Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study.

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl(+) K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 microM with a weekly increase of 0.1 microM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD(+) concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis.

Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

Evaluation of the effects of immunosuppressants on neuronal and glial cells in vitro by multinuclear magnetic resonance spectroscopy.

The use of the undecapeptide cyclosporine and the macrolide tacrolimus as immunosuppressants in transplantation medicine and for the therapy of immune diseases often provokes side effects, among the most important one is neurotoxicity. Changes in the cellular metabolism of glial cells (C6 rat glioma), neuronal cells (N1E-115 mouse neuroblastoma) and primary glia cells (isolated from rats) after addition of cyclosporine and tacrolimus were investigated using 1H-, 13C- and 31P-NMR spectroscopy in vitro. Cells were exposed to various concentrations of the drugs from 3 h to 42 days. The immunosuppressants (cyclosporine IC50 : 55 mumol/l; tacrolimus IC50 : 47 mumol/l) inhibited cell proliferation in a concentration- and time-dependent fashion. Multinuclear NMR studies of PCA extracts of drug-treated cells showed a significant deterioration in the energy status (a decreasing level of PCr : -46 +/- 11%; an increasing NDP/NTP ratio: +136 +/- 4% and an increasing level of Pi : +248 +/- 15%; mean +/- standard deviation). It also showed decreasing concentrations of major cell metabolites like NAA (-59 +/- 12%) in neuroblastoma cells and myo-inositol (-47 +/- 6%) in glia cells compared with untreated controls. Immunosuppressive treatment caused a large reduction of taurine (-36 +/- 12%) and glutamate (-68 +/- 10%) in all cell cultures, whereas intermediates of phospholipid biosynthesis (PE: +59 +/- 13%; PC: +127 +/- 27%;) and breakdown (GPE: +215 +/- 24%; GPC: +245 +/- 17%) increased. No significant differences were observed between the two immunosuppressants. The toxic effects of immunosuppressants on cell cultures are in line with MRI studies of brain oedema observed in patients under immunosuppressive treatment.

Prevention of acute allograft rejection in nonhuman primate lung transplant recipients: induction with chimeric anti-interleukin-2 receptor monoclonal antibody improves the tolerability and potentiates the immunosuppressive activity of a regimen using low doses of both microemulsion cyclosporine and 40-O-(2-hydroxyethyl)-rapamycin.

BACKGROUND: In previous studies of cynomolgus monkey lung allograft recipients, we demonstrated significant immunosuppressive efficacy but reduced tolerability after combined treatment with high doses of microemulsion cyclosporine (CsA) and SDZ RAD (40-O-(2-hydroxyethyl)-rapamycin). The current study was designed to compare efficacy and tolerability of a combination of low-dose CsA and high-dose SDZ RAD (CTL group) to triple therapy using the chimeric anti-interleukin-2 (IL-2) receptor (CD25) monoclonal antibody (mAb) basiliximab (anti-IL-2 receptor mAb) for induction therapy (basiliximab: 5 mg intravenously on days 0 and 4) plus low-dose CsA and low-dose SDZ RAD for maintenance immunosuppression (CD25 group). CsA and anti-IL-2 receptor mAb are drugs that reduce cytokine synthesis and block IL-2-mediated lymphocyte stimulation, respectively. SDZ RAD blocks lymphocyte stimulation by other cytokines (e.g., IL-15) that are not inhibited by anti-IL-2 receptor mAb. METHODS: Twelve unilateral lung transplants were performed. Recipients were observed for 49 days by daily weight assessment, hemograms, blood chemistries, radiographs, and lung biopsies. Monkeys were euthanized before day 49 in the event of excessive weight loss (>25%) or organ failure. Target CsA trough levels were 100-200 ng/ml. Target SDZ RAD trough levels in the CTL group (no mAb) were 20-40 ng/ml, and 10-20 ng/ml in the CD25 group. RESULTS: None of the monkeys in the CD25 group needed to be euthanized early due to signs of drug toxicity. In contrast, four monkeys in the CTL group were sacrificed on days 28-35 as a result of excessive weight loss (n=3) and renal functional impairment (n=1). Three recipients in the CD25 group were euthanized on days 36, 38, and 46 as a result of persistent high fever associated with severe rejection. The median animal survival in the CTL group was 32 vs. 46 days in the CD25 group (P<0.04). The only two long-term survivors in the CTL group showed moderate rejection at day 49. The median rejection scores at day 14 (A0) and day 28 (A2) were identical in the two groups, despite the fact that the mean SDZ RAD trough level was significantly lower in the CD25 group (CTL: 38+/-3 ng/ml, CD25: 18+/-2 ng/ml, P<0.0001). After basiliximab levels fell below the minimum therapeutic level (1 mg/ml) on day 28, the median rejection score at day 49 increased to A4 in the CD25 group. CONCLUSION: This is the first study to combine an anti-IL-2 receptor mAb with a drug from the rapamycin class plus CsA. Our study shows that induction therapy with basiliximab enabled SDZ RAD blood levels to be significantly reduced, which led to improved tolerability without the penalty of increased rejection.

