Dose-related Changes Research Articles

Pulmonary response after exposure to inhaled nickel hydroxide nanoparticles: Short and long-term studies in mice

Short and long-term pulmonary response to inhaled nickel hydroxide nanoparticles (nano-Ni(OH)2, CMD = 40 nm) in C57BL/6 mice was assessed using a whole body exposure system. For short-term studies mice were exposed for 4 h to nominal concentrations of 100, 500, and 1000 μg/m3. For long-term studies mice were exposed for 5 h/d, 5 d/w, for up to 5 months (m) to a nominal concentration of 100 μg/m3. Particle morphology, size distribution, chemical composition, solubility, and intrinsic oxidative capacity were determined. Markers of lung injury and inflammation in bronchoalveolar lavage fluid (BALF); histopathology; and lung tissue elemental nickel content and mRNA changes in macrophage inflammatory protein-2 (Mip-2), chemokine ligand 2 (Ccl2), interleukin 1-alpha (Il-1α), and tumor necrosis factor-alpha (Tnf-α) were assessed. Dose-related changes in BALF analyses were observed 24 h after short-term studies while significant changes were noted after 3 m and/or 5 m of exposure (24 h). Nickel content was detected in lung tissue, Ccl2 was most pronouncedly expressed, and histological changes were noted after 5 m of exposure. Collectively, data illustrates nano-Ni(OH)2 can induce inflammatory responses in C57BL/6 mice.

The effect of nandrolone decanoate on liver of rabbitsusing histological and ultrasound methods

Objective: To compare the effects of nandrolone decanoate on the liver structure in male and female rabbits using histological and ultrasound examinations.Materials and methods: Forty-eight adult rabbits were divided into two groups, male and female groups, each included 24 adult healthy rabbits. The male group was subdivided into 4 subgroups, group A was the control group which received only normal saline, group B was given nandrolone decanoate 2mg/kg body weight, group C was given 4 mg/kg body weight of nandrolone decanoate, while group D was given 6 mg/kg body weight of the drug. The female group was also subdivided into 4 subgroups, group A1 was the control group, group B1 was given 2 mg/kg body weight, group C1 was given 4 mg/kg body weight, group D1 was given 6 mg/kg body weight of nandrolone decanoate.Results: All treated groups of both sexes showed a significant increase in body and liver weights in addition to dose related pathological changes ranging from mild to severe form, including vacuolar degeneration and fatty degenerative changes of hepatocytes. In addition, there was a dilatation and congestion of sinusoids, central venules and portal veins with mononuclear inflammatory cells infiltration. Focal areas of necrosis with chronic inflammatory cells infiltration were also noticed in different hepatic lobules. In spite of these histological changes in the liver no abnormal ultrasound findings of the liver were noticed.Conclusion: These changes indicate that the histological effects of the anabolic androgenic steroid nandrolone decanoate on the liver of adult male and female rabbits is the same in both sexes, but the treated female rabbit groups of medium and high doses (C1 and D1) showed more increase in body weight than male rabbit groups. There is no correlation between histological and ultrasound findingsKeywords: Nandrolone decanoate, liver histology, rabbits.

Phase I Study of Intravenous Vascular Endothelial Growth Factor Trap, Aflibercept, in Patients With Advanced Solid Tumors

Vascular endothelial growth factor (VEGF) Trap (aflibercept) is an angiogenesis inhibitor comprising portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human immunoglobulin G. This phase I study was designed to evaluate the safety, pharmacokinetics, and pharmacodynamics of VEGF Trap administered intravenously (IV) every 2 weeks. Patients with refractory solid tumors or non-Hodgkin's lymphoma with adequate organ function were eligible. Pharmacokinetic/pharmacodynamic markers included measurement of plasma VEGF bound to VEGF Trap and free VEGF Trap. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was incorporated to measure the biologic effects of the drug on tumor vascularity and permeability. The study enrolled 47 patients at doses ranging from 0.3 to 7.0 mg/kg IV every 2 weeks. Dose-limiting toxicities were rectal ulceration and proteinuria at the 7.0 mg/kg dose. Other mechanism-specific toxicities included hypertension. On the basis of these observations and on pharmacokinetics, the recommended phase II dose of VEGF Trap as a single agent is 4 mg/kg every 2 weeks. Three RECIST (Response Evaluation Criteria in Solid Tumors) -defined partial responses were observed, one at the 3.0 mg/kg and two at the 7.0 mg/kg dose level. Maximum plasma concentration of free VEGF Trap increased proportionally with dose. Maximal VEGF-bound VEGF Trap complex levels were reached at doses > or = 2.0 mg/kg. Changes in volume transfer constant measured by DCE-MRI at baseline and at 24 hours after administration indicate a possible dose-related change in this pharmacodynamic marker. IV VEGF Trap was well tolerated at the dose levels tested. Pharmacodynamic and pharmacokinetic markers were indicative of VEGF blockade.

