Novo Nordisk (Germany)

Abstract Hepatocellular carcinoma (HCC) can have viral or non-viral causes 1–5 . Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need 6,7 . Here we report the progressive accumulation of exhausted, unconventionally activated CD8 + PD1 + T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8 + PD1 + T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8 + PD1 + CXCR6 + , TOX + , and TNF + T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 + T cells or TNF neutralization, suggesting that CD8 + T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8 + PD1 + T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology is presented. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.

Yeast Saccharomyces cerevisiae was engineered for de novo production of six different flavonoids (naringenin, liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin) directly from glucose, without supplementation of expensive intermediates. This required reconstruction of long biosynthetic pathways, comprising up to eight heterologous genes from plants. The obtained titers of kaempferol 26.57 ± 2.66 mg L−1 and quercetin 20.38 ± 2.57 mg L−1 exceed the previously reported titers in yeast. This is also the first report of de novo biosynthesis of resokaempferol and fisetin in yeast. The work demonstrates the potential of flavonoid-producing yeast cell factories.

Rapid growth in size and complexity of biological data sets has led to the 'Big Data to Knowledge' challenge. We develop advanced data integration methods for multi-level analysis of genomic, transcriptomic, ribosomal profiling, proteomic and fluxomic data. First, we show that pairwise integration of primary omics data reveals regularities that tie cellular processes together in Escherichia coli: the number of protein molecules made per mRNA transcript and the number of ribosomes required per translated protein molecule. Second, we show that genome-scale models, based on genomic and bibliomic data, enable quantitative synchronization of disparate data types. Integrating omics data with models enabled the discovery of two novel regularities: condition invariant in vivo turnover rates of enzymes and the correlation of protein structural motifs and translational pausing. These regularities can be formally represented in a computable format allowing for coherent interpretation and prediction of fitness and selection that underlies cellular physiology.

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

OBJECTIVE: Both rapid-acting insulin analogs, insulin aspart and lispro, attenuate prandial glucose excursion compared with human soluble insulin. This trial was performed to study the pharmacokinetic and pharmacodynamic profiles of insulin aspart and insulin lispro in type 1 diabetic patients in a direct comparison and to investigate whether the administration of one analog results in favorable effects on prandial blood glucose control. RESEARCH DESIGN AND METHODS: A total of 24 type 1 diabetic patients (age 36 +/- 8 years, 16 men and 8 women, BMI 24.3 +/- 2.6 kg/m(2), diabetes duration 17 +/- 11 years, HbA(1c) 7.9 +/- 0.8%) on intensified insulin therapy were recruited into a single-center, randomized, double-blind, two-period, cross-over, glucose clamp trial. The subjects were given an individual need-derived dose of prandial insulin lispro or aspart immediately before a standard mixed meal. RESULTS: With respect to blood glucose excursions from time 0 to 6 h (Exc(glu(0-6 h))) and from time 0 to 4 h (Exc(glu(0-4 h))), the pharmacodynamic effect of insulin aspart and insulin lispro can be declared equivalent. This was supported by comparison with maximum postprandial blood glucose excursions (C(max(glu))) (estimated ratio aspart/lispro ANOVA [90% CI]: 0.95 [0.80-1.13], 0.97 [0.82-1.17], and 1.01 [0.95-1.07] for Exc(glu(0-6 h)), Exc(glu(0-4 h)), and C(max(glu)), respectively). For pharmacokinetic end points (maximum postprandial insulin excursions and area under the curve for insulin from time 0 to 6 h and from time 0 to 4 h), equivalence was indicated. No difference concerning absorption or elimination for time to maximal insulin concentration, time to half-maximum insulin concentration, and time to decrease to 50% of maximum insulin concentration was observed. CONCLUSIONS: These data suggest that in type 1 diabetic patients, both insulin analogs are equally effective for control of postprandial blood glucose excursions.