Combined immunosuppression with cyclosporine (neoral) and SDZ RAD in non-human primate lung transplantation: systematic pharmacokinetic-based trials to improve efficacy and tolerability.

BACKGROUND: We studied the efficacy and tolerability of combined immunosuppressive therapy with cyclosporine A microemulsion (Neoral) plus the macrolide SDZ RAD 40-0 (2-hydroxyethyl) rapamycin (RAD) in a stringent cynomolgus monkey lung graft model in comparison with cyclosporine or SDZ RAD monotherapy. METHODS: Thirty-nine cynomolgus monkeys received mixed lymphocyte reaction (MLR) mismatched unilateral lung transplants. Immunosuppressants were administered orally as single daily doses. The observation period was 28 days and follow-up included serial trough blood drug concentrations measured by high performance liquid chromatography/mass spectrometry, blood analyses, chest radiographs, open lung biopsies, as well as tissue drug concentrations and graft histology at necropsy. RESULTS: Graft biopsies in monkeys treated with vehicle (n=4), Neoral (day 1-7: 150 mg/kg/day; day 8-28: 100 mg/kg/day; n=6; mean +/- SE trough level (MTL): 292+/-17 ng/ml) or SDZ RAD monotherapy (1.5 mg/kg/day; n=6; MTL: 15+/-1 ng/ml) showed severe rejection. Coadministration in two transplant monkeys of Neoral (150/100 mg/kg/day) and SDZ RAD (1.5 mg/kg/day) caused their early death. In both animals, SDZ RAD blood levels were more than 5-fold higher than under monotherapy (MTL: 82+/-18 ng/ml). Simultaneous administration (n=6) of Neoral (150/100 mg/kg/day; MTL: 217+/-16 ng/ml) and SDZ RAD (0.3 mg/kg/day; MTL: 24+/-2 ng/ml) improved graft outcome (mild rejection). Side effects included renal failure (n=2) and seizures (n=1). Three monkeys survived to day 28. In this group the MTL for cyclosporin was 143+/-13 and for RAD 38+/-3. Staggered treatment completely prevented rejection in four of six grafts. However, five of six monkeys had moderate to severe diarrhea. In a concentration-controlled trial of simultaneously administered Neoral and SDZ RAD in transplant monkeys (target SDZ RAD MTL: 20-40 ng/ml; cyclosporine MTL: 100-200 ng/ml) all six monkeys survived with improved drug tolerability and an average biopsy score of mild rejection. CONCLUSION: Combination of orally administered SDZ RAD and Neoral showed excellent immunosuppressive efficacy in a stringent lung transplant model. The drug interaction and the narrow therapeutic index of this drug combination required careful dose adjustments to optimize tolerability and efficacy.

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain.

Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg(-1) d(-1)), III. sirolimus (3 mg kg(-1) d(-1)), IV. RAD (3 mg kg(-1) d(-1)), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78+/-6% of control; GABA: 67+/-12%; NAD(+): 76+/-3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+/-3% of control, RAD: 69+/-2%; P<0.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+/-4%; GABA: 59+/-8%; NAD(+): 45+/-5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD(+), not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high-energy metabolism and tissue distribution in the rat.

The novel immunosuppressant SDZ-RAD protects rat brain slices from cyclosporine-induced reduction of high-energy phosphates.