Dose-dependent effects of the CRF1 receptor antagonist R317573 on regional brain activity in healthy male subjects

Corticotropin-releasing factor receptor type 1 (CRF(1)) antagonists have been proposed as therapeutic agents in the treatment of mood and anxiety disorders although clinical evidence supporting their development and understanding of a dose-response relationship has been lacking. We tested two doses of the CRF(1) antagonist R317573 for effects on regional cerebral glucose metabolism (rCMglu) using [(18)F] fluoro-2-deoxy-D: -glucose (FDG) positron emission tomography (PET) following single-dose challenges in a double-blind, placebo-controlled, cross-over design, in 12 healthy male volunteers. Single 30- and 200-mg doses of R317573 resulted in dose-related changes in rCMglu. Relative increases in rCMglu were observed in frontal cortical regions while relative decreases occurred in the putamen and right amygdala after both doses. Relative decreases occurred in cerebellum and right parahippocampal gyrus following the higher dose. R317573 appears to produce acute dose-dependent changes in rCMglu. Effects occurred in regions that may be behaviorally relevant to mood and anxiety disorders. In some regions, these effects may be related to the receptor (target) density. Measuring acute effects on rCMglu with FDG-PET may offer a method for defining pharmacologically active doses for central nervous system targets for which selective radiotracers are lacking.

Facilitation of central imidazoline I1‐site/extracellular signal‐regulated kinase/p38 mitogen‐activated protein kinase signalling mediates the hypotensive effect of ethanol in rats with acute renal failure

This study investigated the role of central sympathetic activity and related mitogen-activated protein kinase (MAPK) signalling in the cardiovascular effects of ethanol in a model of acute renal failure (ARF). The effects of pharmacological interventions that inhibit peripheral or central sympathetic activity or MAPK on the cardiovascular actions of ethanol in rats with ARF induced by glycerol were evaluated. Glycerol (50%, 10 mL.kg(-1), i.m.) caused progressive increases and decreases in blood pressure (BP) and heart rate (HR) respectively. Subsequent i.v. ethanol (0.25 or 1 g.kg(-1)) elicited dose-related changes in BP (decreases) and HR (increases). These effects were replicated after intracisternal (i.c.) administration of ethanol. Blockade of nicotinic cholinoceptors (nAChR, hexamethonium, 20 mg.kg(-1)) or alpha(1)-adrenoceptors (prazosin, 1 mg.kg(-1)) attenuated cardiovascular effects of ethanol. Ethanol hypotension was also attenuated after the centrally acting sympatholytic drug moxonidine (selective I(1)-site agonist, 100 microg.kg(-1) i.v.), but not guanabenz (selective alpha(2)-receptor agonist, 30 microg.kg(-1), i.v.), suggesting involvement of central circuits of I(1) sites in ethanol-evoked hypotension. Selective blockade I(1) sites (efaroxan) but not alpha(2) (yohimbine) adrenoceptors abolished the hypotensive response to ethanol. Intracisternal administration of PD98059 or SB203580, inhibitors of extracellular signal-regulated kinase (ERK 1/2) and p38 MAPK, respectively, reduced the hypotensive action of moxonidine or ethanol. When used simultaneously, the two MAPK inhibitors produced additive attenuation of ethanol hypotension. Sympathoinhibitory pathways of central I(1)-sites and downstream ERK/p38 MAPK signalling were involved in the hypotensive action of ethanol in ARF.

