Effects of combined chelation treatment with pyridoxal isonicotinoyl hydrazone analogs and deferoxamine in hypertransfused rats and in iron-loaded rat heart cells

Although iron chelation therapy with deferoxamine (DFO) results in improved life expectancy of patients with thalassemia, compliance with parenteral DFO treatment is unsatisfactory, underlining the need for alternative drugs and innovative ways of drug administration. We examined the chelating potential of pyridoxal isonicotinoyl hydrazone (PIH) analogs, alone or in combination with DFO, using hypertransfused rats with labeled hepatocellular iron stores and cultured iron-loaded rat heart cells. Our in vivo studies using 2 representative PIH analogs, 108-o and 109-o, have shown that PIH analogs given orally are 2.6 to 2.8 times more effective in mobilizing hepatocellular iron in rats, on a weight-per-weight basis, than parenteral DFO administered intraperitoneally. The combined effect of DFO and 108-o on hepatocellular iron excretion was additive, and response at a dose range of 25 to 200 mg/kg was linear. In vitro studies in heart cells showed that DFO was more effective in heart cell iron mobilization than all PIH analogs studied. Response to joint chelation with DFO and PIH analogs was similar to an increase in the equivalent molar dose of DFO alone, rather than the sum of the separate effects of the PIH analog and DFO. This finding was most likely the result of iron transfer from PIH analogs to DFO, a conclusion supported directly by iron-shuttle experiments using fluorescent DFO. These findings provide a rationale for the combined, simultaneous use of iron-chelating drugs and may have useful, practical implications for designing novel strategies of iron chelation therapy.     

Expression of Eya1 and Six1 is decreased in distal airways of rats with experimental pulmonary hypoplasia

Background/Purpose

Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. Eyes absent 1 (Eya1) and sine oculis homebox 1 (Six1) have been identified as essential components of the gene network that regulates foetal lung development. Eya1 and Six1 are expressed in distal epithelial tips of branching airways as well as in surrounding mesenchymal cells, highlighting their important role during branching morphogenesis. Lungs of Eya1^−/− and Six1^−/− knockouts display PH with reduced epithelial branching, appearing to be arrested in the pseudoglandular stage. We hypothesized that Eya1 and Six1 expression is decreased in branching airways of nitrofen-induced PH.

Methods

Time-mated rats received either nitrofen or vehicle on E9.5. Foetal lungs were dissected on E15.5 and divided into control and nitrofen groups, whereas lungs harvested on E18.5 were divided into controls, PH without CDH [PH(−)], and PH with CDH [PH(+)]. Pulmonary gene expression levels of Eya1 and Six1 were analyzed by quantitative real-time PCR. Immunofluorescence staining was performed to investigate Eya1 and Six1 protein expression and localization by confocal laser scanning microscopy (CLSM).

Results

Relative mRNA expression of Eya1 and Six1 was significantly decreased in PH(−) and PH(+) on E18.5 compared to controls. CLSM confirmed markedly diminished immunofluorescence of Eya1 and Six1 in distal airway epithelium as well as in surrounding mesenchymal cells of nitrofen-induced PH on E18.5 compared to controls.

Conclusions

Downregulation of Eya1 and Six1 gene expression in nitrofen-induced PH suggests that decreased Eya1 and Six1 expression during the late pseudoglandular stage may interfere with epithelial branching and distal airway maturation, thus resulting in PH.     

Prenatal retinoic acid increases lipofibroblast expression in hypoplastic rat lungs with experimental congenital diaphragmatic hernia

Background/purpose

Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH.

Methods

Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control + Placebo, Control + ATRA, Nitrofen + Placebo, and Nitrofen + ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining.

Results

Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen + ATRA compared to Nitrofen + Placebo (0.31 ± 0.02 vs. 0.08 ± 0.01; P < 0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen + ATRA compared to Nitrofen + Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen + ATRA compared to Nitrofen + Placebo.

Conclusions

Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development.     

