HPLC separation of ProFAR breakdown products.

<p>The dashed line indicates the trace of stock 1 mM ProFAR used for this assay. The solid line (offset) indicates the trace of 1 mM ProFAR pH 7.5 after incubation at 37°C for 26 hours. Stars indicate unknown break down products. Abbreviations: AICAR, 5-amino-4-imidazolecarboxamide ribonucleotide; ProFAR, 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamide.</p

Growth rates of some <i>hisA</i> mutant strains are increased by exogenous histidine.

<p>Growth rates (in hours⁻¹) are shown and are arranged in descending order by their ability to generate their own histidine (middle data column). All strains were grown in minimal glucose medium at 37°C with adenine and the indicated additions. Thi: thiamine; His: histidine</p>a<p>NG = no growth; growth rate was <0.03 hours⁻¹.</p

Bacterial strains.

a<p>MudJ refers to the Mud1734 transposon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048207#pone.0048207-Castilho1" target="_blank">[9]</a>.</p>b<p>Allele numbers for <i>hisA</i> in the 1400 s were issued by the Salmonella Genetic Stock Center as <i>hsi</i> alleles for historical reasons. For simplicity, we have used the <i>his</i> designation herein.</p>c<p>Tn<i>10d</i>(Tc) refers to the transposition-defective mini-Tn<i>10</i>(Tn<i>10Δ16Δ17</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048207#pone.0048207-Way1" target="_blank">[10]</a>.</p

Possible mechanisms for PRA formation from ProFAR.

<p>General mechanisms for PRA formation from ProFAR are depicted schematically. In pathway I, ProFAR is hydrolyzed to generate R5P by a mechanism that likely requires an enzyme. Ammonia is also released from the non-R5P product and is then available for non-enzymatic formation of PRA. It is possible the R5P and/or the ammonia do not leave the active site of the relevant enzyme. Pathway II depicts the formation of PRA as a direct product and implicates an undefined enzyme-catalyzed mechanism. Abbreviations: ProFAR, 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide; R5P, ribose-5′-phosphate; PRA, phosphoribosylamine.</p

Metabolic flux to ProFAR is required for PurF-independent PRA synthesis.

<p>Growth rates (in hours⁻¹) are shown. Strains were grown in minimal glucose medium with adenine and the indicated additions. His: histidine; Thi: thiamine. Histidine alleles <i>hisA3000, hisI99, hisF109, his-2652</i> (<i>del:CBHAFI</i>) cause a complete loss of function of the relevant gene product(s). Allele <i>hisG1102</i> encodes an enzyme that is insensitive to feedback inhibition by histidine.</p>a<p>NG = no growth; growth rate was <0.03 hours⁻¹.</p

Diverse mutations in <i>hisA</i> allow PurF-independent PRA synthesis.

a<p>From the annotated LT2 genome, NCBI GeneID: 1253299. Numbering starts at the first nucleotide of the coding sequence for HisA. Δ: Deletion</p>b<p>Independent isolates.</p

The biosynthetic pathways for thiamine and histidine in <i>S. enterica</i>.

<p>Panel A shows relevant steps from the thiamine biosynthetic pathway while panel B shows relevant steps in the histidine synthetic pathway, both in <i>S. enterica</i>. The enzymatic steps that lead to the formation of their respective end products are show. Abbreviations: Gln, glutamine; PRPP, phosphoribosyl pyrophosphate; PRA, phosphoribosylamine; Gly, glycine; GAR, glycineamide ribonucleotide; ProFAR, 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamide; PRFAR, 5-[(5-Phospho-1-deoxyribulos-1-ylamino)methylideneamino]-1-(5-phosphoribosyl)imidazole-4-carboxamide.</p

Growth analysis of <i>hisA</i> mutant strain.

<p>Growth curves were performed by monitoring optical density (OD) at 650 nm. Shown is a <i>purF2085 gnd174</i>::MudJ <i>hisA1451</i> (DM10350) strain grown in minimal glucose adenine medium (open triangle), with thiamine (open square), with histidine (filled triangle), and with histidine and thiamine (filled square).</p

<i>In Vivo</i> Dissolution and Systemic Absorption of Immediate Release Ibuprofen in Human Gastrointestinal Tract under Fed and Fasted Conditions

<i>In vivo</i> drug dissolution in the gastrointestinal (GI) tract is largely unmeasured. The purpose of this clinical study was to evaluate the <i>in vivo</i> drug dissolution and systemic absorption of the BCS class IIa drug ibuprofen under fed and fasted conditions by direct sampling of stomach and small intestinal luminal content. Expanding current knowledge of drug dissolution <i>in vivo</i> will help to establish physiologically relevant <i>in vitro</i> models predictive of drug dissolution. A multilumen GI catheter was orally inserted into the GI tract of healthy human subjects. Subjects received a single oral dose of ibuprofen (800 mg tablet) with 250 mL of water under fasting and fed conditions. The GI catheter facilitated collection of GI fluid from the stomach, duodenum, and jejunum. Ibuprofen concentration in GI fluid supernatant and plasma was determined by LC–MS/MS. A total of 23 subjects completed the study, with 11 subjects returning for an additional study visit (a total of 34 completed study visits). The subjects were primarily white (61%) and male (65%) with an average age of 30 years. The subjects had a median [min, max] weight of 79 [52, 123] kg and body mass index of 25.7 [19.4, 37.7] kg/m². Ibuprofen plasma levels were higher under fasted conditions and remained detectable for 28 h under both conditions. The AUC_0–24 and <i>C</i>_max were lower in fed subjects vs fasted subjects, and <i>T</i>_max was delayed in fed subjects vs fasted subjects. Ibuprofen was detected immediately after ingestion in the stomach under fasting and fed conditions until 7 h after dosing. Higher levels of ibuprofen were detected in the small intestine soon after dosing in fasted subjects compared to fed. In contrast to plasma drug concentration, overall gastric concentrations remained higher under fed conditions due to increased gastric pH vs fasting condition. The gastric pH increased to near neutrality after feedingbefore decreasing to acidic levels after 7 h. Induction of the fed state reduced systemic levels but increased gastric levels of ibuprofen, which suggest that slow gastric emptying and transit dominate the effect for plasma drug concentration. The finding of high levels of ibuprofen in stomach and small intestine 7 h post dosing was unexpected. Future work is needed to better understand the role of various GI parameters, such as motility and gastric emptying, on systemic ibuprofen levels in order to improve <i>in vitro</i> predictive models