Single chain des-(B30) insulin

Single chain des-(B30) insulin (SCI) has been synthesized from porcine insulin by trypsin in a medium with a low content of water. Trypsin catalyzes an intramolecular transpeptidation reaction in which the glycine^A1 residue substitutes the alanine^B30 residue, rendering a Lys^B29-Gly^A1 peptide link between the A-and B-chains of insulin. The insulin derivative has been purified by column chromatography and appears to be homogeneous in HPLC and disc electrophoresis. The structure was proven to be B(1–29)–A(1–21) insulin by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degradation. The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form. SCI was devoid of effect in the blood sugar lowering assay in mice, the estimated potency being less than 0.1% of that of insulin.     

Preparation of technetium‐99m bifunctional chelate complexes using a microfluidic reactor: a comparative study with conventional and microwave labeling methods

A series of reactions between the technetium tricarbonyl core, [^99mTc(CO)₃(OH₂)₃]⁺ and both the bifunctional chelate dithiazole valeric acid (DTV) and insulin derivatized with DTV were performed using microfluidic, microwave, and conventional labeling methods. At low concentrations of ligand, the microfluidic reactor resulted in higher yields than both the microwave and conventional reactions. The labeling of DTV at a concentration of 0.01 mg/ml (32.2 µm) and 100 °C did not occur using conventional techniques, whereas the yield after 7.85 min was 61% in the microfluidic reactor and 18% in the microwave reactor. The labeling of a DTV–insulin conjugate (2.1 mg/ml, 330 µm) at 37 °C was conducted using conventional methods producing the desired product in 21% yield in 15.7 min compared with 40% of the desired product in the identically formulated microfluidic reactor. In addition to the higher radiochemical yield, the radiochemical purity was significantly improved in the microfluidic reactor. The microfluidic reactor offers a number of advantages over conventional and microwave methods and is worthy of further exploration as a method to prepare molecular imaging probes derived from Tc‐99m. Copyright © 2011 John Wiley & Sons, Ltd.     

Novel alginate gel microspheres produced by impinging aerosols for oral delivery of proteins

Lysozyme and insulin were encapsulated in alginate gel microspheres using impinging aerosols method. High loadings of around 50% weight/dry microspheres weight were obtained with encapsulation efficiencies of at least 48%. Environmental scanning electron microscopy revealed smooth spherical hydrated microspheres (30–60?µm) in diameter. No lysozyme or insulin release was measured in simulated gastric fluid (HCl, pH 1.2, 37°C). Total insulin release occurred in simulated intestinal fluid (SIF; phosphate buffer saline, pH 7.4, 37°C) in 8?h following 2?h incubation in SGF and was found to retain 75% activity using the ARCHITECT® assay. Lysozyme was released completely in SIF in 10?h following 2?h incubation in SGF and was found to exhibit at least 80% bioactivity using the Micrococcus lysodeikticus assay. The absence of protein release in HCl and the retention of high levels of biological activity demonstrate the potential of alginate gel microspheres, for improving oral delivery of biopharmaceuticals.     

The role of the C-terminus of the insulin B-chain in modulating structural and functional properties of the hormone

