A Proton Pump Inhibitor in the Reformulation Setting: Bioequivalence and Potential Implications for Long-Term Safety.

Proton pump inhibitors (PPIs) have become known for both their therapeutic effect and good safety profile. An application was submitted to the US Food and Drug Administration for approval of a reformulated PPI product that failed bioequivalence testing, but was submitted on the basis of the long history of PPI use as a "surrogate" for equivalence. This review evaluates the safety data for PPIs, discuss variability of pharmacokinetic parameters of PPIs in the reformulation setting, and potential implications of those changes for long-term safety.

Complex frameshift mutations mediated by plasmid pKM101: mutational mechanisms deduced from 4-aminobiphenyl-induced mutation spectra in Salmonella.

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in approximately 2,400 4-aminobiphenyl (4-AB) +S9-induced revertants of the -1 frameshift allele hisD3052 and of the base-substitution allele hisG46 of Salmonella typhimurium. Most of the mutations occurred at sites containing guanine, which is the primary base at which 4-AB forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100 and 99.9%, respectively, of the reversion events at the hisD3052 allele in the pKM101 plasmid-minus strains TA1978 (uvr+) and TA1538 (delta uvrB). In strain TA98 (delta uvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, approximately 85% of the revertants also contained the hotspot deletion; the remaining approximately 15% contained one of two types of mutations: (1) complex frameshifts that can be described as a -2 or +1 frameshift and an associated base substitution and (2) deletions of the CC or GG sequences that flank the hotspot site (CCGCGCGCGG). We propose a misincorporation/slippage model to account for these mutations in which (1) pKM101-mediated misincorporation and translesion synthesis occurs across a 4-AB-adducted guanine; (2) the instability of such a mispairing and/or the presence of the adduct leads to strand slippage in a run of repeated bases adjacent to the adducted guanine; and (3) continued DNA synthesis from the slipped intermediate produces a frameshift associated with a base substitution. This model readily accounts for the deletion of the CC or GG sequences flanking the hotspot site, indicating that these mutations are, in fact, complex mutations in disguise (i.e., cryptic complex frameshifts). The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G.C-->T.A transversions) is consistent with the finding that 4-AB induced primarily G.C-->T.A transversions at the hisG46 base-substitution allele. The model also provides a framework for understanding the different relative mutagenic potencies of 4-AB at the two alleles in the various DNA repair backgrounds of Salmonella.

Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds.

To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev. The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein. Only 0.4-3.9% were true rev. Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions. The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101). The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions. Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times. The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101. Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions. Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated. The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins.

Molecular analysis of mutations induced at the hisD3052 allele of Salmonella by single chemicals and complex mixtures.

More single chemicals and complex environmental mixtures have been evaluated for mutagenicity at the hisD3052 allele of Salmonella, primarily in strain TA98, than in any other mutation assay. The development of colony probe hybridization procedures and the application of the polymerase chain reaction and direct DNA sequencing has permitted rapid molecular access to this allele. We discuss these techniques and the resulting mutation spectra that have been induced by a variety of environmental mutagens and complex mixtures. A common GC or CG deletion within a hot-spot region of the sequence dominates most of the spectra. In addition to this two-base deletion, we have recovered about 200 other types of mutations within the 72-base target for reversion of the hisD3052 allele. These include a variety of deletions (as large as 35 bases), duplications (as large as 46 bases), and complex mutations involving base substitutions. The quasipalindromic nature of the target sequence and its potential to form DNA secondary structures and slippage mismatches appear to be an important basis for the mutability of this allele.

Safety and efficacy of long term esomeprazole therapy in patients with healed erosive oesophagitis.

