Regulatory Peptides 139 (2007) 122 127 www.elsevier.

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Synthesis and evaluation of a glibenclamide glucose-conjugate: A potential new lead compound for substituted glibenclamide derivatives as islet imaging agents
S. Schneider a,, S. Ueberberg a , A. Korobeynikov b , W. Schechinger a , C. Schwanstecher c , M. Schwanstecher c , H.H. Klein a , E. Schirrmacher d
Medical Department I, Division of Endocrinology and Diabetology, University Hospital Bergmannsheil, Bochum, Germany b Institute of Nuclear Medicine, University of Mainz, Mainz, Germany c Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig, Germany Sir Mortimer B. Davis Jewish Hospital, Lady Davis Institute for Medical Research, 3755 Cote Ste Catherine, F-223, Montreal, Canada Received 24 September 2006; received in revised form 7 November 2006; accepted 7 November 2006 Available online 10 January 2007
a

Abstract The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands. 2006 Elsevier B.V. All rights reserved.
Keywords: Glibenclamide glucose-conjugate; Pharmacodynamic; Insulin secretion; Binding to SUR1

1. Introduction The non-invasive assessment of the pancreatic islet cell mass (ICM) in vivo would be of great medical interest but there is currently no islet cell-specific ligand available. In Type 1 diabetic patients, the chronic and progressive loss of the insulin producing pancreatic islet cells due to autoimmune destruction has led to concerted efforts to prevent further loss of -cells by autoantigen-specific immunotherapy of pre-diabetic patients [1]. With a non-invasive technique, the effect of different intervention strategies could be easily monitored. Furthermore, the longitudinal monitoring of the ICM of patients with Type 2
Corresponding author. Tel.: +49 234 302 3469; fax: +49 234 302 6403. E-mail address: Stephan.Schneider@rub.de (S. Schneider). 0167-0115/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.004

diabetes mellitus under different antidiabetic therapies (e.g. GLP-agonists) may lead to the development of new strategies for treatment. Insulin secretion is regulated by the membrane potential of the -cell, which depends on the activity of ATP-sensitive K+ channels (KATP channels) in the plasma membrane [2]. Closure of KATP channels due to a rise of the cytoplasmic ATP/ADP ratio results in a depolarization of the membrane and in opening of voltage-sensitive Ca2+ channels. The increase in cytoplasmic Ca2+ stimulates the exocytosis of insulin. KATP channels are composed of a small inwardly rectifying K+ channel subunit (Kir6.1 or Kir6.2) plus a sulfonylurea receptor (SUR1, SUR2A or SUR2B) belonging to the ATP-binding cassette super family [3]. SURs represent the target for hypoglycaemic sulfonylureas such as glibenclamide, but it is not yet fully clarified which

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structural features determine the binding to SUR and pharmacological behaviour. Especially bulky derivatisation of the original glibenclamide structure has not been reported. It is well documented that the introduction of the structure element 5chloro-N-ethylen-2-methoxy benzamide resulted in an increase of the hyperglycaemic activity of 1000 in comparison to tolbutamide [4,5] and that even small changes at the benzenamide moiety can alter the biological activity significantly [4] (Fig. 1). So far, three sulfonylurea receptors have been cloned: SUR1, SUR2A and SUR2B. While SUR1 is expressed in a very high density at the internal face of the plasma membrane of the pancreatic islet cells (glucagon-secreting - and insulin secreting -cells) but not in the exocrine part of the pancreas [6,7], the SUR2 isoforms are predominant in cardiac, skeletal and smooth muscle cells [8]. Therefore, it was proposed that a radioactively labelled glibenclamide derivative could be a feasible tracer for visualizing the ICM in humans by accumulation at the internal face of the plasma membrane of pancreatic islet cells due to binding to SUR1 and estimation of the specific radioactivity over regions of interest (e.g. pancreas) using positron emission tomography (PET) or single photon emission computed tomography (SPECT). But the original glibenclamide molecule has been proven to be unsuitable for islet imaging, due to a high lipophilicity which in turn leads to a high plasma protein binding [9]. Therefore the search for novel glibenclamide derivatives originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity to the receptor. It has been previously described, that the imaging quality of a ligand could be substantially improved by conjugation with carbohydrates [10]. Due to a markedly increased hydrophilicity after conjugation with carbohydrates, the ligands were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine. Therefore, in this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity and drug solubility. Furthermore, the presented study will provide important additional insight into structure activity relationships. 2. Materials and methods 2.1. Chemistry The synthesis started from commercially available 1,2,3,4,6penta-O-acetyl-D-glucose (1), which was easily converted to 2,3,4,6-tetra-O-acetyl-D-glucose, deprotonated with sodiumhydrid in CH2Cl2 at 20 C and reacted with tert.-butylbromoacetate to yield (O-tert.-butyl carboxymethyl) 2,3,4,6-tetra-O-acetyl--Dglucopyranoside (2). The tert.-butyl protecting group was removed with trifluoracetic acid in a CH2Cl2/water mixture and gave carboxymethyl 2,3,4,6-tetra-O-acetyl--D-glucopyranosid in quantitative yield. For the conjugation to the amino moiety of 4-amino-5-chloro-2-methoxybenzoic acid, the carboxy moiety was converted quantitatively into the corresponding acid chloride (3) by treatment with thionyl chloride at 50 C. Compound 3 was coupled to the aromatic amino function over a period of 4 d but the desired 2-chloro-4-carboxy-5-methoxyphe-

