First application of a transcutaneous optical single-port glucose monitoring device in patients with type 1 diabetes mellitus.

The combination of continuous glucose monitoring (CGM) and continuous subcutaneous insulin infusion can be used to improve the treatment of patients with diabetes. The aim of this study was to advance an existing preclinical single-port system for clinical application by integrating the sensors of a phosphorescence based CGM system into a standard insulin infusion set. The extracorporeal optical phase fluorimeter was miniaturised and is now comparable with commercial CGM systems regarding size, weight and wear comfort. Sensor chemistry was adapted to improve the adhesion of the sensor elements on the insulin infusion set. In-vitro tests showed a linear correlation of R2=0.998 between sensor values and reference glucose values in the range of 0-300mg/dl. Electrical and cytotoxicity tests showed no negative impact on human health. Two single-port devices were tested in each of 12 patients with type 1 diabetes mellitus in a clinical set-up for 12h. Without additional data processing, the overall median absolute relative difference (median ARD) was 22.5%. For some of the used devices the median ARD was even well below 10%. The present results show that individual glucose sensors performance of the single-port system is comparable with commercial CGM systems but further improvements are needed. The new system offers a high extent of safety and usability by combining insulin infusion and continuous glucose measurement in a single-port system which could become a central element in an artificial pancreas for an improved treatment of patients with type 1 diabetes mellitus.

Open flow microperfusion: pharmacokinetics of human insulin and insulin detemir in the interstitial fluid of subcutaneous adipose tissue.

AIMS: To compare the time profile of insulin detemir and human insulin concentrations in the interstitial fluid (ISF) of subcutaneous adipose tissue during constant i.v. infusion and to investigate the relationship between the pharmacokinetics of both insulin molecules in plasma and the ISF of subcutaneous adipose tissue. METHODS: During a 6-h hyperinsulinaemic-euglycaemic clamp (plasma glucose level 8 mmol/l) human insulin (21 and 42 pmol/min/kg) or insulin detemir (209 and 417 pmol/min/kg) were infused i.v. in eight rats per dose level. Open flow microperfusion (OFM) was used to continuously assess interstitial insulin concentrations in subcutaneous adipose tissue. RESULTS: At the lower infusion rate, insulin detemir appeared significantly later in the ISF than in the plasma (p < 0.05) and also appeared later in the ISF relative to human insulin (p < 0.005). CONCLUSIONS: By using OFM we were able to monitor albumin-bound insulin detemir directly in the ISF of subcutaneous tissue and confirm its delayed transendothelial passage to a peripheral site of action.

Clinical applicability of dOFM devices for dermal sampling.

BACKGROUND: Sampling the dermal interstitial fluid (ISF) allows the pharmacokinetics and pharmacodynamics of dermatological drugs to be studied directly at their site of action. Dermal open-flow microperfusion (dOFM) is a recently developed technique that can provide minimally invasive, continuous, membrane-free (thus unfiltered) access to the dermal ISF. Herein, we evaluate the clinical applicability and reliability of novel wearable dOFM devices in a clinical setting. METHODS: Physicians inserted 141 membrane-free dOFM probes into the dermis of 17 healthy and psoriatic volunteers and sampled dermal ISF for 25 h by using wearable push-pull pumps. The tolerability, applicability, reproducibility, and reliability of multiple insertions and 25 h continuous sampling was assessed by pain scoring, physician feedback, ultrasound probe depth measurements, and 25 h-drift and variability of the sodium relative recovery. RESULTS: Insertion pain was moderate and decreased with each additional probe. Probe insertion was precise, although slightly deeper in lesional skin. The wearable push-pull pump enabled uninterrupted ISF sampling over 25 h with low variability. The relative recovery was drift-free and highly reproducible. CONCLUSION: dOFM sampling devices are tolerable and reliable for prolonged continuous dermal sampling in a multiprobe clinical setting. These devices should enable the study of a wide range of drugs and their biomarkers in the skin.

Comparison of open-flow microperfusion and microdialysis methodologies when sampling topically applied fentanyl and benzoic acid in human dermis ex vivo.