In memoriam Howard S. Tager Within the scope of structure-function studies on the proteohormone insulin, the role of the C-terminal segment B26-B30 for self-association and receptor interaction was analyzed. Insulin derivatives with modifications in the region B26-B30 were synthesized by trypsin-catalyzed coupling reactions of des-(B23-B30)-insulin with synthetic peptides. The peptides were obtained by Fmoc solid-phase peptide synthesis. Insulins with multiple amino acid → glycine substitutions were examined to distinguish between the influence of the side chains and the influence of the main chain in positions B27-B30 on the self-association of the hormone. The analogues [Gly^{B27,B28,B29,B30}]insulin and [Gly^B27,B28,B30]insulin exhibit relative receptor affinities of 80% and self-associate. The successive extension of [Ala^B26]des-(B27-B30)-insulin-B26-amide (relative receptor binding 273%) with amino xids corresponding to the native sequence B27-B30 showed the influence of the length of the B-chain on receptor affinity: the extension by B27-threonine amide reduces receptor binding to 71%, all further prolongations have only small effects on the binding. The effect of the B28-side chain on main-chain conformation, self-association and receptor binding was examined with [X^B28]des-(B29-B30)-insulin-B28-amides (X = Phe, Gly, D-Pro). While the glycine and D-proline analogues (relative binding 104 and 143%, respectively) retain the self-association properties typical of insulin, [Phe^B28]des-(B29-B30)-insulin-B28-amide (relative binding 50%) shows diminished self-association. The backbone-modified insulin derivative [Sar^B26]des-(B27-B30)-insulin-B26-amide (sarcosine =N-methylglycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26-amide hydrogen of the native hormone is not important for receptor binding. © Munksgaard 1995.     

NBl-C16-Insulin: Site-Specific Synthesis,Purification, and Biological Activity

Purpose. To develop a simple and efficient method for the synthesis and purification of NB1-lipid-modified-insulin without the use of protecting agents. Methods. Bovine insulin was allowed to react with cis-9-hexadecenal in an aqueous-organic medium in the presence of NaBH₃CN at room temperature overnight. HPLC and ESI LC/MS coupled with dithiothreitol and trypsin treatment were employed for product identification and optimization. The product was purified by a differential C18 solid-phase extraction. The biological effects of the modified insulin were evaluated by receptor binding assay and hypoglycemic effect measurement. Results. NB1-cis-9-hexadecenyl insulin was synthesized by a one-step reductive alkylation in sodium salicylate and isopropanol solution in high yield (80%). The site selectivity and yield of the reaction were found to be affected by pH, medium, and insulin-to-aldehyde ratio. After solid phase extraction, the purity was found to be approximately 98%. This derivative showed a Kd to the insulin receptor of 5.72 X 10 ^{– 9} M and a significantly slower glucose lowering rate than insulin. Conclusions. NB1-hexadecenyl insulin was synthesized by reductive alkylation without the use of protective agents in high yield. NB1-hexadecenyl insulin retained significant binding affinity to insulin receptor and showed a pronounced hypoglycemic effect.     

Enhancement of Insulin Transport Across Primary Rat Alveolar Epithelial Cell Monolayers by Endogenous Cellular Factor(s)

Purpose To characterize factor(s) contained in apical medium of primary cultured rat alveolar epithelial type II cell-like monolayers (RAECM-II) that enhance insulin absorption across alveolar epithelial cells. Materials and Methods Primary rat alveolar epithelial cell monolayers cultured on Transwells in the presence and absence of 10 ng/ml keratinocyte growth factor for 6 days were dosed from the apical compartment with radiolabeled insulin in: newborn bovine serum-containing medium (SM), conditioned medium from apical compartment of rat alveolar epithelial type I cell-like monolayers (RAECM-I) (CMI), or conditioned medium from apical compartment of RAECM-II (CMII). At the end of 2 h incubation, basolateral medium was collected and amounts of transported radiolabeled insulin were determined using a gamma counter. In order to determine the molecular size range of the enhancing factor(s), CMII was centrifuged in 50 kDa molecular weight cut-off Centricon tubes, and both retentate and filtrate were used as separate dosing solutions. Heat denaturation and ammonium sulphate precipitation were used to determine if the involved factor(s) represent proteins or other smaller soluble factors. Transalveolar transport rates of a paracellular marker, ¹⁴C-mannitol, and fluid-phase marker, horseradish peroxidase, were determined in the presence and absence of the factors. Effects of temperature (4, 16 and 37°C) on radiolabeled insulin fluxes were also measured. Results Conditioned medium obtained from the apical compartment of RAECM-II, CMII, increased transport of insulin across the monolayers when compared to SM or CMI. The enhancing effect of CMII was retained in the precipitate following ammonium sulfate treatment and in the retentate after Centricon filtration. The enhancing effect of CMII was significantly decreased when heated at 80°C for 15 min. CMII did not affect the transport of ¹⁴C-mannitol or HRP, while its effect on insulin transport was decreased by 87% when temperature was lowered to 4°C from 37°C. Conclusions Conditioned medium from type II cell-like monolayer cultures appears to contain protein factor(s) which seem to be involved in facilitating active transcellular transport of insulin across primary cultured RAECM-II.     

