Mitigation of the hematopoietic and gastrointestinal acute radiation syndrome by octadecenyl thiophosphate, a small molecule mimic of lysophosphatidic acid.

We have previously demonstrated that the small molecule octadecenyl thiophosphate (OTP), a synthetic mimic of the growth factor-like mediator lysophosphatidic acid (LPA), showed radioprotective activity in a mouse model of total-body irradiation (TBI) when given orally or intraperitoneally 30 min before exposure to 9 Gy γ radiation. In the current study, we evaluated the effects of OTP, delivered subcutaneously, for radioprotection or radiomitigation from -24 h before to up to +72 h postirradiation using a mouse TBI model with therapeutic doses at around 1 mg/kg. OTP was injected at 10 mg/kg without observable toxic side effects in mice, providing a comfortable safety margin. Treatment of C57BL/6 mice with a single dose of OTP over the time period from -12 h before to +26 h after a lethal dose of TBI reduced mortality by 50%. When administered at +48 h to +72 h postirradiation (LD50/30 to LD100/30), OTP reduced mortality by ≥34%. OTP administered at +24 h postirradiation significantly elevated peripheral white blood cell and platelet counts, increased crypt survival in the jejunum, enhanced intestinal glucose absorption and reduced endotoxin seepage into the blood. In the 6.4-8.6 Gy TBI range using LD50/10 as the end point, OTP yielded a dose modification factor of 1.2. The current data indicate that OTP is a potent radioprotector and radiomitigator ameliorating the mortality and tissue injury of acute hematopoietic as well as acute gastrointestinal radiation syndrome.

Combined mitigation of the gastrointestinal and hematopoietic acute radiation syndromes by an LPA2 receptor-specific nonlipid agonist.

Pharmacological mitigation of injuries caused by high-dose ionizing radiation is an unsolved medical problem. A specific nonlipid agonist of the type 2 G protein coupled receptor for lysophosphatidic acid (LPA2) 2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl]benzoic acid (DBIBB) when administered with a postirradiation delay of up to 72 hr reduced mortality of C57BL/6 mice but not LPA2 knockout mice. DBIBB mitigated the gastrointestinal radiation syndrome, increased intestinal crypt survival and enterocyte proliferation, and reduced apoptosis. DBIBB enhanced DNA repair by augmenting the resolution of γ-H2AX foci, increased clonogenic survival of irradiated IEC-6 cells, attenuated the radiation-induced death of human CD34(+) hematopoietic progenitors and enhanced the survival of the granulocyte/macrophage lineage. DBIBB also increased the survival of mice suffering from the hematopoietic acute radiation syndrome after total-body irradiation. DBIBB represents a drug candidate capable of mitigating acute radiation syndrome caused by high-dose γ-radiation to the hematopoietic and gastrointestinal system.

Novel quinic acid derivative KZ-41 prevents retinal endothelial cell apoptosis without inhibiting retinoblastoma cell death through p38 signaling.

PURPOSE: To determine whether a novel NF-κB inhibitor, KZ-41, can inhibit melphalan's actions on retinal endothelial cell (REC) inflammation and apoptosis, without eliminating the chemotherapeutic efficacy of melphalan on cell death of retinoblastoma cells (Y79). METHODS: RECs were cultured in M131 medium supplemented with growth factors and antibiotics. Once cells reached confluence, they were treated with or without 10 µM KZ-41, following treatment with 4 µg/mL melphalan. Cell proteins were extracted and analyzed for intracellular adhesion molecule 1 (ICAM-1) levels and Cell Death ELISA. RECs were also transfected with or without NF-κB siRNA or treated with SB202190 (p38 [mitogen activated protein kinase] MAPK inhibitor) before melphalan treatment to determine the involvement of NF-κB and p38 MAPK in REC apoptosis and ICAM-1 levels. We also cultured retinoblastoma cells (Y79) in RMPI-1640 medium supplemented with 20% fetal bovine serum and performed a Cell Death ELISA after melphalan + KZ-41 treatment to determine if the treatments altered melphalan's ability to promote cell death of Y79 cells. RESULTS: KZ-41 inhibited melphalan-stimulation of ICAM-1 levels and REC apoptosis, whereas KZ-41 did not alter melphalan's effects on Y79 cells. KZ-41's protective effects on REC were mediated through p38 MAPK activation. Although KZ-41 blocked both NF-κB- and p38 MAPK-dependent ICAM-1 stimulation; the p38 MAPK/ICAM-1 pathway appears to be the primary pathway involved in melphalan-induced REC apoptosis. CONCLUSIONS: KZ-41 protects REC against melphalan-induced upregulation of ICAM-1 and apoptosis through p38 MAPK-dependent pathways.