Downregulation of p300 gene expression in airway mesenchyme of nitrofen-induced hypoplastic lungs

Purpose

Congenital diaphragmatic hernia (CDH) is a relatively common developmental abnormality causing life-threatening respiratory distress at birth. The nitrofen model has been widely used to investigate the pathogenesis of hypoplastic lungs associated with CDH. Embryos lacking p300 and CBP genes are significantly smaller in lung formation. We hypothesized that pulmonary gene expression of p300 and CBP is downregulated during late gestation in the nitrofen-induced CDH model.

Methods

Time-pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 (n = 8 at each time point). Pulmonary gene expression of p300 and CBP was analyzed by quantitative real-time PCR. Immunohistochemistry was performed to investigate expression and localization of pulmonary p300 and CBP proteins.

Results

Relative mRNA expression levels of p300 were significantly decreased in nitrofen-induced hypoplastic lungs on D18 compared to controls (3.00 ± 0.20 vs. 3.76 ± 0.14; p = 0.0039), while CBP levels were not altered. p300 immunoreactivity was markedly diminished in surrounding mesenchymal compartments and nuclei of proximal and distal airway cells, while CBP expression was not altered.

Conclusion

Downregulation of p300 gene expression during the early canalicular stage may disrupt epithelial–mesenchymal signaling interactions, contributing to the development of hypoplastic lungs in the nitrofen-induced CDH model.     

Disruption of THY-1 signaling in alveolar lipofibroblasts in experimentally induced congenital diaphragmatic hernia

Purpose

Pulmonary hypoplasia (PH), characterized by alveolar immaturity, remains the main cause of neonatal mortality and long-term morbidity in infants with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) are critically important for normal alveolar development. Thymocyte antigen 1 (Thy-1) is a highly expressed cell-surface protein in this specific subset of lung fibroblasts, which plays a key role in fetal alveolarization by coordinating the differentiation and lipid homeostasis of alveolar LIFs. Thy-1 increases the lipid content of LIFs by upregulation of adipocyte differentiation-related protein (ADRP), a lipogenic molecular marker characterizing pulmonary LIFs. Thy-1 ^?/? mice further show impaired alveolar development with reduced proliferation of pulmonary LIFs, resulting in a PH-similar phenotype. We hypothesized that pulmonary Thy-1 signaling is disrupted in experimentally induced CDH, which may has an adverse effect on the lipid content of alveolar LIFs.

Methods

Timed-pregnant Sprague–Dawley rats were treated with either 100 mg nitrofen or vehicle on embryonic day 9.5 (E9.5). Fetuses were killed on E21.5, and lungs were divided into controls (n = 14) and CDH-associated PH (n = 14). Pulmonary gene expression levels of Thy-1 and ADRP were assessed by quantitative real-time PCR. ADRP immunohistochemistry and oil-red-O staining were used to localize alveolar LIF expression and lipid droplets. Immunofluorescence double staining for Thy-1 and oil-red-O was performed to evaluate Thy-1 expression and lipid content in alveolar LIFs.

Results

Radial alveolar count was significantly reduced in CDH-associated PH with significant downregulation of pulmonary Thy-1 and ADRP mRNA expression compared to controls. ADRP immunoreactivity and lipid droplets were markedly diminished in alveolar interstitial cells, which coincided with decreased alveolar LIF expression in CDH-associated PH compared to controls. Confocal laser scanning microscopy confirmed markedly decreased Thy-1 expression and lipid content in alveolar LIFs of CDH-associated PH compared to controls.

Conclusion

Our study provides strong evidence that disruption of pulmonary Thy-1 signaling results in reduced lipid droplets in alveolar LIFs and may thus contribute to PH in the nitrofen-induced CDH model. Treatment modalities aimed at increasing lipid content in alveolar LIFs may therefore have a therapeutic potential in attenuating CDH-associated PH.     

