Gastrointestinal safety across the albiglutide development programme.

Gastrointestinal (GI) adverse events (AEs) are the most frequently reported treatment-related AEs associated with glucagon-like peptide-1 receptor agonists (GLP-1RAs) in the treatment of type 2 diabetes mellitus. The GI safety of albiglutide, a once-weekly GLP-1RA, was assessed using data from five phase III studies. In a pooled analysis of four placebo-controlled trials, the most common GI AEs were diarrhoea (albiglutide, 14.5% vs. placebo, 11.5%) and nausea (albiglutide, 11.9% vs. placebo, 10.3%), with most patients experiencing 1-2 events. The majority were mild or moderate in intensity and their median duration was 3-4 days. Vomiting occurred in 4.9% of patients in the albiglutide vs. 2.6% in the placebo group. For both albiglutide and placebo, serious GI AEs (2.0% vs. 1.5%) and withdrawals attributable to GI AEs (1.7% vs. 1.5%) were low. In a 32-week trial of albiglutide 50 mg weekly versus liraglutide 1.8 mg daily, nausea occurred in 9.9% of patients in the albiglutide group vs. 29.2% in the liraglutide group. Vomiting occurred in 5.0% in the albiglutide vs. 9.3% in the liraglutide group. In conclusion, albiglutide has an acceptable GI tolerability profile, with nausea and vomiting rates slightly higher than those for placebo but lower than those for liraglutide.

The effects of dutasteride, tamsulosin, and the combination on storage and voiding in men with benign prostatic hyperplasia and prostatic enlargement: 2-year results from the Combination of Avodart and Tamsulosin study.

This article reports the results of a post hoc analysis of the multicenter, randomized, double-blind Combination of Avodart and Tamsulosin (CombAT) study, which aimed to investigate the effects of dutasteride (0.5 mg), tamsulosin (0.4 mg), and their combination on storage and voiding symptoms in 4844 men aged > or =50 years with moderate-to-severe lower urinary tract symptoms (International Prostate Symptom Score > or =12), prostate volume (PV) > or =30 cm(3) and PSA 1.5-10 ng ml(-1). After 24 months, combination treatment achieved significantly greater mean reductions in both voiding and storage symptoms than either monotherapy, in each of the three baseline PV tertiles (30 to <42, 42 to <58, > or =58 cm(3)). Dutasteride was as effective as tamsulosin for control of storage symptoms, but provided significantly greater relief of voiding symptoms than tamsulosin.

Efficacy and safety of dutasteride, tamsulosin and their combination in a subpopulation of the CombAT study: 2-year results in Asian men with moderate-to-severe BPH.

Although ethnicity-based differences in prostate size and physiology have been reported, results of benign prostatic hyperplasia (BPH) treatment trials in predominantly Caucasian patients are assumed to be applicable to non-Caucasian populations. This post hoc analysis investigated whether an Asian subpopulation of men with moderate-to-severe BPH in the CombAT study achieves treatment responses in line with those of the overall study population. In this double-blind, randomized, parallel-group trial, 325 Asian men were assigned to treatment with 0.5 mg dutasteride once daily, 0.4 mg tamsulosin once daily or the combination. Decrease in international prostate symptom score (IPSS) at month 24 from baseline (the primary endpoint) was significantly greater with combination treatment compared with tamsulosin (P<0.05), and numerically, but not statistically significantly, greater compared with dutasteride. Mean IPSS was reduced from baseline by 7.5 (+/-0.84) in the combination group, by 6.3 (+/-0.86) in the dutasteride group and by 4.5 (+/-0.78) in the tamsulosin group, resulting in respective mean IPSS at months 24 of 11.4 (+/-0.60), 12.7 (+/-0.70) and 14.3 (+/-0.74). The adverse event profile was similar to that observed in the overall CombAT population, and drug-related adverse events were more common with combination therapy (26%) than with tamsulosin (15%) or dutasteride (9%). No unexpected adverse events emerged. In conclusion, in Asian men with moderate-to-severe lower urinary tract symptoms and an enlarged prostate, combination therapy achieved significantly greater improvements from baseline BPH symptoms, flow rate, quality of life, reduced prostate volume and improved treatment satisfaction compared with tamsulosin monotherapy.

The proximity between helix I and helix XI in the melibiose carrier of Escherichia coli as determined by cross-linking.