Chlorpyrifos induced reproductive toxicity in male mice

The reproductive toxicity of the insecticide chloropyrifos was studied in male mice. Adult male mice were treated by gavage with chloropyrifos at doses of 0, 5, 15, and 25 mg/kg-d for 4 weeks before mating with untreated females. Signs of cholinergic effects were observed in the 15 and 25 mg/kg-d treated groups. Brain and skeletal muscle acetylcholinesterase activity was inhibited in the same groups. Chloropyrifos treatment was associated with a decreased number of live fetuses, and an increased number of dead fetuses at 25 mg/kg-d. The number of early resorption was decreased in both 15 and 25 mg/kg-d treated groups. The percent morphologically normal spermatozoa were affected in 15 and 25 mg/kg-d dose groups; however, sperm motility and count were decreased in the same treated groups compared to the control. Histologic examination of brain revealed histological abnormalities in the middle and high groups. Dose related histologic changes, including degeneration of muscle fibers, were observed in the muscles of male mice treated with all the doses of chloropyrifos. The current study demonstrated adverse effects of male chloropyrifos exposure on pregnancy outcome with effects on sperm parameters at 15 and 25 mg/kg-d.

Reproductive and developmental toxicity evaluation of a purified Arabinogalactan-Protein (AGP) composition in Wistar rats

A purified Arabinogalactan-Protein composition (LL-4218) was prepared from the leaves of Argemone mexicana to treat psoriasis. The effect of (LL-4218) was evaluated on reproductive (male and female fertility) and developmental toxicity in rats. LL-4218 was administered orally at the doses of 250, 500 and 1000 mg kg − 1 . The results showed that LL-4218 did not produce any significant dose related changes in reproductive and developmental toxicity studies. Therefore, it is concluded that LL-4218 did not produce any significant toxic effect on reproduction and developmental parameters of rats and NOAEL for reproductive and developmental toxicity studies in rats was 1000 mg kg − 1 .

28-Day Oral Toxicity of Cadmium Selenide in Sprague-Dawley Rats.

This study was performed to evaluate the toxicity of cadmium selenide for a period of 28 days in Sprague-Dawley rats. Each of 10 healthy male and females rats per group received daily oral administration for 28-day period at dosage levels 30, 300 and 1,000 mg/kg of body weight. Mortality and clinical signs were checked, and body weight, water intake and food consumption were also recorded weekly. There were no dose-related changes in food consumption or urine volume. All animals survived to the end of study with no clinical signs or differences in body weight gain observed when compared with the control group. At the end of study, all animals including control group, were subjected to necropsy. Blood samples were collected for hematology tests including coagulation time and biochemistry analysis. Blood coagulation time and relative organ weight were unaffected by all received doses. White Blood Cell (WBC) counts significantly increased in the 300 mg/ kg administered male animal group when compared to the control. Monocyte (MO) value were also increased significantly in both 300 and 1,000 mg/kg male animal group. However, Mean Corpuscular Volume (MCV) were significantly decreased compared with the control in the 1,000 mg/kg dose groups for male and female animals. Mean Corpuscular Hemoglobin (MCH) decreased significantly for female in the 300 and 1,000 mg/kg group compared to the control. Blood biochemical values of Inorganic phosphorus (IP) were significantly increased in both the 300 and 1,000 mg/kg dose groups in male animals when compared to the control. Creatinine (CRE) levels indicated significant increase in kidney function for the female, 30 mg/kg dose group when compared with control. There was a significant decrease in thymus absolute organ weight in the female, 1,000 mg/kg dose group when compared with control. Histopathological findings revealed no evidence of injury related to cadmium selenide except for one case of focal hepatic inflammation in the high dose (1,000 mg/kg) group. One case of lung inflammation was also seen in the control group. Basis on these result, the No Observable Adverse Effect Level (NOAEL) of cadmium selenide was determined to be more than 1,000 mg/kg/day for male and female rats under conditions in this study.

