Pharmacokinetics and Pharmacodynamics of a Novel U500 Insulin Aspart Formulation: A Randomized, Double-Blind, Crossover Study in People With Type 1 Diabetes.

OBJECTIVE: To evaluate the pharmacokinetics, pharmacodynamics, and safety of a novel U500 insulin aspart formulation (AT278 U500) compared with insulin aspart (IAsp U100). RESEARCH DESIGN AND METHODS: This single-center, randomized, double-blind study was conducted in 38 men with type 1 diabetes (body weight ≤100 kg and total insulin dose <1.2 units/kg/day). Participants received a single dose of either AT278 U500 or IAsp U100 (0.3 units/kg s.c.) in a crossover design, followed by an 8-h euglycemic clamp in the absence of basal insulin. RESULTS: With AT278 U500, onset of appearance in serum was 6 min earlier (P < 0.0001) and reached 50% of maximum concentration 23 min faster (P < 0.0001). Insulin exposure with AT278 U500 was 4.0-fold higher within the first 30 min (95% CI 3.29, 4.90), 1.5-fold higher within the first 60 min (95% CI 1.35, 1.76), and statistically superior up to 90 min postdose (P < 0.05). With AT278 U500, onset of action was 10 min earlier (P < 0.0001) and reached 50% of maximum glucose infusion rate 20 min faster (P < 0.0001). The glucose-lowering effect with AT278 U500 was 8.9-fold higher within the first 30 min (95% CI 5.96, 17.46), 2.4-fold higher within the first 60 min (95% CI 1.92, 3.22), and statistically superior up to 2 h postdose (P < 0.0001). Overall insulin exposure and glucose-lowering effect were comparable. No significant safety findings were observed. CONCLUSIONS: AT278 U500 offers rapid-acting characteristics in a reduced dose volume, with accelerated absorption and onset of action compared with IAsp U100 in the studied population.

Hepatic Response of Magnesium-Restricted Wild Type Mice.

Magnesium-deficiency is implicated in many metabolic disorders, e.g., type 2 diabetes and metabolic syndrome, representing risk factors for non-alcoholic fatty liver disease (NAFLD). This study aims to investigate the contribution of magnesium-restriction to the development of NAFLD. Magnesium-deficiency was induced in C57BL/6 mice by feeding a magnesium-deficient-diet. Metabolic markers as well as markers of inflammation and liver function were assessed. Furthermore, liver tissue was examined histopathologically and compared with specimens from high-fat-diet fed and control mice. Finally, the hepatic inflammatory response was quantified by determining hepatic IL-6, TNFα, and MCP-1. Magnesium-restriction resulted in at least a 2-fold significant reduction of serum magnesium levels compared to the high-fat-diet fed and control mice, whereas the hepatic magnesium content was decreased due to high-fat-diet feeding. No changes in metabolic markers in magnesium-restricted mice were observed, while the cholesterol content was elevated in high-fat-diet fed mice. Magnesium-restricted mice additionally featured inflammation and enlarged hepatocytes in liver histology. Furthermore, magnesium-restricted and high-fat-diet fed mice exhibited elevated hepatic TNFα levels compared to control mice. Accordingly, our data suggest that magnesium is involved in hepatic inflammatory processes and hepatocyte enlargement, key histological features of human NAFLD, and may therefore contribute to development and progression of the disease.

Pharmacokinetics and Pharmacodynamics of Three Different Formulations of Insulin Aspart: A Randomized, Double-Blind, Crossover Study in Men With Type 1 Diabetes.