CHEMICAL SYNTHESIS OF [DES(TETRAPEPTIDE B27–30), TYR(NH2)26-B] AND [DES(PENTAPEPTIDE B26–30), PHE(NH2)25-B] BOVINE INSULINS*

Two analogs of bovine insulin, [des(tetrapeptide B^27–30), Tyr(NH₂)²⁶-B] and [des(pentapeptide B^26–30), Phe(NH₂)²⁵-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B^27–30), Tyr(NH₂)²⁶-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B^26–30), Phe(NH₂)²⁵-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.     

Comparative reduction/oxidation studies with single chain des-(B30) insulin and porcine proinsulin

The single chain des-(B30) insulin molecule (SCI) has been reduced and reoxidized together with porcine proinsulin (PPI). Yields of correctly folded and reoxidized SCI and PPI were analyzed by HPLC. The concentrations of both proteins were 10^-3M during reduction and 10^-5M during oxidation. The pH during reoxidation was varied from 8.6 to 9.2 and the temperature from 4 to 37°. Under all conditions tested, the recovery of SCI was substantially higher than that of PPI. The recoveries peaked after 24–72 h. It is suggested that the “miniproinsulin” SCI folds correctly up more efficiently than porcine pro-insulin, resulting in higher yields of reoxidized SCI.     

Chemical and Thermal Stability of Insulin: Effects of Zinc and Ligand Binding to the Insulin Zinc-Hexamer

Purpose To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands.Materials and Methods The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry.Results An excellent correlation between the thermal stabilization by ligand binding and the deamidation of Asn^B3 was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37°C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated.Conclusions Thermal stability can be used to predict the rate of Asn^B3 deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.     

Simple and high radiochemical yield synthesis of 2′‐Deoxy‐2′‐[18F]fluorouridine via a new nosylate precursor

We synthesized 2'‐deoxy‐2'‐[¹⁸F]fluorouridine ( 7 ) as a radiotracer for positron emission tomography from a new nosylate precursor ( 6 ). This new precursor was synthesized from uridine in four steps. The overall synthetic yield was 9.4% and we have high stability of >98% purity up to 6 months at 4°C. The optimal manual [¹⁸F]fluorination conditions were 30 mg of the precursor 6 in 500 µl of acetonitrile at 145°C for 15 min with 370 MBq of [¹⁸F]fluoride. The [¹⁸F]fluorination yield was 76.5±2.7% (n = 3). After hydrolysis of protecting groups with 1 N HCl and purification by HPLC, the overall radiochemical yield and purity were 26.5±1.4% and 98.2±2.5%, respectively. The preparation time was 70.0±10.5 min (n = 3 for each result). We also developed an automated method with a radiochemical yield and purity of 24.0±2.8 and 98.0±1.5% (n = 10) using a GE TracerLab MX chemistry module. This new nosylate precursor for 2'‐deoxy‐2'‐[¹⁸F]fluorouridine synthesis showed higher radiochemical yields and reproducibility than previous methods. Copyright © 2006 John Wiley & Sons, Ltd.     