The melibiose carrier of Escherichia coli is a transmembrane protein that comprises 12 transmembrane helices connected by periplasmic and cytoplasmic loops, with both the N- and C-termini located on the cytoplasmic side. Our previous studies of second-site revertants suggested proximity between several helices, including helices XI and I. In this study, we constructed six double cysteine mutants, each having one cysteine in helix I and the other in helix XI: three mutants, K18C/S380C, D19C/S380C, and F20C/S380C, have their cysteine pairs near the cytoplasmic side of the carrier, and the other three, T34C/G395C, D35C/G395C, and V36C/G395C, have their cysteine pairs near the periplasmic side. In the absence of substrate, disulfide formations catalyzed by iodine and copper-(1,10-phenanthroline)(3) indicate that helix I and helix XI are in immediate proximity to each other on the periplasmic side but not on the cytoplasmic side, as shown by protease cleavage analyses. We infer that the two helices are tilted with respect to each other, with the periplasmic sides in close proximity.

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli.

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H(+), Na(+), or Li(+). In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22-100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes.

The effect of modifications of the charged residues in the transmembrane helices on the transport activity of the melibiose carrier of Escherichia coli.

The melibiose transport carrier of Escherichia coli (coded by melB gene) is a cotransport system which couples the transport of a-galactosides to protons, sodium, or lithium ions. The charged amino acid residues in membrane-spanning helices are of considerable interest because many of them have important function in substrate recognition. In most cases changing these charged residue to an uncharged residue (cysteine) results in total loss of activity. In this communication we describe experiments in which the cysteine substitution for a charged residue was chemically changed by sulfhydryl reagents (MTSEA and MTSET to restore a positive charge and MTSES a negative charge) or by iodoacetic acid or through oxidation by hydrogen peroxide so as to regain the original negative charge. In two cases (D55C and D124C) the reconstructed negative charges via the oxidation of the thiol to the sulfinic and/or sulfonic acid resulted in partial recovery of transport: D55C up to 27% of the normal and D124C up to 4% of the normal in melibiose accumulation; D55C up to 36% of the normal and D124 up to 4.5% of the normal in downhill transport. Sulfhydryl reagents and iodoacetic acid failed to recover transport in all cases. We infer that the configurations of the charges as well as the structure of the side chains that carry them are critical in the maintenance of the transport.

Physiological evidence for an interaction between helices II and XI in the melibiose carrier of Escherichia coli.

The melibiose carrier from Escherichia coli is a cation-substrate cotransporter that catalyzes the accumulation of galactosides at the expense of H(+), Na(+), or Li(+) electrochemical gradients. Charged residues on transmembrane domains in the amino-terminal portion of this carrier play an important role in the recognition of cations, while the carboxyl portion of the protein seems to be important for sugar recognition. In the present study, we substituted Lys-377 on helix XI with Val. This mutant carrier, K377V, had reduced melibiose transport activity. We subsequently used this mutant for the isolation of functional second-site revertants. Revertant strains showed the additional substitutions of Val or Asn for Asp-59 (helix II), or Leu for Phe-20 (helix I). Isolation of revertant strains where both Lys-377 and Asp-59 are substituted with neutral residues suggested the possibility that a salt bridge exists between helix II and helix XI. To further test this idea, we constructed three additional site-directed mutants: Asp-59-->Lys (D59K), Lys-377-->Asp (K377D), and a double mutant, Asp-59-->Lys/Lys-377-->Asp (D59K/K377D), in which the position of these charges was exchanged. K377D accumulated melibiose only marginally while D59K could not accumulate. However, the D59K/K377D double mutant accumulated melibiose to a modest level although this activity was no longer stimulated by Na(+). We suggest that Asp-59 and Lys-377 interact via a salt bridge that brings helix II and helix XI close to one another in the three-dimensional structure of the carrier.

Regulation of galactoside transport by the PTS.

Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed. It is now well known that non-phosphorylated IIA(Glc) inhibits the transport via direct binding to the transporter. Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIA(Glc). It is concluded that there are multiple interaction sites for IIA(Glc) in these transporters.

Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli.

The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+). This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS). Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site revertants were isolated from K18C and Y31C. The revertants were found to have mutations in helices I, IV, and VII.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot).

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Sodium-substrate cotransport in bacteria.