OBJECTIVE: To evaluate the safety and tolerability of long term treatment with esomeprazole in patients with healed erosive oesophagitis, and to describe its efficacy in the maintenance of healing. DESIGN AND SETTING: US multicentre, noncomparative, nonblind study. PATIENTS AND PARTICIPANTS: 807 patients with endoscopically confirmed healed erosive oesophagitis. METHODS: Patients received esomeprazole 40 mg once daily for up to 12 months. Adverse events and clinical laboratory tests were assessed over the study period. Endoscopy was performed at the final visit of the antecedent healing trials and at months 6 and 12 of the current safety trial; gastric biopsies were obtained at the initial visit of the healing trials and at the end of the safety trial. RESULTS: 80.9% of patients completed 6 months of treatment; 76.6% completed 12 months of treatment. There were no serious drug-related adverse events. Diarrhoea, abdominal pain, flatulence, and headache were the only treatment-related adverse events reported by >3% of patients. Mean changes in laboratory measures were generally small and not clinically meaningful. Plasma gastrin levels increased, as expected, and reached a plateau after 3 months. No changes in gastric histological scores were noted in the majority of patients. Evaluation of gastric biopsies revealed an overall decline in chronic inflammation and atrophy. Intestinal metaplasia findings remained essentially unchanged. Life table estimates of maintenance of healing were 93.7% [95% confidence interval (CI) 92.0 to 95.5%] at 6 months and 89.4% (95% CI 87.0 to 91.7%) at 12 months. CONCLUSIONS: Daily treatment with esomeprazole 40 mg for up to 1 year in patients with healed erosive oesophagitis was generally well tolerated and effective. No safety concerns arose.

Mutation spectra of Glu-P-1 in Salmonella: induction of hotspot frameshifts and site-specific base substitutions.

We used colony probe hybridization and PCR/DNA sequence analysis to determine the mutations in approximately 1,640 revertants of the -1 frameshift allele hisD3052 and approximately 260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). All of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu-P-1-induced mutations at the frameshift allele in strains TA1978 (uvr+) and TA1538 (delta uvrB) and 99% in TA98 (delta uvrB, pKM101). To explain the induction of these hotspot mutations by Glu-P-1, we describe here a more detailed version of our recently proposed correct incorporation/slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu-P-1-adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu-P-1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu-P-1 at the frameshift allele, it is required by Glu-P-1 to revert the base substitution allele, where Glu-P-1 induces G-C --> T-A transversions (75%) and G-C --> tA-T transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20-30 times less) base substitution mutagenic potency of Glu-P-1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu-P-1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu-P-1-induced rat colon tumors.

Molecular analysis of mutations induced by the intercalating agent ellipticine at the hisD3052 allele of Salmonella typhimurium TA98.

We have used DNA colony hybridization, the polymerase chain reaction (PCR), and direct DNA sequencing to determine the mutations induced by the intercalating agent ellipticine in Salmonella typhimurium TA98 in the presence of S9. Of 400 ellipticine-induced revertants that were selected at a mutant yield that was ninefold over the background, 85.5% contained a GC or CG deletion within a common CGCGCGCG hotspot; this deletion occurred among 47% of the spontaneous revertants. In addition to this hotspot, the ellipticine spectrum contained two deletion warmspots that reside opposite each other in looped-out regions of a possible DNA secondary structure. Ellipticine and its metabolites likely revert Salmonella strain TA98 by forming DNA adducts that promote slippage-mismatches and by stabilizing these slipped mismatched sequences via intercalation. The involvement of these mechanisms, along with a likely role for DNA secondary structures and a possible role for DNA gyrase, may account for the site specificity exhibited by ellipticine in strain TA98.

DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing: application to 1-nitropyrene-induced revertants.

We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.

Studies of temperature-sensitive transfer and maintenance of H incompatibility group plasmids.

The mechanism of temperature-sensitive transfer was studied for plasmids of the H incompatibility group. Transfer depended on the temperature of the mating mixture but the growth temperature of the donor was also important, and donor cells previously grown at 26 degrees C could not facilitate transfer at 37 degrees C. Comparison of transfer characteristics of a non-thermosensitive H plasmid R831b and thermosensitive H plasmids from Salmonella from Ontario during a 2 h mating period showed that the thermosensitive phenotype inhibited transfer by about 200-fold at 37 degrees C and by 10-fold at 42 degrees C. At temperatures between 15 and 30 degrees C, the thermotolerant H plasmid transferred at about the same frequency as the temperature-sensitive plasmid. Elimination of some H plasmids after growth of host cells was also observed and physical evidence of this was obtained. The characteristic of high-temperature elimination (Hte) was limited to plasmids from similar bacterial and geographphical sources. Plasmids from Salmonella spp. isolated in Ontario did not possess this phenotype, whereas plasmids from Serratia marcescens isolated in the United States did. Although the Tra(ts) and Hte phenotypes may both be characteristic of H plasmids, they were shown to be separate and distinct properties. The H plasmids used in this study were isolated and their molecular weights determined by agarose gel electrophoresis. All were large, with molecular weights often exceeding 140 X 10(6). In contrast, the thermostable H plasmid R831b had a molecular weight on only 49 X 10(6).