Insulin aspart (AspB28 human insulin) derivatives formed in pharmaceutical solutions

Purpose. To isolate and identify the main insulin aspart (Asp^B28 human insulin) derivatives formed in pharmaceuticals (pH 7.4 at 5°C), to estimate rates of formation, and to determine their biologic potencies. Methods. Insulin aspart derivatives have been isolated by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by RP-HPLC, peptide mapping, amino acid analysis, mass spectrometry, and N-terminal amino acid sequence analysis. Results. The main derivatives formed were isoAsp^B28, isoAsp^B3, Asp^B3, and desPhe^B1-N-oxalyl-Val^B2 insulin aspart. At 5°C, the rate constants were 0.00028/month for isoAsp^B28 and isoAsp^B3, 0.00024/month for Asp^B3, and 0.00013/month for desPhe^B1-N-oxalyl-Val^B2 derivatives of insulin aspart. Unexpectedly, the rate of isomerization of B28 was high compared to the rate of B3 deamidation at both 5°C and 45°C. The N-terminal and especially the C-terminal of the B-chain are highly flexible, which may explain the high rate of isoAsp^B28 formation and that deamidation of Asn^B3 occurs. All the derivatives had full in vivo biologic potencies. Conclusion. Except for isoAsp^B28 insulin aspart, the main derivatives formed in pharmaceuticals of insulin aspart and human insulin at pH 7.4 are similar. They are all fully active in vivo. In proteins, flexibility of the polypeptide chain seems more important than sequence in the formation of succinimides.     

Radioiodination of balsalazide,bioevaluation, and characterization as a highly selective radiotracer for imaging of ulcerative colitis in mice

This work focuses on tracking ulcerative colitis in mice. High labeling yield and radiochemical purity were achieved for the formation of a [^125/131I]balsalazide radiotracer at optimum conditions of oxidizing agent content (chloramines-T [Ch-T], 75 μg), substrate amount (100 μg), pH of reaction mixture (6), reaction time (30 min), and temperature (37°C), using radioactive iodine-125 (200–450 MBq). The radiolabeled compound, [^125/131I]balsalazide, was stable in serum and saline solution during 24 h. Balsalazide is acting as a peroxisome proliferator-activated receptor (PPARγ). Biodistribution studies were carried in normal and ulcerated colon mice. High uptake of 75 ± 1.90% injected dose/g organ (ID/g) observed in ulcerated mice confirmed the suitability of [¹³¹I]balsalazide as a novel radiotracer for ulcerative colitis imaging in mice.     

Injectable nanospheres from a novel multiblock copolymer: Cytocompatibility,degradation and in vitro release studies

The aim was to develop and characterize nanospheres made from a newly synthesized poly (D, L-lactide-co-ethyleneglycol) (-PLA-PEG-PLA-)_n multi-block copolymer. Nanospheres were prepared under optimized conditions of modified emulsion-solvent evaporation technique in a continuous flow process using rhodamine B as a drug model. They were characterized for size distribution, zeta (ζ) potential, porosity and morphology. Drug loading and yield were also determined. In vitro degradation studies of the copolymer were conducted in phosphate buffer (pH 7.4) at 37°C. The cytotoxic properties of the polymer and vector were analysed by dimethylthiazoldiphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays on the B16 mouse cell line. Release of rhodamine B from the nanospheres was assayed in vitro using a dialysis bag in isotonic phosphate buffer (pH 7.4) at 37°C. Spherical and non-porous nanospheres with mean size less than 800nm could be prepared. The (ζ) potential was neutral. The average yield was approximately 70% with 7% rhodamine loading. A total of 50% of the multiblock underwent initial degradation after 4 weeks, while degradation was complete after 16 weeks. Cellular proliferation was not inhibited as no cytotoxicity was observed with the copolymers and nanospheres. Rhodamine B was released in a stepwise pattern. The initial burst was 20%, and release was prolonged thereafter for 29 days. Thus, injectable nanospheres with prolonged rhodamine B release have been designed and characterized as a potential drug-delivery system.     