AIMS: To compare the pharmacokinetic and pharmacodynamic properties of subcutaneously injected NN304, a novel long-acting insulin analogue, to NPH-insulin during euglycaemic glucose clamps in 11 healthy volunteers. METHODS: On three study days NN304 was injected in three different doses (0.15, 0.3, 0.6 U/kg body weight), while NPH-insulin (0.3 U/kg) was injected in identical dose on two other days. RESULTS: Injection of NN304 resulted in a linear and proportional increase in total NN304 concentrations (AUC0-1440 min: 0.15 U/kg: 344+/-43, 0.3 U/kg: 666+/-82, 0.6 U/kg: 1295+/-210 nmol/l; P<0.001). Maximal concentrations (609+/-140, 1046+/-283, 2033+/-460 pmol/l; P<0.001) were reached after 4-6 h. The metabolic response (expressed as maximal glucose infusion rates (GIR)) induced by subcutaneous injection of NN304 did not show the pronounced peak seen with NPH-insulin in an identical dose: GIRmax 3.2+/-1.1 vs. 4.4+/-1.8 mg/kg/min (P<0.05 for 0.3 U/kg NN304 vs. NPH-insulin; mean of both study days with NPH-insulin, all others not significant). NN304 also showed a slower onset of action, as indicated by a significantly higher tmax (446+/-162 vs. 359+/-175 min) and lower AUC0-240min (0.5+/-0.3 vs. 0.8+/-0.4 g/kg/240min; P<0.05, respectively). The three different doses of NN304 induced a significantly different glucose consumption in the first 720 min after injection (AUC0-720 min 1.1+/-0.6, 1.9+/-0.8, 1.7+/-0.8 g/kg; P<0.05 for 0.15 U/kg), but not over the whole study period (AUC0-1440 min 1.8+/-1.1, 3.1+/-1.3, 2.8+/-1.4 g/kg). CONCLUSIONS: Injection of NN304 at different doses resulted in an increase in total NN304 concentration in a linear dose-response effect and a more even metabolic effect than NPH-insulin. However, we found no clear dose-response in its metabolic effect.

Salmonella strains are traditionally classified into serovars based on their surface antigens. While increasing availability of whole-genome sequences has allowed for more detailed subtyping of strains, links between genotype, serovar, and host remain elusive. Here we reconstruct genome-scale metabolic models for 410 Salmonella strains spanning 64 serovars. Model-predicted growth capabilities in over 530 different environments demonstrate that: (1) the Salmonella accessory metabolic network includes alternative carbon metabolism, and cell wall biosynthesis; (2) metabolic capabilities correspond to each strain's serovar and isolation host; (3) growth predictions agree with 83.1% of experimental outcomes for 12 strains (690 out of 858); (4) 27 strains are auxotrophic for at least one compound, including L-tryptophan, niacin, L-histidine, L-cysteine, and p-aminobenzoate; and (5) the catabolic pathways that are important for fitness in the gastrointestinal environment are lost amongst extraintestinal serovars. Our results reveal growth differences that may reflect adaptation to particular colonization sites.

Traditional oleochemical industry chemically processes animal fats and plant oils to produce detergents, lubricants, biodiesel, plastics, coatings, and other products. Biotechnology offers an alternative process, where the same oleochemicals can be produced from abundant biomass feedstocks using microbial catalysis. This review summarizes the recent advances in the engineering of microbial metabolism for production of fatty acid-derived products. We highlight the efforts in engineering the central carbon metabolism, redox metabolism, controlling the chain length of the products, and obtaining metabolites with different functionalities. The prospects of commercializing microbial oleochemicals are also discussed.

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

Glycosyltransferases catalyse the transfer of a glycosyl moiety from a donor to an acceptor. Members of this enzyme class are ubiquitous throughout all kingdoms of life and are involved in the biosynthesis of countless types of glycosides. Family 1 glycosyltransferases, also referred to as uridine diphosphate-dependent glycosyltransferases (UGTs), glycosylate small molecules such as secondary metabolites and xenobiotics. In plants, UGTs are recognised for their multiple functionalities ranging from roles in growth regulation and development, in protection against pathogens and abiotic stresses and in adaptation to changing environments. In this study, we review UGT-mediated glycosylation of phytohormones, endogenous secondary metabolites, and xenobiotics and contextualise the role this chemical modification plays in the response to biotic and abiotic stresses and plant fitness. Here, the potential advantages and drawbacks of altering the expression patterns of specific UGTs along with the heterologous expression of UGTs across plant species to improve stress tolerance in plants are discussed. We conclude that UGT-based genetic modification of plants could potentially enhance agricultural efficiency and take part in controlling the biological activity of xenobiotics in bioremediation strategies. However, more knowledge of the intricate interplay between UGTs in plants is needed to unlock the full potential of UGTs in crop resistance.