1. SDZ-RAD, 40-O-(2-hydroxyethyl)-rapamycin, is a novel macrolide immunosuppressant. Because of its synergistic interaction, SDZ-RAD is under clinical investigation as immunosuppressant in combination with cyclosporine after organ transplantation. Neurotoxicity is a critical side-effect of cyclosporine. 2. We studied the effect of SDZ-RAD and its combination with cyclosporine on high-energy phosphates, phosphocreatine (PCr) and nucleoside triphosphates (NTP), in brain slices using 31P-magnetic resonance spectroscopy (MRS). 3. Cyclosporine significantly reduced high-energy phosphates after 2 h in a dose-dependent manner (100 micrograms l-1: 93 +/- 3% of control (NTP), 91 +/- 3% (PCr); 500 micrograms l-1: 84 +/- 2% (NTP), 73 +/- 2 (PCr); 5000 micrograms l-1: 68 +/- 3% (NTP), 55 +/- 5% (PCr); n = 6; P < 0.02). 4. In contrast, after perfusion for 2 h, SDZ-RAD (500 micrograms l-1 and 5000 micrograms l-1) significantly increased high-energy phosphate concentrations in the brain slices (P < 0.02). Even at the lowest concentration, SDZ-RAD protected brain energy metabolism against cyclosporine toxicity: 100 micrograms l-1 SDZ-RAD + 5000 micrograms l-1 cyclosporine: 86 +/- 3% (NTP), 83 +/- 7% (PCr), n = 3, P < 0.03 compared to cyclosporine alone. 5. As evaluated using an algorithm based on Loewe isobolograms, the effects of SDZ-RAD/cyclosporine combinations on brain energy reduction were antagonistic. Both drugs were found in mitochondria using h.p.l.c-MS analysis. 6. We conclude that cyclosporine inhibits mitochondrial high-energy phosphate metabolism, which can be antagonized by SDZ-RAD.

Metabolite concentrations in human term placentae and their changes due to delayed collection after delivery.

The present study was designed to quantify the major cellular metabolites in human placentae and their changes due to the confounding effect of time and subsequent hypoxia during sample collection using magnetic resonance spectroscopy ((1)H-and (31)P-MRS). The absolute placental concentrations of lactate, glucose, major amino acids and cellular volume/osmo-regulators, glutathione, high-energy phosphates, fatty acids, phospholipids, triglycerols, and cholesterol are reported. There were no spatial differences in metabolism or protein expression throughout the placenta. The most significant temporal changes, due to the collection time (from 1 to 25 min after delivery), were increased concentrations of lactate (r=0.996, statistically significant P< 0.01 after 11 min) and decreased concentration of glucose and ATP (r=-0.963 and -0.97, respectively, P< 0.01 after 11 min). The placental samples from the later collection groups (16-24 min) had also significantly lower level of NAD(+) (r=-0.95, P< 0.01). Only the latest collection group (21-24 min) had increased lipid peroxidation and changes in lipid metabolites (P< 0.01). We conclude that the optimal window for collecting placental tissue without incurring metabolic artifacts due to hypoxic conditions is within 9 min of placental delivery.

[Genetic control of purine biosynthesis in Pichia methanolica yeast. The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase]. / Geneticheskii kontrol' biosinteza purinov u drozhzhei Pichi methanolica. Gen ADE5 (PUR4), kontroliruiushchii 5'-fosforibozilformilglitsinamidsintetazu.

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for the P. methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P. methanolica by the ade6 mutation complementation in the recipient S. cerevisiae strain.

[Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis]. / Izoliatsiia i pervichnaia kharakteristika mutantov po biogenezu peroksisom u metilitrofnykh drozhzhei Hansenula polymorpha.

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.

[Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources]. / Toksichnost' merkaptoétanola dlia mutantnykh linii drozhzhei Pichia methanolica, rastushchikh na razlichnykh istochnikakh ugleroda.

Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.

Synergistic activity of histone deacetylase and proteasome inhibition against pancreatic and hepatocellular cancer cell lines.