Synthesis of [N‐methyl‐11C]‐3‐[(6‐dimethylamino)pyridin‐3‐yl]‐2,5‐dimethyl‐N,N‐dipropylpyrazolo[1,5‐a]pyrimidine‐7‐amine: A potential PET ligand for in vivo imaging of CRF1 receptors

A convenient synthesis of [N‐methyl‐¹¹C]‐3‐[(6‐dimethylamino)pyridin‐3‐yl]‐2,5‐dimethyl‐N,N‐dipropylpyrazolo[1,5‐a]pyrimidine‐7‐amine (R121920), a highly selective CRF₁ antagonist has been developed as a potential PET ligand. 3 ‐ [(6 ‐ methylamino)pyridin ‐ 3 ‐ yl]‐2,5‐dimethyl‐N,N‐dipropylpyrazolo [1,5‐a]pyrimidine‐7‐amine ( 7 ), the precursor for radiolabelling was synthesized through a novel palladium catalyzed Suzuki coupling of aryl bromide 5 with heteroaryl boronate ester 4 . The requisite boronate ester 4 was synthesized in four steps from 2‐amino‐4‐bromopyridine in 50% overall yield. Although the synthesis of cold R121920 proceeded in 93% yield by sodium hexamethyl‐disilazide (NaHMDS) mediated N‐methylation of the desmethylamine 7 at ?78°C, the attempted radiosynthesis under various conditions using conventional bases were not successful. However, the radiolabeling of [¹¹C]R121920 was successfully carried out with [¹¹C]MeOTf in acetone at ?20°C in the absence of added basic reagents. The radiotracer was purified by RP‐HPLC followed by RP‐solid phase extraction. The yield of the reaction was 5% (at EOB) and the specific activity was >1000 Ci/mmol (at EOB) with a radiochemical purity >99%. Copyright © 2003 John Wiley & Sons, Ltd.     

Radiosynthesis of [11C]SL25.1188 via [11C]CO2 fixation for imaging monoamine oxidase B

Carbon‐11‐labelled (S)‐5‐methoxymethyl‐3‐[6‐(4,4,4‐trifluorobutoxy)benzo[d]isoxazol‐3‐yl]oxazolidin‐2‐[¹¹C]‐one ([¹¹C]SL25.1188), a promising reversibly binding radiotracer for imaging central monoamine oxidase B, was rapidly prepared via an intramolecular cyclization reaction in an automated one‐pot procedure directly from [¹¹C]CO₂, thereby precluding the use of [¹¹C]COCl₂. Formulated [¹¹C]SL25.1188 was isolated in 12 ± 1% uncorrected radiochemical yield, based on starting [¹¹C]CO₂, with a specific activity of 37 ± 2 GBq/µmol at the end of synthesis (30 min; n = 3). Radiochemical and enantiomeric purities were both >99%. The methodology described herein offers an efficient production of [¹¹C]SL25.1188 at ambient temperature and is suitable for human imaging studies.     

Pharmacological evaluation and chemical stability of 2‐benzylthioether‐5′‐O‐(1‐thiotriphosphate)‐adenosine,a new insulin secretagogue acting through P2Y receptors

Activation of P2Y receptors on pancreatic β‐cells by extracellular ATP bring about amplification of glucose‐induced insulin secretion, which has been shown to reduce hyperglycemia in vivo. A new P2 receptor ligand, 2‐benzylthio‐ATP‐α‐S, was synthesized based on a combination of modifications of the ATP skeleton, as a potential insulin secretagogue. The two diastereoisomers of this ligand were separated and are designated A and B. The effects of these compounds on insulin secretion and vascular resistance in the rat isolated and perfused pancreas were evaluated in the presence of a slightly stimulating glucose concentration (8.3 mM) and were compared with ATP‐α‐S and ATP. Both isomers of 2‐benzylthio‐ATP‐α‐S (0.015–1.5 μM) induced a concentration‐dependent increase in glucose‐induced insulin release. The potency of isomer A was not significantly different from that of isomer B, and both were approximately 100‐fold more potent than ATP. ATP‐α‐S induced a similar pattern of insulin response; however, it was only approximately 10‐fold more potent than ATP. These compounds also induced vascular effects: ATP‐α‐S induced a vasodilatation and was transiently vasoconstrictor only at a high concentration, whereas the C2‐substituted derivative constantly induced a vasoconstriction. The chemical stability of these ligands was evaluated under physiological conditions and gastric juice pH. Hydrolysis of 2‐benzylthio‐ATP‐α‐S has been studied both in pH 7.4 and pH 1.4 at 37°C using ³¹P nuclear magnetic resonance (NMR) spectroscopy and high‐performance liquid chromatography. This compound exhibited high chemical stability with respect to hydrolysis of the glycosidic bond and desulfurization of the phosphorothioate moiety. Hydrolysis of the phospho ester bond, which was the only detectable degrading reaction under the investigation conditions (pH 7.4, 37°C), was slow, with a half‐life of 264 h. Moreover, even at gastric juice conditions (pH 1.4, 37°C), hydrolysis of the terminal phosphate was the only detectable reaction, with half‐life of 17.5 h. In conclusion, both isomers of 2‐benzylthio‐ATP‐α‐S are soluble in water and highly chemically stable. These compounds are highly potent and effective insulin secretagogues; however, they increase pancreatic vascular resistance. Drug Dev. Res. 53:33–43, 2001. © 2001 Wiley‐Liss, Inc.     