Insulin-like growth factor binding protein-3 inhibits monocyte adhesion to retinal endothelial cells in high glucose conditions.

PURPOSE: Insulin-like growth factor binding protein-3 (IGFBP-3) is cytoprotective in the retina. The goal of this study was to investigate whether IGFBP-3 inhibits monocyte-endothelial cell adhesion associated with hyperglycemia. METHODS: Human retinal vascular endothelial cells (RECs) were grown in normal (5 mM), medium (15 mM), or high glucose medium (25 mM) for 72 h. After 48 h, cells were transfected with endothelial-cell-specific, non-IGF binding IGFBP-3 plasmid DNA (IGFBP-3NB) at 1 µg/ml for 24 h. Cells were serum starved for 16 h and treated with tumor necrosis factor-alpha (TNF-α; 10 ng/ml) for 4 h. Cell proteins were extracted and analyzed for intercellular adhesion molecule-1 (ICAM-1) expression with enzyme-linked immunosorbent assay. Additional RECs were plated onto attachment factor-coated slides, grown to 90% confluence in high glucose medium, and transfected with IGFBP-3 NB plasmid DNA or ICAM-1 small interfering RNA before treatment with or without TNF-α (10 ng/ml) for 4 h. Slides were then mounted in a parallel-plate flow chamber and subjected to a continuous flow of U937 human monocytes (10(5)/ml) in culture medium at shear stresses of 2 dynes/cm(2), with continual exposure to TNF-α. RESULTS: In high ambient glucose, overexpression of IGFBP-3 in RECs significantly decreased ICAM-1 expression when compared to the TNF-α-treated samples, whereas TNF-α increased monocyte-endothelial cell adhesion. IGFBP-3 significantly decreased monocyte adhesion to RECs in the high glucose condition. RECs transfected with ICAM-1 siRNA also had a decreased number of monocytes attached compared with the scrambled siRNA control. CONCLUSIONS: Data suggest that IGFBP-3 reduces monocyte-endothelial cell adhesion through decreased ICAM-1 levels in a hyperglycemic environment. This is the first demonstration of the role of IGFBP-3 in inhibiting monocyte-endothelial cell adhesion.

Compound 49b prevents diabetes-induced apoptosis through increased IGFBP-3 levels.

PURPOSE: To determine whether Compound 49b, a novel PKA-activating drug, can prevent diabetic-like changes in the rat retina through increased insulin-like growth factor binding protein-3 (IGFBP-3) levels. METHODS: For the cell culture studies, we used both human retinal endothelial cells (REC) and retinal Müller cells in either 5 mM (normal) or 25 mM (high) glucose. Cells were treated with 50 nM Compound 49b alone of following treatment with protein kinase A (PKA) siRNA or IGFBP-3 siRNA. Western blotting and ELISA analyses were done to verify PKA and IGFBP-3 knockdown, as well as to measure apoptotic markers. For animal studies, we used streptozotocin-treated rats after 2 and 8 months of diabetes. Some rats were treated topically with 1 mM Compound 49b. Analyses were done for retinal thickness, cell numbers in the ganglion cell layer, pericyte ghosts, and numbers of degenerate capillaries, as well as electroretinogram and heart morphology. RESULTS: Compound 49b requires active PKA and IGFBP-3 to prevent apoptosis of REC. Compound 49b significantly reduced the numbers of degenerate capillaries and pericyte ghosts, while preventing the decreased retinal thickness and loss of cells in the ganglion cell layer. Compound 49b maintained a normal electroretinogram, with no changes in blood pressure, intraocular pressure, or heart morphological changes. CONCLUSIONS: Topical Compound 49b is able to prevent diabetic-like changes in the rat retina, without producing systemic changes. Compound 49b is able to prevent REC apoptosis through increasing IGFBP-3 levels, which are reduced in response to hyperglycemia.

Intra-ophthalmic artery chemotherapy triggers vascular toxicity through endothelial cell inflammation and leukostasis.

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity.

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth-factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4-tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues block ATX activity with K(i) values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed-mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose-dependent manner. The average terminal half-life for compound 22 is 10±5.4 h and it causes a long-lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The 4-substituted benzylphosphonic acids and 6-substituted naphthalen-2-ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX-LPA-LPAR axis both in vitro and in vivo.

Treating retinoblastoma in tissue culture and in a rat model with a novel isoquinoline derivative.