AIM: To determine the phenotypic effects of belinostat (bel) and bortezomib (bor) against pancreatic cancer (PC) and hepatocellular cancer (HCC) cell lines. MATERIALS AND METHODS: Antiproliferative effects were assessed using a sulforhodamine B assay. Synergy was evaluated using the Chou and Talalay method. Apoptosis was measured by caspase-3/-7 activity and PARP cleavage. Downstream effector proteins were detected via immunoblotting. Quantitative nuclear magnetic resonance (NMR)-based metabolomics analysis was performed. RESULTS: There were single-agent antiproliferative effects against PC and HCC cell lines; the combination of bel and bor (bel+bor) had a synergistic effect. There was up to a 45-fold induction of apoptosis over the control. Post-treatment cell death was associated with p21 up-regulation, more pronounced with treatment with bel+bor. Treatment with bel+bor enhanced hyperacetylation of histone H3 over single-agent bel. A metabolic signature was established for treatments with bor and bel+bor. CONCLUSION: The combination of bel+bor displayed significant antiproliferative activity against PC and HCC cell lines, with exhibiting synergistic antiproliferative and proapoptotic patterns even at suboptimal single-agent doses.

Controlled aggregation of superparamagnetic iron oxide nanoparticles for the development of molecular magnetic resonance imaging probes.

A method for synthesizing superparamagnetic iron oxide (SPIO) multi-nanoparticle aggregates as molecular magnetic resonance imaging (MRI) contrast agents is described. The approach utilizes organic acid/base interactions in the colloid to induce highly controllable nanoparticle aggregation. Monodisperse aggregates with diameters as large as 100 nm are synthesized by manipulating the interfacial surface chemistry of the SPIO nanoparticles in tetrahydrofuran solvent. Subsequent phospholipid micelle encapsulation yields micellar multi-SPIO (mmSPIO) aggregates with enhanced T(2) relaxivity (368.0 s(-1) mmol(-1) Fe) as compared to micellar single particle SPIO (302.0 s(-1) mmol(-1) Fe). mmSPIO conjugated to anti-CA125 monoclonal antibodies were incubated with ovarian carcinoma cell lines to demonstrate targeted in vitro molecular MRI, resulting in a 66% shortening in T(2) time for CA125 positive NIH:OVCAR-3 cells and a less than 3% change in T(2) time for CA125 negative SK-OV-3 cells. The controllable aggregation of mmSPIO shows potential for the development of molecular MRI contrast agents with optimal sizes for specific diagnostic imaging applications.

Assessment of the mechanism of astrocyte swelling induced by the macrolide immunosuppressant sirolimus using multinuclear nuclear magnetic resonance spectroscopy.

The toxic effect of the macrolide immunosuppressant sirolimus on cell metabolism of primary astrocytes was studied by multinuclear NMR spectroscopy of viable cells and perchloric acid (PCA) extracts and compared to the effects of the immunosuppressant cyclosporine. The addition of 5 mg/L sirolimus (5.5 mumol/L) induced swelling of primary astrocytes to 110% of the original volume. Alteration in astrocyte volume in the presence of sirolimus was accompanied by reduction of the following important cell osmolytes and amino acid metabolites: myo-inositol, -58 +/- 12% (mean +/- standard deviation, n = 5); taurine, -44 +/- 5%; glutamine, -13 +/- 2%; compared with control. Sirolimus altered glucose metabolism and partially inhibited the tricarboxylic acid (TCA) cycle: sigma TCA/sigma glycolyse = 1.36 +/- 0.09 (control, n = 3), 0.96 +/- 0.08 (with sirolimus). The increased concentration of phosphodiesters by sirolimus addition (glycerophosphoethanolamine, 52 +/- 18%; glycerophosphocholine, 61 +/- 14%; compared with control, n = 5) indicated disorders in phospholipid metabolism of cellular membranes. Addition of sirolimus led to a decline of the energy state in astrocytes: the concentration of phosphocreatine (PCr) decreased to 75% of control value within 60 min of perfusion with sirolimus and the nucleotide triphosphate (NTP) concentration to 85% within 90 min (n = 3). The effect of sirolimus on the cell metabolism of astrocytes equals that of the immunosuppressants cyclosporine and tacrolimus, the neurotoxicity of which is well-established in clinical studies. The results of this in vitro study indicate that sirolimus possesses neurotoxic potential as well.