OBJECTIVE: To investigate the pharmacokinetic and pharmacodynamic properties and safety of a novel formulation of insulin aspart (AT247) versus two currently marketed insulin aspart formulations (NovoRapid [IAsp] and Fiasp [faster IAsp]). RESEARCH DESIGN AND METHODS: This single-center, randomized, double-blind, three-period, crossover study was conducted in 19 men with type 1 diabetes, receiving single dosing of trial products (0.3 units/kg) in a random order on three visits. Pharmacokinetics and pharmacodynamics were assessed during a euglycemic clamp lasting up to 8 h. RESULTS: Onset of insulin appearance was earlier for AT247 compared with IAsp (-12 min [95% CI -14; -8], P = 0.0004) and faster IAsp (-2 min [-5; -2], P = 0.0003). Onset of action was accelerated compared with IAsp (-23 min [-37; -15], P = 0.0004) and faster IAsp (-9 min [-11; -3], P = 0.0006). Within the first 60 min, a higher exposure was observed for AT247 compared with IAsp by the area under the curve (AUC) glucose infusion rate (GIR) from 0 to 60 min (AUCAsp0-60min: treatment ratio vs. IAsp 2.3 [1.9; 2.9] vs. faster IAsp 1.5 [1.3; 1.8]), which was underpinned by a greater early glucose-lowering effect (AUCGIR,0-60min: treatment ratio vs. IAsp 2.8 [2.0; 5.5] vs. faster IAsp 1.7 [1.3; 2.3]). Furthermore, an earlier offset of exposure was observed for AT247 compared with IAsp (-32 min [-58; -15], P = 0.0015) and faster IAsp (-27 min [-85; -15], P = 0.0017), while duration of the glucose-lowering effect, measured by time to late half-maximum effect, did not differ significantly. CONCLUSIONS: AT247 exhibited an earlier insulin appearance, exposure, and offset, with corresponding enhanced early glucose-lowering effect compared with IAsp and faster IAsp. It therefore represents a promising candidate in the pursuit for second-generation prandial insulin analogs to improve postprandial glycemic control.

Comparison of cerebral Open Flow Microperfusion and Microdialysis when sampling small lipophilic and small hydrophilic substances.

BACKGROUND: Assessment of drug concentration in the brain interstitial fluid (ISF) is crucial for development of brain active drugs, which are mainly small, lipophilic substances able to cross the blood-brain barrier (BBB). We aimed to compare the applicability of cerebral Open Flow Microperfusion (cOFM) and Microdialysis (MD) to sample the lipophilic substance amitriptyline (AMI), its metabolites Hydroxyamitriptyline (HYA), Nortriptyline (NOR), Amitriptyline-N-Oxide (ANO), deuterated water (D2O) and the hydrophilic substance sodium fluorescein (Naf) in brain ISF. NEW METHOD: cOFM has been refined to yield increased spatial resolution and performance. COMPARISON OF COFM AND MD AND RESULTS: Performance of cOFM and MD was assessed by in vivo AUC ratios of probe samples (AUCCOFM/AUCMD) and the in vivo relative recovery of D2O (RRvv,D2O). Adsorption of AMI and Naf to MD and cOFM was assessed by the in vitro relative recovery (RRvt) prior to the in vivo experiments. The in vivo AUC ratio of AMI and RRvv,D2O was about two times higher for cOFM than for MD (AUCOFM/AUCMD = 2.0, RRvv,D2O(cOFM)/RRvv,D2O(MD) = 2.1). cOFM detected all investigated AMI metabolites except NOR. MD did not detect HYA, NOR, ANO and Naf. In vitro adsorption of AMI and Naf to the MD membrane was strong (RRvt,AMI = 4.4%, RRvt,Naf = 1.5%) but unspecific adsorption to cOFM was negligibly small (RRvt,AMI = 98% and RRvt,Naf = 98%). CONCLUSIONS: cOFM showed better performance when sampling AMI and its metabolites, Naf and D2O, and had an about two times higher RRvv,D2O than MD. MD did not detect HYA, NOR, ANO and Naf, most likely due to membrane adsorption.

Assessment of blood-brain barrier function and the neuroinflammatory response in the rat brain by using cerebral open flow microperfusion (cOFM).