Fig. 1. Influence of structural components determining the structureactivity relationship: A) unknown region of structural modification; B) halogen atoms enhance binding affinity and insulin secretion; C) methoxy group as well as larger groups increase binding affinity and insulin secretion; D) acid amide moiety enhances binding affinity by the factor 1000 in comparison to tolbutamide; E) acidic sulfonylurea group is essential for binding; F) cyclohexyl moiety increases lipophilicity, probably responsible for effective cell internalisation.

nyl O-(2,3,4,6-tetra-O-acetyl--D-glucopyranoside)-acetamid (4) was obtained in 27% yield only, probably due to the low nucleophilicity of the amino group. The formation of the amide bond using p-(2-aminoethyl)benzenesulfonamide was performed following an analogous procedure [9] and 4-[2-(2,3,4,6-tetra-Oacetyl--D-glucopyranoside)-O acetoyl]amino-N-{2-[4-(aminosulfonyl)phenyl]ethyl}-5-chloro-2-methoxybenzamide (5) was purified by column chromatography on silica gel. Formation of the sulfonylurea moiety of 4-[2-(2,3,4,6-tetra-O-acetyl--Dglucopyranoside)-O-acetoyl]amino-N-{2-[4 ({[cyclohexylamino) carbonyl]amino}sulfonyl)phenyl]ethyl}-5-chloro-2-methoxybenzamide (acetyl protected (6)) was achieved by reacting 5 with cyclohexylisocyanate under Cu(I)-catalysis. The acetyl protected sulfonyl urea was obtained in high yields of 90% and characterized using 1H NMR, 13C NMR and ESI mass spectroscopy. To obtain the final glucose-conjugate 4-[2-(-Dglucopyranoside)- O-acetoyl]amino- N-{2-[4-{[(cyclohexylamino)carbonyl]-amino}sulfonyl)phenyl]ethyl}-5-chloro-2-methoxy bezamide (6) of glibenclamide, deacetylation was performed under Zempln conditions (NaOMe, MeOH) following a similar procedure. Conjugate 6 was purified using column chromatography in 75% yield and characterized by 1H NMR, 13C NMR and ESI mass spectroscopy (Fig. 2). 2.2. Estimation of the lipophilicity The logD values of the original glibenclamide and its glucoseconjugate were estimated according the OECD Guideline for testing of chemicals with the HPLC-method [11]. 2.3. Binding affinity to human SUR1 To assess binding affinities of the glibenclamide glucoseconjugate for human SUR1, [3H]glibenclamide (0.3 nM) displacement assays were performed with membranes from COS-1 cells transiently expressing human SUR1. Transfections and membrane preparations were performed as described [12,13]. Briefly, COS-1 cells cultured in DMEM HG (10 mM glucose), supplemented with 10% fetal calf serum (FCS), were plated at a density of 5 105 cells per dish (94 mm) and allowed

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Fig. 2. De-novo synthesis of the glibenclamide glucose-conjugate.