Tissue distribution and clinical monitoring of the novel macrolide immunosuppressant SDZ-RAD and its metabolites in monkey lung transplant recipients: interaction with cyclosporine.

We report the tissue distribution and clinical monitoring of the novel macrolide immunosuppressant SDZ-RAD ¿40-O-(2-hydroxyethyl)-rapamycin and its metabolites in monkey lung transplant recipients as well as its interaction with cyclosporine as the Neoral formulation. After left unilateral lung transplantation, cynomolgus monkeys received by oral administration either 1) 1.5 mg/kg/day SDZ-RAD (n = 4); 2) 100 mg/kg/day cyclosporine (n = 4); 3) 0.3 mg/kg/day SDZ-RAD + 100 mg/kg/day cyclosporine (n = 6); 4) 1.5 mg/kg/day SDZ-RAD + 50 mg/kg/day cyclosporine (n = 5); or 5) SDZ-RAD and cyclosporine doses adjusted according to trough blood concentration measurements (n = 6). At the end of the observation period (usually 29 days after transplantation), and 24 h after the last doses, tissue samples were collected and analyzed with HPLC/mass spectrometry. Gall bladder, pancreas, the transplant lung, cerebellum, kidneys, and spleen had the highest SDZ-RAD concentrations. Coadministration of cyclosporine increased SDZ-RAD concentrations in most tissues as well as tissue-to-blood distribution coefficients. In contrast, SDZ-RAD had only a small effect on cyclosporine blood and tissue concentrations. Rejection in lung grafts in monkeys treated with either of the cyclosporine/SDZ-RAD combinations was significantly less than in the monotherapy groups (P <.002). Histological rejection scores were inversely correlated with SDZ-RAD concentrations in blood (r = -0. 68; P <.001; n = 24), lymph nodes (P = -0.58; P <.003; n = 24), thymus (r = -0.63; P <.001; n = 23) and transplant lung tissue (r = -0.58; P <.003; n = 24). We conclude that, in addition to the synergistic pharmacodynamic interaction, a pharmacokinetic interaction resulting in higher SDZ-RAD tissue concentrations contributed to the significantly better immunosuppressive efficacy when both drugs were combined compared with monotherapy.

Greater vascularity, lowered HIF-1/DNA binding, and elevated GSH as markers of adaptation to in vivo chronic hypoxia.

Vascularity is increased in placentas from high- compared with low-altitude pregnancies. An angiogenic response to hypoxia may protect an organ from further hypoxic insult by increasing blood flow and oxygen delivery to the tissue. We hypothesized that increased placental vascularity is sufficient to adapt to high altitude. Therefore, indexes of hypoxic stress would not be present in placentas from successful high-altitude pregnancies. Full-thickness placental biopsies were 1) collected and frozen in liquid nitrogen within 5 min of placental delivery and 2) fixed in formalin for stereologic analyses at high (3,100 m, n = 10) and low (1,600 m, n = 10) altitude. Hypoxia-inducible transcription factor (HIF-1) activity was analyzed by ELISA. Western blot analyses were used to evaluate HIF-1alpha, HIF-1beta, HIF-2alpha, von Hippel-Lindau protein, VEGF, Flt-1, enolase, and GAPDH. Magnetic resonance spectroscopy was used to evaluate endogenous metabolism. The ratio of placental capillary surface density to villous surface density was 70% greater at high compared with low altitude. HIF-1 activity and HIF-1-associated proteins were unchanged in placentas from high- vs. low-altitude pregnancies. Placental expression of HIF-1-mediated proteins VEGF, Flt-1, enolase, and GAPDH were unchanged at high vs. low altitude. Succinate, GSH, phosphomonoesters, and ADP were elevated in placenta from high compared with low altitude. Placentas from uncomplicated high-altitude pregnancies have greater vascularity and no indication of significant hypoxic stress at term compared with placentas from low altitude.

Evaluation of individual and combined neurotoxicity of the immunosuppressants cyclosporine and sirolimus by in vitro multinuclear NMR spectroscopy.