PURPOSE. To investigate the effectiveness of a novel isoquinoline derivative, EDL-155, in killing retinoblastoma in vitro and in vivo. METHODS. Dose-response curves were generated in which Y79 retinoblastoma cells tagged with luciferase (Y79-Luc) were treated with serial concentrations of EDL-155. Electron microscopy was used to evaluate the ultrastructural morphology of EDL-155-treated Y79 cells. To determine whether autophagy was induced in EDL-155-treated Y79-Luc cells, staining with acridine orange and LC-3 immunoblot analysis was performed. To evaluate the efficacy of EDL-155 in vivo, Y79-Luc retinoblastoma cells were injected into the vitreous cavity of newborn rats, followed by periocular injections of EDL-155 (20 mg/kg/day) or an equivalent dosage of saline. RESULTS. EDL-155 appeared to destroy the retinoblastoma cells in vitro with an EC(50) of 9.1 micriM. EDL-155-treated retinoblastoma cells displayed a lack of viable mitochondria and the presence of autophagosomes wrapped in the characteristic double membranes. Acridine orange staining of EDL-155-treated retinoblastoma cells demonstrated the accumulation of vacuoles, and the immunoblots displayed a shift in molecular weight of LC-3, indicative of incorporation into autophagosome vesicles. In the retinoblastoma animal model, four doses of EDL-155 were delivered over 4 days, which was sufficient to see a significant decrease (P = 0.01) in viable intraocular tumors. Seven of the 25 rats treated with EDL-155 had no detectable living tumor. No significant decrease in viable tumor was observed in control animals. CONCLUSIONS. EDL-155 appears to eliminate retinoblastoma cells by disrupting mitochondria and inducing autophagy. Local delivery of EDL-155 may be an effective therapy for some types of ocular cancers.

Macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations.

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms (single-nucleotide polymorphisms (SNPs)) (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3' end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.

Effect of goldenseal (Hydrastis canadensis) and kava kava (Piper methysticum) supplementation on digoxin pharmacokinetics in humans.

Phytochemical-mediated modulation of P-glycoprotein (P-gp) and other drug transporters may give rise to many herb-drug interactions. Serial plasma concentration-time profiles of the P-gp substrate, digoxin, were used to determine whether supplementation with goldenseal or kava kava modified P-gp activity in vivo. Twenty healthy volunteers were randomly assigned to receive a standardized goldenseal (3210 mg daily) or kava kava (1227 mg daily) supplement for 14 days, followed by a 30-day washout period. Subjects were also randomized to receive rifampin (600 mg daily, 7 days) and clarithromycin (1000 mg daily, 7 days) as positive controls for P-gp induction and inhibition, respectively. Digoxin (Lanoxin, 0.5 mg) was administered p.o. before and at the end of each supplementation and control period. Serial digoxin plasma concentrations were obtained over 24 h and analyzed by chemiluminescent immunoassay. Comparisons of area under the curve (AUC)((0-3)), AUC((0-24)), C(max,) CL/F, and elimination half-life were used to assess the effects of goldenseal, kava kava, rifampin, and clarithromycin on digoxin pharmacokinetics. Rifampin produced significant reductions (p < 0.01) in AUC((0-3)), AUC((0-24)), CL/F, t(1/2), and C(max), whereas clarithromycin increased these parameters significantly (p < 0.01). With the exception of goldenseal's effect on C(max) (14% increase), no statistically significant effects on digoxin pharmacokinetics were observed following supplementation with either goldenseal or kava kava. When compared with rifampin and clarithromycin, supplementation with these specific formulations of goldenseal or kava kava did not appear to affect digoxin pharmacokinetics, suggesting that these supplements are not potent modulators of P-gp in vivo.

P-glycoprotein modulates aldosterone plasma disposition and tissue uptake.

Aldosterone plays an important role in the pathophysiology of numerous cardiovascular disorders including heart failure and hypertension. Because aldosterone's actions are primarily mediated by its interaction with an intracellular mineralocorticoid receptor, factors affecting the cellular uptake and distribution of aldosterone may be important determinants of the hormone's activity. P-glycoprotein (P-gp) is an ATP-binding cassette efflux transporter encoded by the ABCB1 (also known as MDR1) gene in humans. P-gp is expressed on the luminal membrane of the capillary endothelial cells of tissues that are targets for aldosterone, including the brain and heart, where it attenuates cellular uptake of substrates. Recent in vitro evidence indicates P-gp transports aldosterone. Therefore, in this study we tested the hypothesis that P-gp modulates the uptake of aldosterone into the brain and heart by comparing the plasma and tissue distribution of [3H]-aldosterone in wild-type and P-gp-deficient [mdr1a/1b (-/-)] mice. Compared with wild-type mice, [3H]-aldosterone activity in the plasma, brain, and heart was significantly (P < 0.05) higher in the mdr1a/1b (-/-) animals. The area under the plasma or tissue concentration-time curves in the mdr1a/1b (-/-) mice was 2.0, 1.6, and 1.6-fold higher in the brain, heart, and plasma, respectively, than in wild-type controls. Our results demonstrate that P-gp plays an important role in aldosterone plasma disposition and modestly limits its uptake into the brain. The increased exposure of the brain and heart to aldosterone in the absence of P-gp suggests P-gp may play a key role in modulating aldosterone's effects in these organs.

