Safety and pharmacokinetics of single doses of (+)-calanolide a, a novel, naturally occurring nonnucleoside reverse transcriptase inhibitor, in healthy, human immunodeficiency virus-negative human subjects.

(+)-Calanolide A is a novel, naturally occurring, nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase first isolated from a tropical tree (Calophyllum lanigerum) in the Malaysian rain forest. Previous studies have demonstrated that (+)-calanolide A has specific activity against the reverse transcriptase of HIV-1 and a favorable safety profile in animals. In addition, (+)-calanolide A exhibits a unique HIV-1 resistance profile in vitro. The safety and pharmacokinetics of (+)-calanolide A was examined in four successive single-dose cohorts (200, 400, 600, and 800 mg) in healthy, HIV-negative volunteers. In this initial phase I study, the toxicity of (+)-calanolide A was minimal in the 47 subjects treated. Dizziness, taste perversion, headache, eructation, and nausea were the most frequently reported adverse events. These events were not all judged to be related to study medication nor were they dose related. While 51% of subjects reported mild and transient dizziness, in many cases this appeared to be temporally related to phlebotomy. Calculation of the terminal-phase half-life (t(1/2)) was precluded by intrasubject variability in the 200-, 400-, and 600-mg dose cohorts but was approximately 20 h for the 800-mg dose group. (+)-Calanolide A was rapidly absorbed following administration, with time to maximum concentration of drug in plasma (T(max)) values occurring between 2.4 and 5.2 h postdosing depending on the dose. Plasma levels of (+)-calanolide A at all dosing levels were quite variable; however, both the mean concentration in plasma (C(max)), and the area under the plasma concentration-time curve increased proportionately in relation to the dose. Although raw plasma drug levels were higher in women than in men, when doses were normalized for body mass, the pharmacokinetic profiles were virtually identical with those observed for males. In general, levels of (+)-calanolide A in human plasma were higher than would have been predicted from animal studies, yet the safety profile remained benign. In conclusion, this study demonstrated the safety and favorable pharmacokinetic profile of single doses of (+)-calanolide A in healthy, HIV-negative individuals.

Expression of the INO2 regulatory gene of Saccharomyces cerevisiae is controlled by positive and negative promoter elements and an upstream open reading frame.

The INO2 gene encodes a transcriptional activator of the phospholipid biosynthetic genes of Saccharomyces cerevisiae. Complete derepression of phospholipid biosynthetic gene expression in response to inositol/choline deprivation requires both INO2 and INO4. Ino2p dimerizes with Ino4p to bind the upstream activating sequence (UAS)INO element found in the promoters of the target genes. We have demonstrated previously that transcription from the INO2 promoter is autoregulated 12-fold in a manner identical to that of the target genes. Here, we show that this regulation occurs at the levels of transcription and translation. Transcription accounts for fourfold regulation, whereas translation accounts for an additional threefold regulation. Regulation of transcription requires a UAS(INO) element. Additional promoter elements include an upstream essential sequence (UES) located upstream of the UAS(INO) element and a negative regulatory element in the vicinity of the UAS(INO) element. Regulation of translation is dependent on an upstream open reading frame (uORF) in the INO2 leader. These data support the model that regulatory gene promoters may display unusual organizations and may be subject to multiple levels of regulation. We have shown previously that the UME6 gene positively regulates INO2 expression. Here, we limit the UME6-responsive region of the INO2 promoter to nucleotides -217 to -56.

Glutathione content of colonic mucosa: evidence for oxidative damage in active ulcerative colitis.

Oxidative stress appears to play a role in the tissue damage of active ulcerative colitis, and it has been suggested that a defect in mucosal antioxidant defenses is a etiological factor in the disease. This study was undertaken to investigate the mucosal content and oxidation state of glutathione in ulcerative colitis in the active and inactive states and to examine the relationship between glutathione content and disease activity in this patient population. Endoscopic biopsies of colon mucosa were collected from normal subjects, from macroscopically normal tissue of patients with inactive and active ulcerative colitis, and from inflamed tissue of patients with active ulcerative colitis. The mucosal contents of GSH and GSSG were determined by liquid chromatography. We found no significant differences in tissue contents of reduced glutathione among the four groups. The median tissue level of oxidized glutathione in inflamed mucosa from patients with active ulcerative colitis was increased 1.7-fold (P = 0.017) over that of patients with inactive disease. The oxidized glutathione content of the mucosa also showed significant positive correlations with clinical and histological indices of disease severity among ulcerative colitis patients. In conclusion, a change in the redox status of mucosal glutathione is associated with inflammation and disease activity in ulcerative colitis. This change appears to be a consequence of inflammation rather than a pathogenic factor for the disease.

The effect of a selective 5-lipoxygenase inhibitor, zileuton, on tissue damage in acute colonic inflammation in rats.