BACKGROUND: No medications are currently approved for the treatment of nonmonogenic, nonsyndromic obesity in children younger than 12 years of age. Although the use of liraglutide has been shown to induce weight loss in adults and adolescents with obesity, its safety and efficacy have not been established in children. METHODS: In this phase 3a trial, which consisted of a 56-week treatment period and a 26-week follow-up period, we randomly assigned children (6 to <12 years of age) with obesity, in a 2:1 ratio, to receive either once-daily subcutaneous liraglutide at a dose of 3.0 mg (or the maximum tolerated dose) or placebo, plus lifestyle interventions. The primary end point was the percentage change in the body-mass index (BMI; the weight in kilograms divided by the square of the height in meters). The confirmatory secondary end points were the percentage change in body weight and a reduction in BMI of at least 5%. RESULTS: A total of 82 participants underwent randomization; 56 were assigned to the liraglutide group and 26 to the placebo group. At week 56, the mean percentage change from baseline in BMI was -5.8% with liraglutide and 1.6% with placebo, representing an estimated difference of -7.4 percentage points (95% confidence interval [CI], -11.6 to -3.2; P<0.001). The mean percentage change in body weight was 1.6% with liraglutide and 10.0% with placebo, representing an estimated difference of -8.4 percentage points (95% CI, -13.4 to -3.3; P = 0.001), and a reduction in BMI of at least 5% occurred in 46% of participants in the liraglutide group and in 9% of participants in the placebo group (adjusted odds ratio, 6.3 [95% CI, 1.4 to 28.8]; P = 0.02). Adverse events occurred in 89% and 88% of participants in the liraglutide and placebo groups, respectively. Gastrointestinal adverse events were more common in the liraglutide group (80% vs. 54%); serious adverse events were reported in 12% and 8% of participants in the liraglutide and placebo groups, respectively. CONCLUSIONS: Among children (6 to <12 years of age) with obesity, treatment with liraglutide for 56 weeks plus lifestyle interventions resulted in a greater reduction in BMI than placebo plus lifestyle interventions. (Funded by Novo Nordisk; SCALE Kids ClinicalTrials.gov number, NCT04775082.).

Recombinant activated coagulation factor VII (rFVIIa) (NovoSeven®) was developed for treatment of bleeding in hemophilia patients with inhibitors (antibodies) against factors VIII or IX. rFVIIa initiates the coagulation cascade by binding to tissue factor at the site of injury and causes the formation of sufficient amounts of thrombin to trigger coagulation. Patients with a variety of other coagulation deficiencies than hemophilia characterized by an impaired thrombin generation and life-threatening bleeding have been reported as successfully treated with rFVIIa. Data are now entered into clinical registries established to further monitor this experimental treatment with NovoSeven®. rFVIIa is produced free of any added human protein. The amino acid sequence of rFVIIa is identical to plasma-derived FVIIa (pdFVIIa). Posttranslational modifications (i.e., γ-carboxylations, N- and O-glycosylations) are qualitatively identical in pdFVIIa and rFVIIa although some quantitative differences exist. The activities of rFVIIa and pdFVIIa are indistinguishable. Manufacturing of rFVIIa involves expression in baby hamster kidney (BHK) cells followed by purification, including three ion-exchange and one immunoaffinity chromatography steps. The last anion-exchange chromatography step ensures completion of the autoactivation of recombinant factor VII (rFVII) to rFVIIa. This review describes the mechanism of action, characterization, manufacturing, and preclinical and current clinical evidence for the efficacy and safety of rFVIIa. KEYWORD Factor VIIa - rFVIIa - recombinant expression - in vitro activation - biological activity