PURPOSE: The purpose of this study is to compare two sampling methods--dermal Open-Flow Microperfusion (dOFM) and dermal Microdialysis (dMD) in an international joint experiment in a single-laboratory setting. We used human ex-vivo skin and sampled topically administered Fentanyl and Benzoic Acid. The second purpose was to provide guidance to researchers in choosing the most efficient method for a given penetrant and give suggestions concerning critical choices for successful dermal sampling. METHODS: The dOFM and dMD techniques are compared in equal set-ups using three probe-types (one dOFM probe and two dMD probe-types) in donor skin (n = 9)--27 probes of each type sampling each penetrant in solutions applied in penetrationchambers glued to the skin surface over a time range of 20 h. RESULTS: Pharmacokinetic results demonstrated concordance between dOFM and dMD sampling technique under the given experimental conditions. The methods each had advantages and limitations in technical, practical and hands-on comparisons. CONCLUSION: When planning a study of cutaneous penetration the advantages and limitations of each probe-type have to be considered in relation to the scientific question posed, the physico-chemical characteristics of the substance of interest, the choice of experimental setting e.g. ex vivo/in vivo and the analytical skills available.

The propeptide of yeast cathepsin D inhibits programmed necrosis.

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast.

Insulin glulisine, insulin lispro and regular human insulin show comparable end-organ metabolic effects: an exploratory study.

AIMS: To compare the end-organ metabolic effects of insulin glulisine (glulisine), insulin lispro (lispro) and regular human insulin (RHI) in patients with type 1 diabetes mellitus. METHODS: Eighteen patients with type 1 diabetes mellitus (mean age 36.9 +/- 8.6 years, BMI 23.6 +/- 2.8 kg/m(2), haemoglobin A(1c) 7.4 +/- 0.9%) were randomized in this single-centre, double-blind, three-period cross-over, standard Latin-square, euglycaemic glucose clamp trial. Patients received sequential, primed stepwise intravenous infusions of glulisine, lispro or RHI (infusion rates were increased in a stepwise manner from an initial rate of 0.33 [180 min] to 0.66 [180 min] and 1.00 [180 min] mU/kg/min). The primary variables were the suppression of endogenous glucose production (S(EGP)) and glucose uptake (GU). RESULTS: Mean basal endogenous glucose production (EGP) was 1.88, 2.12 and 2.12 mg/kg/min for glulisine, lispro and RHI respectively. Mean (+/-s.e.) maximum absolute S(EGP) (adjusted for basal EGP) was -1.64 +/- 0.06, -1.72 +/- 0.05 and -1.56 +/- 0.05 mg/kg/min respectively. Mean (+/-s.e.) maximum absolute increase in GU (adjusted for basal GU) was 6.46 +/- 0.26, 6.23 +/- 0.24 and 6.72 +/- 0.24 mg/kg/min respectively. There were no clinically relevant differences between the three insulin treatments with respect to serum insulin, free fatty acid (FFA), glycerol or lactate levels. No serious adverse events and no episodes of severe hypoglycaemia were reported. CONCLUSIONS: This study shows that glulisine, lispro and RHI have similar effects on S(EGP), GU, FFA, glycerol and lactate levels, providing evidence for similar end-organ metabolic effects.

Proportional dose-response relationship and lower within-patient variability of insulin detemir and NPH insulin in subjects with type 1 diabetes mellitus.

AIMS: This study was conducted to evaluate the dose ratio of insulin detemir and neutral protamine Hagedorn (NPH) insulin over a range of therapeutically relevant subcutaneous doses. METHODS: The study was a randomized, double-blind, crossover 24-h-iso-glycemic clamp trial in 12 C-peptide-negative type 1 diabetic patients. Each subject received, by an incomplete block design selection, two of three possible doses of insulin detemir (0.15, 0.3, 0.6 U/kg) and NPH insulin (0.15, 0.3, 0.6 IU/kg), respectively. A detailed assessment of endogenous glucose production (EGP) and glucose uptake was performed, by use of stable isotopic labeled glucose tracer (D-[6,6- (2)H (2)] glucose). RESULTS: Dose proportionality was observed within the tested dose range. Regarding unit dose ratio, 0.68 U insulin detemir equals 1 IU NPH insulin (95% CI [0.35; 1.30]). There was no statistically significant difference in effect on the area under the curve (AUC) of glucose infusion rate (GIR) (AUC (GIR)) and the maximal GIR (GIR (max)) values, when comparing U (insulin detemir) to IU (NPH insulin). The pharmacodynamic within-subject profile was lower with insulin detemir in regard to AUC (GIR 0-24 h), GIR (max) and duration of action ( P<0.05). There was a tendency for a greater reduction of EGP with insulin detemir than with NPH insulin in regard to the area over the curve (AOC) of EGP in 24 hours (AOC (EGP 0-24 h)) ( P=0.07) and minimal EPG (EGP (min)) ( P=0.02). CONCLUSIONS: These data show that insulin detemir is dose-proportional to NPH insulin in type 1 diabetic patients at clinically relevant doses. The data indicate that insulin detemir has a lower degree of within-subject variability.