Though the mechanism of tissue damage induced by colonic inflammation in ulcerative colitis is unknown, it has been established that the inflammatory mediator and potent neutrophil (PMN) chemotaxin, leukotriene B4(LTB4), is present in elevated amounts in the inflamed mucosa. The unique role of 5-lipoxygenase in the production of leukotrienes has made it a target for inhibition. This study used a rat model of acute colonic inflammation induced by a single IP injection of Mitomycin-C to test the efficacy of a specific and potent 5-lipoxygenase inhibitor zileuton in the treatment of colonic inflammation. We hypothesized that after inducing colitis in rats with mitomycin-C, the administration of oral zileuton would inhibit leukotriene production, thus preventing PMN infiltration and subsequent tissue damage. Zileuton decreased colonic tissue damage as measured by Histological score. However, zileuton did not significantly decrease neutrophil infiltration measured by mucosal PMN or myeloperoxidase (MPO) levels. Although zileuton was successful in significantly decreasing the frequency of severe colitis in our model, the fact that the decrease in PMN count and MPO level was not statistically significant suggests that another mechanism may be involved in its anti-inflammatory effect.

Mucosal reactive oxygen species production in oesophagitis and Barrett's oesophagus.

Reactive oxygen species (ROS) produced by inflammatory cells can contribute to tissue destruction. ROS have been implicated in various gastrointestinal abnormalities, including the acid related peptic diseases. Although the development of oesophagitis and Barrett's columnar epithelium is associated with prolonged reflux of gastric acid, the exact mechanism by which tissue damage occurs is not known. To discover if ROS are involved in damage to the oesophageal mucosa, this study measured in vitro the mucosal ROS concentrations of biopsied mucosal samples taken from patients with reflux oesophagitis using luminol enhanced chemiluminescence (LECL). Mucosal biopsy specimens were taken from 83 patients: 19 with normal oesophageal mucosa (group I); 20 with macroscopic oesophagitis (group II); 20 with biopsy confirmed Barrett's epithelium without macroscopic oesophagitis (group III); and 24 with Barrett's epithelium with macroscopic oesophagitis (group IV). The mucosa from patients exhibited significantly higher LECL values than the mucosa from controls. But, there were no significant differences between groups II, III, and IV. Addition of the myeloperoxidase inhibitor, azide, or the hydrogen peroxide scavenger, catalase, to the tissue suspension caused a decrease in LECL values of 32% and 45%, respectively, suggesting that neutrophils--although important--are not the only source of mucosal LECL. These data are consistent with the proposal that ROS play an important part in the tissue injury associated with oesophagitis and Barrett's columnar epithelium.

Modulation of neutrophil function by novel colonic factors: possible role in the pathophysiology of ulcerative colitis.

Tissue damage in acute ulcerative colitis (UC) may be triggered by neutrophils (PMNs) and their inflammatory mediators such as reactive oxygen species (ROS). Because circulating PMNs appeared normal in subjects with UC, we hypothesized that the critical abnormality that attracts and activates PMNs in UC is a local colonic factor. Accordingly, the colonic milieu was sampled by using in vivo rectal dialysis (mol wt < or = 12 kd). Normal PMNs were exposed in vitro to rectal dialysates (RD) from control subjects (cRD) or subjects with active UC (aRD) or inactive UC (IRD). PMN-derived ROS were measured by chemiluminescence. cRD did not increase ROS production by unstimulated PMNs; aRD significantly and concentration-dependently increased ROS; IRD gave intermediate results. cRD inhibited the PMN-stimulating effects of both the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA). aRD and IRD blunted the effect of fMLP and PMA significantly less than did cRD. Rectal dialysates from 44% of subjects with active UC exaggerated the fMLP effect, whereas potentiation occurred for only 13% of cRDs and 18% of iRDs. cRD preconditioned with either activated or nonactivated PMNs was not significantly different than unconditioned cRD. We thus infer the existence of colonic factors in UC that (1) can trigger PMNs to produce ROS and (2) have a proinflammatory modulatory effect on bacterial peptide-induced, PMN-mediated ROS production, thereby initiating or perpetuating inflammation and eventually causing tissue damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated breath ethane levels in active ulcerative colitis: evidence for excessive lipid peroxidation.

OBJECTIVE: Reactive oxygen species (ROS) may be a major cause of tissue injury in ulcerative colitis (UC). One possible mechanism for ROS-mediated tissue injury is lipid peroxidation. Breath ethane and pentane excretion are noninvasive means for measuring peroxidation of omega-3 and omega-6 polyunsaturated fatty acid, respectively. Hence, we measured breath ethane in 17 subjects with active UC and correlated the results with disease severity. METHODS: Breath samples for ethane and pentane analysis were collected every 2 weeks, and rectal biopsies were obtained monthly to assess for chemiluminescence, a marker of ROS. Ethane and pentane concentrations (nmol/L) were measured by gas chromatography, and mucosal chemiluminescence was measured spectrophotometrically. Data were compared to control values (C) from healthy subjects. Disease activity was assessed both clinically and endoscopically. RESULTS: Ethane excretion was significantly elevated in patients (UC, 0.45 +/- 0.04; C, 0.33 +/- 0.06; p = 0.013). Ethane excretion was positively correlated with endoscopic score (r = 0.45; p < 0.05), symptom score (r = 0.34; p < 0.05), disease activity (r = 0.36, p < 0.05), and chemiluminescence (r = 0.65; p < 0.001). Pentane levels did not correlate with any of the clinical measurements. Chemiluminescence in the rectal tissue was positively correlated with endoscopic score (r = 0.71; p < 0.05) and disease activity (r = 0.61; p < 0.01). CONCLUSIONS: Tissue damage in UC may be ROS-induced lipid peroxidation. Disease activity can be assessed noninvasively by breath ethane excretion.