NN304 is a long-acting insulin analogue that is acylated with a 14-C-fatty acid chain. Protraction of action of this novel insulin analogue is due not to slow absorption after subcutaneous administration but to reversible binding to albumin. We investigated the pharmacokinetic and pharmacodynamic properties of insulin analogue NN304 (0.3 and 0.6 U/kg) in comparison to NPH insulin (0.3 and 0.6 IU/kg) in 10 healthy volunteers performing a randomised, double-blind, cross-over, placebo-controlled glucose clamp study. During the observation period of 24 hours the areas under the insulin curve for NPH[0.3 IU/kg] vs. NPH[0.6 IU/kg] were 60 vs. 102 nmol min l(-1) (p<0.01) and for insulin analogue NN304[0.3 U/kg] vs. NN304[0.6 U/kg] 490 vs. 932 nmol min l(-1) (p <0.001), suggesting a clear dose-response relationship for both NPH insulin and NN304. The amount of disposed glucose (area under the curve of glucose infusion) differed with statistical significance between the five treatments and was highest with NPH[0.6 IU/kg] (2671 mg/kg) and lowest with placebo (265 mg/kg). However, area under the curve of glucose infusion after treatment with NN304 was only 36% (dose of 0.3 U/kg) and 24% (dose of 0.6 U/kg) of that observed with corresponding doses of NPH insulin. Moreover, increasing dosages of NN304 failed to demonstrate a significant dose-response with regard to the area under the curve of glucose infusion. This study demonstrates that the principle of protracted insulin action of NN304 by reversible binding to albumin is effective in humans albeit at a much lower rate of glucose utilisation when compared to NPH insulin. Thus, in contrast to animal studies NN304 and NPH insulin can not be considered equipotent in humans.

INTRODUCTION: The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. OBJECTIVES: Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient's infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). METHODS: High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. RESULTS: The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74-0.91) and 0.76 (CI 0.67-0.86). CONCLUSION: Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

BACKGROUND: It is unclear whether postoperative B-type natriuretic peptides (i.e., BNP and N-terminal proBNP) can predict cardiovascular complications in noncardiac surgery. METHODS: The authors undertook a systematic review and individual patient data meta-analysis to determine whether postoperative BNPs predict postoperative cardiovascular complications at 30 and 180 days or more. RESULTS: The authors identified 18 eligible studies (n = 2,051). For the primary outcome of 30-day mortality or nonfatal myocardial infarction, BNP of 245 pg/ml had an area under the curve of 0.71 (95% CI, 0.64-0.78), and N-terminal proBNP of 718 pg/ml had an area under the curve of 0.80 (95% CI, 0.77-0.84). These thresholds independently predicted 30-day mortality or nonfatal myocardial infarction (adjusted odds ratio [AOR] 4.5; 95% CI, 2.74-7.4; P < 0.001), mortality (AOR, 4.2; 95% CI, 2.29-7.69; P < 0.001), cardiac mortality (AOR, 9.4; 95% CI, 0.32-254.34; P < 0.001), and cardiac failure (AOR, 18.5; 95% CI, 4.55-75.29; P < 0.001). For greater than or equal to 180-day outcomes, natriuretic peptides independently predicted mortality or nonfatal myocardial infarction (AOR, 3.3; 95% CI, 2.58-4.3; P < 0.001), mortality (AOR, 2.2; 95% CI, 1.67-86; P < 0.001), cardiac mortality (AOR, 2.1; 95% CI, 0.05-1,385.17; P < 0.001), and cardiac failure (AOR, 3.5; 95% CI, 1.0-9.34; P = 0.022). Patients with BNP values of 0-250, greater than 250-400, and greater than 400 pg/ml suffered the primary outcome at a rate of 6.6, 15.7, and 29.5%, respectively. Patients with N-terminal proBNP values of 0-300, greater than 300-900, and greater than 900 pg/ml suffered the primary outcome at a rate of 1.8, 8.7, and 27%, respectively. CONCLUSIONS: Increased postoperative BNPs are independently associated with adverse cardiac events after noncardiac surgery.