to attach overnight. 200 g of pECE-human SUR1 complementary DNA (GenBank NP_000343) were used to transfect 10 plates. For transfection the cells were incubated 4 h in a Trisbuffered salt solution containing DNA (510 g/ml) plus DEAEdextran (1 mg/ml), 2 min in HEPES-buffered salt solution plus dimethyl-sulfoxide (10%) and 4 h in DMEM-HG plus chloroquine (100 M). Cells were then returned to DMEM-HG plus 10% FCS and were used 6072 h post transfection to prepare membranes as described [12]. To measure binding to membranes from COS-cells the resuspended fraction (final protein concentration 550 g/ml) was incubated in Tris-buffer (50 mM, pH 7.4) containing [3H]glibenclamide (final concentration 0.3 nM, non-specific binding defined by 1 M glibenclamide) and glibenclamide glucose-conjugate. Incubations were carried out for 1 h at room temperature and were terminated by rapid filtration through Whatman GF/B filters. Half-maximally inhibitory drug concentrations (IC50 values) and Hill coefficients (n) were estimated by fitting the function B = 1 / (1 + ([drug] / IC50)n) to the data of each single displacement experiment. KDs were calculated from IC50 values as described [12]. 2.4. Insulin secretion studies For testing the in vitro function of the de-novo synthesized glibenclamide glucose-conjugate a standardized batch stimulation was performed according to a protocol established in our laboratory [14]. Adult rat islets were isolated by collagenase digestion and purified by a density gradient. Briefly, CD rats (Central Animal Facility, University of Mainz), 68 weeks old and with a body weight of 250270 g, were used as islet donors. Rats were anaesthetized by intraperitoneal pentobarbital administration (60 mg/kg). A midline abdominal incision was performed and the pancreas was exposed and injected via the pancreatic duct

with Hanks' balanced salt solution (HBSS; Gibco BRL, Long Island, NY) containing 1.7 mg/ml collagenase (Serva PanPlus, Heidelberg, Germany). After the death of the animals, the pancreatic tissue was surgically removed and incubated for 10 min at 37 C in the collagenase solution. Mechanical disruption of the digested pancreatic tissue was achieved by further incubation at 37 C for 10 min in collagenase solution, interrupted every 2 min by shaking for 30 s. The digestion process was stopped by the addition of cooled HBSS plus 10% fetal calf serum (4 C). Islet purification was achieved using a discontinuous three-phase Ficoll density gradient (densities: 1.090, 1.077 and 1.040). Islets were cultured in complete medium (RPMI 1640 medium, Biochrom KG, Berlin, Germany) at 37 C in a humidified atmosphere containing 5% CO2. The RPMI 1640 medium was supplemented with 5 mM glucose, 10 mM HEPES (Greiner Laboratories, Frickenhausen, Germany), 0.2 g/l Glutamax (GibcoBRL, Paisley, Scotland), 10% fetal calf serum (FCS; Greiner Laboratories, Frickenhausen, Germany) and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin; GibcoBRL, Paisley, Scotland and 10 g/ml Ciprobay; Aventis, Frankfurt, Germany). For each sample eight islets were picked (equal in size and shape) in a culture-insert with a membrane of 12 m pore size (Millicell PCF, Millipore, France) and incubated in a 24-well culture-plate (Falcon Multiwell, Becton Dickinson, USA). First, basal insulin secretion was tested by incubating the islets with normoglycaemic culture media (RPMI 1640+ 5 mM D-glucose + 10% FCS) for 1 h at 37 C+ 5% CO2. After the culture period the media were collected and stored at 20 C. The inserts with islets were transferred to normoglycaemic culture-medium containing 50 nmol/l glibenclamide or its glucose-conjugate and incubated for a stimulation period of 1 h. As a positive control several inserts with islets were cultured in hyperglycaemic culturemedium (RPMI 1640+ 15 mM D-glucose + 10% FCS) only. For

