (BGL-ASP and RMP). We conclude from our studies that the immunogenicity of BGL-ASP is similar and comparable to RMP and the observed immunogenicity in terms of anti-insulin Aspart antibody levels had no impact on the biological activity of insulin.

Expression of recombinant human insulin precursor by Pichia pastoris in a 10 liter bioreactor

Recombinant insulin is a vital medicine for diabetic patients. This hormone is produced by microbes such as Pichia pastoris that carry the recombinant gene of a human insulin precursor (HIP). Large-scale protein production involves a bioreactor to promote the optimal condition for the yeast to express the protein target. In order to obtain a large amount of insulin, the cultivation of recombinant P.pastoris/pD902-IP carrying human insulin precursor gene in a bioreactor 10 Liter was developed. The isolate was cultivated in a half concentration of basal salt media for 124.5 hours. Induction of the protein was done by continual methanol feeding. The fermentation condition was set to have a temperature at 28°C, agitation at 300 rpm, aeration at 2 L/min and a pH value of around 5. Dry cell weight (DCW) was measured, and HPLC quantified the content of HIP, glycerol and methanol. This work’s DCW and HIP concentrations were 46.5 g/L and 928 mg/L, respectively. The results can be higher by increasing the number of cells in the culture or extending the cultivation time so that the HIP concentration may exceed 1 g/L.

Development and Characterization of Guinea Pig Anti-Insulin Polyclonal Antibody.

Development and characterization of guinea pig anti-insulin polyclonal antibody against a target-specific insulin antigen. In India, an insulin immunogenicity kit for detecting insulin antibodies (neutralizing Nab) is an unmet medical need for diabetic patient's routine diagnosis. Type 1 diabetics rely on insulin injections daily basis for survival; if the body develops anti-insulin antibodies and neutralizes the exogenous recombinant insulin, glucose control is lost, and the patient eventually dies. Antibodies are excellent diagnostic reagents due to the specificity and sensitivity they provide in recognizing specific and unique target antigens. The paper describes the use of insulin as a target antigen and the development of target (insulin) specific antibodies in guinea pigs for use as a positive control for immunogenicity kit validation. Anti-insulin polyclonal antibody was raised against insulin in the Dunkin Hartley guinea pigs host. Anti-insulin antibody titer of all bleeds from four animals was tested using an indirect ELISA assay format. All four animals responded to the target-specific antigen but only one animal (#4) responded with a high-affinity antibody titer. The hyperimmune sera were purified using a protein A column. The purified anti-insulin antibody was characterized through SDS Page and western blot. The specificity, reactivity, and antibody binding efficiency were confirmed through immunoassays. Guinea pig anti-insulin polyclonal antibody developed in this study showed good specificity, reactivity, and efficiency in the immunoassays. This paper describes the development and characterization of anti-insulin antibodies for use as a control in developing a user-friendly insulin immunogenicity kit.

Insulin Tregopil: An Ultra-Fast Oral Recombinant Human Insulin Analog: Preclinical and Clinical Development in Diabetes Mellitus.

Insulin therapy is indispensable for achieving glycemic control in all patients with type 1 diabetes mellitus and manypatients with type 2 diabetes mellitus. Insulin injections are associated with negative connotations in patients owing to administration discomfort and adverse effects such as hypoglycemia and weight gain. Insulin administered orally can overcome these limitations by providing a convenient and effective mode of delivery with a potentially lower risk of hypoglycemia. Oral insulin mimics the physiologic process of insulin secretion, absorption into the portal circulation, and subsequent peripheral delivery, unlike the subcutaneous route that results in peripheral hyperinsulinemia. Insulin tregopil (IN-105), a new generation human recombinant insulin, methoxy (polyethylene glycol) hexanoyl human recombinant insulin, is developed by Biocon as an ultra-fast onset short-acting oral insulin analog. This recombinant oral insulin is a single short-chain amphiphilic oligomer modified with the covalent attachment of methoxy-triethylene-glycol-propionyl moiety at Lys-β29-amino group of the B-chain via an amide linkage. Sodium caprate, an excipient in the insulin tregopil formulation, is a permeation enhancer that increases its absorption through the gastrointestinal tract. Also, meal composition has been shown to non-significantly affect its absorption. Several global randomized, controlled clinical trials have been conducted in type 1 and type 2 diabetes patients towards the clinical development of insulin tregopil. The formulation shows post-prandial glucose control that ismore effective than placebo throughout the meal period; however, compared withan active comparator insulin aspart, the post-prandial control ismore effective mainly in the early post-meal period. It shows a good safety profile with a lower incidence of clinically significant hypoglycemia. This review covers the overall clinical development of insulin tregopil establishing it as an ultra-fast onset, short-acting oral insulin analog for optimizing post-prandial glucose.