Characterization of a plasmid mutation affecting maintenance, transfer and elimination by novobiocin.

We have isolated a mutant H group plasmid temperature-sensitive for plasmid maintenance. Unlike the wild type plasmid (pSD114), the mutant (pDT4) was eliminated at 37 degrees C and also at 30 degrees C after novobiocin treatment. The mutant plasmid interfered with host cell growth at the non-permissive temperature. Conjugative transfer of the mutant was reduced at 30 degrees C compared to the wild-type plasmid. Introduction of a coumermycin-novobiocin resistance DNA gyrase (cou) mutation into Escherichia coli prevented pDT4 elimination by novobiocin but did not affect the temperature-sensitive phenotype. The evidence indicates that the mutant plasmid used bacterial DNA gyrase for replication. Models to account for the behaviour of this unusual mutant are discussed.

Microbiological analysis of sepsis complicating non-surgical biliary drainage in malignant obstruction.

Cholangitis with sepsis is a common complication of non-surgical biliary drainage and represents a difficult management problem. Microbiological data for 18 septic episodes in 15 patients following endoscopic biliary drainage (EBD) and 14 septic episodes in 14 patients following percutaneous transhepatic drainage (PTD) were reviewed. In the EBD group, all 18 patient episodes of sepsis had blood cultures and 10 had bile cultures obtained. In total, 17 gram-negative isolates and 5 gram-positive isolates were grown in the EBD sepsis group. All of the 14 PTD patients had bile cultures and 13 had blood cultures. In total, 23 gram-negative isolates and 26 gram-positive isolates were grown in the PTD sepsis group. When PTD cultures were compared with EBD cultures, there were significantly more gram-positive cultures (26 vs. 5) associated with PTD sepsis than EBD sepsis (p less than 0.0005). Analysis of the antibiotic sensitivities revealed that a combination of ticarcillin clavulanate with gentamicin covered 20 of the 22 (91%) EBD organisms and 38 of the 49 (78%) PTD organisms. Based on this data, ticarcillin clavulanate with gentamicin is the therapy of choice for EBD and PTD sepsis at our institution.

Mutation spectrum of cigarette smoke condensate in Salmonella: comparison to mutations in smoking-associated tumors.

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in approximately 1600 revertants of Salmonella induced by cigarette smoke condensate (CSC) in the presence of S9. CSC induced approximately 80% GC-->TA transversions and approximately 20% GC-->AT transitions at the base-substitution allele (hisG46) in strain TA100. This spectrum was similar to those of the polycyclic aromatic hydrocarbon (PAH) benzo[alpha]pyrene and various aromatic amines such as 4-aminobiphenyl and Glu-P-1, all of which are present in CSC. This spectrum was also similar to that produced by PAHs in other bacteria, mammalian cells, and rodents as well as to that of the p53 gene in lung tumors from smokers. The results in Salmonella are consistent with a role for the PAH component of cigarette smoke in the base-substitution specificity found in the p53 gene of smoking-associated lung tumors. At the frameshift allele in strains TA1538 and TA98, CSC induced only a hotspot 2-base deletion, which is a mutation spectrum that is identical to that induced by the heterocyclic amine pyrolysate products of amino acids, such as Glu-P-1. This is consistent with bioassay-directed fractionation studies showing that aromatic amines account for most of the frameshift specificity of CSC in Salmonella. Rodent and human studies indicate that aromatic amines are responsible for smoking-associated bladder cancer. Repeated freezing and thawing of the CSC samples changed the chemical composition of the mixtures as evidenced by the production of an altered mutation spectrum. This emphasizes the necessity of proper storage and handling of labile complex mixtures. This study (i) confirms our previous studies showing that the mutation spectrum of a complex mixture reflects the dominance of one or a few classes of chemical mutagens within the mixture, and (ii) illustrates the potential of bioassay-directed molecular analysis for identifying the chemical classes in a complex mixture that are responsible for specific classes of mutation and tumor types produced by the mixture.