Dose-related changes in retinal function and PKC-alpha expression in rabbits on vigabatrin medication

To investigate, in a rabbit model, the effect of two different doses of vigabatrin (VGB) on retinal function and morphology. Twenty-nine rabbits of mixed strain were divided into two groups, receiving either high-dose (n = 15) or low-dose (n = 14) oral VGB treatment (cumulative dose 29.8 +/- 2.9 g and 14.2 +/- 0.6 g respectively). Ten rabbits receiving water served as control animals. The rabbits underwent three baseline ff-ERG measurements before initiation of VGB medication and two ff-ERG registrations during treatment, after 8 and 12-14 weeks respectively. At the end of the study, the expression of protein kinase C-alpha (PKC-alpha), gamma amino butyric acid (GABA) A receptors, vimentin, glial fibrillary acidic protein (GFAP) and peanut agglutinin (PNA) was examined in retinal sections from all rabbits. In animals of the high-dose group, the ff-ERG measurements revealed a significant decrease of isolated rod b-wave amplitudes, combined rod-cone b-wave amplitudes and amplitudes of oscillatory potentials (OPs); OP1, OP2 and OP3. In the low-dose group, the b-wave amplitudes of combined rod-cone responses as well as OP2 and OP3 were significantly reduced. PKC-alpha labeling demonstrated a dose-related translocation of the enzyme in rod bipolar cells, also revealing a significant decline of the number of PKC-alpha labeled rod bipolar cells in treated animals. Vimentin labeling showed a dose-related deviant labeling pattern of Müller cells, with strikingly low labeling intensity in the outer parts of the cells; in the outer limiting membrane (OLM) as well as the outer nuclear layer (ONL). Labeling with antibodies against GABA A receptors and GFAP, as well as PNA staining, revealed no differences between treated animals and controls. In this study, VGB medication was associated, in a dose-related manner, with a decrease of ff-ERG amplitudes as well as with altered protein expression in rod bipolar cells and Müller cells, suggesting alterations of inner retinal function. The dose-related morphological and electrophysiological changes indicate a retinal pathology that may explain the constricted visual fields seen in some patients treated with VGB.

Immunohistochemical study of postnatal neurogenesis after whole-body exposure to electromagnetic fields: evaluation of age- and dose-related changes in rats.

It is well established that strong electromagnetic fields (EMFs) can give rise to acute health effects, such as burns, which can be effectively prevented by respecting exposure guidelines and regulations. Current concerns are instead directed toward the possibility that long-term exposure to weak EMF might have detrimental health effects due to some biological mechanism, to date unknown. (1) The possible risk due to pulsed EMF at frequency 2.45 GHz and mean power density 2.8 mW/cm(2) on rat postnatal neurogenesis was studied in relation to the animal's age, duration of the exposure dose, and post-irradiation survival. (2) Proliferating cells marker, BrdU, was used to map age- and dose-related immunohistochemical changes within the rostral migratory stream (RMS) after whole-body exposure of newborn (P7) and senescent (24 months) rats. (3) Two dose-related exposure patterns were performed to clarify the cumulative effect of EMF: short-term exposure dose, 2 days irradiation (4 h/day), versus long-term exposure dose, 3 days irradiation (8 h/day), both followed by acute (24 h) and chronic (1-4 weeks) post-irradiation survival. (4) We found that the EMF induces significant age- and dose-dependent changes in proliferating cell numbers within the RMS. Our results indicate that the concerns about the possible risk of EMF generated in connection with production, transmission, distribution, and the use of electrical equipment and communication sets are justified at least with regard to early postnatal neurogenesis.

