Safety, tolerance, and pharmacokinetic studies of OPT-80 in healthy volunteers following single and multiple oral doses.

Current therapies for Clostridium difficile infection (CDI) are encumbered by treatment failures and recurrences. Due to its high in vitro activity against C. difficile but low activity against the typical intestinal flora, minimal absorption, and durable cure in the hamster model of C. difficile infection, OPT-80 was considered for clinical development as a therapy for CDI. This trial consisted of two phases. Four single oral doses of OPT-80 (100, 200, 300, and 450 mg) were administered in a crossover manner to 16 healthy volunteers in a double-blind, placebo-controlled phase 1A study; a 1- to 2-week washout interval separated the treatments. In the double-blind phase 1B study, 24 healthy subjects were randomized to receive OPT-80 (150, 300, or 450 mg) or placebo for 10 days. In both studies, OPT-80's safety and tolerability were evaluated and the concentrations of OPT-80 and its primary metabolite (OP-1118) in plasma and feces were determined. OPT-80 levels in the urine were also analyzed for the phase 1A study. In both the single-dose and the multiple-dose studies, OPT-80 was well tolerated by all subjects in all dose groups. Maximal plasma concentrations were near or below the limit of quantification (5 ng/ml) across the dose range; urine concentrations were below the detection limit. The fecal total recovery of OPT-80 plus its major metabolite, OP-1118, approximated 100%. The tolerability, high fecal concentration, and low systemic exposure data from these studies support the further clinical development of OPT-80 as an oral therapy for CDI.

The effect of subclinical ketosis in early lactation on reproductive performance of postpartum dairy cows.

Data generated from 796 Holstein cows enrolled in a clinical trial to investigate the health effect of a monensin controlled release capsule were analyzed to investigate the association between circulating serum beta-hydroxybutyrate (BHBA) concentration in the peri-parturient period and subsequent reproductive performance. Overall, accounting for both repeated measures within cow and clustering at the herd level, non-pregnant cows after first insemination tended to have increased circulating BHBA concentrations from 3 wk before calving to 9 wk after calving relative to pregnant cows. Including the interaction between the week of sample collection and pregnancy outcome, non-pregnant cows had higher circulating BHBA concentrations in the second week after calving than cows diagnosed pregnant after first artificial insemination. Within individual weeks, cows with circulating BHBA concentrations > or =1,000 micromol/L in the first week postpartum were less likely to be diagnosed pregnant after first insemination. In the second week postpartum, the cows with circulating BHBA concentrations > or =1,400 micromol/L were significantly less likely to be pregnant after first artificial insemination. A dose response relationship was found when a comparison of the probability of pregnancy after first insemination and duration of elevated circulating ketone bodies was investigated. The probability of pregnancy was reduced by 20% in cows diagnosed subclinically ketotic in either the first or second week postpartum. Nevertheless, cows above the subclinical ketosis threshold in both the first and second week postpartum were 50% less likely to be pregnant after first insemination. Similarly, the median time to pregnancy increased in cows experiencing elevated BHBA concentrations in either (124 d) or both (130 d) the first and second week postpartum relative to cows never experiencing elevated BHBA concentrations (108 d). To further investigate this, the effect of elevated circulating BHBA was permitted to vary with time. The effect decreased with time, such that the daily probability of pregnancy increased similar to nonsubclinically ketotic cows by approximately 160 d in milk. From this analysis, both the relative circulating concentration of BHBA and the duration of elevated circulating BHBA were negatively associated with the probability of pregnancy at first service.

Synchronization of estrus and pregnancy risk in anestrous dairy cows after treatment with a progesterone-releasing intravaginal device.