Blood-brain barrier (BBB) impairment in systemic inflammation leads to neuroinflammation. Several factors including cytokines, chemokines and signal transduction molecules are implicated in BBB dysfunction in response to systemic inflammation. Here, we have adopted a novel in vivo technique; namely, cerebral open flow microperfusion (cOFM), to perform time-dependent cytokine analysis (TNF-alpha, IL-6 and IL-10) in the frontal cortex of the rat brain in response to a single peripheral administration of lipopolysaccharide (LPS). In parallel, we monitored BBB function using sodium fluorescein as low molecular weight reporter in the cOFM sample. In response to the systemic LPS administration, we observed a rapid increase of TNF-alpha in the serum and brain, which coincides with the BBB disruption. Brain IL-6 and IL-10 synthesis was delayed by approximately 1 h. Our data demonstrate that cOFM can be used to monitor changes in brain cytokine levels and BBB disruption in a rat sepsis model.

Polyamines in biological samples: rapid and robust quantification by solid-phase extraction online-coupled to liquid chromatography-tandem mass spectrometry.

Polyamines are ubiquitous active biogenic amines which contribute to basic cellular functions. Hence, their quantification in samples of diverse biological origins is essential for understanding how they function, especially in disease-relevant conditions. We present here a robust, high-throughput solid-phase extraction online coupled to a liquid chromatography-tandem mass spectrometry (SPE-LC/MS/MS) approach for the simultaneous quantification of eight polyamines in various biological samples. The polyamines include 1,3-diaminopropane, putrescine, cadaverin, N-acetyl-putrescine, spermidine, spermine, N(1)-acetyl-spermine, and l-ornithine. The novelty of the work is the use of two SPE columns online coupled to LC/MS/MS, which minimizes the sample pretreatment to a single derivatization step. The analysis is complete within 4min, making the method highly suitable for routine clinical analysis and high throughput screenings. The method was fully validated with serum samples. Dynamic ranges were 0.03 to 15µg/ml for ornithine and 1 to 500ng/ml for other polyamines, which cover physiological concentrations in serum samples. Lower limits of quantification (LLoQ) were found to be between 0.1 and 5ng/ml. As a proof of concept, we investigated gender differences in polyamine levels by analyzing the serum levels of 102 subjects.

Cerebral open flow microperfusion: a new in vivo technique for continuous measurement of substance transport across the intact blood-brain barrier.

The blood-brain barrier (BBB) limits substance transport to the brain and is therefore the major hurdle to overcome when developing neuroactive drugs. Herein, we report on cerebral open flow microperfusion (cOFM) as a new membrane-free technique for measuring substance transport across the intact BBB. The cOFM technique is based on a probe that is inserted into the brain, rupturing the BBB. The BBB is re-established within 15 days, which then allows sampling of interstitial brain fluid under physiological conditions. The aims of the present proof-of-concept study were to: (i) determine the time between cOFM probe insertion and BBB re-establishment; and (ii) demonstrate the ability of cOFM to sample the interstitial cerebral fluid with an intact BBB. The cOFM probe was inserted into the frontal lobe of Sprague-Dawley rats, resulting in BBB rupture. Re-establishment of the BBB was determined using Evans blue (EB) dye, which is an established marker for BBB intactness because it does not cross the intact BBB. Evaluating EB levels in the brain tissue indicated that the BBB was healed 11 days after probe insertion. To demonstrate transport across the healed BBB, we used sodium fluorescein (Naf), a sensitive, low molecular weight marker that can cross the intact BBB and can be used to monitor changes in BBB permeability. Significantly increased Naf levels were found in the interstitial fluid when hyperosmolar mannitol (known to open the BBB) was introduced via cOFM, which indicated partial opening of the BBB surrounding the cOFM probe. In conclusion, we show herein that cOFM allows monitoring of BBB permeability, which should be useful for measuring pharmacokinetics across the BBB and pharmacodynamics in the brain.

Development and validation of a low cost blood filtration element separating plasma from undiluted whole blood.