Incidence of plasmid DNA in Salmonella strains isolated from clinical sources in Ontario, Canada, during 1979 and 1980.

Gel electrophoresis of DNA from 70 clinical strains of Salmonella revealed a heterogenous plasmid population. Plasmid DNA, ranging in molecular weight from 1.4 X 10(6) to 145 X 10(6), was demonstrated in 26 of 32 antibiotic-resistant strains. Several resistant strains carried up to six plasmids; however, of these, five strains which were multiply resistant contained a single plasmid of molecular weight 54 X 10(6) to 145 X 10(6). Only one incompatibility group H2 (IncH2) plasmid (pDT28) was detected in a strain of S. heidelberg; thus, this represents a reduction in the prevalence of these plasmids in Ontario Salmonella strains since 1974. The pDT28 plasmid resembled other IncH2 plasmids by its high molecular weight (145 X 10(6) ) and by virtue of its temperature-sensitive mode of transfer, resistance to tellurium, and inhibition of coliphage development. Of the 38 antibiotic-susceptible Salmonella strains, approximately half contained plasmids, ranging in molecular weight from 1.4 X 10(6) to 60 X 10(6). The plasmid-containing antibiotic-susceptible strains carried either a group of two to four small plasmids, with molecular weights less than 4.5 X 10(6), or a single large plasmid of molecular weight 23 X 10(6) or 60 X 10(6).

Genetic studies of H group plasmids by bacteriophage P1 transduction.

Bacteriophage P1 transduction was used to study the incompatibility group H1 plasmid pRG1251, molecular weight 120 x 10(6), and the incompatibility group H2 plasmid pSD114, molecular weight 166 x 10(6). The order of resistance (R) determinants on pSD114 was deduced from transduction and segregation experiments to be chloramphenicol-tetracycline-kanamycin-streptomycin. Resistance to tellurium and to coliphages, which are properties also encoded by many H2 plasmids, were not transduced with the other markers. On pRG1251, the ampicillin and tetracycline resistance markers appear to be located together, as do the chloramphenicol, streptomycin, and sulfamethoxazole resistance markers. Frequently, blocks of R determinants were transposed to the P1 genome or to the Escherichia coli chromosome. P1 DNA was isolated which carried the chloramphenicol, streptomycin, and sulfamethoxazole markers from pRG1251 and had a molecular weight of 64 x 10(6). Other P1 prophages carried R determinants from pSD114 and had molecular weights of 86 x 10(6). A plasmid of molecular weight 124 x 10(6) was also isolated which contained incompatibility determinants from P1 (incompatibility group Y) and from the H2 group plasmid. The mechanism of formation of these unusual plasmid species is discussed.

Esomeprazole-based Helicobacter pylori eradication therapy and the effect of antibiotic resistance: results of three US multicenter, double-blind trials.