Lactating Holstein cows (located in 4 dairy herds) that had failed to display estrus as defined by increased pedometer activity by 63 +/- 3 d in milk, were enrolled to investigate the effect of a progesterone-releasing intravaginal device (PRID, n = 268) relative to a placebo intravaginal device (PID, control, n = 266) on days from device removal to artificial insemination (AI), the probability of pregnancy at first AI, and days from device removal to pregnancy. Cows were assigned randomly to receive a PRID or PID for 7 d and an injection of PGF2alpha at device removal. Upon device removal, a vaginitis score was assigned and AI occurred at observed estrus. Cows failing to display estrus within 14 d of device removal were subjected to a subsequent reproductive exam and were treated with PGF2alpha. Two percent of PRID-treated cows and 11% of control cows displayed estrus during the 7-d exposure period. Among the remaining cows, 93% of the devices were present at the scheduled removal. Cows treated with the PRID were 60% less likely to have purulent debris on the device than control cows. Vaginal reaction, however, was not associated with any of the reproductive outcomes. Investigation of the reproductive outcomes revealed a treatment x parity interaction. Progesterone-treated primiparous cows were inseminated 17 d earlier, with no significant change in the probability of pregnancy at first AI (30.3 vs. 42.0%), and no difference in median time from device removal to pregnancy (52 vs. 53 d) relative to control primiparous cows. Conversely, PRID-treated multiparous cows were inseminated 8 d earlier, with no change in probability of pregnancy at first AI (24.6 vs. 18.8%); however, median time from device removal to pregnancy was reduced by 20 d (67 vs. 87 d). These results support the efficacy of a PRID to induce estrus in previously anestrous cows. The reason, however, for the variable response between primiparous and multiparous cows was not clear.

The effect of a progesterone releasing intravaginal device (PRID) on pregnancy risk to fixed-time insemination following diagnosis of non-pregnancy in dairy cows.

The objective was to compare the probability of pregnancy after fixed-time insemination in cows diagnosed as non-pregnant and re-inseminated following the Ovsynch protocol, with or without exogenous progesterone. Cows (n=415) used in this study originated from 25 farms. Upon diagnosis of non-pregnancy between 30 and 60 days after AI, cows were randomly assigned to receive either a progesterone releasing intravaginal device (PRID; n=208) or a placebo intravaginal device (PID; n=207). All cows received GnRH at enrollment (Day 0), PGF(2alpha) concurrent with intravaginal device removal 7 days later, GnRH on Day 9 and fixed-time insemination 16h later (Day 10). Cows observed in estrus prior to Day 7, had the device removed and were inseminated. Ovaries were examined by transrectal palpation at the time of enrollment and the prominent structures were assessed and recorded. Body condition score, lameness status, interval from previous insemination, and times bred at enrollment were recorded. At intravaginal device removal, the occurrence and intensity of vaginitis was determined according to the amount of debris on the device. Overall, the intravaginal device retention rate was 91%. A total of 5.2% of PID-treated cows and 2.9% of PRID-treated cows were detected in estrus within the 7 days treatment period. Pregnancy status was diagnosed between 30 and 56 days after insemination and all cows were followed for a minimum of 150 days after enrollment. Approximately 28% of cows had evidence of mild vaginitis in response to the intravaginal device, whereas 6% of cows had copious debris associated with the intravaginal device at removal. The probability of pregnancy after fixed-time insemination was 43.8% versus 34.9% in PRID-treated versus PID-treated animals. Exogenous progesterone provided through an intravaginal device to non-pregnant cows that had not displayed estrus improved the probability of pregnancy after fixed-time AI.

Prevalence and risk factors for postpartum anovulatory condition in dairy cows.