Role of the orbitofrontal cortex and dorsal striatum in regulating the dose-related effects of self-administered cocaine

Little is known regarding which neural systems regulate dose-related changes in responses maintained by self-administered cocaine. This empirical question is important because elucidating neural systems engaged in this process could provide clues for effectively treating cocaine addiction. It has been suggested that different cocaine doses represent reinforcers of differing magnitudes, implicating the dorsal striatum or orbitofrontal cortex as important. Rats were trained to self-administer 1.0 mg/kg cocaine under a fixed-interval based second-order schedule. Next, cocaine unit doses (0.1–3.0 mg/kg) were each non-systematically available for a 5-day block of sessions. Tests (1 h) were conducted on day 3 (vehicle) and day 5 (100 μg lidocaine) of each block. Lidocaine inactivation of the lateral dorsal striatum had no effect on dose-related responding or cocaine intake. In contrast, when doses along the ascending limb were available for self-administration, lidocaine inactivation of the lateral orbitofrontal cortex caused reductions in responding and cocaine intake, resulting in overall flattening of dose–response curves. This included reductions during the entire 1-h test sessions and during the interval immediately following the first cocaine infusion of test sessions. Lidocaine inactivation of the lateral orbitofrontal cortex did not alter responding during the first cocaine-free interval of test sessions, but increased the latency to the first infusion. Collectively, the findings suggest that when the amount of experience with different cocaine unit doses is limited to a few sessions, the lateral orbitofrontal cortex regulates the dose-related effects of self-administered cocaine, likely by processing information pertaining to the reinforcing value of each unit dose.

Dose-related immunohistochemical and ultrastructural changes after oral methylphenidate administration in cerebrum and cerebellum of the rat

Methylphenidate is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity disorder of children and young adults. Our aim is to investigate dose-dependent dopamine-2 receptor and glial fibrillary acidic protein expression and ultrastructural changes of the rat brain, to demonstrate possible toxicity of the long-term and high dose use of the methylphenidate. In this study, 27 female prepubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) were treated orally with methylphenidate dissolved in saline solution for 5 days per week during 3 months. At the end of the third month, tissues were removed and sections were collected for immunohistochemical and ultrastructural studies. We believe that methylphenidate causes dose-related activation of the dopaminergic system in several brain regions especially in ventral tegmental area and also causing neuronal degeneration and capillary wall structural changes such as basal membrane thickness and augmentation of the pinostatic vesicle in the endothelial cells. Also, increased dose of Ritalin is inducing astrocytes hypertrophy especially astrogliosis in pia-glial membrane and this is the result of the degenerative changes in prefrontal cortex region due to high dose methylphenidate administration. The dose-related accumulation of the astrocytes in capillary wall might well be a consequence of the need for nutrition of the neuronal tissue, due to transport mechanism deficiency related to neuronal and vascular degeneration. Thus, we believe that the therapeutic dose of methylphenidate must be kept in minimum level to prevent ultrastructural changes.

Assessment of General and Cardiac Toxicities of Astemizole in Male Cynomolgus Monkeys: Serum Biochemistry and Action Potential Duration.

Toxicology screening following treatment with astemizole, a histamine receptor antagonist, at oral doses of 0, 10, 30 and 60 mg/kg was carried out in male cynomolgus monkeys (Macaca fascicularis). No dose-related changes in mortality, clinical signs, body weight changes, food consumption, or urine analysis occurred in any animal compared to the vehicle control. However, the high-dose group showed a decrease in BUN and ALP compared to vehicle control group. In addition, the levels of TG, AST, ALP and CK increased. Although astemizole did not produce significant toxicological changes at any dose tested, we predict that it can cause toxicological changes of the liver and heart based on the changes in the serum parameters related to the heart and liver. The Action Potential Duration (APD) was prolonged in the heart of 60 mg/kg treatment group compared to the control group. The APD increase in 60 mg/kg treatment group along the other related changes in toxicological parameters imply that astemizole has major cardiotoxic effects in the cynomolgus monkey. This study is a valuable assessment for predicting the general toxicity and cardiotoxic effects of antihistamine drugs using nonhuman primates.