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hypermodification of cognate tRNAs, leading to the exchange of G34 by preQ1 at the wobble position in the anticodon loop. Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium due to inefficient translation of a virulence protein mRNA. Herein, we describe the discovery of a ligand with an unexpected binding mode. On the basis of this binding mode, three slightly deviating pharmacophore hypotheses have been derived. Virtual screening based on this composite pharmacophore model retrieved a set of potential TGT inhibitors belonging to several compound classes. All nine tested inhibitors being representatives of these classes showed activity in the micromolar range, two of them even in the submicromolar range.

A process termed "restitution" enables rapid repair of the respiratory epithelium by migration of neighbouring cells. Mucin-associated TFF-peptides (formerly P-domain peptides or trefoil factors) are typical motogens enhancing migration of cells in various in vitro models mimicking restitution of the intestine. The human bronchial epithelial cell line BEAS-2B was used as a model system of airway restitution. The motogenic activities of recombinant human TFF2 as well as porcine TFF2 were demonstrated by in vitro wound healing assays of BEAS-2B cells. TFF2 did not induce phosphorylation of the epidermal growth factor (EGF) receptor. EGF was capable of enhancing the motogenic effect of human TFF2 at a concentration of 3 x 10(-10) M whereas EGF itself (i.e., in the absence of TFF2) did not stimulate migration at this low concentration. Furthermore, TFF2 as well as monomeric and dimeric forms of TFF3 enhanced migration of BEAS-2B cells in Boyden chambers. Motogenic activity of TFF2 was also shown for normal human bronchial epithelial (NHBE) cells in Boyden chambers. These results suggest that TFF-peptides act as motogens in the human respiratory epithelium triggering rapid repair of damaged mucosa in the course of airway diseases such as asthma.

OBJECTIVE: The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain. METHODS: Using single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of mice in the fed and fasted states. RESULTS: Of these, 8,910 were neurons that group into 30 clusters, with 4,289 from mice fed ad libitum and 4,621 from overnight fasted mice. A total of 7,124 nuclei were from non-neuronal cells, including oligodendrocytes, astrocytes, and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL, and CALCR, which bind GLP1, GIP, GDF15, and amylin, respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC-expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2, or both, implying that melanocortin peptides are likely produced by these neurons. CONCLUSION: We provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds and also prove useful in the continued search for other novel therapeutic targets.

AIMS: In intensified insulin therapy, the recent development of short-acting insulin analogues with a very rapid onset of action forces a new discussion in terms of the optimal injection-meal interval. This study evaluated prandial glycaemia in patients with Type 1 diabetes following the subcutaneous injection of soluble human insulin (HI) and the insulin analogue insulin aspart (IAsp) at different injection-meal intervals and investigated whether administration of IAsp after the meal might provide satisfactory metabolic control. METHODS: In a randomized, double-blind, double-dummy, four-period crossover study, 20 Type 1 diabetic patients were investigated. Prandial insulin was administered 15 min before the start of the meal (HI(-15min)), immediately before the meal (HI(0min); IAsp(0min)) and 15 min after the start of the meal (IAsp(+15min)). RESULTS: Plasma glucose excursions from baseline levels during the 4 h (PGexc) were highest with HI(0min) (17.9 mmol.l(-1).h; P < 0.05 vs. other treatments) and were not statistically different for HI(-15min), IAsp(0min) and IAsp(15min) (13.6, 11.9 and 14.2 mmol.l(-1).h, respectively). Maximum concentration of plasma glucose (PGmax) was lowest with IAsp(0min) (11.2 mmol/l; P < 0.05 vs. other treatments). PGmax was comparable with HI(-15min), HI(0min) and IAsp(+15min) (13.3, 14.1 and 13.2 mmol/l, respectively). CONCLUSIONS: With regard to prandial glycaemia IAsp(+15min) is as effective as HI(-5min) and superior to HI(0min). Thus, post-prandial dosing of the insulin analogue IAsp offers an attractive and feasible therapeutic option for well-controlled patients with Type 1 diabetes mellitus.