Neurotoxicity, a crucial side effect of immunosuppressive therapy with cyclosporine, also has been demonstrated in vitro for sirolimus, a novel macrolide immunosuppressant, which is under clinical investigation in combination with cyclosporine. NMR spectroscopy was used to study the separate and combined effects of cyclosporine and sirolimus on cerebral metabolism, both in brain cells and in perfused rat brain slices. The high-energy phosphate metabolism was already affected significantly at cyclosporine concentrations as low as 100 micrograms/liter: phosphocreatine was reduced by 10 +/- 2% [half-maximal inhibition concentration (IC50) = 1850 +/- 600 micrograms/liter], and nucleoside triphosphate was reduced by 11 +/- 5% (IC50 = 1110 +/- 420 micrograms/liter; n = 4, P <.05). At 500 micrograms/liter cyclosporine, N-acetylaspartate and glutamate were decreased by 13 +/- 7% (IC50 = 1100 +/- 330 micrograms/liter) and 22 +/- 9% (IC50 = 360 +/- 220 micrograms/liter; n = 4, P <.05), respectively. As evaluated using an algorithm based on Loewe isobolograms, combination of cyclosporine and sirolimus resulted in a synergetic reduction of high-energy phosphate metabolites. Addition of sirolimus to the perfusion medium increased brain slice concentrations of cyclosporine. It is concluded that cyclosporine significantly reduced high-energy phosphate metabolism in brain tissue at in vivo relevant concentrations. Combination with sirolimus resulted in synergism, which, in part, is explained by a greater distribution of cyclosporine into the brain tissue in the presence of sirolimus.

Automated, fast and sensitive quantification of drugs in blood by liquid chromatography-mass spectrometry with on-line extraction: immunosuppressants.

We developed a universal LC-mass spectrometry assay with automated online extraction (LC/LC-MS) to quantify the immunosuppressants cyclosporine, tacrolimus, sirolimus and SDZ-RAD alone or in combination in whole blood. After protein precipitation, samples were loaded on a C18 extraction column, were washed and, after activation of the column-switching valve, were backflushed onto the C8 analytical column. [M+Na]+ ions were detected in the selected ion mode. For tacrolimus, sirolimus and SDZ-RAD, the assay was linear from 0.25 to 100 microg/l and for cyclosporine from 7.5 to 1250 microg/l (all r2>0.99). Analytical recovery was >85% and, in general, inter-day, intra-day variability for precision and accuracy were <10%.

Efficacies of sirolimus (rapamycin) and cyclosporine in allograft vascular disease in non-human primates: trough levels of sirolimus correlate with inhibition of progression of arterial intimal thickening.

We investigated the efficacies of sirolimus (rapamycin) and cyclosporine for inhibition of graft vascular disease (GVD) in cynomolgus monkey recipients of aortic allografts. Increases in arterial intimal thickening in the midgraft (six consecutive cross-sections) after transplantation were quantified by serial intravascular ultrasound (IVUS) from day 21 to day 105. These data enabled correlations between changes in intimal indexes [II = (intimal area/vessel area) x 100] and trough levels of sirolimus and cyclosporine to be determined. Eighteen recipients received no immunosuppression for 6 weeks to allow alloimmune injury to occur. On day 45, monkeys were treated daily with sirolimus (n = 6) or cyclosporine (n = 6); six monkeys remained untreated. II increased significantly from day 63 to day 105 in untreated monkeys and monkeys treated with cyclosporine, whereas monkeys treated with sirolimus did not have a significant increase in II (P = 0.008, P = 0.006, P = NS; paired t-test). The change in II from days 63 to 105 was significantly greater in untreated monkeys compared to sirolimus-treated monkeys (P = 0.13; one-way ANOVA, P = 0.012 Tukey's post hoc test); other post hoc pairwise comparisons were not significant. Mean sirolimus and cyclosporine levels +/- SEM were 43 +/- 7 ng/ml and 562 +/- 20 ng/ml, respectively. Sirolimus trough levels, but not cyclosporine levels, correlated inversely with changes in II from day 42 to 105 (r2 = 0.73, P = 0.03). This non-human primate study shows that inhibition of intimal thickening by sirolimus depends on trough levels and provides the rationale for clinical trials of sirolimus for the control of GVD in organ transplant recipients.