Pharmacokinetics and pharmacodynamics of a premixed formulation of soluble and protamine-retarded insulin aspart

Objective: With the aim to obtain a premixed rapid-acting insulin with a serum insulin profile more closely resembling the endogenous meal-stimulated serum insulin profiles, a 30/70 (rapid/intermediate-acting) premixed suspension of the rapid-acting insulin analogue insulin aspart (BIAsp30) was compared with a similar premixed suspension of biphasic human insulin 30/70 (BHI30) after a single subcutaneous injection. Methods: The study had a randomised, double-blind, two-period crossover design. Twenty-four healthy male subjects received a single subcutaneous dose of either 0.2 U · kg⁻¹ bodyweight of BIAsp30 or BHI30 on two study days. Results: BIAsp30 was absorbed faster than BHI30, as reflected in the area under the insulin concentration-time curve from 0 to 90 min after dosing [AUC_{(0–90 min)}]. This was significantly larger for BIAsp30 than for BHI30 (1403 ± 372 versus 752 ± 191 mU · l⁻¹ · min⁻¹ [mean ± SD]; P < 0.0001). Furthermore, the time to maximum serum insulin concentration (t_max) of BIAsp30 was approximately half the t_max of BHI30 (60 [45–70] versus 110 [90–180] min [median, interquartile range]; P=0.0001) and the maximum insulin concentration (C_max) was significantly higher for BIAsp30 than for BHI30 (23.4 ± 5.3 versus 15.5 ± 3.7 mU · l⁻¹ [mean ± SD]; P < 0.0001). The serum glucose profiles showed a significantly earlier onset of the glucose-lowering effect following BIAsp30 than following BHI30. Conclusions: The improved absorption properties of soluble insulin aspart in its premixed formulation provide a basis for a more efficient meal-related glucose control and immediate pre-meal delivery when compared with a similar human premixed insulin in the treatment of diabetes mellitus. Received: 22 November 1999 / Accepted in revised form: 7 April 2000     

Effects of flavoxate hydrochloride on voltage-dependent Ba<Superscript>2+</Superscript> currents in human detrusor myocytes at different experimental temperatures