A variety of sodium-substrate cotransport systems are known in bacteria. Sodium enters the cell down an electrochemical concentration gradient. There is obligatory coupling between the entry of the ion and the entry of substrate with a stoichiometry (in the cases studied) of 1:1. Thus, the downhill movement of sodium ion into the cell leads to the accumulation of substrate within the cell. The melibiose carrier of Escherichia coli is perhaps the most carefully studied of the sodium cotransport systems in bacteria. This carrier is of special interest because it can also use protons or lithium ions for cotransport. Other sodium cotransport carriers that have been studied recently are for proline, glutamate, serine-threonine, citrate and branched chain amino acids.

Sugar recognition mutants of the melibiose carrier of Escherichia coli: possible structural information concerning the arrangement of membrane-bound helices and sugar/cation recognition site.

Melibiose carrier mutants, isolated by growing cells on melibiose plus the non-metabolizable competitive inhibitor thiomethyl-beta-galactoside (TMG), were studied to determine sugar and cation recognition abnormalities. Most of the mutants show good transport of melibiose but have lost the recognition of TMG. In addition, most mutants show little or no transport of lactose. Cation recognition is also affected as all of these mutants have lost the ability to transport protons with melibiose. The amino acids causing these mutations were determined by sequencing the melB gene on the plasmid. The mutations were located on helices I, IV, VII, X and XI. We propose that these five helices are in proximity with each other and that they line the sugar/cation transport channel.

The melibiose carrier of Escherichia coli: cysteine substitutions for individual residues in helix XI.

The melibiose carrier from Escherichia coli is a sugar-cation cotransport system. Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374-396, which includes all of the residues in the proposed helix XI. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS). Studies were carried out on both intact cells and inside out vesicles. Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C). PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C). Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS. It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel.

Mutants of the lactose carrier of Escherichia coli which show altered sugar recognition plus a severe defect in sugar accumulation.

Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog beta-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K(m) for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V(max)) of normal. All of the mutants accumulated methyl-alpha-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli.

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.

Arg-52 in the melibiose carrier of Escherichia coli is important for cation-coupled sugar transport and participates in an intrahelical salt bridge.

Arg-52 of the Escherichia coli melibiose carrier was replaced by Ser (R52S), Gln (R52Q), or Val (R52V). While the level of carrier in the membrane for each mutant remained similar to that for the wild type, analysis of melibiose transport showed an uncoupling of proton cotransport and a drastic reduction in Na(+)-coupled transport. Second-site revertants were selected on MacConkey plates containing melibiose, and substitutions were found at nine distinct locations in the carrier. Eight revertant substitutions were isolated from the R52S strain: Asp-19-->Gly, Asp-55-->Asn, Pro-60-->Gln, Trp-116-->Arg, Asn-244-->Ser, Ser-247-->Arg, Asn-248-->Lys, and Ile-352-->Val. Two revertants were also isolated from the R52V strain: Trp-116-->Arg and Thr-338-->Arg revertants. The R52Q strain yielded an Asp-55-->Asn substitution and a first-site revertant, Lys-52 (R52K). The R52K strain had transport properties similar to those of the wild type. Analysis of melibiose accumulation showed that proton-driven accumulation was still defective in the second-site revertant strains, and only the Trp-116-->Arg, Ser-247-->Arg, and Asn-248-->Lys revertants regained significant Na(+)-coupled accumulation. In general, downhill melibiose transport in the presence of Na(+) was better in the revertant strains than in the parental mutants. Three revertant strains, Asp-19-->Gly, Asp-55-->Asn, and Thr-338-->Arg strains, required a high Na(+) concentration (100 mM) for maximal activity. Kinetic measurements showed that the N248K and W116R revertants lowered the K(m) for melibiose, while other revertants restored transport velocity. We suggest that the insertion of positive charges on membrane helices is compensating for the loss of Arg-52 and that helix II is close to helix IV and VII. We also suggest that Arg-52 is salt bridged to Asp-55 (helix II) and Asp-19 (helix I).

Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2.

The melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose. Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of melibiose and its associated cation through the carrier.

Mutants of Citrobacter freundii that transport and utilize melibiose.

We have isolated mutants of Citrobacter freundii that can grow on melibiose. Inducible alpha-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized with melB (the gene for the melibiose transporter) DNA of Escherichia coli in the chromosomal DNA of wild-type C. freundii. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-beta-D-thiogalactoside) transport in the mutant cells.

The construction of a cysteine-less melibiose carrier from E. coli.

The melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less melibiose carrier had no significant decrease in ability to accumulate melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the melibiose carrier.