Evaluation of norbornene- beta-cyclodextrin-based monomers and oligomers as chiral selectors by means of nonaqueous capillary electrophoresis.

Norbornen-5-yl carboxylic acid and norbornen-5-ylmethylsilyl ether-based beta-cyclodextrins (beta-CDs) containing up to three norbornene ester and up to five norbornene silyl ether units have been prepared from beta-CD and norbornen-5-carboxylic chloride and norbornen-5-ylmethyldichlorosilane, respectively. Oligomers (n = 2-4) were prepared therefrom using ring-opening metathesis polymerization (ROMP). Monomeric and oligomeric substituted beta-CDs were evaluated as chiral selectors in nonaqueous capillary zone electrophoresis using 35 mM sodium bicarbonate in N-methylformamide (NMF) as background electrolyte. Both monomeric and oligomeric norbornene ester- and norbornene silyl ether-type selectors showed good enantioresolution for dansylated (DNS-) amino acids using concentrations of the chiral selector of up to 4% w/v. A significant improvement in resolution was observed upon the introduction of up to five norbornene silyl ether units into a beta-CD molecule, whereas higher degrees of substitution with norbornen-5-yl-carboxyl groups lead to a reduction in enantioresolution of DNS-amino acids. Thus, pentakis(norbornen-5-ylmethylhydroxysiloxyl)-beta-CD turned out to be superior to mono(norbornen-5-ylmethylhydroxysiloxyl)-beta-CD in terms of enantioresolution. Moreover, norbornene silyl ether-type selectors were found to be more efficient than norbornene ester-type selectors. Finally, oligomeric selectors were found to possess superior or at least comparable enantioselectivity in the separation of DNS-amino acids compared to the parent monomers. A maximum in enantioresolution was obtained with oligo(pentakis(norbornen-5-ylmethylhydroxysiloxyl)beta-CD).

Chiral beta-cyclodextrin-based polymer supports prepared via ring-opening metathesis graft-polymerization.

A series of norborn-2-ene-derivatized beta-cyclodextrins (beta-CDs), 6-O-(norborn-2-ene-5-carboxyl)-beta-CD (1), tetrakis(6-O-norborn-2-ene-5-carboxyl)-beta-CD (2), (3), 6-O-(6-norborn-2-ene-5-car-6-O-(7-oxanorborn-2-ene-5-carboxyl)-beta-CD bonylaminohexoyl)-beta-CD (4), 6-O-(norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (5), tris(6-O-norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (6), tetrakis(6-O-norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (7) and hexakis(6-O-norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (8), have been synthesized. Compounds 1-3 were prepared via reaction of beta-CD with norborn-2-ene-5-carboxylic chloride and 7-oxanorborn-2-ene-5-carboxylic chloride, respectively; compounds 5-8 were synthesized from norborn-2-ene-5-yl-methyldichlorosilane and beta-CD, respectively. Compound 4 was accessible by reaction of norbom-2-ene-5-carboxylaminohexoyl chloride with beta-CD. Compounds 1-8 were surface grafted onto norborn-2-ene-derivatized silica-based supports using ring-opening metathesis polymerization employing the ruthenium-based initiator bis(tricyclohexylphosphino)benzylideneruthenium dichloride [Cl2Ru(CHC6H5)(PCy3)2, Cy=cyclohexyl, 9]. Generally speaking, the resulting chiral stationary phases (CSPs) I-VIII may be prepared with high reproducibility and may be used within a pH of 2-10. Thus, relative standard deviations (sigman-1) of the mean resolution (Rs) are <7%. The CSPs were used for the enantioselective separation of beta-blockers, N-dansyl-, N-3,5-dinitrobenzoyl- and Fmoc-protected amino acids and were characterized in terms of chemical stability, selectivity (alpha') and resolution (Rs). Additionally, the role of the spacer as well as influences of capacity and the degree of substitution of the beta-CD moiety on the separation characteristics were determined.