Effect of milk thistle (Silybum marianum) and black cohosh (Cimicifuga racemosa) supplementation on digoxin pharmacokinetics in humans.

Phytochemical-mediated modulation of P-glycoprotein (P-gp) and other drug transporters may underlie many herb-drug interactions. Serial serum concentration-time profiles of the P-gp substrate, digoxin, were used to determine whether supplementation with milk thistle or black cohosh modified P-gp activity in vivo. Sixteen healthy volunteers were randomly assigned to receive a standardized milk thistle (900 mg daily) or black cohosh (40 mg daily) supplement for 14 days, followed by a 30-day washout period. Subjects were also randomized to receive rifampin (600 mg daily, 7 days) and clarithromycin (1000 mg daily, 7 days) as positive controls for P-gp induction and inhibition, respectively. Digoxin (Lanoxicaps, 0.4 mg) was administered orally before and at the end of each supplementation and control period. Serial digoxin serum concentrations were obtained over 24 h and analyzed by chemiluminescent immunoassay. Comparisons of area under the serum concentration time curves from 0 to 3 h (AUC(0-3)), AUC(0-24), Cmax, apparent oral clearance of digoxin (CL/F), and elimination half-life were used to assess the effects of milk thistle, black cohosh, rifampin, and clarithromycin on digoxin pharmacokinetics. Rifampin produced significant reductions (p < 0.01) in AUC(0-3), AUC(0-24), and Cmax, whereas clarithromycin increased these parameters significantly (p < 0.01). Significant changes in digoxin half-life and CL/F were also observed with clarithromycin. No statistically significant effects on digoxin pharmacokinetics were observed following supplementation with either milk thistle or black cohosh, although digoxin AUC(0-3) and AUC(0-24) approached significance (p = 0.06) following milk thistle administration. When compared with rifampin and clarithromycin, supplementation with these specific formulations of milk thistle or black cohosh did not appear to affect digoxin pharmacokinetics, suggesting that these supplements are not potent modulators of P-gp in vivo.

Effects of grapefruit juice on intestinal P-glycoprotein: evaluation using digoxin in humans.

STUDY OBJECTIVES: To determine the effects of grapefruit juice on the pharmacokinetics of oral digoxin, a P-glycoprotein substrate not metabolized by cytochrome P450 3A4, in healthy volunteers, and to assess whether polymorphic multidrug-resistance-1 (MDR1) expression contributes to interindividual variability in digoxin disposition. DESIGN: Prospective, open-label, unblinded, crossover study. SETTING: University research center. SUBJECTS: Seven healthy adult volunteers (four men, three women). INTERVENTION: Each subject received a single oral dose of digoxin 1.0 mg with water or grapefruit juice with at least a 2-week washout between treatments. During the grapefruit juice phase, juice was administered 3 times/day for 5 days before digoxin administration to maximize any effect on P-glycoprotein. MEASUREMENTS AND MAIN RESULTS: Digoxin pharmacokinetics in the presence and absence of grapefruit juice were compared. The MDR1 exon 26 C3435T genotype was determined by real-time polymerase chain reaction. Compared with water, grapefruit juice significantly reduced the digoxin absorption rate constant (3.0 +/- 2.4 to 1.2 +/- 1.0 hr(-1), p<0.05) and increased absorption lag time (0.32 +/- 0.12 to 0.53 +/- 0.34 hr, p<0.05). Grapefruit juice did not affect digoxin maximum concentration (Cmax), area under the curve (AUC), elimination half-life, or renal clearance. The effect of grapefruit juice on digoxin Cmax (-45% to +41%) and AUC(0-4) (-29% to +25%) varied substantially among subjects and was inversely correlated with the values during the water phase. Trends toward higher digoxin Cmax AUC, and absorption rate constant during the water phase were found in CC homozygotes compared with subjects carrying a T allele. CONCLUSION: Inhibition of intestinal P-glycoprotein does not appear to play an important role in drug interactions involving grapefruit juice. Interindividual variability in response to grapefruit juice may be related to the balance of intestinal drug uptake and efflux transport.