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negative control normoglycaemic culture-medium (RPMI 1640 + 5 mM D-glucose+ 10% FCS) lacking glibenclamide or its glucose-conjugate was used. The insulin content of each probe was quantified by a rat-insulin ELISA (Mercodia, Uppsala, Sweden). Insulin secretion was expressed as insulin release per ng/islet/h. The stimulation index was calculated by dividing insulin output during stimulation (15 mM D-glucose or 5 mM Dglucose + test substance) by insulin secretion during basal incubation (5 mM D-glucose). 2.5. Determination of the intravenous potency of the compounds Glibenclamide (10 mg/ml) or its glucose-conjugate (15 mg/ ml) was prepared as stock solution in DMSO. The drugs were added to the final concentration in a 1:1 mixture of phosphatebuffered saline and 0.9% saline. The volume of the administered dose was 4 ml/kg. The highest concentration of DMSO in the injected solution was 2.5%, and the control solution was prepared with the same amount of the solvent. CD rats (Central Animal Facility, University of Mainz) were allocated into groups of 47 individuals and anaesthetized as described [15]. A cannula (22G) was inserted in a jugular vein of each animal for intravenous injection of either glibenclamide or its glucoseconjugate (1.0 mg/kg). 10 min and 120 min after injection of the drug blood and urine samples were taken for later determinations of plasma D-glucose and drug concentrations. Blood glucose levels were determined by enzymatic analysis (EBIO 6666, Eppendorf, Hamburg, Germany) and the plasma drug concentrations (mg/ml) by HPLC-technique as described previously [16]. 2.6. Statistics Values are given if not stated otherwise as Mean standard error of mean (SEM). Statistical significance of differences was calculated with an unpaired Student's t-test (two-sided). 3. Results 3.1. Determination of the lipophilicity The de-novo synthesized glibenclamide glucose-conjugate showed an approximately 12-fold lower lipophilicity (LogD 0.43) compared to glibenlamide (LogD 1.74). 3.2. Binding affinities for human SUR1 Competition binding experiments were performed to assess the binding affinity of the glucose-conjugate towards human SUR1. The results clearly showed that the glucose-conjugate induced a complete monophasic inhibition curve with a Hill coefficient of 1.04 which was comparable to that of glibenclamide (0.94). As shown by the Hill coefficient both compounds bind in a non-cooperative fashion to SUR1. The glucose-conjugate showed a rather high binding affinity with a dissociation constant (KD) of 2.08 nM, which was decreased to some extent compared to glibenclamide (KD 0.38 nM; Fig. 3).

3.3. Evaluation of the insulin-stimulating capacity using a static incubation challenge Six groups of ten islets were incubated for 60 min with either 50 nmol/l of glibenclamide or its glucose-conjugate. The insulin secretion after stimulation with the glucose-conjugate increased from basal to 4.2 1.6 ng/islet/h. In contrast insulin secretion after stimulation with glibenclamide increased to 7.1 3.1 ng/ islet/h (p b 0.05). Concordant with these findings, the estimated stimulation index was also slightly reduced for the glucoseconjugate (2.3 1.2 vs. 3.5 1.4; p b 0.05). In conclusion, consistent with the binding affinity data the conjugation of glibenclamide with glucose leads to a slightly reduced insulinstimulating capacity in vitro. 3.4. In vivo hypoglycaemic effects and pharmacodynamics of the compounds Fig. 4 illustrates the blood glucose profiles of normoglycaemic rats at different times (0210 min) after intravenous application of glibenclamide, its glucose-conjugate or solvent only. As indicated all groups showed the same blood glucose concentration at start of the experiment (time zero). But the subsequent blood sugar decrease after intravenous application was faster (30 vs. 60 min) and more pronounced in the glibenclamide compared to the glucose-conjugate group (nadir 37 mg/dl vs. 70 mg/dl). Furthermore, the hypoglycaemic action of the glucose-conjugate was of significantly shorter duration than that of glibenclamide (150 min vs. 270 min). In accordance with these findings, the pharmacodynamics of glibenclamide and its glucose-conjugate were vastly different from one another. 10 min after drug administration, the plasma concentration of the glucose-conjugate was slightly higher compared to glibenclamide (glucose-conjugate: 0.005 0.006 mg/ml vs. glibenclamide: 0.0008 0.0006 mg/ ml). In contrast 120 min after drug administration the plasma concentration of the glucose-conjugate decreased substantially (0.0007 0.0005 mg/ml) whereas the glibenclamide concentration was unchanged (0.001 0.0006 mg/ml). This could be explained

Fig. 3. Binding affinity of glibenclamide and its glucose-conjugate towards human SUR1. Results are Mean SEM. 46 experiments.