Endotoxin Alters Early Fetal Lung Morphogenesis

The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Gestational day 15 (E15) fetal rat lung explants (term = 22 d) were treated with either 200 ng/mL or 2 microg/mL lipopolysaccharides (LPS) with controls and examined daily by phase microscopy. After 5 d, explants were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 5 mum in the coronal plane. Immunohistochemical analysis was performed with platelet endothelial cell adhesion molecule (PECAM) to define endothelial cells, vascular endothelial growth factor (VEGF) to examine endothelial mitogenesis, and COX-2 antibodies as a marker for prostaglandin synthesis. Real-time PCR examined inducible nitric oxide synthase (iNOS), FGF9, FGF10, and FGFr2 gene expression. Air space fraction and airway epithelium were analyzed with Image J software. Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas. By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VEGF expression was unaltered. LPS increased iNOS, but decreased FGF9, FGF10, and FGFr2 gene expression. These data support evidence for an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that proinflammatory stimuli may ultimately lead to abnormal pulmonary development via fibroblastic growth factor (FGF)-directed mechanisms that affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.

Prostaglandin E2 glycerol ester, an endogenous COX‐2 metabolite of 2‐arachidonoylglycerol, induces hyperalgesia and modulates NFκB activity

Recombinant cyclooxygenase-2 (COX-2) oxygenates 2-arachidonoylglycerol (2-AG) in vitro. We examined whether prostaglandin E2 glycerol ester (PGE2-G), a COX-2 metabolite of 2-AG, occurs endogenously and affects nociception and immune responses. Using mass spectrometric techniques, we examined whether PGE2-G occurs in vivo and if its levels are altered by inhibition of COX-2, monoacylglycerol (MAG) lipase or inflammation induced by carrageenan. We also examined the effects of PGE2-G on nociception in rats and NFkappaB activity in RAW264.7 cells. PGE2-G occurs endogenously in rat. Its levels were decreased by inhibition of COX-2 and MAG lipase but were unaffected by carrageenan. Intraplantar administration of PGE2-G induced mechanical allodynia and thermal hyperalgesia. In RAW264.7 cells, PGE2-G and PGE2 produced similar, dose-related changes in NFkappaB activity. PGE2-G was quickly metabolized into PGE2. While the effects of PGE2 on thermal hyperalgesia and NFkappaB activity were completely blocked by a cocktail of antagonists for prostanoid receptors, the same cocktail of antagonists only partially antagonized the actions of PGE2-G. Thermal hyperalgesia and immunomodulation induced by PGE2-G were only partially mediated by PGE2, which is formed by metabolism of PGE2-G. PGE2-G may function through a unique receptor previously postulated to mediate its effects. Taken together, these findings demonstrate that 2-AG is oxygenated in vivo by COX-2 producing PGE2-G, which plays a role in pain and immunomodulation. COX-2 could act as an enzymatic switch by converting 2-AG from an antinociceptive mediator to a pro-nociceptive prostanoid.

Open-Label Comparative Clinical Study of Chlorproguanil−Dapsone Fixed Dose Combination (Lapdap™) Alone or with Three Different Doses of Artesunate for Uncomplicated Plasmodium falciparum Malaria