Chemical Modification of the Amino Groups of Human Insulin: Investigating Structural Properties and Amorphous Aggregation of Acetylated Species.

The efficacy of human recombinant insulin can be affected by its aggregation. Effects of acetylation were observed on insulin structure, stability, and aggregation at 37 and 50°C and pH of 5.0 and 7.4 with the use of spectroscopy, circular dichroism (CD), dynamic light scattering (DLS), and atomic force microscopy (AFM). Raman and FTIR results were indicative of structural changes in AC-INS, and CD analyses showed a slight increase in β-sheet content in AC-INS. Melting temperature (Tm) measurements indicated an overall more stable structure and spectroscopic assessment showed a more compact one. Formation of amorphous aggregates was followed over time and kinetics parameters showed a longer nucleation phase (higher t* amount) and lower aggregates amount (lower Alim) for acetylated insulin (AC-INS) compared to native (N-INS) in all tested conditions. The results of amyloid-specific probes approved the formation of amorphous aggregates. Size particle and microscopic analysis suggested that AC-INS was less prone to form aggregates, which were smaller if formed. In conclusion, this study has demonstrated that controlled acetylation of insulin may lead to its higher stability and lower propensity toward amorphous aggregation and has provided insight into the result of this type of post-translational protein modification.

A Brief Atlas of Insulin.

Insulin is an essential factor for mammalian organisms: a regulator of glucose metabolism and other key signaling pathways. Insulin is also a multifunctional hormone whose absence can cause many diseases. Recombinant insulin is widely used in the treatment of diabetes. Understanding insulin, biosimilars, and biobetters from a holistic perspective will help pharmacologically user-friendly molecules design and develop personalized medicine-oriented therapeutic strategies for diabetes. Additionally, it helps to understand the underlying mechanism of other insulindependent metabolic disorders. The purpose of this atlas is to review insulin from a biotechnological, basic science, and clinical perspective, explain nearly all insulin-related disorders and their underlying molecular mechanisms, explore exogenous/recombinant production strategies of patented and research-level insulin/analogs, and highlight their mechanism of action from a structural perspective. Combined with computational analysis, comparisons of insulin and analogs also provide novel information about the structural dynamics of insulin.

Efficacy and Safety of Enteral Human Recombinant Insulin to Reduce the Time to Full Enteral Feeding in Preterm Infants: A Meta-Analytical Study.

Recombinant human insulin plays an important role in the gut maturation of preterm infants. This meta-analysis was carried out to assess the efficacy and safety of enteral recombinant human insulin in decreasing the time to full enteral feeding in preterm infants. The pooling of data from four clinical trials yielded a significant decrease in the time to full enteral feeding in preterm infants under both low (Mean difference [MD] -3.43 days; 95% CI: -6.18 to -0.69 days; I2 = 48%) and high doses of insulin (MD -7.10 days; 95% CI: -10.02 to -4.18 days; I2 = 0%). These findings require confirmation by further large trials that evaluate the efficacy and safety of enteral insulin, especially at supraphysiological doses.

In Vitro Biological Characterization of Recombinant Insulin Aspart from Biogenomics and Originator Insulin Aspart.

Bioassays are used to identify the pharmacological activity of new or chemically unknown compounds, as well as their undesirable effect, including toxicity. Biological assays are also required to ensure the quality, safety, and efficacy of recombinant biologics to confirm its biosimilarity to its originator. In the present study, analytical similarity between the biosimilar and its innovator is established by in vitro bioassays. The objective of this study was to show the comparative in vitro characterization of the recombinant insulin aspart from BioGenomics with its originator insulin aspart, using relevant biological assays. In vitro assays such as receptor binding, receptor autophosphorylation, glucose uptake, and mitogenic potential were analyzed for biological characterization of BioGenomics recombinant insulin aspart (BGL-ASP) manufactured by BioGenomics Limited and NovoRapid® as the reference medicinal product (RMP) manufactured by NovoNordisk. Insulin receptor binding was studied by a state-of-the-art method, surface plasmon resonance (SPR) for biomolecular interactions. The receptor autophosphorylation assay measures the phosphorylated insulin receptor in cell lysates. The glucose uptake assay measures the uptake of glucose by 3T3-L1 cells in the presence of insulin. Lipogenesis was studied in treated 3T3-L1 cells by detecting the accumulation of lipid droplets in the cells. Mitogenic effect was studied by cell proliferation assay using MCF-7 cells. A rabbit bioidentity test was performed by measuring the sudden decrease in blood glucose in the presence of insulin. The binding studies showed thatthe affinity of BGL-ASP was highly comparable to NovoRapid®. Insulin receptor autophosphorylation, glucose uptake, and lipogenesis demonstrated high similarity to the RMP. The mitogenic assay for BGL-ASP did not show any proliferative effect and was comparable to the RMP. The in vivo bioidentity test showed that the BGL-ASP is highly similar to the innovator, NovoRapid®. The biological characterization studies of BGL-ASP demonstrated high binding and functional similarity to NovoRapid®.