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Fig. 4. In vivo hypoglycaemic effects of glibenclamide and its glucose-conjugate. Glibenclamide (1.0 mg/kg; black circles; n = 4), its glucose-conjugate (1.0 mg/kg; white circles; n = 7) or solvent only (black triangles; n = 5) was administered as a single intravenous bolus injection. Results are Mean SEM. p b 0.001 glibenclamide vs. solvent; #p b 0.05 glucose-conjugate vs. solvent.

by a faster renal clearance of the glucose-conjugate, as shown by a significant higher urine concentration after 120 min (glucoseconjugate: 0.002 0.001 mg/ml vs. glibenclamide: 0.0001 0.0001 mg/ml; p b 0.05). 4. Discussion Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high islet/background ratios and thus signal intensity in diagnostic imaging by positron emission tomography (PET). As published by Schottelius et al. [10] introduction of carbohydrates is a powerful tool to increase the hydrophilicity of a tracer and to improve the signal-to-noise ratio. This becomes true because a markedly increased hydrophilicity leads to an excretion predominantly via the kidneys and a decrease in activity accumulation in liver and intestine. Therefore, in this study the conjugation of glibenclamide with glucose was performed. As expected glucose conjugation of glibenclamide leads to a significantly increased hydrophilicity (12-fold). Although glibenclamide is well known for more than 30 years it is not yet fully clarified which structural features determine the binding to the SUR1 and its pharmacological behaviour. Therefore it was interesting to note that the glucose-conjugate showed slightly decreased but nevertheless a high binding affinity toward human SUR1 (KD of 2.08 nM). Concerning the use of glycosylated glibenclamide derivatives as imaging agents these results are encouraging, because the aim to develop a hydrophilic compound with high affinity binding toward SUR1 could be realized. Furthermore, the hydrophilic character of the glucoseconjugate leads to a completely different pharmacodynamic profile in vivo. Due to its hydrophilic character the compound was cleared much faster from the blood stream via the kidneys, which indicates a significant reduced plasma protein binding. These results are promising because they provide for the first time

evidence that glycosylation at the benzamide structure is possible with substantial effects on the excretion profile. We assume that the excretion predominantly via the kidney could lead to an increased signal-to-noise ratio and a better detection of the pancreas signal in vivo. But more work is needed to prove if a radiolabelled glibenclamide glucose-conjugate will really be suitable for non-invasive imaging of the pancreatic islet cell mass. Optimal control of blood glucose is one of the main goals of therapy to avoid secondary diabetic complications. In this context, drug therapy includes compounds of different mechanisms of action [1]. Among those, sulfonylureas as insulin secretagogues are most widely prescribed. To date those drugs are often associated with clinical relevant side effects such as severe hypoglycaemic episodes, due to their long duration of action, predominantly in the elderly. Therefore, intensive research on the synthesis of novel short acting oral antidiabetic drugs is still performed. Concerning this issue the obtained results are interesting. Due to the hydrophilic character of the glucose-conjugate the hypoglycaemic action profile was of significant shorter duration compared to the original glibenclamide (150 min vs. 270 min). This is promising, but despite high affinity binding toward human SUR1 the insulin secretory capacity in vitro was diminished by about 40% compared to glibenclamide. In accordance, the hypoglycaemic capacity in vivo was also significantly weaker. Furthermore, it could be assumed that the hydrophilic character of the derivative will lead to a complete different resorption profile after oral application, due to a reduced intestinal uptake. Therefore, it seems only in part realistic that this compound may lead to the development of novel short acting oral antidiabetics. In conclusion, in this study the de-novo synthesis of a glibenclamide glucose-conjugate as a potential lead compound for the design of novel substituted glibenclamide derivatives as islet imaging agents is reported. Glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with a complete different excretion profile. Acknowledgements This work was supported by research grants from the Wissenschaftskomission of the University Hospital Bergmannsheil and the Dr. Brger Bsing Stiftung to S.S. The authors wish to thank M. Piel for the determination of the logD value. Furthermore, S.S. was awarded for this work with the KarlOberdisse Preis 2006 of the Nordrhein-Westflische Gesellschaft fr Endokrinologie und Diabetologie. References
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