The objective of this study was to determine the appropriate dose of artesunate for use in a fixed dose combination therapy with chlorproguanil−dapsone (CPG−DDS) for the treatment of uncomplicated falciparum malaria.MethodsOpen-label clinical trial comparing CPG−DDS alone or with artesunate 4, 2, or 1 mg/kg at medical centers in Blantyre, Malawi and Farafenni, The Gambia. The trial was conducted between June 2002 and February 2005, including 116 adults (median age 27 years) and 107 children (median age 38 months) with acute uncomplicated Plasmodium falciparum malaria. Subjects were randomized into 4 groups to receive CPG–DDS alone or plus 4, 2 or 1 mg/kg of artesunate once daily for 3 days. Assessments took place on Days 0−3 in hospital and follow-up on Days 7 and 14 as out-patients. Efficacy was evaluated in the Day 3 per-protocol (PP) population using mean time to reduce baseline parasitemia by 90% (PC90). A number of secondary outcomes were also included. Appropriate artesunate dose was determined using a pre-defined decision matrix based on primary and secondary outcomes. Treatment emergent adverse events were recorded from clinical assessments and blood parameters. Safety was evaluated in the intent to treat (ITT) population.ResultsIn the Day 3 PP population for the adult group (N = 85), mean time to PC90 was 19.1 h in the CPG−DDS group, significantly longer than for the +artesunate 1 mg/kg (12.5 h; treatment difference −6.6 h [95%CI −11.8, −1.5]), 2 mg/kg (10.7 h; −8.4 h [95%CI −13.6, −3.2]) and 4 mg/kg (10.3 h; −8.7 h [95%CI −14.1, −3.2]) groups. For children in the Day 3 PP population (N = 92), mean time to PC90 was 21.1 h in the CPG−DDS group, similar to the +artesunate 1 mg/kg group (17.7 h; −3.3 h [95%CI −8.6, 2.0]), though the +artesunate 2 mg/kg and 4 mg/kg groups had significantly shorter mean times to PC90 versus CPG−DDS; 14.4 h (treatment difference −6.4 h [95%CI −11.7, −1.0]) and 12.8 h (−7.4 h [95%CI −12.9, −1.8]), respectively. An analysis of mean time to PC90 for the Day 14 PP and ITT populations was consistent with the primary analysis. Treatment emergent, drug-related adverse events were experienced in 35.3% (41/116) of adults and 70.1% (75/107) of children; mostly hematological and gastroenterological. The nature and incidence of adverse events was similar between the groups. No dose-related changes in laboratory parameters were observed. Nine serious adverse events due to any cause occurred in five subjects including two cases of hemolysis believed to be associated with drug treatment (one adult, one child). One adult died of anaphylactic shock, not associated with investigational therapy.ConclusionsCPG–DDS plus artesunate demonstrated advantages over CPG–DDS alone for the primary efficacy endpoint (mean time to PC90) except in children for the 1 mg/kg artesunate dose. Based on a pre-defined decision matrix, the primary endpoint in the child group supported an artesunate dose of 4 mg/kg. Secondary endpoints also supported a 4 mg/kg artesunate dose to take forward into the remainder of the development program.Trial RegistrationClinicalTrials.gov NCT00519467

QS144. Endotoxin Alters Branching Morphogenesis and Matrix Deposition of The Fetal Lung

Background: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Methods: Gestational day 15 (E15) fetal rat lung explants (term=22 d) at the pseudoglandular stage were incubated at 37°C in 95% air/5% CO2 as submersion cultures on inserts in serum-free BGJb media supplemented with antibiotics and ascorbic acid. Explants were treated with either 200 ng/mL or 2 μg/mL LPS with controls and examined daily by phase microscopy. After 5 days, explants were fixed in 4% formaldehyde, paraffin embedded and sectioned at 5 μm in the coronal plane. Immunohistochemical analysis was performed with PECAM to define endothelial cells, VEGF to examine endothelial mitogenesis and COX-2 antibodies as a marker for prostaglandin synthesis. Air space fraction was analyzed with Image J software. Results: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas (graph). By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VGEF expression was unaltered. Conclusions: This is the first evidence of an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that the proinflammatory stimuli that may ultimately lead to abnormal pulmonary development affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.

Toxicological Comparisons of Three Styles of a Commercial U.S. Cigarette (Marlboro®) with the 1R4F Reference Cigarette

Toxicological comparisons were made of three commercial cigarettes, namely Marlboro® full flavor, Marlboro Lights, and Marlboro Ultra Lights, with the 1R4F reference cigarette. The main comparison was a 90-d inhalation study with mainstream smoke at 150 mg total particulate matter per cubic meter, in Sprague-Dawley rats using 6 h/d and 7 d/w exposures. The principal endpoint was histopathology of the respiratory tract, along with examinations of free lung cell counts after broncho-alveolar lavage. Additional studies on mainstream smoke included Salmonella mutagenicity, cytotoxicity of particulate and gas/vapor phases, and analytical chemistry. The exposures produced effectively the same responses in each of the four groups, and the histopathology results in the commercial cigarette groups were also effectively the same. The 1R4F was also tested at 75 and 200 mg/m3, and most of the histopathology results obtained here showed dose-response relationships. The free lung cell responses were similar in the 1R4F/commercial cigarette comparison, and there were dose-related changes in the 1R4F groups, most notably for neutrophils. Most of the changes produced in the 90–d of exposure were resolved in a 42-d post-inhalation period. Responses in the in vitro and analytical assays for the four cigarettes were in general similar, when data were expressed either per mg TPM or per mg tar yield. There were judged to be no toxicologically meaningful differences between the profiles evaluated at similar smoke concentrations for the three commercial cigarettes and for the 1R4F using these assays.