The objectives of this research were to determine the prevalence of the anovulatory condition within a temperate region of North America and identify cow-level and herd-level risk factors for this condition. A total of 1,341 cows from 18 herds were classified as cycling or anovular based on skim milk progesterone concentration determined at 46 and 60 +/- 7 d in milk. Calving history, periparturient disease incidence, body condition score, milk ketone concentration in the first 2 wk of lactation, and first 305-d mature-equivalent milk projections were recorded. Reproductive and culling information was retrieved monthly from the Dairy Herd Improvement Association. The cow-level prevalence of anovulation was 19.5%, with a herd-specific range from 5 to 45%. Accounting for the effect of clustering at the herd level, cows experiencing a difficult calving, cows with twin calvings, displaced abomasum, and cows with subclinical ketosis in the first week after calving were at greater risk for diagnosis of anovulation. Anovular cows within herds using ovulation synchronization programs were inseminated at the same time postpartum with a 6-percentage point reduction in the probability of pregnancy relative to cycling herdmates (29.7 vs. 35.9%, respectively), whereas anovular cows in herds breeding based on observed estrus were inseminated 8 d later and suffered a 10-percentage point reduction in the probability of pregnancy at first insemination (20.3 vs. 30.5). Time to pregnancy was delayed in anovular cows by 30 d (156 vs. 126 d). Using survival analysis, the impact of anovulation decreased with time. The daily probability of pregnancy (hazard ratio) was similar to cycling cows by 165 d in milk. The results underline the important associations of peripartum health with reproductive function and performance.

Functional studies of yeast glucokinase.

Glucose phosphorylation capacity is known to be in excess of glucose flux in Saccharomyces cerevisiae wild type but not in a mutant strain lacking the two hexokinases but still having glucokinase. Nonetheless, we show here that in the latter strain, as in the wild type, the internal concentration of glucose is apparently low during growth on glucose and that additional glucokinase activity does not increase glucose flux. The glucokinase-dependent strain accumulates substantial amounts of glucose internally in batch culture after exhaustion of glucose, as well as from maltose. In both of these situations, low concentrations of radioactive glucose provided to the medium are used with incomplete, if any, mixing with the internal pool. Furthermore, in contrast to activity of hexokinase and other enzymes, little glucokinase activity is revealed by toluene treatment of cells. These results may point to a connection between glucose entry and its phosphorylation by glucokinase, but separate explanations for the various findings are also possible.

Saccharomyces cerevisiae null mutants in glucose phosphorylation: metabolism and invertase expression.

A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.

Cloning of genes that complement yeast hexokinase and glucokinase mutants.

Genes complementing the glucose-negative fructose-negative Saccharomyces cerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13.

DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left.

Tn10 is 9,300 base pairs long and has inverted repeats of an insertion sequence (IS)-like sequence (IS10) at its ends. IS10-right provides all of the Tn10-encoded functions used for normal Tn10 transposition. IS10-left can also provide these functions but at a much reduced level. We report here the complete nucleotide sequence of IS10-right and a partial sequence of IS10-left. From our analysis of this information, we draw the following conclusions. (i) IS10-right is 1,329 base pairs long. Like most IS elements, it has short (23-base pair) nearly perfect inverted repeats at its termini. We can divide these 23-base pair segments into at least two functionally distinct parts. IS10-right also shares with other elements the presence of a single long coding region that extends the entire length of the element. Genetic evidence suggests that this coding region specifies an essential IS10 transposition function. A second, overlapping, coding region may or may not be important. (ii) The "outside" end of IS10-right contains three suggestively positioned internal symmetries. Two of these (A1 and A2) are nearly identical in sequence. Symmetry A1 overlaps the terminal inverted repeat; symmetry A2 overlaps the promoter shown elsewhere to be responsible for expression of IS10 functions and lies very near a second characterized promoter that directs transcription outward across the end of IS10. Symmetries A1 and A2 may play a role in modulation of Tn10 activity and are likely to function at least in part as protein recognition sites. We propose that the third symmetry (B) acts to prevent fortuitous expression of IS10 functions from external promoters. The transcripts from such promoters can assume a stable secondary structure in which the AUG start codon of the long coding region is sequestered in a region of double-stranded mRNA formed by pairing between the two halves of symmetry B. (iii) IS10-left differs from IS10-right at many nucleotide positions in both the presumptive regulatory region and the long coding region. The available evidence suggests that Tn10 may be older than other analyzed drug-resistance transposons and thus have had more time to accumulate mutational changes.