OBJECTIVES: To determine the efficacy of once-daily esomeprazole plus antibiotics for eradication of Helicobacter pylori, to assess the effect of antibiotic resistance on eradication rate, and to define the rate of emergent resistance. METHODS: Three separate randomized trials were performed in H. pylori-positive patients with a duodenal ulcer or history of documented duodenal ulcer within 5 yrs: 1) esomeprazole (40 mg once daily), amoxicillin (1 g b.i.d.), and clarithromycin (500 mg b.i.d.; this combination will be referred to as EAC) versus esomeprazole (40 mg once daily) plus clarithromycin (500 mg twice daily; this combination will be referred to as EC); 2) EAC versus esomeprazole (40 mg once daily; E); and 3) EC versus E. Therapy was given for 10 days. Endoscopy and biopsies for CLOtest, histology, and culture with susceptibility testing were done at baseline and 4 wk after completion of therapy. RESULTS: Per-protocol and intent-to-treat eradication rates, respectively, were as follows. For EAC versus EC in study 1 (N = 448), 84 versus 55% and 77 versus 52% (p < 0.001); for EAC versus E in study 2 (N = 98), 85 versus 5% and 78 versus 4% (p < 0.001); for EC versus E in study 3 (N = 66), 50% versus 0 and 46% versus 0 (p < 0.05). The 15% of patients in the combined studies with baseline clarithromycin resistance had significantly lower rates of eradication than those with susceptible strains (EAC: 45 vs. 89%; EC: 13 vs. 61%). Emergent resistance was less common after treatment with EAC [2/6 (33%)] than with EC (23/27 [85%]). CONCLUSIONS: Ten-day triple therapy with once-daily esomeprazole plus twice-daily amoxicillin and clarithromycin achieves an eradication rate virtually identical to that of the twice-daily proton pump inhibitor-based triple therapies. Baseline clarithromycin resistance, present in 15% of patients, predicts a markedly decreased rate. Use of an amoxicillin-containing regimen may decrease emergence of clarithromycin resistance.

Efficacy and safety of esomeprazole compared with omeprazole in GERD patients with erosive esophagitis: a randomized controlled trial.

OBJECTIVE: In patients with gastroesophageal reflux disease (GERD), esomeprazole, the S-isomer of omeprazole, has demonstrated pharmacological and clinical benefits beyond those seen with the racemic parent compound. This study was designed to further evaluate the efficacy and tolerability of esomeprazole relative to that of omeprazole in healing erosive esophagitis and resolving accompanying symptoms of GERD. METHODS: Esomeprazole 40 mg was compared with omeprazole 20 mg once daily in 2425 patients with erosive esophagitis (Helicobacter pylori negative by serology) in an 8-wk, multicenter, randomized, double-blind, parallel-group study conducted in 163 centers throughout the US. The primary efficacy endpoint was the proportion of patients with healed esophagitis at wk 8. Secondary endpoints were the proportion of patients healed at wk 4, resolution of heartburn at wk 4, time to first resolution and sustained resolution of heartburn, and proportion of heartburn-free days and nights. Safety and tolerability were also assessed. RESULTS: Significantly more patients were healed with esomeprazole versus omeprazole at wk 8 (93.7% vs 84.2%, p < 0.001; life table estimates, intention-to-treat analysis). Healing rates at wk 4 were 81.7% and 68.7%, respectively. Esomeprazole was superior to omeprazole for all secondary measures and had a similar safety profile. The most common adverse events in both treatment groups were headache, diarrhea, and nausea. CONCLUSIONS: Esomeprazole demonstrates significantly greater efficacy than omeprazole in the treatment of GERD patients with erosive esophagitis. The tolerability and safety of esomeprazole are comparable to that of omeprazole. (Am

Budesonide enema for the treatment of active, distal ulcerative colitis and proctitis: a dose-ranging study. U.S. Budesonide enema study group.

BACKGROUND & AIMS: Budesonide is a highly potent topical glucocorticosteroid that is characterized by low systemic availability as a result of high first-pass hepatic metabolism. The aim of this study was to evaluate the efficacy and safety of three doses of an enema preparation of budesonide in patients with active distal ulcerative colitis/proctitis. METHODS: In a double-blind multicenter trial, 233 patients were randomized to receive either a placebo enema or budesonide enema at a dose of 0.5 mg/100 mL, 2.0 mg/100 mL, or 8.0 mg/100 mL. The primary efficacy variables were an improvement of sigmoidoscopic inflammation grade, total histopathology score, and remission rates. Effects on cortisol concentrations were also assessed. RESULTS: After 6 weeks of treatment, there was significant improvement in sigmoidoscopy and histopathology scores in the budesonide 2.0-mg and 8.0-mg dose groups compared with placebo. Remission was achieved in 19% of patients in the 2.0-mg budesonide group (P