Early gene expression in human lymphocytes after gamma-irradiation–a genetic pattern with potential for biodosimetry

Purpose: Identification of early radiation response genes (ERG) in human lymphocytes after γ-irradiation by using the whole-human-genome DNA-microarrays and the evaluation of their possible role in rapid radiation biodosimetry by applying real-time quantitative polymerase chain reaction (RT-qPCR) methodology for validation in a small group of human individuals.Materials and methods: Whole blood from a healthy human donor was exposed at 37°C to 137Cs γ-radiations (absorbed dose: 1–4 Gy). Fifteen minutes following irradiation the lymphocytes were isolated from the blood (for 2 h at 20°C) and their gene expression was investigated using the DNA-microarrays. Subsequently, 14 genes were selected and validated using the TaqMan™ probes based upon the RT-qPCR assay within a group of 6 human donors.Results: A dose-related relative change in quantitative gene expression using the DNA-microarray assay was demonstrated in 24 of 102 genes. Up-regulation of expression was observed in 15 genes: CD69 (CD69 molecule), CDKN1A (cyclin-dependent kinase inhibitor 1A), EGR1 (early growth response 1), EGR4 (early growth response 4), FLJ35725 (chromosome 4 ORF 23), hCG2041177 (hCG – human Celera® Genome), hCG1643466.2, IFN-γ (interferon-γ), ISG20L (interferon stimulated exonuclease gene 20 kDa – like 1), c-JUN (jun oncogene), MDM2 (mouse double minute 2), MUC5B (mucine), PLK2 (polo-like kinase 2), RND1 (rho-family GTPase 1) and TNFSF9 (tumour necrosis factor superfamily member 9). Down-regulation of expression was found in the remaining nine genes: GRIK3 (glutamate receptor ionotropic kainate 3), hCG1985174, hCG1998530, hCG2038519, OCLN (occludin), RPL10A (ribosomal protein L10a), SERHL2 (serine hydrolase-like 2), SGK3 (serum/glucocorticoid regulated kinase 3) and STARD13 (START domain containing 13).Conclusion: A significant correlation between absorbed radiation dose and change in relative gene expression was particularly evident for EGR1, EGR4, IFN-γ, c-JUN and TNFSF9 (p ≤ 0.05). Results warrant the further investigation of these ERG as potential biodosimetric markers.

EEG power spectrum analysis for monitoring depth of anaesthesia during experimental surgery

The first attempts to introduce computerized power spectrum analysis of the electroencephalogram (EEG) as an intraoperative anaesthesia monitoring device started approximately 30 years ago. Since that time, the effects of various anaesthetic agents, sedative and analgesic drugs on the EEG pattern have been addressed in numerous studies in human patients and different animal species. These studies revealed dose-dependent changes in the EEG power spectrum for many intravenous and volatile anaesthetics. Moreover, EEG responses evoked by surgical stimuli during relative light levels of surgical anaesthesia have been classified as 'arousal' and 'paradoxical arousal' reaction, previously referred to as 'desynchronization' and 'synchronization', respectively. Contrasting reports on the correlation between quantitative EEG (QEEG) variables derived from power spectrum analysis (i.e. spectral edge frequency, median frequency) and simultaneously recorded clinical signs such as movement and haemodynamic responses, however, limited the routine use of intraoperative EEG monitoring. In addition, the appearance of EEG burst suppression pattern and isoelectricity at clinically relevant concentrations/doses of newer general anaesthetics (i.e. isoflurane, sevoflurane, propofol) may have weakened the dose-related EEG changes previously reported. Despite these findings, the EEG power spectrum analysis may still provide valuable information during intraoperative monitoring in the individual subject. The information obtained from EEG power spectrum analysis may be further supplemented by newer EEG indices such as bispectral index and approximate entropy or other neurophysiological monitors including auditory evoked potentials or somatosensory evoked potentials.