The inhibitory effects of flavoxate hydrochloride (piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba²⁺ currents (I _Ba) in human detrusor myocytes were investigated at different temperatures using conventional whole-cell patch-clamp techniques. When the bath-solution temperature was increased from 22°C to 30°C, I _Ba peak amplitude was enhanced by approximately twice at several test potentials. Neither the I _Ba threshold nor the membrane potentials for the I _Ba maximum peak amplitude was affected by the temperature change. The concentration-response curves of flavoxate at both 30°C (K _i  = 5.1 μM) and 37°C (K _i  = 4.6 μM) were slightly shifted to the left in comparison with that at 22°C (K _i  = 10.3 μM). Similar results were also obtained in the presence of nifedipine (K _i  = 14 nM at 22°C vs. K _i  = 2.5 nM at 30°C and K _i  = 2.1 nM at 37°C). Altering the bath-solution temperature from 22°C to 30°C shifted the steady-state inactivation curve of I _Ba at −90 mV to the left. At 30°C, the steady-state inactivation curve of I _Ba in the presence of flavoxate was also shifted to the left in comparison with that in the absence of flavoxate. Either 3-isobutyl-1-methylxanthine (IBMX) or theophylline, a phosphodiesterase inhibitor, caused little effects on I _Ba, although cyclic nucleotides (dibutyryl cAMP and 8-Br-cGMP) inhibited I _Ba. These results suggest that the inhibitory actions of flavoxate on I _Ba in human detrusor myocytes were slightly changed at different experimental temperatures and that flavoxate directly blocked voltage-dependent L-type Ca²⁺ channels, not through the inhibition of phosphodiesterase activity pathway.     

Didemnin B Conformation and dynamics of an antitumour and antiviral depsipeptide studied in solution by 1H and 13C. n.m.r. spectroscopy

The solution conformation of didemnin B, the most potent member of a family of depsipeptides that shows antitumour, antiviral, and immunosuppressive activity, has been studied in chloroform solution using n.m.r. spectroscopy. ¹H and ¹³C spectra have been assigned from analysis of a number of two-dimensional homonuclear and heteronuclear chemical shift correlation experiments which confirm the recently corrected primary structure of the molecule. The conformation of the peptide has been deduced from measurements of the temperature dependence of the NH chemical shifts, analysis of coupling constant data and primarily through NOE effects observed in the rotating frame. Interproton distance bounds determined from a quantitative analysis of the ROE data provide 41 constraints from which a family of closely related structures were calculated using distance geometry algorithms. A type II β-turn involving residues Thr⁶, Leu⁷, and Pro⁸ is well represented in the computed conformers as is a hydrogen bonding interaction between the NH of Leu³ and the carbonyl oxygen of Thr⁶. This latter interaction causes the linear portion of the structure to fold back over the depsipeptide ring, imparting to it a degree of structural stability as well as giving the molecule a somewhat globular character. Only one transannular hydrogen bond, between 1st¹ NH and Leu³ carbonyl, stabilizes the conformation of the depsipeptide, which has an irregular non-planar configuration. The small temperature coefficients (< 2.0 × 10^?3ppm/°C) for the NHs of 1st¹ and Leu³ are consistent with their involvement in these hydrogen bonding interactions. We find that many of the structural features observed in the crystalline form of didemnin B are conserved in solution. Analysis of the ¹³C spin-lattice relaxation rates of the protonated carbons reveals small variations in effective correlation times at specific sites in the molecule. The data suggests that the peptide segment encompassing residues Leu³through to Thr⁶ is in a more motionally restricted part of the structure.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues

The incubation of a solution of the human growth hormone releasing factor analog, [Leu²⁷] hGRF(1-32)NH₂ at pH 7.4 and 37°, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ and [α-Asp⁸, Leu²⁷] hGRF(1-32)NH₂, produced by deamidation of the Asn⁸ residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was estimated to be 400-500 times less potent than the parent Asn⁸ peptide, while [2-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was calculated to be 25 times less potent than the parent Asn⁸ peptide. Three additional analogs of [Leu²⁷] hGRF(1-32)NH₂ containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37°). Based on disappearance kinetics, [Leu²⁷] hGRF(1-32)NH₂ had a half-life of 202 h while the other analogs had the following half-lives: [Leu²⁷, Asn²⁸] hGRF(1-32)NH_: (150h); [Ser⁸, Leu²⁷, Asn²⁸] hGRF(l-32)NH₂ (746h); and [Ser⁸, Leu²⁷] hGRF(1-32)NH₂ (1550 h). After 14 days, incubated samples of the Asn⁸ analogs lost GH releasing potency, while the Ser⁸ analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.