AGAP2-AS1 affects TNM staging and prognosis of lung cancer patients by acting on SLC7A11 mRNA stability and ferroptosis*

Abstract Objective The initiation and progression of lung carcinomas are critically regulated by long non-coding RNAs (lncRNAs). However, the role of lncRNAs in the pathways causing lung cancer remains unknown. Methods Cell morphology was regularly observed using an inverted phase-contrast microscope. Cell viability was assessed using CCK-8 according to the manufacturer’s instructions. Total RNA was retrotranscribed from each specimen using the RNAiso Plus Kit. The RT-PCR data were calculated using the Ct approach for comparison. Flow cytometric analyses were prepared by Click-iT™ Plus TUNEL Assay for In Situ apoptosis detection, with Alexa Fluor™ 594 dye, as instructed. RNA immunoprecipitation assays were used to determine RNA concentration. Results Activated natural killer cells repeat and PH domain-containing protein 2 antisense RNA 1 (AGAP2-AS1) levels in cancerous tissues were significantly correlated with cancerous tumor node metastasis (TNM) stage, with cancerous AGAP2-AS1 levels being higher in cancerous tissues than healthy tissues. Patients withelevated AGAP2-AS1 levels had considerably worse outcomes than those with reduced AGAP2-AS1 levels, regardless of the progression-free or overall survival. Functionally, AGAP2-AS1 downregulation represseslung cancer cell growth. AGAP2-AS1 elimination induces erastin-mediated ferroptosis in lung cancer cells.However, the ferritin inhibitor FERSINT-1 negated this result, whereas ERASTIN induced lung cancer cellmortality. After AGAP2-AS1 silencing, erastin-treated lung cancer cells showed a remarkable decrease inGSH levels. These results indicated that AGAP2-AS1 enhanced the stabilization of SLC7A11 mRNA via Recombinant Insulin Like Growth Factor Binding Protein 2(IGF BP2). Patients with elevated AGAP2-AS1 had considerably worse outcomes. Down-regulating AGAP2-AS1 was able to repress lung cancer cell growth and induce greater Erastin-mediated ferroptosis. Lungcancer cells treated with Erastin exhibited a remarkable decrease inglutathione (GSH) levels. The mechanical findingsindicated that AGAP2-AS1 enhanced the stabilization of SLC7A11 mRNA via the IGF2BP2. Conclusion We identified a novel effect of AGAP2-AS1 on TNM staging and the prognosis of patientswith lungcancer by modulating SLC7A11 mRNA stability and ferroptosis.

Human growth hormone and insulin, from tissue extracts to recombinant DNA technology, their biosimilars and analogues in treatment of Endocrine Disorders

Forty years ago, recombinant DNA technology caused a revolution in the way growth disorders and diabetes could be treated. Bacteria or human cells were modified so as to be able to produce human hormones instead of relying on cadaveric material in the case of growth disorders and porcine insulin in the case of diabetics. The new technology also removed the risk of contracted hepatitis or fatal Creutzfeldt Jacob disease. A signal peptide was used to transport the new protein into the periplasmic space giving rise to an exact, correctly folded version of monomeric human growth hormone. More eligible patients could be treated, resulting not only in a large number of clinical trials. Daily tiny subcutaneous injections were found to be much more effective than twice or thrice weekly in the treatment of children and adults, The progress for recombinant human insulin turned out to be somewhat different. Insulin is a more complicated protein, with an Alpha and Beta chain joined by two disulphide bridges, but is produced in the human pancreas as a single chain, proinsulin, the C peptide is removed to make the active molecule. Recombinant insulin development was in a way the opposite to growth hormone since human insulin acts faster than porcine. Short fast and long-acting analogues have been developed all to improve the treatment regimens for both type 1 and type 2 diabetics, A similar development is now taking place in the growth hormone field. Depot preparations for weekly administration are under development to achieve better compliance. One recently approved depot uses an analogue, modified in one amino acid of the peptide chain to permit acylation and albumin binding. In a way this is a retrograde step and it remains to be seen if the patient’s growth and metabolic responses are comparable to the daily administration of tiny amount of recombinant natural sequence hormone over several years of treatment.