Clinical point of care testing often needs plasma instead of whole blood. As centrifugation is labor intensive and not always accessible, filtration is a more appropriate separation technique. The complexity of whole blood is such that there is still no commercially available filtration system capable of separating small sample volumes (10-100 µl) at the point of care. The microfluidics research in blood filtration is very active but to date nobody has validated a low cost device that simultaneously filtrates small samples of whole blood and reproducibly recovers clinically relevant biomarkers, and all this in a limited amount of time with undiluted raw samples. In this paper, we show first that plasma filtration from undiluted whole blood is feasible and reproducible in a low-cost microfluidic device. This novel microfluidic blood filtration element (BFE) extracts 12 µl of plasma from 100 µl of whole blood in less than 10 min. Then, we demonstrate that our device is valid for clinical studies by measuring the adsorption of interleukins through our system. This adsorption is reproducible for interleukins IL6, IL8, and IL10 but not for TNFα. Hence, our BFE is valid for clinical diagnostics with simple calibration prior to performing any measurement.

Dermal PK/PD of a lipophilic topical drug in psoriatic patients by continuous intradermal membrane-free sampling.

BACKGROUND: Methodologies for continuous sampling of lipophilic drugs and high-molecular solutes in the dermis are currently lacking. We investigated the feasibility of sampling a lipophilic topical drug and the locally released biomarker in the dermis of non-lesional and lesional skin of psoriatic patients over 25h by means of membrane-free dermal open-flow microperfusion probes (dOFM) and novel wearable multi-channel pumps. METHODS: Nine psoriatic patients received a topical p-38 inhibitor (BCT194, 0.5% cream) on a lesional and a non-lesional application site once daily for 8 days. Multiple dOFM sampling was performed for 25 h from each site on day 1 and day 8. Patients were mobile as dOFM probes were operated by a novel light-weight push-pull pump. Ultrasound was used to verify intradermal probe placement, cap-LC-MS/MS for BCT194 and ELISA for TNFα analysis. RESULTS: dOFM was well tolerated and demonstrated significant drug concentrations in lesional as well as non-lesional skin after 8 days, but did not show significant differences between tissues. On day 8, TNFα release following probe insertion was significantly reduced compared to day 1. CONCLUSIONS: Novel membrane-free probes and wearable multi-channel pumps allowed prolonged intradermal PK/PD profiling of a lipophilic topical drug in psoriatic patients. This initial study shows that dOFM overcomes limitations of microdialysis sampling methodology, and it demonstrates the potential for PK/PD studies of topical products and formulations in a clinical setting.

Microdialysis based device for continuous extravascular monitoring of blood glucose.

Glycemic control of intensive care patients can be beneficial for this patient group but the continuous determination of their glucose concentration is challenging. Current continuous glucose monitoring systems based on the measurement of interstitial fluid glucose concentration struggle with sensitivity losses, resulting from biofouling or inflammation reactions. Their use as decision support systems for the therapeutic treatment is moreover hampered by physiological time delays as well as gradients in glucose concentration between plasma and interstitial fluid. To overcome these drawbacks, we developed and clinically evaluated a system based on microdialysis of whole blood. Venous blood is heparinised at the tip of a double lumen catheter and pumped through a membrane based micro-fluidic device where protein-free microdialysate samples are extracted. Glucose recovery as an indicator of long term stability was studied in vitro with heparinised bovine blood and remained highly stable for 72 h. Clinical performance was tested in a clinical trial in eight healthy volunteers undergoing an oral glucose tolerance test. Glucose concentrations of the new system and the reference method correlated at a level of 0.96 and their mean relative difference was 1.9 +/- 11.2%. Clinical evaluation using Clark's Error Grid analysis revealed that the obtained glucose concentrations were accurate and clinically acceptable in 99.6% of all cases. In conclusion, results of the technical and clinical evaluation suggest that the presented device delivers microdialysate samples suitable for accurate and long term stable continuous glucose monitoring in blood.