A MODEL BASED ASSESSMENT OF REDISTRIBUTION DEPENDENT ELIMINATION AND BIOAVAILABILITY OF RIFABUTIN

The autoinduction characteristic of rifabutin (RIF) following multiple oral dosing was investigated via pharmacokinetic modeling. A two-compartment model with first-order absorption was fit to plasma RIF data obtained from a study conducted in healthy normal volunteers following both a single and multiple oral doses. Parameter estimates showed an elimination rate constant (k₁₀) of about 0·12--0·14 h⁻¹ which was independent of the single or multiple-dosing condition. The lower-than-expected drug accumulation following multiple dosing seems to suggest that prolonged dosing perturbs the linear kinetic system. However, this analysis has shown no significant changes (p>0·05) in the rate constants describing RIF absorption, tissue distribution/redistribution, and elimination. The mean rate of drug redistribution from the tissue compartment (k₂₁; 0·04--0·06 h⁻¹) was twofold to threefold lower than k₁₀, and, with a large steady-state distribution volume (V_ss/F after a single dose, 1630 L), RIF elimination appears to be dependent on drug redistribution. This hypothesis was further supported by a significant correlation (p<0·01) BETWEEN RIF TISSUE REDISTRIBUTION (k₂₁) and terminal disposition phase rate (λ_z) constants. The redistribution dependent elimination of RIF also helps explain the stability of the terminal half-life under both single and multiple-dosing paradigms. Urinary excretion of RIF and its 25-O-deacetyl metabolite totalled less than 7% of the oral dose following single dosing, and decreased to about 4% after multiple dosing. For individual patients, the decrease in urinary recovery of the 25-O-deacetyl metabolite was directly proportional to the decrease in urinary RIF recovery. In addition, both estimates of the model intercepts (A and B) were lower following multiple dosing. Further analyses revealed a linear relationship between A and B intercepts, and also between the urinary RIF recovery and the B intercept. These relationships, in conjunction with the lack of significant increase in the rate of elimination, indicate that induction of presystemic extrahepatic metabolism and/or decrease in the extent of oral absorption may be the primary causes for the lower-than-expected systemic RIF plasma levels after multiple oral dosing.     

A highly selective pyridoxal-based chemosensor for the detection of Zn(ii) and application in live-cell imaging; X-ray crystallography of pyridoxal-TRIS Schiff-base Zn(ii) and Cu(ii) complexes

In a simple, one-step reaction, we have synthesized a pyridoxal-based chemosensor by reacting tris(hydroxymethyl)aminomethane (TRIS) together with pyridoxal hydrochloride to yield a Schiff-base ligand that is highly selective for the detection of Zn(ii) ion. Both the ligand and the Zn(ii) complex have been characterized by ¹H & ¹³C NMR, ESI-MS, CHN analyses, and X-ray crystallography. The optical properties of the synthesized ligand were investigated in an aqueous buffer solution and found to be highly selective and sensitive toward Zn(ii) ion through a fluorescence turn-on response. The competition studies reveal the response for zinc ion is unaffected by all alkali and alkaline earth metals; and suppressed by Cu(ii) ion. The ligand itself shows a weak fluorescence intensity (quantum yield, Φ = 0.04), and the addition of zinc ion enhanced the fluorescence intensity 12-fold (quantum yield, Φ = 0.48). The detection limit for zinc ion was 2.77 × 10⁻⁸ M, which is significantly lower than the WHO''s guideline (76.5 μM). Addition of EDTA to a solution containing the ligand–Zn(ii) complex quenched the fluorescence, indicating the reversibility of Zn(ii) binding. Stoichiometric studies indicated the formation of a 2 : 1 L₂Zn complex with a binding constant of 1.2 × 10⁹ M⁻² (±25%). The crystal structure of the zinc complex shows the same hydrated L₂Zn complex, with Zn(ii) ion binding with an octahedral coordination geometry. We also synthesized the copper(ii) complex of the ligand, and the crystal structure showed the formation of a 1 : 1 adduct, revealing 1-dimensional polymeric networks with octahedral coordinated Cu(ii). The ligand was employed as a sensor to detect zinc ion in HEK293 cell lines derived from human embryonic kidney cells grown in tissue culture which showed strong luminescence in the presence of Zn(ii). We believe that the outstanding turn-on response, sensitivity, selectivity, lower detection limit, and reversibility toward zinc ion will find further application in chemical and biological science.

The synthesis, characterization, X-ray crystallography, and live-cell imaging of pyridoxal-TRIS Schiff-base ligand which is selective as a luminescence sensor to detect Zn(ii) ion, and the corresponding Zn(ii) and Cu(ii) complexes are described.